Note: Descriptions are shown in the official language in which they were submitted.
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VIRAL VECTORS WITH LATE TRANSGENE EXPRESSION
BACKGROUND OF THE INVENTION
The genome of the adenovirus has been well characterized. This information
has been used to design recombinant adenoviruses capable of acting as vectors
for the
introduction of exogenous DNA into target cells. Many of these viral vectors
contain
modifications to the early gene products to endow the vectors with specific
activities.
For example, the early genes E1 and E2, are transcribed early after infection.
The
products of these genes are responsible for the suppressing the ability of the
infected
cell to respond to the infection and to promote viral replication in the host.
In particular, the immediate early gene Ela is transcribed rapidly following
infection. The Ela product can immortalize primary cells in vivo. Ela has also
been
show to bind a cellular protein p105 RB, the product of the retinoblastoma
gene. The
Elb gene is not capable of transforming cells on its own, but cooperates with
Ela to
stably transform cells. Both Ela and Elb are necessary for the full
transformation and
tumor formation in animals. The Ela gene has a variety of functions. The full
scope of
activity of the adenovirus Ela region is described in Bayley, S. and Mymryk,
J. (1994)
Intl. J. of Oncology 5:425-444.
The Elb genes are known to interact with host cell proteins. In particular two
proteins are produced by differential splicing of the Elb sequence, p19 and
p55. The
p55 protein has been well characterized as interacting with the p53 gene
product. p53 is
a well characterized protein and has been shown to activate programmed cell
death
(PCD) apoptotic pathways when present in sufficient intracellular
concentration. p53
induced apoptosis has been shown to take place in response to a wide variety
of cell
injury including radiation, DNA damaging agents, etc. By binding to p53, p55
prevents the formation of the active p53 phosphorylated tetramer. Since p53 is
sequestered by p55, a primary apoptotic pathway is never initiated and the
infected cell
undergoes uncontrolled replication in combination with Rb activity in response
to the
Ela 12S and 13S proteins.
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Ela and Elb cooperate in their activities. In particular Ela has been shown to
induce p53 dependent and p53 independent apoptosis. In addition to inducing
other
early viral genes, Ela drives quiescent cell into S-phase which is required
for viral
replication. It is believed that this early pressure to enter S-phase by the
virus on the
quiescent cell induces a apoptotic response from the cell. The adenovirus
responds by
the expression of the Elb SSK protein to bind to p53 and delay the induction
of '
apoptosis so that the virus is able to replicate prior to the death of its
host. Adenovirus
also produced the E1B19K protein which prevents or delays Ela mediated
apoptosis as
well.
Alternative to this type of selectively replicating vector is the employment
of a
replication deficient adenoviral vector containing extensive elimination of E1
function.
In particular, vectors containing elimination of El, E3 and partial E4
deletions have
been employed to delivery exogenous transgenes. Such vectors have been
employed 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.
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, by exploiting the natural ability to the virus to
propagate under
particular conditions enables these vectors to spread to surrounding tumor
cells. Since
the vectors themselves are able to replicate, a lower initial dose of such
vectors is
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required. This is favorable from an immunological perspective as well as for
economic
reasons in the manufacture of such agents.
In order to facilitate the understanding of the present invention, a brief
overview
of the life cycle of a typical virus used for delivery of exogenous
transgenes, the
adenovirus, is offered. The adenoviral replicative cycle in human cells can be
divided
into the early and late phase which are punctuated by the onset of viral DNA
replication.
The early phase beings when viral particles attach to cells through
interaction between
the virion fiber domain and cell surface receptors. The virion moves into the
cell by
either endocytosis or direct penetration of the cytoplasmic membrane and is
transported
to the nucleus where most of the capsid is shed. In the nucleus, the virion
core proteins
are removed yielding viral chromosomes that are almost entirely devoid of
virion
proteins. Expression of the viral genome is temporally coordinated and begins
with the
Ela region about one hour after infection. The other early genes Elb, E2, E3
and E4
are first express soon after EIa at the 1.5-2.0 hours post infection, A number
of the
protein products encoded by the early genes are required for viral DNA
replication,
while other prepare the DNA synthesis machinery of the infected cell for
efficient viral
DNA replication. Some early virally encoded proteins have been associated with
protecting infected cells from immune surveillance.
The late phase of infection with onset of DNA replication at about 7 hour post
infection. In the native adenovirus, the messenger RNAs for all late gene
products are
spliced from a primary RNA which is transcribed from the major late promoter
{MLP).
The MLP is located at position 16.5 on the r-strand. Although the major late
promoter
is active to a limited extent in the early phase of infection, the
transcription does not
proceed past map position 39. During the late phase of the viral life cycle,
the MLP is
fully activated and continues to map position 99. Each late primary RNA
transcript is
processed into one of five different mRNAs, L1-LS. These mRNAis all contain a
common tripartite leader sequence of 203 nucleotides. Late mRNAs encode capsid
components and proteins required for assembly of virions and packaging of the
viral
chromosome. Viral DNA replication requires the terminal protein for initiation
and
proceed by a semi-conservative mechanism. With the onset of replication,
efficient
transcription of the late gene families from the major late promoter begins
and attains a
maximal level approximately 18 hours post infection. During the late phase
viral
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proteins block cellular DNA and protein synthesis, presumably so that maximum
viral
macromolecular synthesis can occur. Intermediate gene expression, which
actually
begins during the early phase, reaches a maximum between 8 -12 hour post
infection.
Assembly of the virion and packaging of the viral genome begins at about 24
hours
after infection. Infected cells are killed because of attrition and lyre
yielding
approximately 10,000 virions per cell.
SUMMARY OF THE INVENTION
The present invention provides a conditionally replicating recombinant virus
containing a therapeutic transgene under control of a late regulatory element.
The
. invention further provides pharmaceutical formulations and methods of use of
same.
The present invention further provides recombinant producer cells capable of
complementing the packaging genes defective or deleted in the vector. The
present
invention also provides method of making such vectors and formulations.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is an autoradiogram of a time course study comparing the levels of
p53
expression of a replication competent MLP-p53 (AdSdElbMLPp53) virus in
comparison to the replication deficient p53 vector (ACN53) at two different
concentrations (1.8 x 108 particleslml (upper panel) and 1.8 x 109
particles/ml (lower
panel) in MRC9 cells according to the procedures of Example 2.a. herein. As
can be
seen from the data presented, the replication competent MLP-p53 construct
resulted in
expression later in time than the replication deficient CMV-p53 virus (ACN53).
The
data further illustrates that the levels of p53 expression produced from the
replication
competent MLP-p53 virus are significantly greater than the replication
deficient CMV-
p53 virus.
Figure 2 presents the results of an experiment substantially similar to that
presented~in Figure 1 except that the experiment was conducted in SK-HEPl
hepatocellular carcinoma cells according to the procedures of Example 2.b.
herein.
