Note: Descriptions are shown in the official language in which they were submitted.
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
RECOMBINANT ElA DELETED ADENOVIRAL VECTORS
BACKGROUNDS ~'HE INVENTION
The adenovirus El region, which encodes the immediate early gene Ela and the
early gene Elb, plays a key role in the adenovirus life cycle and is
responsible for
interfering with the ability of the infected cell to regulate cell cycle
progression,
differentiation and programmed cell death (apoptosis). The Ela gene products
stimulate
infected cells, which are normally differentiated and quiescent, to progress
into the S-
phase of the cell cycle in order that viral DNA replication can occur. Normal
cells
usually respond to unscheduled stimulation of cell cycle progression (by Ela
or other
mitogenic factors) by activation of p53-dependent apoptosis. However, in the
context
of a viral infection the Ela products do not stimulate apoptosis because the
protein
products of the El region gene, Elb, are effective inhibitors of apoptosis.
Therefore,
during the early stage of viral infection the Ela and Elb gene products
cooperate to
bring about a quasi tumorigenic state in the infected cell which is required
for efficient
viral DNA replication and a productive infection cycle. The full scope of
activity of the
adenovirus Ela region is descri' . j in Bayley, S. and Mymryk, J. (1994) Intl.
J. of
Oncology 5:425-444. For a comprehensive review of the adenovirus biology see
Shenk, T. (1996), Fields Virology, 3r°Edition: p2111-2148.
The E1 genomic sequence is located at the extreme right end of the 3bkb
adenoviral genome. The primary Ela mRNA is differentially spliced during the
early
phase of replication into two prominent mRNAs, called 13S and 125, which give
rise
. to .2898 and 243 AA proteins, respectively (see Figure 9 of the attached
drawings).
The 2898 and 2438 proteins differ only by an internal sequence of 46 amino
acids that
is unique to the larger protein. Although the Ela primary transcript is
spliced into to 3
other mRNAs called 11S, lOS, and 9S, which encode for proteins of 2178, 1718
and
SSR respectively, these messages and not made efficiently in the early phase
of
infection and it is likely that the 2898 and 2438 proteins carry out the
primary functions
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
of Ela during the viral life cycle. TheElb gene transcript is also
differentially spliced
to yield mRNAs of 22S, l4.Ss, 14S, and 13S each of which contains two open
reading
frames. One of these open-reading frames is common to all of the messages and
encodes a protein of 1798. Depending on the mRNA, the other open reading
frames
give rise to proteins of 84R, 93R, 1558 and 4968. Of the Elb proteins the 1768
and
4968 proteins, which are also referred to as Elb 19K and Elb SSK respectively,
are
the most prominent and best characterized.
The Ela and Elb gene products play critical roles in the productive infection
cycle to prepare the infected cell for viral replication and to regulate viral
specific
processes. The Ela and Elb products do not contain intrinsic enzymatic
activities, but
are thought to carry out their functions by interacting with a number of
cellular proteins.
The Ela proteins associate with a wide range of cellular proteins including
p400, p300,
cAMP-responsive transcription binding protein (CBP), p130, p107, pRb, cyclin
A,
cdk2 and TATA-binding protein (TBP). Mapping studies have been used to compare
cellular protein binding domains and functional domains in the Ela proteins
(see Figure
10). Stimulation of cell cycle progression by Ela has been mapped to three
regions in
the common amino terminal domain of the 2438 and 2898 proteins (Howe et al.,
(1990) PNAS 87:5883-5887). These regions are commonly referred to as the amino-
terminal domain, conserved region 1 (CR1) and conserved region 2 (CR2). The
amino-terminal domain and CRl are required to bind a number of proteins
including
p300/CBP which are thought to be co-activators of gene transcription that have
been
implicated in regulation of cell proliferation and differentiation. The third
ElA region
required for cell cycle regulation by Ela is CR2 which is required for
association with
the know members of the pRb family of related cell cycle regulators including
pRb,
p 107 and p 130.
The pRb family members regulate the cell cycle by binding to a the E2F class
of
transcription factors that in turn regulate expression of genes that are
required for cell
cycle phase transitions. Binding of p300 and the pRb family members appears to
inactivate the ability of these proteins to suppress cell cycle progression
and this appears
to be the major mechanism by which Ela induces resting cells to progress into
the cell
cycle. A large body of evidence has accumulated to support this hypothesis.
For
_2_
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/Z1451
example, by associating with pRb the Ela proteins disrupt E2F-pRb complexes
which
frees E2F to stimulate gene expression that allows progression into the S-
phase of the
cell cycle. It is not precisely known how p300 regulates cell proliferation,
but p300 is
known to regulate expression of genes that are required to maintain a
differentiated
phenotype and that inhibition of p300 can block terminal differentiation. In
addition, it
is known that Ela-mutants that associate with p300, but are defective for
binding pRb,
are nevertheless able to stimulate phosphorylation of pRb which leads to
disruption of
pRb-E2F complexes and cell cycle progression (Wang et al., (1991) Mol. Cell.
Bio.
11, 4253-4265 ).
Induction of unscheduled DNA synthesis by Ela is a cellular stress that is
sensed by the infected host cell. The infected cell responds by inducing
apoptosis
which is normally mediated by p53. P53 is activated in response to a wide
variety of
cellular stresses including DNA damage, hypoxia and expression of mitogenic
oncogenes including Ela. Productive viral infection cannot occur if the
infected cell
commits programmed cell death and therefore the virus has evolved to inhibit
apoptosis,
at least early during the infectious cycle, by production of the Elb 19K and
55K
products( for a review of regulation of apoptosis by Elb see White, E., 1998,
Seminars in Virology 8, 505-513). The Eib 19K is considered to be the primary
inhibitor of Ela-induced apoptosis because Elb 19K alone blocks Ela induced
apoptosis more efficiently than Elb 55K alone. The Elb 19K inhibits apoptosis
by two
different mechanisms. First, E1B 19K associates with ;?~e proapoptotic Bcl-2
family
members Bax, NbkBik and BNIP3, and inhibit the ability of these proteins to
induce
apoptosis. Second, the E1B 19K protein can inhibit apoptosis by interacting
with
factors such as FADD and CED4 which normally act to activate caspases for
apoptosis.
The Elb 55K protein binds to inhibits the ability of p53 to act as an
activator of
transcription and can therefore augment the ability of the E1B 19K protein to
inactivate
p53-dependent apoptosis.
In addition to stimulating cell cycle progression and suppressing apoptotic
pathways the Ela and Elb proteins also play important viral specific roles
during the
replication cycle of the virus. The Ela protein s initiate the coordinated
expression of
the viral genome by stimulating expression of promoters for the Elb gene in
addition to
-3-
CA 02345211 2001-04-04
WO 00122136 PCT/US99/21451
the other early gene regions E2, E3 and E4. The 2898 Ela protein is primarily
responsible for the transactivation of the early adenoviral promoters and
mapping
studies have shown that the 46 amino acid unique domain of the 2898 protein
plays the
major role in activation of the early viral promoters. The Elb 55K protein
also carries
5 out critical viral specific roles during the productive infection cycle.
Adenovirus Elb
55K mutants are defective for late viral protein production and shutoff of
host cell
protein synthesis, consequently these mutant viruses are defective for growth
on a
number of human cell lines (Babiss and Ginsberg, (1984) J.Virol. 50:202-212;
Babiss
et al., 1985 Mol. Cell. Biol. 52552-2558; Pidler et al.,(1986)
Mol.Cell.Bio1.6:470-476;
10 Yew et al.,(1990) Virology 179:795-805). More recently it has been
suggested that the
E1b55K may alter cell cycle controls in infected cells (Goodrum and Ornelles,
1997 J.
Virol. 71, 548-561) and in addition E1b55K may influence viral DNA replication
(Ridgway et al, (1997) Virology 237:404-4I3).
Attempts have been made to exploit the ability of the Elb 55K protein to bind
15 p53 in the design of adenoviruses that selectively replicate in and kill
p53 deficient cells
by the elimination of Elb 55K function. See McCormick, United States Patent
No.
5,677,178 issued October 14, 1997. The vectors have been under commercial
development by ONYX Pharmaceuticals. A particular vector, ONYX-015 contains a
deletion in the p55 coding sequence. This prevents the expression of a E1b55K
20 product capable of binding p53 and is claimed to result in preferential
replication of the
virus in p53 deficient tumor cells. However, a number of reports, in ~~dditaon
to data
presented below, have brought the replication specificity of the Elb 55K
viruses for
p53 deficient tumor cells into question. Goodrum and Ornelles (1997) J. Virol.
71,
548-561 have suggested that the Elb 55K proteins relieve growth restrictions
imposed
25 on viral replication by the cell cycle and that the ability of E1b55K
mutant viruses to
replicate is not mediated by the status of p53. In addition, other studies
have suggested
that the interaction between p53 and Elb 55K may be required for efficient
vifal
replication (Ridgway et al. (1997) Virology 237:404-413). Data presented below
extend
these observation by demonstrating that E1b55K mutant viruses are defective
for viral
30 growth in normal cells, but also in a variety of tumor cell lines
regardless of the status
of the p53 gene. Together these observations suggest that Elb 55K mutant
viruses are
-4-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
growth defective in all cell types and do not target p53 defective tumor cells
for selective
cell killing. Therefore there is a need for replication competent adenovirus
vectors that
target tumor cells specifically.
Alternative to the idea of selectively replicating vector is the employment of
a
replication deficient adenoviral vector containing extensive elimination of El
function.
In particular, vectors containing elimination of E1, E2, E3 and partial E4
deletions have
been employed to delivery exogenous transgenes. Such vectors have been emphyed
to
deliver the p53 gene to target cells. It has been demonstrated that the
expression of an
exogenously administered wild type p53 in a p53 deficient (p53 mutated or p53
null)
tumor cell is capable of inducing p53 mediated apoptosis in the tumor cell.
Such viral
vectors for the delivery of p53 are currently under development Schering
Corporation
and Introgen Corporation. Again these vectors have demonstrated acceptable
toxicology profiles and therapeutic efficacy for human therapeutic
applications and are
in Phase II clinical trials in man for the treatment of p53 related
malignancies.
Replication deficient and selectively replicating vectors have, at least in
theory,
design drawbacks which are of concern to clinicians. Because the replication
deficient
vectors will not propagate uncontrollably in the patient, they have a more
theoretically
appealing safety profile. However, as effective tumor elimination requires the
infection
of the substantial majority of the tumor cells being infected, a substantial
molar excess
of vector is commonly used to insure therapeutic effectiveness. Selectively
replicating
vectors are viewed as being more of an issue from a safety perspective because
of their
ability to replicate and potentially mutate to form fully replication
competent vectors in
the patient. However, exploiting the natural ability to the virus to propagate
under
particular conditions enables these vectors to spread to surrounding tumor
cells. Si::ce
the vectors themselves are able to replicate, a lower initial dose of such
vectors is
required. This is favorable from an immunological perspective as well as for
economic
reasons in the manufacture of such.agents.
Therefore, there is a need in the art for a selectively replicating vector
that
addresses the perceived safety problems while providing the increased
therapeutic
index. The present invention solves these problems by providing a selectively
replicating adenovirus vector containing particular Ela modifications such
that the
-5-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
vector replicates preferentially in rapidly growing non-differentiated or
dedifferentiated
cells. The present invention also provides pharmaceutical formulations
comprising
such vectors. The present invention also provides methods of eliminating tumor
cells
from a population of normal cells by using such vectors.