Again, the time course experiment demonstrates the temporal and greater
expression of
p53 in the replication competent MLP-p53 construct.
Figure 3 presents the results of an experiment substantially identical to that
presented in Figure 1 except that the experiment was conducted in NCI H358
breast
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cancer cells according to the procedures of Example 2.c. herein. Again, the
time course
experiment demonstrates the temporal expression of p53 in the replication
competent
MLP-p53 construct.
Figure 4 presents the results of an experiment similar to that presented in
Figure
3 except that a replication competent E1bd155k-CMV-p53 virus was included for
comparison and only a single dose was administered according to the procedures
of
Example 2.d. herein. This time course experiment demonstrates the temporal
expression of p53 in the replication competent MLP-p53 construct as compared
to the
substantially similar construct wherein the p53 gene was under control of the
constituitive CMV promoter. This data confirms that the MLP-p53 construct is
indeed
expressing the p53 in a temporal manner late in the viral replication cycle.
Figure 5 is a digest of viral DNA from SK-BR3 cells infected with a one hour
pulse of the indicated viruses at a concentration of I.8 x 109 particles/ml
and harvested
approximately 48 hours later according to the procedures of Example 2.d.
herein. The
results presented demonstrate that replication competent wild-type Ad5
(AdSWT),
replication competent E1Bd155K (ZAZA) virus and replication competent E1Bd155K-
MLP-p53 (SSK/1VB.P53) virus all replicate their viral DNA well while the
replication
deficient adenovirus control (rAdcon) and the replication deficient vector
encoding p53
(FTCB) does not.
Figure 6 is a graphical representation of the data obtained in vivo in a PC-3
mouse tumor model. Tumor volume is plotted on the vertical axis and days
following
administration is plotted on the horizontal axis. As can be seen from the data
presented,
the replicating virus ElBd,~55K-MLP-p53 (cFAMA) was able to produce tumor
regression in an in vivo mouse model of human cancer. The replication
competent
CMV driven p53 virus also replicates its viral DNA, but to a lesser extent.
pETAILED DESCRIPTION OF THE INVENTION
The present invention provides a replication competent recombinant virus
containing a therapeutic transgene operably linked to a late regulatory
element.
I Re~ication Comaetent Recombinant Virus
The term "replication competent" is made in reference to a virus which is
capable of replicating its genome and packaging the replicated viral genome
into
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infectious particles in mammalian cells. It should be noted that the term
replication
competent does not generally apply to virus that can only be grown in cells
which have
been modified to provide deleted viral functions L traps.
The term "recombinant virus" refers to any of the obligate intracellular
parasites
having no protein-synthesizing or energy-generating mechanism.capable of
infecting a
mammalian cell whose genomes have been modified by conventional recombinant
DNA
techniques. The viral genome may be RNA or DNA contained with a coated
structure
of protein of a Iipid membrane. The terms viruses) and viral vectors) are used
interchangeably herein. The viruses useful in the practice of the present
invention
include recombinantly modified enveloped or non-enveloped DNA and RNA viruses,
preferably selected from baculoviridiae, parvoviridiae, picornoviridiae,
herpesveridiae;
poxviridae, adenoviridiae, or picornaviridiae. Chimeric viral vectors which
exploit
advantageous elements of each of the parent vector properties (See e.g., Feng,
et
al.(1997) Nature Biotechnology x:866-870) may also be useful in the practice
of the
present invention. Minimal vector systems in which the viral backbone contains
only
the sequences need for packaging of the viral vector and may optionally
include a
transgene expression cassette may also be produced according to the practice
of the
present invention. 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,
equine
herpes virus vectors for human gene therapy are described in W098/27216
published
August 5, 1998. The vectors are described as useful for the treatment of
humans as the
equine virus is not pathogenic to humans. Similarly, ovine adenoviral vectors
may be
used in human gene therapy as they are claimed to avoid the antibodies against
the
human adenoviral vectors. Such vectors are described in WO 97/06826 published
April
10, 1997.
In the preferred practice of the invention, the virus is an adenovirus. The
term
"adenovirus" is synonomous with the term "adenoviral vector" and refers to
viruses
of the genus adenoviridiae. 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
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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 (AdI lA and Ad l IP),
12,
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, 41, 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
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 practice of the invention, the recombinant adenoviral vector
is
derived from genus adenoviridiae. Particularly preferred viruses are derived
from the
human adenovirus type 2 or type 5. In the preferred practice of the invention
as
exemplified herein, the preferred vector is derived from the human
adenoviridiae. More
preferred are vectors derived from human adenovirus subgroup C. Most preferred
are
adenoviral vectors derived from the human adenovirus serotypes 2 and 5. In the
most
preferred practice of the invention the virus is derived human adenovirus Type
5 41309.
41327, 41520 or wild-type adenovirus.
II. Late Regulator3r Element
The term "late regulatory element" refers to regulatory element which drives
transcription of the therapeutic transgene a point later in time than the
element which
induces initial viral replication. Characteristically, these promoter elements
are found
driving expression of packaging proteins and other proteins late in the viral
life cycle.
An example of such late regulatory element when the parent vector is
adenovirus is the
adenovirus major late promoter (MLP). Other viral vector systems also possess
late
temporally regulated promoters. For baculoviral vectors, the AcNPV basic gene
promoter and the polyhedrin gene promoters may be employed (Sridhar, et al.
(1993)
FEBS Lett. 315:282-286. For herpes simplex viruses, the Latent Activated
Promoters
may be employed. See, e.g. Rivera-Gonzalez, et al. (1994) Virology 202:550-564
and
Imbal, et al. (1992) J. Virol. 66:5453-5463. For human papilloma viruses, the
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HPV3Ib promoter may be employed (Ozbun and Meyers (1998) J. Virol. 72:2715-
22).
For parvoviruses, the P39 promoter. For vaccinia virus, the pl l promoter. In
the
preferred practice of the invention, the virus is derived from the genus
adenoviridiae
and the late regulatory element is the adenoviral Major Late Promoter. The
Major Late
Promoter is well characterized in the art and resides at approximately map
position 16.5
of the adenoviral Type 2 genome.
III. Onerably Linked:
The term "operably linked" refers to a linkage of polynucleotide elements in a
functional relationship. A nucleic acid sequence is "operably linked" when it
is placed
into a functional relationship with another nucleic acid sequence. For
instance, a
promoter or enhancer is operably linked to a coding sequence if it affects the
transcription of the coding sequence. Operably linked means that the
nucleotide
sequences being linked are typically contiguous. However, as enhancers
generally
function when seperated from the promoter by several kilobases and intronic
sequences
may be of variable lengths, some polynucleotide elements may be operabIy
linked but
not directly flanked and may even function in traps from a different allele or
chromosome.
IV. Therapeutic Tran~gene:
The term "therapeutic transgene" refers to a nucleotide sequence the
expression
of which in the target cell produces a therapeutic effect. The term
therapeutic transgene
includes but is not limited to tumor suppressor genes, antigenic genes,
cytotoxic genes,
cytostadc genes, pro-drug activating genes, apoptotic genes, pharmaceutical
genes or
anti-angiogenic genes. The vectors of the present invention may be used to
produce
one or more therapeutic transgenes, either in tandem through the use of IRFS
elements
or through independently regulated promoters.
A. Tumor Suppressor Gene
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
and/or inducing apoptosis. Examples of tumor suppressor genes useful in the
practice
of the present invention include the p53 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 .
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(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
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.
B . Antigenic Genes
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), p53 (as described in Levine, A. PCT
International
Publication No. W094/02I67 published February 3, 1994). In order to facilitate
immune recognition, the antigenic gene may be fused to the MHC class I
antigen.
C. C~rtotoxic Genes
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.
D. C~ostatic Genes
The term "cytostatic gene" refers to nucleotide sequence, the expression of
which in a cell produces an arrest in the cell cycle. Examples of such
cytostatic genes
include p21, the retinoblastoma gene, the E2F-Rb gene, genes encoding cyclin
dependent kinase inhibitors such as P16, p15, p18 and p19, the growth arrest
specific
homeobox (GAS gene as described in Branellec, et al . (PCT Publication
W097/16459 published May 9, 1997 and PCT Publication W096/30385 published
October 3, 1996) .
E. Cvtokine Gene
The term "cytokine gene" refers to a nucleotide sequence, the expression of
which in a cell produces a cytokine. Examples of such cytokines include GM-
CSF, the
interleukins, especially IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20,
interferons of the
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alpha, beta and gamma subtypes especially interferon a-2b and fusions such as
interferon a-2a-1.
F . Chemolgne Gene
The term "chemokine gene" refers to a nucleotide sequence, the expression of
which in a cell produces a cytokine. The term chemokine refers to a group of
structurally related low-molecular cytokines weight factors secreted by cells
are
structurally related having mitogenic, chemotactic or inflammatory activities.
They are
primarily cationic proteins of 70 to 100 amino acid residues that share four
conserved
cysteine residues. These proteins can be sorted into two groups based on the
spacing
of the two amino-terminal cysteines. In the first group, the two cysteines are
separated
by a single residue (C-x-C), while in the second group, they are adjacent (C-
C).
Examples of member of the'C-x-C' chemokines include but are not limited to
platelet
factor 4 (PF4), platelet basic protein (PBP), interleukin-8 (IL.-8), melanoma
growth
stimulatory activity protein (MGSA), macrophage inflammatory protein 2 (MIP-
2),
mouse Mig (m119), chicken 9E3 (or pCEF-4}, pig alveolar macrophage chemotactic
factors I and I (AMCF-I and -II), pre-B cell growth stimulating factor (PBSF),
and
IP10. Examples of members of the'C-C' group include but are not limited to
monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-
2),
monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-
4),
macrophage inflammatory protein 1 a (MIP-1-a), macrophage inflammatory protein
I
~i (MIP-1-(3}, macrophage inflammatory protein 1 y (MIP-1-y), macrophage
inflammatory protein 3-a (MIP-3-a, macrophage inflammatory protein 3 ~i (MIP-3-
(3),
chemokine (ELC), macrophage inflammatory protein 4 (M)P-4), macrophage
inflammatory protein 5 (MIP-5), LD78 Vii, RANTES, SIS-epsilon (p500), thymus
and
activation-regulated chemokine (TARC), eotaxin, I-309, human protein HCC-1/NCC-
2, human protein HCC-3, mouse protein C 10.
G Pharmaceutical Protein Genes
The term "pharmaceutical protein gene" refers to nucleotide sequence, the
expression of which results in the production of protein have pharmaceutically
effect in
the target cell. Examples of such pharmceutical genes include the proinsulin
gene and
analogs (as described in PCT International Patent Application No. W098/31397,
growth hormone gene, dopamine, serotonin, epidermal growth factor, GABA, ACTH,
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NGF, VEGF (to increase blood perfusion to target tissue, induce angiogenesis,
PCT
publication W098/32859 published July 30, 1998), thrombospondin etc.
H. Pro-ApQptotic Genes:
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 p16 gene is also apoptotic in Rb
positive,
p16 negative, p53 wild type tumors. (FrizelIe, et al . (1998) Oncogene 16:3087-
95 and
Sandig, et al. (1997) Nature Medicine 3:313)
~ I. Pro-Drug~Activating Genes:
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 (5-FC) to 5-fluorouracil (5-Fin, 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 necessity 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.
J. Anti-Angiogenic Genes:
The term "anti-angiogenic" genes refers to a nucleotide sequence, the
expression
of which results in the extracellular secretion of anti-angiogenic factors.
Anti-
angiogenesis factors include angiostatin, inhibitors of vascular endothelial
growth factor
(VEGF) such as Tie 2 (as described in PNAS(USA)(1998) 95:8795-8800), and
endostatin.
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K Modifications of Therapeutic Genes:
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.
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 reference to the p53 gene includes not only the wild type
protein but also modified p53 proteins, allelic variations thereof, or
proteins derived
from other mammalian species. The wild-type p53 sequence is well known in the
art.
Other mammalian p53 molecules are also known in the art and may be
incorporated into
the practice of the present invention such as marine p53, porcine p53, equine
p53,
bovine p53, canine p53, etc. The term "modified p53 proteins" refers to
modifications
to the primary, secondary, tertiary, or quaternary structure of the p53
protein which
retains the function of p53 proteins. Examples of such modified p53 proteins
include
modifications to p53 to increase nuclear retention, deletions such as the DI3-
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). Alternatively, the p53
sequence
may be modified to replace the endogenous tetramerization domains with a
leucine
zipper oligermization domain.
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 transciptionally active proteins (e.g. p53) as the
fusion
proteins are efficiently transported to the nuclei of the surrounding cells.
See,
e.g.ElIiott, G. & O'Hare, P. Cell. 88:223-233:1997; Marshall, A. & Castellino,
A.
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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.
V Additional Modifications to the Virus'
The present invention also provides recombinant adenoviruses containing
additional modifications to the viral genome such as targeting modifications,
modifications to make the vectors replicate selectively in particular cell
types or
phenotypic states, controlled expression characteristics, suicide genes or
additional
modifications to enhance cytotoxicity. However, this is not meant to imply
that other
modifications to the viral genome may not also be included or that multiple
such
modifications may not be employed.