~CT1VSMARY OF THE INVE_N_TION
The present invention provides a recombinant adenoviral vector containing
modifications to the Ela coding sequence so as to eliminate the ability of the
Ela gene
product to bind to the p300/CBP andlor related proteins, and the Rb protein
family,
while retaining the transactivating function of the ElA CR3 domain. The ElA
proteins
associate with p300/CBP, in addition to pRb, to down regulate differentiation
and cell
growth control pathways in the normally quiescent differentiated cells that
are infected
by wild type adenovirus. Binding of these cellular regulatory proteins by ElA
results
in a quasi-tumorigenic state in the infected cell that is required for
productive adenoviral
infection. This invention is intended to target rapidly dividing
dedifferentiated cells
(such as tumor cells), as opposed to growth arrested and/or differentiated
cells, by
attenuating ElA functions that deregulate cellular regulatory path nays. These
constructs
will preferentially replicate in cell types, such as tumor cells, in which
cellular growth
and differentiation pathways are disrupted. The invention further provides
pharmaceutical formulations and methods of use of same. The present invention
also
provides method of making such vectors and formulations.
BRIEF DESCRIPTION OF THE FIGURES
Figure i is a microscopic (100X) view of C33A cells were infected with the
indicated viral constructs at a concentration 1.8 x 109 particles per ml and
stained with
crystal violet.
Figure 2 represents experimental results similar to those presented in Figure
1
except that the viral concentration was 1.8 x 10$ particles per ml.
Figure 3 represents experimental results similar to those presented in Figure
1
except that the viral concentration was 1.8 x 10' particles per ml.
Figure 4 is a microscopic (100X) view of MRC9 cells were infected with the
indicated viral constructs at a concentration 1.8 x 109 particles per ml and
stained with
crystal violet.
-6-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
Figure 5 represents experimental results similar to those presented in Figure
4
except that the viral concentration was 1.8 x 10g particles per ml.
Figure 6 represents experimental results similar to those presented in Figure
4
except that the viral concentration was 1.8 x 108 particles per ml.
Figure 7 is a histogram which illustrates that the production of virus at 48
hours
post infection in various cell lines as indicated. Cells were infected at a
particle
concentration 1.8 x 109 particles per ml.
Figure 8 represents the experimental results similar to those presented in
Figure
7 except that the viral concentration was 1.8 x 10$ particles per ml.
Figure 9 is a map illustraring the differential splicing of the ElA message.
Figure 10 is a graphical representation of the various mutations in the Ela
coding sequence and the phenotypes associated with the respective mutants.
Figure 11 is a graphical presentation of data relating to the effective dose
required to achieve cell killing in normal lung endothelial cells (Panel A)
and the A549
lung endothelial tumor cells (Panel B). The vertical axes represent the
percent of
uninfected controls and the horizontal axes represent the viral dose in
particles per
milliliter. The experiments were performed by exposing a culture of each cells
o six
different concentrations of virus from 106 to 10'° viral particles. The
viruses used in
these experiments were the d1309 virus ( filled squares ), the d101/07/309
virus (filled
circles), the E1Bd155K virus ( open circles ), and control virus (open
squares)
containing a deletion of the Ela and E1B regions and referred to as the A/C/N
virus
described in Wills, et al. (1994) Human Gene Therapy. The cells were exposed
to the
virus for a period of one hour, the excess virus washed and the percent of
viable cells at
six days following infection was determined the MTS assay (Promega, Madison
WI) in
substantial accordance with the manufacturer's instructions. The horizontal
dotted line
represents the level at which SO°lo of the cells remained viable. The
intersection of the
curves generated by the data and the horizontal dotted line is a measure of
the EDT of
the virus.
Figure 12 is a microscopic (100X) view of MRC-9 transformed normal cells
(Panel A) and DLD-1 tumor cells (Panel B) infected with viral constructs at
varying
concentration (1.8 x 106 to 1.8 x 109 for DLD-1 cells and 1.8 x 10' to 1.8 x
10'° for
normal cells) of virus as indicated and stained with crystal violet. The first
column in
each panel is the d1309 virus. The second column in each panel is the
d101/07/309
_7_
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
virus. The third column in each panel is the d11101 virus. The fourth column
in each
panel is the d11107 virus. The fifth column in each panel is the E1bd155K
virus. The
sixth column in each panel is the control virus (A/C/N). :In Panel A the top
row
represents a dose of 1.8 x 10'° particles/ml, the second row represents
a dose of 1.8 x
109particles/ml, the third row represents a dose of 1.8 x 108 particles/ml,
the bottom
row represents a dose of 1.8 x 10' particles/ml. ). In Panel B the top row
represents a
dose of 1.8 x 109 particles/ml, the second row represents a dose of 1.8 x 10a
particles/ml, the third row represents a dose of 1.8 x 10' particles/ml, the
bottom row
represents a dose of 1.8 x 106 particles/ml.
Figure 13 is a graphical representation of data obtained from a nude mouse
tumor model study to evaluate the anti-tumor effects of intratumoral
administration of
viruses. On Day 1 of the study, nude mice were injected subcutaneously with
5x106
DLD-1 tumor cells and tumors allowed to form for approximately 12 days until
tumors
reach a volume of approximately 200 mm3. Mice were injected intratumorally
with a
dose of 2.5x109 viral particles in a volume of 100 microliters of each of the
viruses
rAd-Con (filled triangles); ElBd155K (open squares), d1309 (open circles) and
d101/07/309 (filled squares) and vPBS (filled circles) as a buffer control on
days
12,13,14,15, and 16. The tumor sizes were evaluated on days 17, 25 and 32 of
the
study. The vertical axis represents mean tumor volume ire cubic millimeters
and the
horizontal axis represents the day of the study.
Figure 14 is a graphical representation of data obtained in a nude mouse model
study to evaluate the anti-tumor effects of intravenous systemic
administration of
viruses. On Day 1 of the study, nude mice were injected subcutaneously with
5x106
PC-3 (prostate carcinoma) tumor cells and tumors allowed to form for
approximately
seven days until tumors reached a volume of approximately 50-60 mm'. Mice were
injected intratumorally with a dose of 1 x 10'° viral particles in a
volume of 200
microliters of each of the viruses ElBdI55K (open circles), d1309 (filled
squares) and
d101/07/309 (filled circles) and vPBS (open squares) as a buffer control on
days 7, 8,
24, 25, and 16 of the study. The tumor sizes were evaluated on days 7, 21, 29,
35 and
42 of the study. The vertical axis represents mean tumor volume in cubic
millimeters
and the horizontal axis represents the day of the study.
_g_
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
DETAjLED DESCRIPTION OF THE INVENTION
The present invention provides a method of ablating neoplastic cells in a
population of normal cells contaminated by said neoplastic cells by the
administration to
said population of cells a selectively replicating recombinant adenovirus
which contains
modifications to the Ela coding sequence so as to produce Ela gene products
which are
deficient in binding to one or more p300 protein family members and one or
more Rb
protein family member protein but expresses a modified 2898 protein retaining
the
transactivating function of the Ela CR3 domain.
The term "neoplastic cell" is a cell displaying an aberrant growth phenotype
characterized by independence of normal cellular growth controls. As
neoplastic cells
are not necessarily replicating at any given time point, the term neoplastic
cells comprise
cells which may be actively replicating or in a temporary non-replicative
resting state
(G1 or GO). Localized populations of neoplastic cells are referred to as
neoplasms.
Neoplasms may be malignant or benign. Malignant neoplasms are also refenred to
as
cancers. The term cancer is used interchangeably herein with the term tumor.
Neoplastic transfonmation refers the conversion of a normal cell into a
neoplastic cell,
often a tumor cell.
The term "selectively replicating" refers to a recombinant adenoviral vector
capable of preferential replication in a cell in one phenotypic state relative
to another
phenotypic state. Examples of different phenotypic states would include the
neoplastic
phenotype versus a normal phenotype in a given cell type. A vector which is
"selectively replicating" will replicate in and kill neoplastic cells at least
10 fold more
efficiently than the same virus in a non-transformed normal cell of the same
tissue cell
type at a dosage level which is sufficient to induce substantial cell death in
tumor cells
but not normal cells. The effect of dosage is an important consideration when
determining whether a given recombinant adenovirus results in preferential
neoplastic
cell killing because at a sufficiently high dose almost any adenovirus,
regardless of the
degree to which its genome has been modified, will be cytotoxic due simply to
the
effects of the presence of the viral proteins such as hexon which is known to
be
cytotoxic. Similarly, even though the scientific literature may refer to an El
mutant
adenovirus as "replication defective" (suggesting that the virus is absolutely
incapable
-9-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
of replication in the absence of a cell line capable of complementing the El
defect), such
viruses are more accurately described as "attenuated for replication" because
even
viruses containing a deletion of the entire El region will replicate to some
degree,
particularly in cycling or rapidly dividing cells. As Mulligan observed (1990,
Science
260:926-932):
Although the expression of the El region has been shown to affect the
expression of other viral gene products necessary for replication (citing
Horwitz, M. in Virology, B.N. Fields Ed. (Raven, New York, 1990)
Chapter 60)), the required of El gene expression for viral replication
10 does not appear to be absolute. The early characterization of E1-
deficient viruses demonstrate that at high multiplicities of infection, the
E1 region was dispensable for replication 1',citing Jones and Shenk
(1979) PNAS(LJSA) 76(8):3665-3669).
Consequently, the effect of viral dose cannot be ignored when determining
whether or
not a virus is truly a selectively replicating virus. A virus containing
extensive deletions
of E1 may appear to possess selectivity because they will replicate under
certain
conditions and certain dosages. For example, the X1312 adenovirus (Jones and
Shenk,
1979, PNAS(LTSA) 76(8):3665-3669) contains a deletion of nucleotides 448-1349
which results in elimination of all E1 a functions. The sequences encoding the
2898
20 protein begins at nucleotide 560 and ends at approximately nucleotide 1542.
The
nucleotide sequence encoding the entire 2898 protein including the CR3
transactivation
domain is completely absent in this construct. As Jones and Shenk observed
when
d1312 was used to infect HeLa cells (which are transformed cervical carcinoma
cells),
"no RNA species corresponding to early regions 2, 3 or 4 were detected"
clearly
25 indicating that the transactivation function of the Ela 2898 protein which
is responsible
for transactivating the E2, E3 and E4 genes was absent. However, at a higher
dose
(multiplicity of infection), replication of this virus was seen. Additionally,
it should be
remembered that HeLa cells are cycling neoplastically transformed cells which,
although genotypically pasitive for pRb105, possess the HPV 18 E6 and E7
functions
30 which degrade pRb105 thus rendering HeLa cells phenotypically negative for
p105Rb.
Furthermore, in order to determine if an E 1 mutant adenovirus is truly
selective, it is necessary to evaluate the ability of the virus to replicate
in tumor cells
as compared to normal cells of the same tissue cell type and not transformed
or
immortalized cell lines which are already cycling. This is clear based on our
35 understanding of the function of Ela adenoviral proteins. The primary
purpose of the
-10-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
E 1 a proteins is to force the normalcy quiescent cell into the cell cycle.
This first step
following viral infection is essential in for viral replication in quiescent
cells because
adenoviruses require factor present only in S-phase to achieve efficient viral
replication. However, if a cell has already entered the cell cycle (such as an
immortalized or transformed cell line), the effect of the E1 a deletions will
be, to some
degree, obscured. Thus, in order to truly assess neoplastic cell selectivity,
it is
necessary that the comparison be made with normal cells versus neoplastic
cells.