A. Selectivel3r Replicating
The term "selectively replicating" refers to a recombinant viral vector
capable of
preferential replication in one cell type versus another cell type, in a cell
in one
phenotypic state relative to another phenotypic state, or in a given cell type
in response
to an external stimuli. Selective replication is achieved by the use of
replication control
elements. The term "replication control elements" refers to DNA sequences
inserted
into the viral genome or modifications to the viral genome in order to produce
recombinant viral vectors which selectively replicate in one cell type versus
another cell
type, in a cell in one phenotypic state relative to another phenotypic state
(cell state
specific), or in a given cell type in response to an external stimuli
(inducible).
Examples of such replication control elements include cell-type specific
promoter, cell
state specific promoters, and inducible promoters.
1 Cell Type Specific Promoters:
Cell type specific replication may be achieved by the linkage of a cell type
specific promoter to an early viral gene such as the El, Ela, E2 or E4 gene
when the
virus is selected from the adenovirus genome. 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 promoters or pathway
responsive
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promoters. In the preferred practice of the invention, the cell type specific
promoter is
linked to the E4 gene rather than the El gene because factors such as NF-IL6
can
substitute for Ela in regulating Ela responsive promoters in the adenovirus in
the
absence of Ela function (Spergel, et al. (1992) J. Virol. 66:1021-1030).
a. Cell Cycle Regulatory Gene 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}.
b. Tissue Specific Promoters:
Tissue specific promoters are well known in the art and include promoters
active
preferentially in smooth muscle (a-actin promoter), pancreas specific
(Palmiter, et
al.(1987} CeII 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 specif"rc
(United
States Patent 5,698,443, Henderson, et al. issued December 16, 1997}, etc.
c. Pathwa,~ponsive Promoters
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
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
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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.
i . Examples Of Pathway Promoters:
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-
27779), the cytokine pathway-responsive promoter, the glucocorticoid pathway-
responsive promoter (Lange, et al.(1992) J Biol. Chem. 267:15673-80), IL1 and
II,6
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 (TREs), TGF-(3 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
pathway-responsive promoters are well known in the an and can be identified in
the
Database of Transcription Regulatory Regions on Eukaryotic Genomes accessible
through the Internet at http:l/www.eimb.rssi.ru/TRRD.
ii. Preferred Pathway Promoters:
In the preferred practice of the invention as exemplified herein, the vector
comprises a synthetic TGF-~i 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-~i 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
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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
be generated by annealing complimentary oligonucleotides encoding the Smad-4
binding sequences and cloning in plasmid pGL#3 - promoter luciferase vector
(commercially available from ProMega).
1, p53 Pathway Promoters:
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 may be 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 p53 response element constructed by
insertion of two synthetic p53 consensus DNA binding sequences (as described
in
Funk, et al. (1992) Mol.Cell. Biol.12_: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
responsive promoters can be constructed by cloning in plasmid pGL3-promoter
luciferase vector (commercially available from ProMega).
d. Tissue Specific Promoters onerably linked to an earl~gene.
Tissue specific promoters are well known in the art and include promoters
active
preferentially in smooth muscle (alpha-actin promoter), pancreas specific
(Palmiter, et
al. (1987) Cell 50:435), liver specific Rovet, et.al. (1992) J. Bial. 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 and prostate
specific
antigen promoter.
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2. Cell State Specific:
Examples of different phenotypic states would include the neoplastic phenotype
versus a normal phenotype in a given cell type. Selective replication is
achieved by the
use of viral replication control elements. The term viral replication control
element
refers to a DNA sequence engineered into the vector of the present invention
such that
the virus is preferentially enabled to replicate the viral genome in a
particular type of
target cell. Again, these cell state specific promoters may be linked to an
early gene
such as E1, E2, or preferably E4 to achieve selective replication in response
to specific
phenotypic states.
a. Tumor Specific:
In order to achieve expression of the adenovirus in tumor cells, one may
employ
a tumor specific promoter to drive expression of an early gene. The term
"tumor
specific promoters" refers to promoters which are active in tumor cells and
inactive in
cells which are not transformed. Examples of tumor specific promoters include
the
alpha-fetoprotein promoter, the tyrosinase promoter. The use of tumor specific
promoters to achieve conditional replication of adenoviral vectors is
described in co-
pending United States Patent Application 08/433,798 filed May 3, 1995 and
International Patent Application No. PCT/LTS96/06199 published as
International
Publication No. WO 96/34969 on November 7, 1996 the entire teaching of which
is
herein incorporated by reference.
For example, the alpha-fetoprotein promoter could be used to replace the
endogenous E4 promoter and achieve greater selectivity in conditional
replication .
Other factors such as NF-IL6 can substitute for Ela in regulating Ela
responsive
promoters in the adenovirus in the absence of Ela function (Spergel, et al.
(1992) J.
Viroll 66:1021-1030) and this can be avoided by substitution of the E4
promoter with a
tumor specific promoter.
b. Repressor of Viral Replication:
Although one may use the pathway responsive promoter to drive replication of
the virus in the presence of a functional pathway, alternatively, the one may
use a
pathway responsive promoter to drive expression of a repressor of viral
replication to
control expression. The term "repressor of viral replication" refers to a
protein, if
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expressed in a given cell, substantially represses viral replication. As will
be
appreciated by those of skill in the art, the repressor of viral replication
will be
dependent on the nature of the parent adenoviral vector from which the
recombinant
vector of the present invention is derived. For example, in the case of
adenoviral
vectors or other DNA tumor viruses, 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 heterodimerizations domains of the human
E2F
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-dependent transcription 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 E2F-1
protein
is available from GenBank under accession number M96577 deposited August 10,
1992. The sequences of E2F from other E2F family members of E2F from other
species may be employed when constructing a vector for use in other species.
In the
situation where the recombinant virus is based on adenoassociated virus, the
rep protein
and its derivates is an effective repressor of viral replication in the
absence of
adenovirus infection. In the situation where the virus is derived from herpes
simplex
virus, the ICPO-NX, a deleted form of the immediate early protein ICPO (Liun,
et al.
(1998) J. Virol. 2:7785-7795), protein may be used as an effective repressor
of viral
replication. Similarly, any protein with dominant negative activity can be
used as a
repressor of viral replication.
These modifications may be combined with the 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
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.
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3. Inducible Promoters
The term "inducible promoter" refers to promoters which facilitate
transcription of the therapeutic transgene preferable (or solely) under
certain conditions
and/or in response to external 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)
MoI. Cell. Biol. 17(9):5097-5105; and Dreher, et al . (1997) J. Biol. Chem.
272(46);
29364-29371. Examples of radiation inducible promoters include those induced
by
ionizing radiation such as the Egr-1 promoter (as described in Manome, et al.
(1998)
Human Gene Therapy 9:1409-17; Takahashi, et al . (1997) Human Gene Therapy
8:827-833; Joki. et al. Human Gene Therapy (1995) 6:1507-1513; Boothman, et
al.