Additionally, wild-type vectors may also appear to replicate selectively in
tumor cells
relative to normal cells early after infection because the cell is already
cycling, however
this apparent selectivity diminishes over time once the virus has stimulated
the cell
cycle. Consequently, the time following infection when selectivity is measured
must
sufficient to avoid this initial replication lag in normal cells.
The commonly used parameter EDT (which is defined as the dose sufficient to
induce cell death in 50% of the cells) provides an appropriate basis of
comparison. The
EDT of a virus can readily be determined by typical dose escalation
experiments in
vitro. In order to ensure the most consistent basis of comparison, the EDso is
most
appropriately expressed relative to a viral control to minimize the effects of
variations
of infectivity between the normal and neoplastic cell types and any assay
variations.
Consequently the unitless ratio: EDSO(virus)/EDso(control) is used to express
the
relate : toxicity of the virus in the cell and will be referred to as the
"relative toxicity
index" or "RTL" For purposes of the present invention, the "selectivity index"
of a
given virus is expressed by the ratio: RTI(tumor cells)/RTI(normal cells).
Selectively
:~plicating vectors will have a selectivity index of at least 10 and
preferably much
greater. For example, the selectively replicating vector d101/07 was evaluated
for its
ability to replicate in and kill A549 cells (a lung tumor cell line) and in
normal lung
. endothelial cells with appropriate viral controls. The results of these
experiments are
presented in Figure 11 of the accompanying drawings. The following table
summarizes
the data presented:
-11-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
Table 1. Summary
of RTI and
Selectivity
Indices of
Viruses
Virus RTI RTI Selectivity
(normal cells)(tumor cells) Index
rAd-Con 1.0 1.0 1.0
d1309 450 900 2.0
d101/07/309 1.8 225 125
E1Bd155K 40 30 1.33
As can be seen from the data presented, the d1309 (wild-type) virus possesses
essentially no selectivity. The slight increase in replication shown in tumor
cells relative
to normal cells is expected as the cycling tumor cells will facilitate viral
replication.
Furthermore, the E1Bd155K virus possesses essentially no selectivity. However,
the
01/07/309 virus possesses a high selectivity index (125) demonstrating it
possesses
high selectivity for replication in tumor cells relative to normal cells of
the same
histological type.
In order to achieve a selectively replicating adenovirus, it is essential that
the
ability of the 2898 and 2438 proteins to bind to pRb105 and p300 both be
deleted
while maintaining taw functionality of the CR3 transactivation domain of 2898.
The
CR3 domain is present only in the Ela 2898 polypeptide as the 2438 protein
does not
possess the transactivational activity of the 2898 protein. Retaining the
transactivational activity of the 2898 protein is essential for efficient
viral replication.
While retaining the CR3 domain is essential it is not sufficient on its own.
For example
the d11010 virus described in Whyte, et al (1989) Cell 56:67-75, contains a
deletion of
amino acids 2-150 of the 2898 protein replacing them with a single glycine
residue.
This deletion eliminates the p300 and pRB105 binding domains of the Ela 2898
gene
product and retain the CR3 domain (although the effect of the deletion of
amino acid on
CR3 function may be questioned by virtue of the deletion of the Va1147 and
Pro150 in
view of the pm1120 and pm1122 point mutation viruses described in Jelsma, et
al.
(1988) Virology 163:494-502). However, the d11010 virus will not retain the
tranactivational functions of the CR3 domain because the deletions to the 2898
protein
-12-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
are too extensive. 3elsma, et al. confirm this by testing an Ela mutant
(d11119)
containing a lesser deletion of amino acids 4-138 of the 2898 protein (which
will not
disrupt the Va1147 and Pro150 residues arguably within the CR3 domain) for its
transactivational activity. The results presented in Table 2 of the reference
and the
comments of the authors indicate that this Ela molecule possesses "essentially
no ability
to transactivate." Consequently, large deletions in the Ela region upstream of
the CR3
domain will result in a mutant 2898 gene product which is so conformationaliy
disrupted that even though the CR3 domain is expressed it is not functional.
Similarly, it is essential that the p300 and p105 Rb binding be eliminated as
elimination of each on its own is insufficient to confer selectivity. For
example, a CPE
assay was performed to evaluate the ability of replication competent
adenoviruses
containing deletions in p300 binding {d11101 ) and pRb (dl l 107) were
compared with
the d101/07 adenovirus (which deletes both functions) to kill DLD-1 tumor
cells and a
MRC-9 "normal" cell line. Appropriate control viruses rAd CON (or ZZCB
containing
a deletion of the entire Ela and Elb regions), d1309 (a phenotypically wild-
type virus
containing deletions in the non-essential E3 region) and E1B55K (a recombinant
virus
containing a deletion in the 55K binding region of the Elb55k) were included.
The
results are presented in Figure 12 of the attached drawings. As can be seen
from the
data presented, at an equivalent dose (particle concentration) the d11101 and
d11107
viruses were capable of killing the normal (MRC9) cells essentially as
efficiently as
wild type virus. Similar results were obtained with respect to the tumor {DLD-
1) cells.
However, if one compares the pe~~'ormance of the d101/07 virus in the normal
cell line
and tumor cell line, the d101/07 virus killed tumor cells as efficiently as
the wild-type
virus but was toxic to the normal cells only at dose equivalent to the El
defective
control virus. Furthermore, the d101/07 virus was substantially less toxic to
the normal
cells when compared to the d11101 and d11107 viruses. Consequently,
selectivity is
determined not just by the deletion of Rb 105 binding but also by the
elimination of
p300 binding.
The deletions in the Ela 2898 coding sequence necessary to achieve elimination
of p300 and pRb binding are preferably as minimal as possible to prevent major
disruption of the secondary and tertiary structure of the Ela 2898 protein. In
order to
eliminate p300 binding it is preferred that a mutation be introduced in the
DNA
sequence encoding the p300 binding domains of 2898. Deletions of less than
about 30
amino acids in the C-terminal region to eliminate p300 binding are preferred,
although
-13-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/2145I
smaller modifications are preferred. The deletion of amino acids 4-25 of the
2898
protein are sufficient to disrupt p300 binding without affecting
transactivational
functions of CR3. For example, a deletion of amino acids from about amino acid
30 to
amino acid 49 (dll 103) and more particularly 36 to 49 are alternatively
preferred to
eliminate p300 binding. Point mutations sufficient to disrupt binding p300 are
particularly prefer ed. For example, a point mutation of the second amino acid
from
arginine to glycine (Arg2 -~ Gly2) in the 2898 protein has been demonstrated
to disrupt
p300 binding (See e.g., pm563, Whyte, et al, (1989) Cell 56:67-75). Similarly,
in
regard to eliminating pRb105 binding, minimal modifications are preferred.
Elimination of selective amino acids in the pRb105 binding domain such as
amino acid
111-123 (d11107) and amino acids 124-127 (d11108) are preferred. Deletion of
amino
acids 111-123 (d11107) is particularly preferred in that it retains the p107
binding
activity of the 2898 protein.
The d101/07/309 virus is a particularly preferred embodiment of the present
invention because although it deletes the p300 and pRb105 binding regions of
the Ela
2898 protein, it retains the ability of the 2898 protein to bind to p107.
Elevated levels
of free E2F are the primary factor inducing the cell cycle. By retaining the
p107
binding domain, the 2898 protein will bind to a sequester p107 resulting in
slightly
elevated intracellular levels of E2F. Although this low level of E2F is
insufficient to
initiate cell cycle progression on its own in normal cells, in tumor cells,
the ability of the
2898 protein to bind up p107 produces an elevated level of E2F in excess of
the E2F
threshold level necessary to induce cell cycling thereby enhancing the ability
of the virus
to replicate. This results in enhanced cytetoxicity of the virus in tumor
cells while not
affecting the toxicity of the virus to normal cells.
The term "recombinant adenovirus" is synonymous with the term "recombinant
adenoviral vector" and refers to viruses of the genus adenoviridiae capable to
infecting a
cell whose genomes have been modified through conventional recombinant DNA
techniques. The term adenoviridiae refers collectively to animal adenoviruses
of the
genus mastadenovirus including but no limited to human, bovine, ovine, equine,
canine, porcine, murine and simian adenovirus subgenera. In particular, human
adenoviruses includes the A-F sugenera as well as the individual serotypes
thereof the
individual serotypes and A-F subgenera including but not limited to human
adenovirus
types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (AdllA and Ad 11P), 12,
-14-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
13,14,15,16,17,18,19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 i,
32, 33,
34, 34a, 35, 35p, 36, 37, 38, 39, 40, 4i, 42, 43, 44, 45, 46, 47, 48, and 91.
The
term bovine adenoviruses includes but is not limited to bovine adenovirus
types
1,2,3,4,7, and 10. The term canine adenoviruses includes but is not limited to
canine
5 types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2. The term
equine
adenoviruses includes but is not limited to equine types 1 and 2. The term
porcine
adenoviruses includes but is not limited to porcine types 3 and 4. The term
recombinant
adenovirus also includes chimeric (or even multimeric) vectors, i.e. vectors
constructed
using complementary coding sequences from more than one viral subtype. See,
e.g.
Feng, et al. Nature Biotechnology 15:866-870. In the preferred embodiment of
the
invention, the adenovirus is a human adenovirus of serotype 2 or 5.
The term "modifications" refers to changes in the genomic structure of the
recombinant adenoviral vector. Such modifications include deletions and/or
changes in
amino acid coding sequence so as to produce a protein deficient in binding to
its
substrate. For example, the Rb-105 binding domain of the Ela-12S and 13S
proteins
has been characterized u.; located within amino acids 111-127. The p300
binding
domain of the Ela-12S and 13S proteins has been narrowed to the first 69 amino
acids.
Egan, et al. (1988) Mol. Cell Biol. 8:3955-3959. However, it has been shown
that
amino acids 26 to 35 are not necessary for p300 binding. There are two regions
of
20 p300 binding in the 12S and 13S molecules from approximat -ly amino acids 4-
25 and
amino acids 36-49. Elimination of one or both is sufficient to disrupt p300
binding.
Preferably, the elimination of amino acids in the 4-25 region are employed to
eliminate
the p300 binding function.
The term "deficient in binding" refers to a gene product forming a complex
with
less than 50% of the thermodynamic stability of the complex of the wild type
gene
product to its substrate under physiological conditions. For example, a 13S
gene
product which contains a deletion in the p300 binding domain would bind to
p300
protein with less than 50% of the thermodynamic stability of the wild-type 13S
protein.
The thermodynamic stability of binding can readily be determined by
conventional
assay techniques to determine equilibrium binding constants under
physiological
conditions.
-15-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
The term "Ela gene" refers to the immediate early gene of the adenovirus
genome first transcribed following infection. This genomic sequence represents
at least
the transcription of five mRNAs encoding the 9S, 10S, 11S, 12S and 13S
proteins.
The 12S and 13S proteins are expressed in the early phase following infection
while the
5 9S, lOS and 11S proteins are expressed later in the adenovirus cycle. The
12S and 13S
proteins have 243 and 289 amino acids respectively. There are three conserved
regions
in the Ela genomic sequence referred to as conserved region ("Ck")-1, CR2 and
CR3.
CR1 represents amino acids 41-80.of the 12S and 13S proteins. CR2 represents
amino
acids 12I-139 of the 12S and 13S sequence.
IO The "transactivating function of the CR3 domain" refers to the ability of
the
products of the Ela gene to activate transcription of promoters later in the
viral cycle
such as Elb and E2. The CR3 region is functionally present only in the 13S
protein and
represents amino acids 140 to 188. The transactivating function of the Ela
gene
product is contained in the CR3 region. The transactivating' region is
retained in the
15 vectors of the present invention to permit activation of other viral genes
and improve the
cytotoxicity.
The term "p300 protein family members" refers the proteins which associate
with the amino terminus of Ela including p300 and CBP. In particular p300 co-
activates the activity of the transactivating genes, Myb and C/EBP. Mink, et
al. (1997)
20 Molecular and Cellular Biology 17:6609-6617. The human p300 protein is
known in
the art and is publicly available from the Swiss-Prot database under
:~ccession number
Q09472, its corresponding mRNA is available from GenBank under accession
number
U01877 deposited June 6, 1994 and is described in Eckner, et al. (1994) Genes
Dev.
8:869-884.
25 The term "Rb protein family members" refers to the retinoblastoma gene
product
(pb105), p107 and p130. The retinoblastorna gene is well characterized in the
art. The
amino acid sequence of human Rb is available from GenBank under accession
Number
190959 deposited July 12,1995 and the mRNA sequence is available from GenBank
under accession number M15400 and is described in Lee, et al. (1988) PNAS
(USA)
30 85:6017-6021.
-16-
CA 02345211 2001-04-04
WO 00/22136 PC'f/US99/21451
In order to demonstrate the efficacy of the vectors of the present
invention, the vectors were evaluated first using in vitro experiments. The in
vitro
experiments presented below were designed to determine the potential of the
vectors to
selectively destroy tumor cells as compared to wild type virus and several
other vector
S constructions. The outcome of these experiments would determine the
potential of the
vectors as agents for treatment of hyperproliferative diseases including
cancer. The
vectors were prepared in substantial accordance with the teaching of Example 1
herein.
The vectors used for comparison include: wild type adenovirus type 5 (AdSwt),
d1309
a phenotypic wild type Type S human adenovirus containing modifications to
eliminate
certain E3 functions (as described in Jones and Shenk (1978) Cell 13:181-I88;
and
Jones and Shenk (1979) Cell I7:683-689); a mutant adenovirus (Elb,1~1SSK)
which
contains a deletion of the majority of the Elb~l_SSK gene that eliminates
production of a
functional EIb~ISSK protein prepared in substantial accordance with the
teaching of
Example 2 herein; mutant adenovirus EIb~SSK/309 which contains a double point
1S mutation in the coding region of the EIbSSK gene and therefore also
eliminates
production of the EIbSSK protein {McLorie, et al. !1991) J. Gen. Virol.
72:1467-
1471), the adenovirus based gene therapy vector ACNS3 (Wills, et al. (1994)
Human
Gene Therapy 5:1079-1088) in which the complete EI region is replaced with the
pS3
gene under control of the CMV promoter; and a control vector ACN which
contains a
complete El region deletion but does not encode a therapeutic transgene.
The ability of the vectors to destroy cells in vitro was evaluated in normal
diploid fibroblasts and in several tumor lines by using an the CPE assay to
evaluate the
cell killing by the vector constructions. The CPE assay is described in
Bischoff, et al.
( 1996) Science 274:373-376. This assay involves infecting cells in culture
with a range
2S of viral particle concentrations of the test viral constructs, and then
staining with crystal
violet the infected cells at time points after infection to determine the
number of viable
cells left on the cell culture growth substrate. Cell killing as a result of
replication
competent adenovirus infection results in cell lysis and detachment of the
infected cells
from the growth substrate. Therefore, the number of cells remaining can be
used as a
30 qualitative measure of the cell killing potential of the viral construct.
Cell killing by the
replication incompetent pS3 gene delivery vector ACNS3 can also be evaluated
using
-17-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
this assay. In contrast to the replication competent vectors, cell death as a
result
ACN53 occurs in many tumor cells as a result of p53-dependent apoptosis which
also
leads to detachment of cells from the growth matrix. Therefore it is possible
to use the
assay described above to compare cell death as a result of p53-induced
apoptosis with
cell kilfing by lysis with the replication competent vectors.
Representative results from experiments in which the normal fibroblast cell
line
MRC9 (p53+, pRb+), were infected with the test viral constructions are shown
in Figure
4, S and 6. In MRC9 cells infected at a low concentration of 1.8X10'
particles/ml some
cell detachment was observed only in wtAdS infected MRC9 cells, but not with
the
other constructs. At an infection concentration of 1.8X10$ particles/m I wtAdS
infected
MRC9 cells were efficiently destroyed and there was some evidence of killing
in the
E1B~1_SSK infected cells. Infection at a high concentration of 1.8X109
particles/ml
resulted in almost complete cell killing in MRC9 cells infected with wtAdS and
E1bd155K. Cell killing was also observed with ElAø~,01/07 after infection at
1.8X10$
particles/ml, but at a reduced level compared to the E1bd155K and wtAdS
vectors. The
ACN53 vector for p53 gene delivery had only a nunor toxic effect on the MRC9
cells,
as compared to the empty vector control, and only at the high infection
concentration of
1.8X109 particles/ml.
The test vectors were next used to evaluate their potential for cell killing
in the
cervical carcinoma line C33A (p53~, pRb'). The results of representative
experiments
are shown in Figures 1, 2, and 3. After infection of the C33A cells at 1.8X109
particles/ml alI of the viral constructions, with the exception of the E1-
deficient control
vector ACN, efficiently destroyed the entire monolayer of cells. Infection
with wtAdS
and E1A~01/07 at concentrations of 1.8X108 particles/rnl and 1.8X10' also
resulted in
complete destruction of the C33A monolayers. However, the E1B~SSK vector was
somewhat defective for cell killing after infection at 1.8X108 particles/ml
and
significantly defective at 1.8X10' particles/ml . The ACN53 vector for p53
gene
delivery did not induce significant cell death beyond the high infection level
of 1.8X109
particles/ml as illustrated by the number of C33A cells remaining after
infection at
1.8X108 and 1.8X10' particles/xnl of this construct.
-18-
CA 02345211 2001-04-04
WO 00/Z2136 PCT/US99/21451
To further characterize the vectors for viral growth in vitro a panel of cells
was
infected with the test constructs and viral replication was determined
quantitatively at 48
hours after infection using the procedure of Huyghe et al. (1995) Human Gene
Therapy
ø: 1403-1416. The cell lines evaluated in addition to the normal line MRC9
(p53+,
S pRb'') and the C33A (p53', pRb') tumor line were a lung non-small cell
carcinoma line
H358 (p53"°", pRb'") and a liver carcinoma line SKHep1 (p53+, pRb'").
The results of
representative experiments in which cells were infected either at 1.8x109 or
1.8x108
particles/ml are shown in Figure 3. The results of these experiments show that
Elb~l_SSK was defective for growth in all cell lines as compared to wtAdS, but
was
most defective in the MRC9 normal line, and the lung tumor line H358. The
E1A~01/07 construct was, as expected, was defective for virus production in
the
normal MRC9 cells, but replicated as efficiently as wild type virus and d1309
virus in
the C33A cervical carcinoma line. In both the lung carcinoma H358 (p53nu",
pRb'") and
the hepatocellular carcinoma SKHepl(p53+, pRb+) cells the E1A~01/07 virus
replicated slightly less efficiently than wtAdS and X309, but the construct
consistently
replicated more efficiently than the E1B~SSK virus.
Together the results of the above experiments showed that the E 1 Ad101 /07
vector induced cell killing more efficiently in tumor cell lines than in
normal cells, and
that the status of the tumor suppressor genes pRb and p53 did not appear to
affect
the ability of the virus to replicate in tumor cells. In addition, our results
differ from
those of Bischoff, et al. (1996) Science 274:373-376, who reported that an
E1bd155K
vector replicated as efficiently as wtAdS in p53-defective cell lines,
including the
C33A carcinoma cell line studied here, but was replication defective in p53+
cells
lines. See Bischoff, et al. (1996) Science 274:373-376. Our results show that
E1Bd55K constructs are defective for cell growth in both normal and tumor cell
lines.
In order to demonstrate the efficacy of the vectors of the present invention,
the vectors of the present invention were evaluated in a in vivo model of
cervical
carcinoma. The mouse model was based on the establishment of tumors and then
treated
with the above vectors in substantial accordance with the teaching of Example
3 herein.
The results are shown in Table 2 provided below.
-19-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99121451
Table 2.
Tumor Volume
(mm3) In
Response
to Treatment
With rAd
Vectors
Vector D6 D9 D13 D16 D20 D23 D27 D30 D34
vPBS 128 128 380 463 808 1215 2116 2583 4153
X309 112 112 115 91 75 56 52 73 78
E1A~101/07 114 114 95 70 53 43 45 57 60
E1B~SSk 102 102 114 114 92 78 153 221 299
ACN53 118 118 143 163 229 353 657 1016 1297
As can be seen from the data presented in Table 2, the vectors of the present
invention
as exemplified by ElA~1,01/07, demonstrate effective in vivo anti-tumor
activity.
Additionally, it should be noted that the double deleted E1A~01/07 vector
achieved a
reduction in tumor size approximately equivalent to the wild-type d1309 virus.
This is
in substantial contrast to the ACN53 and E1~155K vectors which merely slowed
the
growth of the tumor.
Additional in vivo studies were performed in nude mice to evaluate the
efficacy
of the vectors of the present invention following both intratumoral and
intravenous
systemic administration of virus. The first study was DLD-1 tumor model to
assess the
efficacy of the intratumorally administered d101/07 virus. The results of the
study are
presented in Figure 13 of the accompanying drawings. As can be seen from the
data
presented, the d101/07/309 virus was effective in preventing tumor growth at
least as (if
not more) effectively than the wild-type virus and significantly better than
the E1bd155K
virus. These results are particularly significant as the DLD-1 tumors are very
aggressive. The second model was designed to assess the efficacy of the
d101/07 virus
when administered systemically by intravenous injection. The results of this
study are
presented in Figure 14 of the accompanying drawings. As can be seen from the
data
presented, the dlOl/07 virus is effective in reducing tumor growth in vivo
following
systemic administration. The fact that the virus was able to find the tumor
following
systemic administration and slow its growth in the absence of any targeting
-20-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
modifications to the virus is particularly relevant. Together, these studies
demonstrate
that selectively replicating viruses containing deletions of 2898 p300 and
pRb105
binding functions are useful in the treatment of tumors when administered
either
intratumorally or systemically.
In the preferred practice of the invention, the recombinant adenoviral vector
is
derived from genus adenoviridiae. Particularly prefer ed viruses are derived
from the
human adenovirus type 2 or type 5. In the preferred practice of the invention,
the vector
is derived from the human adenoviridiae. More preferred are vectors derived
from
human adenovirus subgroup C. Most prefer ed are adenoviral vectors derived
from the
human adenovirus serotypes 2 and 5. In the most preferred practice of the
invention the
virus is derived from human adenovirus Type 5 41309 or 41520.
The vectors of the present contain deletions in the Ela coding sequence to
eliminate p300 and p105-Rb binding sites in the 13S coding sequence. In the
preferred
practice of the invention, the p300 binding deletions are represented by
deletions of
amino acids from about 4 to about 25 or from about 36 to about 49. In the
preferred
practice of the invention, the Rb binding deletions are represented by
elimination of
amino acids from about 111-127, preferably from about 111-123. More preferred
is a
vector wherein said deletion in the Ela-p300 binding domain comprises a
deletion of
the codons for amino acids 4 to 25 of the adenoviral Ela gene product. More
preferred
is a vector wherein deletion in the Ela-Rb binding domain comprises a deletion
of the
codons for amino acids 111-123 of the adenoviral Ela gene product.