(1994) volume 138 supplement pages S68-S71; and Ohno, T (1995) Tanpakushitsu
Kakusan Koso 40:2624-2630), X-ray inducible promoters such as the XRE promoter
(as descrbed in Boothman, et al . (1994) Radiation Research 138(Suppl.l):S68-
S71),
and UV inducible promoters such as those isolated from Clostridium perfringens
(Gamier and Cole (1988) Mol. Microbiol. 2:607-614.
B Alternative Modifications to the Viral C',~enome~
1. E1Bd155K deletion:
As previously indicated the Elb 55K protein binds to p53. Consequently, in
order to enhance the effect of the p53 introduced by the viral vector it is
preferred to
introduce Descriptions of E1Bd155K mutations to eliminate p53 binding
described in
McCormick, United States Patent No. 5,677,178 issued October 14, 1997, the
entire
teaching of which is herein incorporated by reference.
In the preferred practice of the invention as exemplified herein, the vines is
a
recombinant adenoviral vector encoding p53 under control of the MLP promoter
containing a deletion of nucleotides 2247-3272 of the adenoviral genome to
eliminate
the function of the Elb 55K protein.
2. E4 Modifications
Addtionally modfications to increase the potency of the vectors of the present
invention include but are not limited to alterations within Elb. The vectors
of the
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present invention may be modified to introduce mutations in E4 to increase the
cytotoxicity (Muller, et al. (1992) J. Virol. 66:5867-5878) or contain
upregulation of
viral cytopathic proteins such as E4orf4 or E3 11.6K proteins. For example,
the E4
region of the adenovirus genome has been implicated in viral DNA replication,
host
S protein synthesis shut off and viral assembly. E4orf6 is sufficient for DNA
replication and late protein synthesis in immortalized cells. However,
E4orf6/7
appears to be required for replication in non-dividing cells. Consequently
elimination
of E4orf6/7 assists in restricting the replication of the virus in
immortalized (i.e. tumor
cells) and may be incorporated into the vectors of the present invention.
Furthermore, E4 deletions have been shown to reduce the immongenicity of
the vectors (Wang, et al. (1997) Gene Therapy 4:393-400; Dedieu, et al. (1997)
J.
Virol 71:4626-37), but can affect the persistence of transgene expression
depending on
the open reading frames of E4 retained and the promoter used to drive
expresion of the
transgene (Armentano, et al. (1997) J. Virol 71:2408-2416). The E4 region also
encodes a protein (E4orf6) capable of binding to and inactivating the
transciptional
activity of p53 (Dubner, et al. (1996) Science 272:1470-73). Therefore, it may
be
desirable to modify the E4 region to delete those open reading frames with
undesirable
properties for the particular virus construct while retaining those with
desired
properties. For example, for the conditionally replication virus described
herein as the
E1Bd155K-MLP-p53, the E4 orf6 region may be deleted to reduce inactivation of
p53
while retaining E4orf3 to allow continued expression of p53 and replication of
the
virus. In order to preserve replication competency of the virus, either E4orf6
or
E4orf3 should be retained.
Examples of other E4 deleted adenoviral vectors are described in Gregory, et
al.
United States Patent No 5,670,488 issued September 23, 1997.
3 E1 deletions To Achieve Selective Replication in RapidlX
Dividin~~Cells
In order to achieved selective replication of adenovirus in rapidly dividing
cells,
the adenovirus may contain 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
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approximately 219 to approximately 289 of the Ela 2898 protein (or
approximately
amino acids 173 to approximately amino acid 243 of the Ela 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
eliminate of amino acids 124-127 of the Ela 2438 and 2898 proteins.
4. E3 Modifications:
The E3 region of the adenovirus encodes proteins which help adenovirally
infected cells avoid clearance by the immune system (Wold, et al. (1995) Curr.
Top.
Microbiol. Immunol. 199:237-274). Upregulation of this region and subfragments
thereof has been shown to prevent or decrease the immune response to virally
infected
cells, leading to longer term gene expression. (Ilan, et al. (I997) PNAS
94:2587:2592,
Bunder, et al. (1997) J. Virol. 71:7623-28). Therefore, modifications to the
E3 region
(or sub-components thereof) to overexpress their proteins (e.g. by
upregulating the E3
region using a strong constituitive promoter such as CMV) may be desirable to
allow
for a greater degree of viral replication due to its ability to avoid or delay
the immune
mediated clearance of infected cells.
B . Tarl;etin Mg-odificarions:
The present invention provides recombinant viruses 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 viruses
having
characteristically broad infectivities such as adenovinis 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(11):8221-8229 (incorporation of RGD
peptides into
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adenoviral fiber proteins); Arnberg, 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-
4790; 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).
VI. Experimental Results:
A. Vector Constructs:
The ElBd~55K-MLP-p53 (FAMA) vector was prepared in substantial
accordance with the teaching of Example 1 herein. Briefly, the adenovirus
genome was
modified by introducing a deletion of base pairs 2247 to 3272 of the
adenovirus
genome so that the ability of the 55K protein is unable to bind to an inhibit
the
transcriptional activity of p53. In substitution of this deletion was placed a
polyA signal
from adenovirus protein IX for the E1b19K upstream of the deletion and the
cDNA
encoding the Ad2 major late promoter and tripartite leader sequence driving
expression
of p53 from the plasmid pA/M/53 as describe in Wills, et al . (1994) Human
Gene
Therapy 5:1079-88. The remainder of the virus sequence is wild-type AdS.
B . In vitro Experiments:
The following experiments were performed to demonstrate that the major late
promoter functioned in a temporal manner in expressing p53 from a replicating
virus.
Experiments were performed in substantial accordance with the teaching of
Example 2
herein in order to evaluate the time course expression of p53 in various tumor
cell lines.
The data is presented in Figure 1-4 of the attached drawings. The data
presented
demonstrates the temporal expression of the cFAMA vector in a variety of tumor
cell
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lines with varying genotype as well as increased expression levels relative to
replication
deficient viruses expressing p53 (ACN53).
In one experiment, the expression of p53 from the FAMA vector was compared
to a substantially similar vector wherein p53 was operably linked to the CMV
constituitive promoter. The experiment was conducted in substantial accordance
with
the teaching of Example 2.c. herein and the results are presented in Figure 4
of the
attached drawings. his demonstrates that the FAMA virus does replicate in a
temporal
manner compared with the CMV containing FAIC virus.
In an additional expreiment conducted in accordance with Example 2.d., viral
replication from FAMA was compared to the substantially similar FAIC vector
where
the temporal major late promoter was replaced by the constituitive CMV
promoter. By
expressing the p53 protein from the MLP promoter in a temporal manner, a
greater
replication of the virus is achieved relative to a constitutive promoter. The
results are
presented in Figure 5 of the attached drawings.