Alternatively,
pRb binding may be eliminated by the introduction of a mutation to eliminate
amino
acids 124-127 of the ElA 2898 and 2438 proteins. In the most preferred
embodiment
of the present invention as exemplified herein the vector comprises a deletion
of amino
acids 4-25 and 111-123 of the Ela 13S gene product.
The invention further provides a recombinant adenovirus which contains
modifications to the Ela coding sequence so as to produce Ela gene products
which are
deficient in binding to one or more p300 protein family members and one or
more Rb
protein family member protein but retain the transactivating function of the
Ela CR3
domain and a deletion of the amino acids from approximately 219 to
approximately 289
of the Ela 2898 protein (or approximately amino acids 173 to approximately
amino
-21 -
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
acid 243 of the Eia 2438 protein. In the preferred practice of the invention
the deletion
of the binding to the p300 family members is achieved by introducing a
deletion
corresponding to amino acids 4-25 of the Ela 2438 and 2898 proteins or amino
acids
38-60 of the Ela 2438 and 2898 proteins. In the preferred practice of the
invention the
deletion of the binding to the pRb family members is achieved amino acids 111-
123 of
the Ela 2438 and 2898 proteins. Alternatively, deletion of the binding to the
pRb
family members may be achieved by elinunate of amino acids 124-127 of the Ela
2438
and 2898 proteins.
As previously described, the deletions in the adenoviral genome result in
preferential replication in rapidly dividing cells. These desirable features
may be
combined with other elements to provide even greater degrees of selectivity
and/or
cytotoxicity to such cells. For example, the vectors of the present invention
also
include recombinant adenoviruses containing modifications to the viral genome
to
induce preferential replication in particular cell types using cell type
specific promoters
i5 or inducible promoters. The term "cell type specific promoter" refers to
promoters
which are differentially activated in as a result of cell cycle progression or
in different
cell types. Examples of cell-type specific promoters includes cell cycle
regulatory gene
promoters, tissue specific of tumor specific promoters or pathway responsive
promoters. The term "cell cycle regulatory gene promoters" describe promoters
for
genes which are activated substantially upon entry into S-phase. Examples of
such
promoters include the E2F regulated promoters (e.g. DHFR, DNA polymerase
alpha,
thymidylate synthase, c-myc and b-myb promoters). Tissue specific and tumor
specific
promoters are well known in the art and include promoters active
preferentially in
smooth muscle (a-actin promoter), pancreas specific (Palmiter, er al.(1987)
Cell
50:435), liver specific Rovet, et.al. (1992) J. Biol. Chem.. 267:20765;
Lemaigne, et
al. (1993) J. Biol. Chem.. 268:19896; Nitsch, et al. (1993) Mol. Cell. Biol.
13:4494),
stomach specific (Kovarik, et al. (1993) J. Biol. Chem.. 268:9917, pituitary
specific
(Rhodes, et al. (1993) Genes Dev. 7:913, prostate specific (United States
Patent
5,698,443, Henderson, et al.issued December 16, 1997), etc.
The term "pathway-responsive promoter" refers to DNA sequences that bind a
certain protein and cause nearby genes to respond transcriptionally to the
binding of the
-22-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
protein in normal cells. Such promoters may be generated by incorporating
response
elements which are sequences to which transcription factors bind. Such
responses are
generally inductive, though there are several cases where increasing protein
levels
decrease transcription. Pathway-responsive promoters may be naturally
occurring or
synthetic. Pathway-responsive promoters are typically constructed in reference
to the
pathway or a functional protein which is targeted. For example, a naturally
occurnng
p53 pathway-responsive promoter would include transcriptional control elements
activated by the presence of functional p53 such as the p21 or bax promoter.
Alternatively, synthetic promoters containing p53 binding sites upstream of a
minimal
promoter (e.g. the SV40 TATA box region) may be employed to create a synthetic
pathway-responsive promoter. Synthetic pathway-responsive promoters are
generally
constructed from one or more copies of a sequence that matches a consensus
binding
motif. Such consensus DNA binding motifs can readily be determined. Such
consensus sequences are generally arranged as a direct or head-to-tail repeat
separated
by a few base pairs. Elements that include head-to-head repeats (e.g.
AGGTCATGACCT) are called palindromes or inverted repeats and those with tail-
to-
tail repeats are called evened repeats.
Examples of pathway-responsive promoters useful in the practice of the present
invention include synthetic insulin pathway-responsive promoters containing
the
consensus insulin binding sequence (Jacob, et al. (1995). J. Biol. Chem.
270:27773-
277 : ~), the cytokine pathway-responsive promoter, the glucoconicoid pathway-
responsive promoter (Lange, et al.(1992) J Biol. Chem. 267:15673-80), IL1 and
IL6
pathway-responsive promoters (Won K.-A and Baumann H. (1990) Mol.Cell.Biol.
10:
3965-3978), T3 pathway-responsive promoters, thyroid hormone pathway-
responsive
promoters containing the consensus motif: 5' AGGTCA 3.', the TPA pathway-
responsive promoters ('I'REs), TGF-~i pathway-responsive promoters (as
described in
Grotendorst, et al.(1996) Cell Growth-and Differentiation 7: 469-480).
Additionally,
natural or synthetic E2F pathway responsive promoters may be used. An example
of
an E2F pathway responsive promoter is described in Parr, et al. (1997, Nature
Medicine 3:1145-1149) which describes an E2F-1 promoter containing 4 E2F
binding
sites and is reportedly active in tumor cells with rapid cycling. Examples of
other
- 23 -
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
pathway-responsive promoters are well known in the art and can be identified
in the
Database of Transcription Regulatory Regions on Eukaryotic Genomes accessible
through the Internet at http://www.eimb.rssi..
In the preferred practice of the invention as exemplified herein, the vector
comprises a synthetic TGF-~3 pathway-responsive promoter active in the
presence of a
functional TGF-(3 pathway such as the promoter containing SRE and PAI pathway
responsive promoters. PAI refers to a synthetic TGF-(3 pathway-responsive
promoter
comprising sequences responsive to TGF-~i signally isolated from the
plasminogen
activator-I promoter region. The PAI-pathway-responsive promoter may be
isolated as
a 749 base pair fragment isolatable from the plasmid p8001uc (as described in
Zonneveld, et al. (1988) PNAS 85:5525-5529 and available from GenBank under
accession number J03836). SRE refers to a synthetic TGF-~3 response element
comprising a repeat of 4 of the Smad-4 DNA binding sequences (GTCTAGAC as
described in Zawel, et al. (1988) Mol. Cell 1:611-617. The SRE response
element may
1 S be generated by annealing complimentary oligonucleotides encoding the
Smad~i
binding sequences and cloning in plasmid pGL#3 - promoter luciferase vector
(commercially available from ProMega).
Similarly, a "p53 pathway-responsive promoter" refers to a transcriptional
control element active in the presence of a functional p53 pathway. The p53
pathway-
responsive promoter maybe a naturally occurring transcriptional control region
active in
the presence of a functional p53 pathway such as the p21 or mdm2 promoter.
Alternatively, the p53 pathway-responsive promoter may be a synthetic
transcriptional
control region active in the presence of a functional p53 pathway such as the
SRE and
PAI-RE pathway-responsive promoters. p53-CON describes a p53 pathway-
responsive promoter containing a synthetic pS3 response element constructed by
insertion of two synthetic p53 consensus DNA binding sequences (as described
in
Funk, et al. (1992) Mol.Cell Biol. x:2866-2871) upstream of the SV40 TATA box.
RGC refers to a synthetic p53 pathway-responsive promoter using a tandem of
the p53
binding domains identified in the ribosomal gene cluster. p53CON and RGC
response
elements can be constructed by annealing complementary oligonucleotides and
p53
-24-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
responsive promoters can be constructed by cloning in plasmid pGL3-promoter
luciferase vector (commercially available from ProMega)
Altemadvely, the viral genome may be modified to include inducible promoters
which are functional under certain conditions in response to chemical or other
stimuli.
Examples of inducible promoters are known in the scientific literature (See,
e.g.
Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; Iida, et
al.
(1996) J. Virol. 70(9):6054-6059; Hwang, et al. (1997) J. Virol 71(9):7128-
7131; Lee,
et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al. (1997) J.
Biol.
Chem. 272(46); 29364-29371. An example of radiation inducible promoters
include
the EGR-1 promoter. Boothman, et al. (1994) volume 138 supplement pages S68-
S71
The cell type specific (tissue specific, tumor specific, pathway specific,
cell
cycle regulatory promoter) promoter or inducible promoter may be used in lieu
of the
native Ela promoter region in the vectors of the present invention to provide
preferential
expression in particular cell types.
Alternatively, the one may use a pathway responsive promoter to drive
expression of a repressor of viral replication. The term "repressor of viral
replication"
refers to a protein, if expressed in a given cell, substantially represses
viral replication.
In the case of adenoviral vectors, the E2F-Rb fusion construct as described in
European
Patent Application No. 94108445.1 published December 6, 1995 (Publication
number.
0 685 493 A1) may be employed. E2F-Rb fusion protein consists of the DNA
binding
and DP1 heterodimerization do~~:ains of the human E2F-2 transcription factor
protein
(amino acids 95-286 of wild type E2F) fused to the Rb growth suppression
domain
(amino acids 379-928 of the wild type Rb protein). The E2F-Rb fusion protein
is a
potent repressor of E2F trmscription and arrests cells in G1. The DNA binding
domain
is located at amino acids 128-193 and the dimerization domain is located at
194-289.
The sequence of the human E2F2 protein is available fram GenBank under
accession
number 2494288 deposited November 1, 1997 as updated July 15, 1998.
These modifications may be combined with the previously cell cycle regulatory
gene promoters described above. For instance, an E2F pathway responsive
promoter
may be used to drive expression of the modified Ela coding sequence. Using a
p53
pathway responsive promoter driving expression of E2F-Rb fusion protein, one
-25-
CA 02345211 2001-04-04
WO 00122136 PCTNS99/21451
achieves repression of both E1 function and E2 function because the E2F-Rb
fusion
protein will suppress both the E2 and E2F response elements. In p53 deficient
tumor
cells, the p53 response element is inactive and E2F-Rb is not expressed.
Consequently, Ela expression is enhanced by the presence of E2F in the tumor
cell and
the failure to repress E2 promoter enables viral replication to proceed.
As previously described, the deletions in the adenoviral genome result in
preferential replication in rapidly dividing cells. While these viruses will
replicate and
ultimately kill the tumor cells, these viruses may also incorporate a
therapeutic transgene
expression cassette to enhance cytotoxicity. The term "expression cassette" is
used
herein to define a nucleotide sequence containing regulatory elements and a
transgene
coding sequence so as to result in the transcription and translation of a
transgene in a
transduced cell. The term "regulatory element" refers to promoters, enhancers,
transcription terminators, polyadenylation sites, and the like. The regulatory
elements
may be arranged so as to allow, enhance or facilitate expression of the
transgene only in
a particular cell type. For example, the expression cassette may be designed
so that the
transgene is under control of an inducible promoter, tissue specific or tumor
specific
promoter, or temporal promoter. The term "temporal promoters" refers to
promoters
which drive transcription or the therapeutic transgene at a point later in the
viral cycle
relative to the promoter controlling expression of the respanse element and
are used in
conjunction with viral vector systems. Examples of such temporally regulated
promoters include the adenovirus major late t~; ~~moter (NiLP), other late
promoters.
The term "therapeutic transgene" refers to a nucleotide sequence the
expression
of which in the target cell produces a cytotoxic or cytostatic effect. The
term therapeutic
transgene includes but is not limited to r4mor suppressor genes, antigenic
genes,
cytotoxic genes, dendritic cell chemoattractants, cytostatic genes, pro-drug
activating
genes, or pro-apoptotic. The vectors of the present invention may be used to
produce
one or more therapeutic transgenes, either in tandem through the use of IRES
elements
or through independently regulated promoters.
The term "tumor suppressor gene" refers to a nucleotide sequence, the
expression of which in the target cell is capable of suppressing the
neoplastic phenotype
andlor inducing apoptosis. Examples of tumor suppressor genes useful in the
practice
-26-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
of the present invention include the pS3 gene, the APC gene, the DPC-4 gene,
the
BRCA-1 gene, the BRCA-2 gene, the WT-1 gene, the retinoblastoma gene (Lee, et
al.
(1987) Nature 329:642), the MMAC-1 gene, the adenomatous polyposis coli
protein
(Albertsen, et al., United States Patent 5,783,666 issued July 21, 1998), the
deleted in
S colon carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene, nasopharyngeal
carcinoma tumor suppressor gene that maps at chromosome 3p21.3. (Cheng, et al.
1998. Proc. Nat. Acad. Sci. 95:3042-3047), the MTS 1 gene, the CDK4 gene, the
NF-
1 gene, the NF2 gene, and the VHI. gene.
The term "antigenic genes" refers to a nucleotide sequence, the expression of
which in the target cells results in the production of a cell surface
antigenic protein
capable of recognition by the immune system. Examples of antigenic genes
include
carcinoembryonic antigen (CEA), pS3 (as described in Levine, A. PCT
International
Publication No. W094/02167 published February 3, 1994). In order to facilitate
immune recognition, the antigenic gene may be fused to the MHC class I
antigen.
1S The term "cytotoxic gene" refers to nucleotide sequence, the expression of
which in a cell produces a toxic effect. Examples of such cytotoxic genes
include
nucleotide sequences encoding pseudomonas exotoxin, ricin toxin, diptheria
toxin, and
the like.
The term "cytostatic gene" refers to nucleotide sequence, the expression of
which in a cell produces an arrest in the cell cycle. Example of such
eytostatic genes
include p21, the retinoblastoma gene, the E2F-Rb gene, genes encoding cyclin
dependent kinase inhibitors such as P16, plS, p18 and p19, the growth arrest
specific
homeobox (GAX) gene as described in Branellec, et al. (PCT Publication
W097/16459
published May 9, 1997 and PCT Publication W096/30385 published October 3,
1996).
2S The term "dendritic cell chemoattractants" refers to chemotactic chemokines
capable of attracting and/or directing the migration of dendritic cells to a
particular
location. It has been demonstrated that certain chemokines, fMLP
(representative of
fonmyl peptides of bacterial origin), CSa and the C-C chemokines monocyte
chemotactic protein (MCP)-3, macrophage inflammatory protein (MIn)-1 o~/LD78,
and
RAN')'ES, have been involved in the recruitment and chemotactic migration of
dendritic
cells. Sozzani, et al. (1995) J. Immunol. 1995 155(7):3292-S. Xu, et al.
suggest that
-27-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
all C-C chemokines, including MCP-1, MCP-2, MCP-3, MIP1 a, MIP-1 Vii, and
R:~~1VTES, induced migration of DC-enriched cells cultured with or without IL-
4. Xu,
et al.. (1996) J. ieukoc. Biol. 60(3):365-71. Greaves, et al. (1997) J. Exp.
Med.
186(6):837-44, indicate that MIP-3-a specifically interacts with the CC
chemokine
receptor 6 expressed on dendritic cells capable of directing migration of
dendritic cells.
In the preferred practice of the invention, the dendridc cell chemoattractant
is MIP-3-a.
The dendritic cell chemoattractant may be expressed intracellular form where
it is
released upon cell lysis or in secreted form by the use of a signal peptide.
The term "pro-apoptotic gene" refers to a nucleotide sequence, the expression
thereof results in the programmed cell death of the cell. Examples of pro-
apoptotic
genes include p53, adenovirus E3-11.6K, the adenovirus E4orf4 gene, p53
pathway
genes, and genes encoding the caspases.
The term "pro-drug activating genes" refers to nucleotide sequences, the
expression of which, results in the production of protein capable of
converting a non-
therapeutic compound into a therapeutic compound, which renders the cell
susceptible
to killing by external factors or causes a toxic condition in the cell. An
example of a-
prodrug activating gene is the cytosine deaminase gene. Cytosine deaminase
converts 5-
fluorocytosine to 5 fluorouracil, a potent antitumor agent). The lysis of the
tumor cell
provides a localized burst of cytosine deaminase capable of converting 5FC to
5FU at
the localized point of the tumor resulting in the killing of many surrounding
tumor cells.
This results in the killing of a large number of tumor cells without the nP~;.
~sity of
infecting these cells with an adenovirus (the so-called bystander effect").
Additionally,
the thymidine kinase (TK) gene (see e.g. Woo, et al. United States Patent No.
5,631,236 issued May 20, 1997 and Freeman, et al. United States Patent No.
5,601,818 issued February 11, 1997) in which the cells expressing the TK gene
product are susceptible to selective killing by the administration of
gancyclovir may be
employed.
It will be readily apparent to those of skill in the art that modifications
and or
deletions to the above referenced genes so as to encode functional
subfragments of the
wild type protein may be readily adapted for use in the practice of the
present invention.
For example, the reference to the p53 gene includes not only the wild type
protein but
-28-
CA 02345211 2001-04-04
WO 00/2213b PCT/US99/21451
also modified p53 proteins. Examples of such modified p53 proteins include
modifications to p53 to increase nuclear retention, deletions such as the 013-
19 amino
acids to eliminate the calpain consensus cleavage site, modifications to the
oligomerization domains (as described in Bracco, et al. PCT published
application
W097/0492 or United States Patent No. 5,573,925).
It will be readily apparent to those of skill in the art that the above
therapeutic
genes may be secreted into the media or localized to particular intracellular
locations by
inclusion of a targeting moiety such as a signal peptide or nuclear
localization
signal(NLS). Also included in the definition of therapeutic transgene are
fusion
proteins of the therapeutic transgene with the herpes simplex virus type 1
(HSV-1)
structural protein, VP22. Fusion proteins containing the VP22 signal, when
synthesized in an infected cell, are exported out of the infected cell and
efficiently enter
surrounding non-infected cells to a diameter of approximately 16 cells wide.
This
system is particularly useful in conjunction with transcriptionally active
proteins (e.g.
p53) as the fusion proteins are efficiently transported to the nuclei of the
surrounding
cells. See, e.g. Elliott, G. & O'Hare, P. Cell. 88:723-233:1997; Marshall, A.
&
Castellino, A. Research News Briefs. Nature Biotechnology. 15:205:1997;
O'Hare, et
al. PCT publication W097/05265 published February 13, 1997. A similar
targeting
moiety derived from the HIV Tat protein is also described in Vives, et al.
(1997) J.
Biol. Chem. 272:16010-16017.
The present invention provides recombinant adenoviruses which contain
"targeting modifications" in order to achieve preferential targeting of the
virus to a
particular cell type. The term "targeting modification" refers to
modifications to the
viral genome designed to result in preferential infectivity of a particular
cell type. Cell
type specificity or cell type targeting may also be achieved in vectors
derived from
vimses having characteristically broad infectivities such as adenovirus by the
modification of the viral envelope proteins. For example, cell targeting has
been
achieved with adenovirus vectors by selective modification of the viral genome
knob
and fiber coding sequences to achieve expression of modified knob and fiber
domains
having specific interaction with unique cell surface receptors. Examples of
such
modifications are described in Wickham, et al. (1997) J. Virol 71(1 I):8221-
8229
-29-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
(incorporation of RGD peptides into adenoviral fiber proteins); Amberg, et al.
(1997)
Virology 227:239-244 (modification of adenoviral fiber genes to achieve
tropism to the
eye and genital tract); Harris and Lemoine (1996) TIG 12(10):400-405;
Stevenson, et
al. (1997) J. Virol. 71(6):4782-4.790; Michael, et al.(1995) gene therapy
2:660-668
(incorporation of gastrin releasing peptide fragment into adenovirus fiber
protein); and
Ohno, et al. (1997) Nature Biotechnology 15:763-767 (incorporation of Protein
A-igG
binding domain into Sindbis virus). Other methods of cell specific targeting
have been
achieved by the conjugation of antibodies or antibody fragments to the
envelope
proteins (see, e.g. Michael, et al. (1993) J. Biol. Chem. 268:6866-6869,
Watkins, et
al. (1997) gene therapy 4:1004-1012; Douglas, et al. (1996) Nature
Biotechnology 14:
1574-1578. Alternatively, particular moieties may be conjugated to the viral
surface to
achieve targeting (See, e.g. Nilson, et al. (1996) gene therapy 3:280-286
(conjugation
of EGF to retroviral proteins). These recombinantly modified vectors may be
produced
in accordance with the practice of the present invention.
The present invention further provides recombinant adenoviral vectors
comprising a suicide gene. In some instances, it may be desirable to include a
suicide
gene in the viral vector. This provides a "safety valve" to the viral vector
delivery
system to prevent widespread infection due to the spontaneous generation of
replication
competent viral vectors. The term "suicide gene" refers to a nucleic acid
sequence, the
expression of which renders the cell susceptible to killing by external
factors or causes a
toxic condition in the cell. A well known example of a suicide gene is the
thymidine
kinase (TK) gene (see e.g. Woo, et al. United States Patent No. 5,631,236
issued May
20, 1997 and Freeman, et al. United States Patent No. 5,601,818 issued
February 11,
1997) in which the cells expressing the TK gene product are susceptible to
selective
killing by the administration of gancyclovir.
The present invention further provides a pharmaceutically acceptable
formulation of the recombinant adenoviruses in combination with a carrier. The
vectors
of the present invention may be formulated for dose administration in
accordance with
conventional pharmaceutical practice with the addition of carriers and
excipients.
Dosage formulations may include intravenous, intratumoral, intramuscular,
intraperitoneal, topical, matrix or aerosol delivery.
-30-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
The term "carrier" refers to compounds commonly used on the formulation of
pharmaceutical compounds used to enhance stability, sterility and
deliverability of the
therapeutic compound. When the virus is formulated as a solution or
suspension, the
delivery system is in an acceptable Garner, preferably an aqueous Garner. A
variety of
aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0,3%
glycine,
hyaluronic acid and the like. These compositions may be sterilized by
conventional,
well known sterilization techniques, or may be sterile filtered. The resulting
aqueous
solutions may be packaged for use as is, or lyophilized, the lyophilized
preparation
being combined with a sterile solution prior to administration. The
compositions may
contain pharmaceutically acceptable auxiliary substances as required to
approximate
physiological conditions, such as pH adjusting and buffering agents, tonicity
adjusting
agents, wetting agents and the like, for example, sodium acetate, sodium
lactate,
sodium chloride, potassium chloride, calcium chloride, sorption monolaurate,
triethanolamine oleate, etc.