C . In vivo Experiments:
In order to confirm the efficacy of the vectors of the present invention in
vivo in
an animal, the vectors of the present invention were evaluated in a mouse
human
prostate cancer model. The model and experiments were peformed in substantial
accordance with the teaching of Example 3 herein. The data is presented in
Table 1
below and in Figure 6 of the attached drawings.
Table 1. Tumor
Volume Following
In vivo Administration
of FAMA
Avg. Tumor Volume # Animals
V~ (mm') t SD' % T/CZ Tumor Free
Saline control 1266 t 403 100 0/6
Wt Ad 5 16 1.3 5/6
c~ 39 13 3.1 3/6
cFAMA 25 2.0 5/6
cFAIC 16 1.3 5/6
FTCB 215 t 93 17 0/6
~B 702 t 109 55 0/6
Day 27 Post Initiation of Treatment
2 Treated/saline control
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As can be seen from the data presented, the vectors of the present invention
demonstrated antitumor efficacy in vivo. It should be noted that no only did
the tumors
fail to grow, but actually regressed in size such that 5 of 6 animals were
tumor free 27
days post adnunistration. This is particularly surprising in light of the non-
replicating
p53 vector FTCB in which p53 is also expressed from the CMV promoter. Also,
the
effect is not due to merely to the Elb55k deletion (as represented by cZAZA)
which
showed lower efficacy at 27 days post administration.
VII. Pharmaceutical Formulations:
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 Garners and
excipients.
Dosage formulations may include intravenous, intratumoral, intramuscular,
intraperitoneal, topical, matrix or aerosol delivery.
A. Garners:
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 carrier, preferably an aqueous carrier. 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.
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B Delivery Enhancing Agents;
The present invention further provides pharmaceutical formulations of the
vectors of recombinant adenoviruses of the present invention with a carrier
and 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, lysophosphatidylcholine, polyethyleneglycol 400,
polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO.
Bile
salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate,
chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other
astringents 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
(NSAlDS)
like indomethacin, naproxen, diclofenac may be used.
Delivery-enhancing agents includes compounds of the Formula I:
0
X~-IC N-(CHZ)m N-(CHZ)~ N-R
C=O
X2
wherein n is an integer from 2-8, X1 is a cholic acid group or deoxycholic
acid group,
and X2 and X3 are each independently selected from the group consisting of a
cholic
acid group, a deoxycholic acid group, and a saccharide group. At least one of
X2 and
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X3 is a saccharide group. The saccharide group may be selected from the group
consisting of pentose monosaccharide groups, hexose monosaccharide groups,
pentose-pentose disaccharide groups, hexose-hexose disaccharide groups,
pentose-
hexose disaccharide groups, and hexose-pentose disaccharide groups.
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 form
and a
solution for reconstitution of the lyophilized product along with instructions
for use.
Recombinant adenoviruses of the present invention may be lyophilized by
conventional
procedures and reconstituted.
Another example of a delivery enhancing agent which may be employed in the
formulations of present invention include calpain inhibitors. The "calpain
inhibitor"
(abbreviated "Cr') refers to a compound which inhibits the proteolytic action
of calpain-
I, e.g. p.-calpains. The term calpain inhibitors as used herein includes those
compounds having calpain I inhibitory activity in addition to or independent
of their
other biological activities. A wide variety of compounds have been
demonstrated to
have activity in inhibiting the proteolytic action of calpains. Examples of
calpain
inhibitors are useful in the practice of the present invention include N-
acetyl-leu-leu-
norleucinal also known as "calpain inhibitor 1." Additional calpain inhibitors
are
described in the following United States Patents, herein incorporated by
reference,
United States Patent No. 5,716,980 entitled Alcohol or aldehyde derivatives
and their
use; United States Patent No. 5,714,471 entitled Peptide and peptide analog
protease
inhibitors; United States Patent No. 5,693,617 entitled Inhibitors of the 26s
proteolytic
complex and the 20s proteasome contained therein; United States Patent No.
5,691,368 entitled Substituted oxazolidine calpain and/or cathepsin B
inhibitors;
United States Patent No. 5,679,680 entitled.a: substituted hydrazides having
calpain
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inhibitory activity; United States Patent No. 5,663,294 entitled Calpain-
inhibiting
peptide analogs of the kininogen heavy chain; United States Patent No.
5,661,150
entitled Drug for neuroprotection; United States Patent No. 5,658,906 entitled
Cysteine
protease and serine protease inhibitors; United States Patent No. 5,654,146
entitled
Human ice homolog; United States Patent No. 5,639,783 entitled Ketone
derivatives;
United States Patent No. 5,635,178 entitled Inhibition of complement mediated
inflammatory response using monoclonal antibodies specific for a component
forming
the C56-9 complex which inhibit the platelet or endothelial cell activating
function of the
C56-9 complex; United States Patent No. 5,629,165 Neural calcium-activated
neutral
proteinase inhibitors; United States Patent No. 5,622,981 entitled Use of
metabotropic
receptor agonists in progressive neurodegenerative diseases; United States
Patent No.
5,622,967 entitled Quinolone carboxamide Calpain inhibitors; United States
Patent No.
5,621,101 entitled Protein kinase inhibitors for treatment of neurological
disorders;
United States Patent No: 5,554,767 entitled Alpha-mercaptoacrylic acid
derivatives
having calpain inhibitory activity; United States Patent No. 5,550,108
entitled
Inhibition of complement mediated inflammatory response; United States Patent
No.
5,541,290 entitled Optically pure calpain inhibitor compounds; United States
Patent
No. 5,506,243 entitled Sulfonamide derivatives; United States Patent No.
5,498,728
entitled Derivatives of L-tryptophanal and their use as medicinals; United
States Patent
No. 5,498,616 entitled Cysteine protease and serine protease inhibitors;
United States
Patent No. 5,461,146 entitled Selected protein kinase inhibitors for the
treatment of
neurological disorders; United States Patent No. 5,444,042 entitled Method of
treatment of neurodegeneration with calpain inhibitors; United States Patent
No.
5,424,325 entitled aminoketone derivatives; United States Patent No. 5,422,359
entitled a-aminoketone derivatives; United States Patent No. 5,416,117
entitled
Cyclopropenone derivatives; United States Patent No. 5,395,958 entitled
Cyclopropene derivatives; United States Patent No. 5,340,922 entitled Neural
calcium-
activated neutral proteinase inhibitors; United States Patent No. 5,336,783
entitled
Calpain inhibitor cystamidin A and its production; United States Patent No.
5,328,909
entitled Cyclopropenone derivatives; and United States Patent No. 5,135,916
entitled
Inhibition of complement mediated inflammatory response. The uses of calpain
inhibitors in gene therapy protocols is further described in Atencio, et al.,
co-pending
United States Patent Applications Serial Nos. 09/172,685 and 60/104,321 filed
October
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15, 1998. In the preferred practice of the invention, the calpain inhibitor is
N-acetyl-
leu-leu-norcinal (calpain inhibitor-1) commercially available from Boehringer-
Mannheim (Indianapolis, IN).