The present invention further provides pharmaceutical fornnulations of the
vectors of recombinant adenoviruses of the present invention with a carrier an
' a
delivery enhancing agent(s). The terms delivery enhancers" or "delivery
enhancing
agents" are used interchangeably herein and includes one or more agents which
facilitate
uptake of the virus into the target cell. Examples of delivery enhancers are
described in
co-pending United States Patent Application Serial No. filed July 7, 1998.
Examples of such delivery enhancing agents include detergents, alcohols,
glycols,
surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors,
hypertonic salt
solutions, and acetates. Alcohols include for example the aliphatic alcohols
such as
ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols
include
glycerine, propyleneglycol, polyethyleneglycol and other low molecular weight
glycols
such as glycerol and thioglycerol. Acetates such as acetic acid, gluconic
acid, and
sodium acetate are further examples of delivery-enhancing agents. Hypertonic
salt
solutions like 1M NaCI are also examples of delivery-enhancing agents.
Examples of
surfactants are sodium dodecyl sulfate (SDS) and lysolecithin, polysorbate 80,
nonylphenoxypolyoxyethylene, lysophosphatidyicholine, polyethyleneglycol 400,
polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO.
Bile
-31 -
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
salts such as taurocholate, sodium tauro-deoxychoiate, deoxycholate,
chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other
astringents such as silver nitrate may be used. Heparin-antagonists like
quaternary
amines such as protamine sulfate may also be used. Cyclooxygenase inhibitors
such as
sodium salicylate, salicylic acid, and non-steroidal antiinflammatory drug
(NSAmS)
like indomethacin, naproxen, diclofenac may be used.
The term "detergent" includes anionic, cationic, zwitterionic, and nonionic
detergents. Exemplary detergents include but are not limited to taurocholate,
deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride,
Zwittergent3-
14 detergent, CHAPS (3-[(3-Cholamidopropyl) dimethylammoniol]-1-
propanesulfonate hydrate), Big CHAP, Deoxy Big CHAP, Triton-X-100 detergent,
C12E8, Octyl-B-D-Glucopyranoside, PLURONIC- F68 detergent, Tween 20
detergent, and TWEEN 80 detergent (CalBiochem Biochemicals).
Unit dosage formulations of the present invention may be included in a kit of
products containing the recombinant adenovirus of claim 1 in lyophilized fonm
and a
solution for reconstitution of the lyophilized product. Recombinant
adenoviruses of the
present invention may be lyophilized by conventional procedures and
reconstituted.
The present invention provides a method of ablating neoplastic cells in a
mammalian organism in vivo by the administration of a pharmaceutically
acceptable
formulation of the recombinant adenovirus of the present invention. The term
"ablating" means the substantial reduction of the population of viable
neoplastic cells so
as to alleviate the physiological maledictions of the presence of the
neoplastic cells. The
term "substantial" means a reduction in the population of viable neoplastic
cells in the
mammalian organism by greater than approximately 20°l0 of the
pretreatment
population. The term "viable" means having the uncontrolled growth and cell
cycle
regulatory characteristics of a neoplastic cell. _The term "viable neoplastic
cell" is used
herein to distinguish said cells from neoplastic cells which are no longer
capable of
replication. For example, a tumor mass may remain following treatment, however
the
population bf cells comprising the tumor mass may be dead. These dead cells
have
been ablated and lack the ability to replicate, even though some tumor mass
may
remain.
-32-
CA 02345211 2001-04-04
WO 00/22136 PC'T/US99/21451
The tenor "mammalian organism" includes, but is not limited to, humans, pigs,
horses, cattle, dogs, cats. Preferably one employs an adenoviral vector
endogenous to
the mammalian type being treated. Although it is generally favored to employ a
virus
from the species to be treated, in some instances it may be advantageous to
use
vectors derived from different species which possess favorable pathogenic
features.
For example, it is reported (WO 97/06826 published April 10, 1997) that ovine
adenoviral vectors may be used in human gene therapy to minimize the immune
response characteristic of human adenoviral vectors. By minimizing the immune
response, rapid systemic clearance of the vector is avoided resulting in a
greater
duration of action of the vector.
While the present invention provides a method of use of the recombinant
adenoviruses alone, the recombinant adenoviruses of the present invention and
formulations thereof may be employed in combination with conventional
chemotherapeutic agents or treatment regimens. Examples of such
chemotherapeutic
agents include inhibitors of purine synthesis (e.g., pentostatin, 6-
mercaptopurine, 6-
thioguanine, methotrexate) or pyrimidine synthesis (e.g. Pala, azarbine), the
conversion
of ribonucleotides to deoxyribonucleotides (e.g. hydroxyurea), inhibitors of
dTMP
synthesis (5-fluorouracil), DNA damaging agents (e.g. radiation, bleomycines,
etoposide, teniposide, dactinomycine, daunorubicin, doxorubicin, mitoxantrone,
alkylating agents, mitomycin, cisplatin, procarbazine) as well as inhibitors
of
microtubule function (e.g vinca alkaloids and colchicine). Chemotherapeutic
treatment
regimens refers primarily to non-chemical procedures designed to ablate
neoplastic cells
such as radiation therapy. Examples of combination therapy when the
therapeutic gene
is p53 are described in Nielsen, et al. W0/9835554A2 published August 20,
1998.
The immunological response is significant to repeated administration of viral
vectors. Consequently, the vectors of the present invention may be
administered in
combination with immunosuppressive agents. Examples of immunosuppressive
agents
include cyclosporine, azathioprine, methotrexate, cyclophosphamide, lymphocyte
immune globulin, antibodies against the CD3 complex, adrenocorticosteroids,
sulfasalzaine, FK-506, methoxsalen, and thalidomide.
- 33 -
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
The present invention also provides a method of ablating neoplastic cells in a
population of normal cells contaminated by said neoplastic cells ex vivo by
the
administration of a recombinant adenovirus of the present invention to said
population.
An example of the application of such a method is currently employed in ex
vivo
applications such as the purging of autologous stem cell products commonly
known as
bone marrow purging. The term "stem cell product" refers to a population of
hematopoietic, progenitor and stem cells capable of reconstituting the long
term
hematpoietic function of a patient who has received myoablative therapy. Stem
cell
products are conventionally obtained by apheresis of mobilized or non-
mobilized
peripheral blood. Apheresis is conventionally achieved through the use of
known
procedures using commercially available apheresis apparatus such as the COBE
Spectra
Apheresis System, commercially available from COBS International, 1185 Oak
Street,
Lakewood, CO. It is prefen:ed that treatment conditions be optimized to
achieve a "S-
log purge" (i.e. removal of approximately 99.9% of the tumor cells from the
stem cell
produce) and most preferably a "5-log purge" (removal of approximately 99.999%
of
tumor cells from the stem cell product). In the preferred practice of the
invention, a
stem cell product of 100 ml volume would be treated with 1 x 105 to 1 x 109
particles of
the recombinant adenovirus of the present invention for a period of
approximately 4
hours at 37C.
In addition to therapeutic applications described above, the vectors of the
present invention are also useful for diagnostic purposes. For example, the
vectors of
the present invention may incorporate a reporter gene which is expressed upon
viral
replication. The teen "reporter gene" refers to a gene whose product is
capable of
producing a detectable signal alone or in combination with additional
elements.
Examples of reporter genes includes the ~3-galactosidase gene, the luciferase
gene, the
green fluorescent protein gene, nucleotide sequences encoding proteins
detectable by
imaging systems such as.X-rays or magnetic field imaging systems (NIItI).
Alternatively, such vectors may also be employed to express a cell surface
protein
capable of recognition by a binding molecule such as a fluoroescently labeled
antibody.
Alternatively where the response element is used to drive a repressor of viral
replication
(e.g. E2F-Rb) later viral promoters (for example E2 which is turned off by E2F-
Rb)
-34-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
could be used to drive the reporter gene for diagnostic applications where the
response
element is off. These diagnostic constructs may be used for diagnostic
purposes in
vivo or in vitro. Examples of in vivo applications include imaging
applications such as
X-ray, CT scans or Magnetic Resonance Imaging (MRI).
The present invention further provides a method of producing the recombinant
adenovirus comprising the modifications to the Ela gene product domains
described
above, said method comprising the steps of:
a. infecting a producer cell with a recombinant adenovirus of the
present invention,
b. culturing said infected producer cell under conditions so as to
permit replication of the viral genome in the producer cell,
c. harvesting the producer cells, and
d. purifying the recombinant adenovirus.
The term "infecting" means exposing the recombinant adenovirus to the producer
cell
under conditions so as to facilitate the infection of the producer cell with
the
recombinant adenovirus. In cells which have been infected by multiple copies
of a
given virus, the activities necessary for viral replication and virion
packaging are
cooperative. Thus, it is preferred that conditions be adjusted such that there
is a
significant probability that the producer cells are multiply infected with the
virus. An
exa~~~le of a condition which enhances the production of virus in the producer
cell is an
increased virus concentration in the infection phase. However, it is possible
that the
total number of viral infections per producer cell can be overdone, resulting
in toxic
effects to the cell. Consequently, one should strive to maintain the
infections in the
virus concentration in the range of 106 to 10'°, preferably about 109,
virions per ml.
Chemical agents may also be employed to increase the infectivity of the
producer cell
line. For example, the present invention provides a method to increase the
infectivity of
producer cell lines for viral infectivity by the inclusion of a calpain
inhibitor. Examples
of calpain inhibitors useful in the practice of the present invention include
calpain
inhibitor 1 (also known as N-acetyl-leucyl-leucyl-norleucinal, commercially
available
from Boehringer Mannheim). Calpain inhibitor 1 has been observed to increase
the
infectivity of producer cell lines to recombinant adenovirus.
-35-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
The term "producer cell" means a cell capable of facilitating the replication
of the
viral genome of the recombinant adenovirus to be produced. A variety of
mammalian
cell lines are publicly available for the culture of recombinant adenoviruses.
For
example, the 293 cell line (Graham and Smiley (1977) J. Gen. Virol. 36:59-72)
has
been engineered to complement the deficiencies in Ela function and is a
preferred cell
line for the production of the current vectors. Examples of other producer
cells include
A549 cells, HeLa cells, PERC.6 cells (as described in publication WO/97/00326,
application serial No. PCT/NL96/00244.
The term "culturing under conditions to permit replication of the viral
genome"
means maintaining the conditions for the infected producer cell so as to
permit the virus
to propagate in the producer cell. It is desirable to control conditions so as
to maximize
the number of viral particles produced by each cell. Consequently it will be
necessary
to monitor and control reaction conditions such as temperature, dissolved
oxygen, pH,
etc. Commercially available bioreactors such as the CelliGen Plus Bioreactor
(commercially available from New Brunswick Scientific, Inc. 44 Talmadge Road,
Edison, NJ) have provisions for monitoring and maintaining such parameters.
Optimization of infection and culture conditions will vary somewhat, however,
conditions for the efficient replication and production of virus may be
achieved by those
of skill in the art taking into considerations the known properties of the
producer cell
line, properties of ~'oe virus, type of bioreactor, etc. When 293 cells are
employed as
the producer cell line, oxygen concentration is preferably maintained from
approximately 50% to approximately 120°lo dissolved oxygen, preferably
100%
dissolved ox«gen. When the concentration of viral particles (as determined by
conventional methods such as HPLC using a Resource Q column) begins to
plateau, the
reactor is harvested.
The term "harvesting" means the collection of the cells containing the
recombinant adenovirus from the media. This may be achieved by conventional
methods such as diffential centrifugation or chromatographic means. At this
stage, the
harvested cells may be stored or further processed by lysis and purification
to isolate the
recombinant virus. For storage, the harvested cells should be buffered at or
about
physiological pH and frozen at -70C.
-36-
CA 02345211 2001-04-04
WO 00/22136 PCTNS99/21451
The term "lysis" refers to the rupture of the producer cells. Lysis may be
achieved by a variety of means well known in the art. When it is desired to
isolate the
viral particles from the producer cells, the cells are lysed, using a variety
of means well
known in the art. For example, mammalian cells may be lysed under low pressure
(100-200 psi differential pressure) conditions or conventional freeze thaw
methods.
Exogenous free DNA/RNA is removed by degradation with DNAse/RNAse.
The term "purifying" means the isolation of a substantially pure population of
recombinant virus particles from the lysed producer cells. Conventional
purification
techniques such as chromatographic or differential density gradient
centrifugation
methods may be employed. In the preferred practice of the invention, the virus
is
purified by column chromatography in substantial accordance with the process
of
Huyghe et al. (1995) Human Gene Therapy ø: 1403-1416 as described in Shabram,
et
al., United States Patent 5,837,520 issued November 17, 1998, the entire
teaching of
which is herein incorporated by reference.
Additional methods and procedures to optimize production of the recombinant
adenoviruses of the present invention are described in co-pending United
States Patent
Application Serial No. 09/073,076, filed May 4, 1998.
It will be apparent to those of skill in the art to which the invention
pertains, the
present invention may be embodied in forms other than those specifically
disclosed
below without departing from tire spirit or essential characteristics of the
invention. The
particular embodiments of the invention described below, are therefore to be
considered
as illustrative and not restrictive. In the following examples, "g" means
grams, "ml"
means milliliters, "mol" means moles, "°C" means degrees Centigrade,
"min." means
minutes, "FBS" means fetal bovine serum, and "PN" specifies particle number.
~~~~le iConstruction of Recombinant Adenovirus ElAd101/07
The in-frame deletion mutations x,1101 and ~1I07, as described in Jelsma et
al., ( 1998) Virology ,~,, 494-502, were constructed using the oligonucleotide
site
directed technique of Zoeller and Smith (1984) DNA ~, 479-488, as modifed by
Kunkel (1985) PNAS $?, 488-492. All of the reagents, bacterial strains, and
M13
vectors used for mutagenesis were provided in the Muta-Gene in vitro
mutagenesis kit
-37-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
(commercially availble from Bio Rad, Hercules, CA). The M13 template DNA, used
for mutagenesis of the ElA region, contained Ad5 sequences from nucleotide
positions
22-1339 inserted between the BamHl and Xbal restriction enzyme sites in the
multiple
cloning sequence of M13mp19. The bacteriophage construct produced, named
Mi3mp19ElA, was then propagated in dut ung E. coli bacterial strain CJ236
which
results in an occasional incorporation of uracil in place of thymidine in the
newly
synthesized DNA. The oligonucleotides, for construction of the El mutants,
were
synthesized to consist of sequences of either 11 or 12 nucleotides of Ad5
sense DNA
on either side of the sequence that was to be removed.
For the mutagenesis reaction, the mutagencic oligonucleotides were first
phosphorylated at the 5' end, and then annealed to uracil containing
M13mp11ElA
single-stranded template DNA. The annealed primer/template reactions were
incubated
with T4 DNA polymerase, T4 DNA ligase and deoxyribonucleotides (dATP, dCTP,
dGTP and dTTP) to synthesize a complementary strand containing the ElA
mutation of
interest. The complementary strand synthesis reaction were then
transfom.~.d into the ung'" wild type host bacterial strain, MV1190. After
transformation
the parental M13mp19ElA DNA strand, which contains uracil, cannot be
replicated
efficiently in MV 1190. Therefore the replicative form double strand DNA
containing
the ElA mutation of interest is enriched. M13mp19ElA phage DNA from potential
ElA mutants was first screened by restriction Pnzyme analysis and then by DNA-
sequencing, in both strands, to confirm she desired ElA-mutations.
Construction of the ElA~i101/07adenovirus was carried out by using
homologous recombination in the adenovirus E1-region containing 293 cell line
by the
method of McGory, et al., (1988) Virology 163, 614-617. This method requires
two
plasmids, one a viral plasmid containing the entire wtAdS genome, and the
other a
transfer plasmid containing an ElA gene with the ~O1/07 double ElA-mutant .
The
viral plasmid used for this work was pJMl7, a non-infectious 40 kb plasmid,
containing the entire Ad5~11309 genome in which the 4.4kb piasmid, pBX, is
inserted in
the unique Xbal site. The transfer plasmid used, pLE2 contains wtAdS sequences
from 22-1774 cloned in the tetracycline gene of pBR322 Jelsma et al.,
(1988)Virology
~, 494-502. For transfer of the ElA X1101 and X1107 Ela-mutants from the
-38-
CA 02345211 2001-04-04
WO 00/22136 PGT/US99/21451
M13mp19 background in which they were constructed, wild ElA restriction enzyme
fragments in pLE2 were replaced with cognate mutated ElA fragments from
M13mp19E1,~1101 and M13mp19E1A~1107 to create pLE2ElA~01/07. For
recombination to produce adenovirus ElA~],O1/07 the viral plasmid, pJMl7 and
S pLE2ElAø~O1/07 were cotransfected into 293 cells by calcium phosphate
mediated
transfection. After 5 hours the precipitate was rinsed and the cells were
overlayed with
growth medium containing agarose to isolate viral plaques. At 7-10 days after
the initial
transfection viral plaques were isolated,.plaque purified two times, and
subsequently
viral DNA was screened using restriction enzyme analysis and DNA sequencing.
Viral
stocks were purified by double cesium chloride gradients and quantitated by
column
chromatography as described in Huyghe, et al. (1995) Human Gene Therapy 6:1403-
1416.
Exam~~2. Construction of El,' dIB SSK
The E1Bd155K adenovirus was prepared by using oligonucleotide site directed
mutatgenesis in substantial accordance with the teaching of Example 1 above.
This
procedure was used to introduce restriction enzyme nuclease cleavage sites in
the E1B
SSK coding region. The first site was introduced by modifying positions 2247
and
2248 of the wild type Ad5 genome wherein a guanine2~" was replaced with a
thymidine
and thymidine'~ replaced with cytosine (respectively) to introduce a EcoRl
cleavage
site. This results in a modification of the E1B coding sequence at position 77
from
valine to serine. A second resriction site was introduced a~ position 3272
wherein
thymidine'2'2 was replaced with cytosine site (silent mutation) to introduce
an XhoI
site. The new restriction enzyme sites were used in a restriciton enzyme
digest with
EcoRI and Xhol. The EcoRI and XhoI sites were rejoined with a small polylinker
cassette to introduce the polyiinker isolated from the pBlueScriptSK
(Stratagene, San
Diego, CA). The resulting E1B mutation results in a coding sequence encoding
the first
76 amino acids of the E1BSSK proteinwfollowed by 18 missense amino acids
resulting
in a non-functional deleted E1B protein.
Example 3. In Vivo Mouse Model
On day 0, 30 athymic nude-nu mice (Harlan-Sprague-Dawley, Indianapolis IN)
were injected in each flank with approximately 1x10'C33A cervical carcinoma
cells in
-39-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
200 microliters of Dulbecco's Modified Eagle Medium (DMEM). C33A cells are
derived from human cervical carcinoma tissue and possess a p53 negative, Rb
negative
genotype and were obtained from the American Type Culture Collection. The
tumors
were allowed to grow for 6 days at which time they had reached a palpable size
of
approximately 100 mm'. The animals were randomized by tumor size into 6 groups
of
5 animals each. Each animal was intratumorally injected with 2.5x109 particles
of each
different adenovirus construct in 60 microliters of PBS on Day i, 8, 9, 10,
and 11
following C33A administration. Each injection was divided among the four tumor
quadrants. Tumors size was determined on Day 6, 9, 13, 16, 20, 23, 27 30 and
34
following C33A injection.
The recombinant adenovirus ElAd101/08 is prepared in substantial accordance
with the teaching of Example 1 above except that the transfer plasmid
incorporates an
in-frame deletion mutant to eliminate amino acids 124-127 of the ElA 2898 and
2438
proteins.
~g~5. Construction of E1 d,A 43/08
The recombinant adenovirus E1A~43/08 is prepared in substantial accordance
with the teaching of Example 1 above except that the transfer plasmid
incorporates an
in-frame deletion mutant to eliminate amino acids 38-60 and amino acids 124-
127 of the
ElA 2898 and 2438 proteins.
Examg~e 6. Construction of E2F-ElAd101 X07
The recombinant adenovirus E2F-ElAd101/07 describes a virus where an E2F
pathway responsive promoter is used to drive expression of the modified Ela
coding
sequence. The base vector (ElAd101/07) is prepared in accordance with the
teaching of
Example 1 above. However, the transfer plasmid is modified to replace the Ela
promoter function with the E2F responsive promoter described in Parr, et al.
(1997,
Nature Medicine 3:1145-1149).
example ~, Construction of E2~~-ElAd101/07 n53CON-E2F-RB
The recombinant adenovims E2F-ElAd101/07 p53CON-E2F-RB is the E2F-
ElAd101/07 vector prepared in substantial accordance with the teaching of
Example 9
above and further comprises an expression cassette encoding a p53 pathway
responsive
-40-
CA 02345211 2001-04-04
WO 00/22136 PCT/US99/21451
promoter driving expression of an inhibitor of viral replication {E2F-Rb). The
expression cassette encoding a p53 pathway responsive promoter driving
expression of
an inhibitor of viral replication (E2F-Rb) is prepared as follows. A synthetic
DNA
sequence encoding a fusion protein comprising amino acids 95-286 of wild type
E2F
and amino acids 379-928 of the wild type Rb protein as described in European
Patent
Application No. 94108445.1 published December 6, 1995 (Publication number. 0
685
493 A1), with appropriate restriction sites is prepared by conventional
techniques. The
p53 pathway responsive promoter 53-CONis a synthetic p53 pathway responsive
promoter constructed by insertion of two synthetic p53 consensus DNA binding
sequences (as described in Funk, et al. (1992) Mol.Cell Biol. ,2:2866-2871)
upstream
of the SV40 TATA box. p53CON can be constructed by annealing compliementary
oligonucloeitides and p53 responsive promoters can be constructed by cloning
in
plasmid pGL3-promoter luciferase vector (commercially available from ProMega).
This sequence is introduced into the E3 region of the d1309 adenvirus by
homomogous
recombination.
Ex~;p~ , I~ a 8. E?F-ElAd101/07 -SRE-E2F-Rb
VectorE2F-ElAd101/07-SRE-E2F-Rb is prepared in substantial accordance
with the teaching of Example 10 above. However, in place of the p53 pathway
responsive promoter p53CON, a TGF-beta pathway respoonsive promoter (SRE) is
used to drive expression of the E2F-Rb fusion protein. SRE refers to a
synthe~ic
TGF-(3 response element comprising a repeat of 4 of the Smad-4 DNA binding
sequences (GTCTAGAC as described in Zawel, et al. (1988) Mol. Cell 1:611-617.
The SRE response element may be generated by annealing complimentary
oligonucleotides encoding the Smad-4 binding sequences and cloning in plasmid
pGL#3 - promoter luciferase vector (commercially availabe from ProMega).
-41 -