IIX. Therapeutic Applications:
A. Anti-Neo lash tic Applications:
The present invention provides a method of eliminating neoplastic cells in a
mammalian organism by the administration of a recombinant viral vector of the
present
invention to a neoplastic cell. 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 referred to as cancers. The term cancer is used
interchangeably
herein with the term tumor. Neoplastic transformation refers the conversion of
a
normal cell into a neoplastic cell, often a tumor cell. The term "mammalian
organism"
includes, but is not limited to, humans, pigs, horses, cattle, dogs, cats.
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 described above. The term "ablating"
means
the substantial reduction of the population of viable neoplastic cells so as
to alleviate the
physiological maladictions 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% 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
hereing 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 of 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.
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In the preferred practice of the invention as exemplified herein, a
recombinant
adenovirus containing a deletion of the E1B-55K gene function and expressing a
tumor
suppressor gene from the adenoviral major late promoter is formulated with a
pharmaceutically acceptable carrier for administration by intravenous,
intraperitoneal, or
intratumor injection. The appropriate dose and method of administration of the
vector
to be administered to the mammalian organism in need of treatment will be
determined
by the skilled artisan taking into account the extent of metastatis of the
primary tumor,
the delivery enhancer(s) included in the formulation, the extent to which the
immunological response is suppressed, etc. Each of these latter factors will
decrease
the dosage of the vector provided to the mammalian organism in need of
treatment. In
the preferred practice of the invention, a dosage of approximately 1 x 105 to
1 x 10'3
particles (preferably 1 x 106 to 1 x 10" particles, most preferably x 107 to 1
x 10'°
particles) will be administered to the mammalian organism in one or more
dosages in a
treatment regimen. The typical course of treatment will be the daily
administration of a
pharmaceutically acceptable formulation of the vector of the present invention
over a
period of three to ten days, preferably five to eight days. In a preferred
embodiment,
the tumor suppressor gene is the wild-type p53 gene. In another preferred
embodiment, the tumor suppressor gene encodes is a VP22-p53 fusion protein.
In a further preferred practice of the invention, the pharmaceutically
acceptable
Garner contains a delivery enhancing agent. In a further preferred practice of
the
invention, the delivery enhancing agent is a calpain inhibitor. In the most
preferred
practice of the invention as exemplified herein, the recombinant adenoviral
vector E1B-
d155K-MLP-p53 is formulated in a Garner solution further comprising the
calpain
inhibitor n-acetyl-leu-leu-norcinal (calpain inhibitor 1) at a concentration
of from
approximately 1 to 50 micromolar. In such instances, the daily dosage may be
reduced
as compared to a formulation absent such delivery enhancing agents by a factor
of one
to two logs.
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
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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,
6thioguanine, 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) and farnesyl protein
transferase inhibitors. Chemotherapeutic treatment regimens refers primarily
to non-
chemical procedures designed to ablate neoplastic cells such as radiation
therapy. In
such instances, the daily dosage in the course of treatment is reduced in
comparison to
those dosages provided absent such chemotherapeutic agents.
It has been observed that the immune system is capable of recognizing and
eliminating recombinant viral vectors. As this would effectively reduce the
amount of
adenovirus reaching the target cell, it is preferable in many instances to
administer the
compounds of the present invention in combination with immunosuppressive
agents
such as etoposide. In the preferred practice of the invention, the
immunosuppressive
agent is administered in advance, preferably for about a week in advance of
the
introduction of the recombinant viral vector of the present invention to
eliminate the
humoral immune response to the viral particles. In the preferred practice of
the
invention, a pharmaceutically acceptable formulation of the vector of the
present
invention is administered intratumorally following the administration of a
immunosuppressive agent for a period of from about one day to about two weeks
in
advance of administration of the vector. The vector is preferably an
adenoviral vector
and further contains a deletion of the E1B-55K protein and contains an
expression
cassette expressing the p53 tumor suppressor gene from an adenoviral major
late
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promoter. In the preferred practice of the invention, the immunosuppressive
agent is
etoposide and is administered daily for a period of from about 1 to 7 days
(preferably 3-
7 days) prior to administration of the vector. In such instances, the daily
dosage in the
course of treatment is reduced in comparison to those dosages provided absent
such
immunosuppressive agents.
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 functionvof 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 COBE International, 1185 Oak
Street,
Lakewood, CO. It is preferred that treatment conditions be optimized to
achieve a "3-
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 approximatley 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 at a concentration of from
about
1xI06 to 1x10'° particles/ml of the recombinant adenovirus of the
present invention for
a period of approximately 4 hours at 37°C.
B . Recruitment of Dendritic Cells:
The present invention provides a recombinant viral vectors capable of
recruiting
immature dendritic cells to a tumor site and exposing the dendritic cells to a
localized
high concentration of tumor antigens characteristic of the tumor present in
the patient.
The vectors of the present invention are specifically engineered to induce
killing of
tumor cells. The lysed tumor cell (or the apoptotic bodies produced by an
apoptosed
tumor cell) provides a rich localized concentration of tumor specific
proteins. By
introducing a gene encoding a dendritic cell chemoattractant, immature
dendritic cells
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capable engulfing tumor antigens are recrutited to the site of the lysed tumor
cells
thereby engulfing tumor antigents and presenting these antigens to the immune
system.
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 formyl
peptides of
bacterial origin), C5a and the C-C chemokines monocyte chemotactic protein
(MCP)-3,
macrophage inflammatory protein (MIP)-1 alphalLD78, and RANTES, have been
involved in the recruitment and chemotactic migration of dendritic cells.
Sozzani, et al .
(1995) J. Immunol. 1995 155(7):3292-5. Xu, et al. suggest that all C-C
chemokines,
including MCP-1, MCP-2, MCP-3, MIP1 alpha, MIP-1 beta, and RANTES, induced
migration of DC-enriched cells cultured with or without IL-4. Xu, et al.
(1996) J.
Leukoc. Biol. 60(3):365-71. Greaves, et al . (1997) J. Exp. Med. 186(6):837-
44,
indicate that MIP-3-alpha 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 dendritic cell chemoattractant is MIP-
3-alpha.
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.
Upon
expression of the dendritic cell chemoattractant, the dendritic cells then
engulf the tumor
antigens or apoptotic bodies, mature and migrate through existing pathways to
the
lymph and present the tumor antigens to the T-cells. The resulting T-cells are
then
capable of recognizing and killing tumor cells. The appropriate dosage regimen
in such
instances is in accordance with the typical anti-neoplastic dosage formulation
and
regimen as described above.
IX. Diagnostic Applications
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 in place of the
therapeutic gene
which is expressed upon viral replication. The term "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 beta-
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
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systems (MRI). Alternatively, such vectors may also be employed to express a
cell
surface protein capable of recognition by a binding molecule such as a
fluorescently
labelled antibody. Examples of in vivo applications include imaging
applications such
as X-ray, CT scans or Magnetic Resonance Imaging (MRI).
X Method of Making The Comnositions~
The present invention further provides a method of producing the recombinant
adenovirus comprising the modifications to packaging domains described above,
said
method comprising the steps of:
a. infecting a producer cell with a recombinant virus;
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 adenovin,~s to the
1 S producer cell under conditions so as to facilitate the infection of the
producer cell with
the recombinant virus. 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
example 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).
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The term "producer cell" means a cell capable of facilitating the replication
of the
viral genome of the recombinant adenovirus to be produced and capable of
complementing the packaging defects of the recombinant adenovirus. 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 Elfunction and is a
preferred cell line
for the production of the current vectors. In a similar manner, cell lines may
be
developed incorporating viral sequences stably integrated into the viral
genome. For
example, Cunningham and Davidson ((1997, Virol 197:116-I24) demonstrate that
overlapping cosmids may be used to complement deleted viral functions for
herpes viral
vectors. A similar approach may be employed to complement adenoviral and other
viral
elements. Additional genes, such as those encoding drug resistance, can be
included to
allow selection or screening for the presence of the recombinant complementing
vector.
Such additional genes can include, for example, genes encoding neomycin
resistance,
multi-drug resistance, thymidine kinase, beta-galactosidase, dihydrofolate
reductase
(DHFR), and chloramphenicol acetyl transferase. Examples of other producer
cells
parent cell lines which may be employed include HeLa cells, PERC.6 cells (as
described in publication WO/97/00326, application serial No. PCT/NL96/00244
and
the A549-E1 cell line (as described in International Patent Application No.
PCT/US97/8I0039 published February 23, 1998 as International Publication No.
W098/US3473.
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 e~cient 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 the virus, type of bioreactor, etc. When 293 cells are
employed as
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the producer cell line, oxygen concentration is preferably maintained from
approximately 50% to approximately 120% dissolved oxygen, preferably 100%
dissolved oxygen. 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.
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 DNAselRNAse.
The term "purifying" means the isolation of a substanially 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 C: 1403-1416 as described in co-
pending
United States Patent application Serial No. 08/400,793 filed March 7, 1995.
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.
The purified virus is then admixed with appropriate excipients and carriers or
delivery enhancing agents. The solution is sterilized for individual packaging
and
vialed for storage.
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Alternatively, the virus may be lyphophilized for storage and reconsituted in
a
solution containing delivery enhancing agents, buffers, preservatives,
cryoprotectants
and/or carriers. The lyophilized virus and the reconsitution solution may be
packaged
together as a kit for consumption by the end user along with instructions for
appropriate
handling and administration.
EXAMPLES
As will be apparent to those skilled 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 the 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. The scope of the present
invention is as set
forth in the appended claims rather than being limited to the examples
provided below.
Example 1. Construction of E1Bd155K-MLP- 53 cFAMA)
The E1B55K-MLP-p53 (cFAMA) adenovirus was prepared using the
oligonucleotide site directed mutatgenesis technique of Deng and Nickoloff
(1992)
Anal. Biochem ~, 81-88. All of the reagents, bacterial strains,.and vectors
used for
mutagenesis were provided in the Transformer Site-Directed mutagenesis kit
(commercially availble from Clontech, Palo Alto, CA). The Ad5 region
containing the
sequence to be mutated was inserted into Clontech plasmid pEGFP-1 and named
pXB-
ElB. Two primers were designed to anneal to: (1} Ad5 sequence 2236 to 2260 and
modify 62247 to thymidine and T2248 to cytosine and (2) Ad5 sequence 3255 to
3284
and modify T3272 to cytosine. A third primer was designed to eliminate the
Hindi
site within the original vector sequence, and provide a method for selection.
For the mutagenesis reaction, the mutagenic oligonucleotides were first
phosphorylated at the 5' end, and then annealed to denatured pXB-E1B 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 mutant strand. The complementary strand synthesis reaction was
then
transfomed into the bacterial strain, BMH 71-18 mutS. This bacterial strain is
defective
for mis-match repair, preventing undesired repair of the mutant strand. The
transformants were digested with HindlII to cut any parental plasmid strands,
and
retransformed into DHSa for amplification. Potential E1B mutants were then
screened
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by restriction enzyme analysis to confirm the desired mutations.
This procedure was used to introduce restriction enzyme nuclease cleavage
sites
in the E1B 55K coding region. The first site was introduced by modifying
positions
2247 and 2248 of the wild type Ad5 genome wherein a guanine' was replaced with
a
thymidine and thymidine2'''s 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 at position
3272 wherein
thymidine3an was replaced with cytosine site (silent mutation) to introduce an
XhoI
site. The new restriction enzyme sites were used in a restriction enzyme
digest with
EcoRI and Xhol.
A cassette containing the p53 coding sequence, under control of the adenovirus
Major Late Promoter and tripartite leader sequence, was removed by EcoRl and
partial
Xhol digestion from the plasmid, pAd-MLP-p53. This plasmid is based on the
pBR322 derivative pML2 (pBR322 deleted for base pairs 1140-2490) and contains
an
adenovirus type 5 sequences extending from base pair 1 to 5788 except that it
is deleted
for adenovirus type 5 base paris 357-3327. At the site the Ad5 357-3327
deletion, a
transciption unit is inserted iwhich is comprised of the adenovirus type 2
major late
promoter, the adenovirus type 2 tripartite leader DNA and human p53 cDNA. This
EcoRI/Xhol fragment is inserted into the EcoRl and Xhol sites introduced into
the
E1B55k coding region. The polyA sequence that follows wtAdS pIX was amplified
by
PCR (Ad5 sequence 4001-4368). The primers used for amplification included
sequences to introduce an EcoRl site at the 5' end of the Ad5 sequence, and a
SacII site
at the 3' end of the Ad5 sequence. This fragment was then inserted into the
EcoRl-
SacII sites immediately following the E1B 19k coding sequence. The SacII site
was
included in the MLP-p53 cassette inserted previously, and was upstream of the
MLP
promoter sequence. The resulting E1B mutation results in a sequence encoding
the first
76 amino acids of the E1B55K protein followed by 11 missense amino acids
resulting
in a non-functional deleted EIB protein.
Construction of the E1Bd155K-MLP-p53 adenovirus was carried out by using
homologous recombination in the adenovirus El-region containing 293 cell line
by the
method of McGory, et al., (1988) Virology 163, 614-617. This method requires
two
fragments of DNA, one a transfer plasmid containing the E1B55k deleted/NIL,P-
p53
cassette and the other Ad5 viral DNA containing the wtAdS genome ("Ad5 large
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fragment"), from Ad5 bp918 to the 3' ITR (bp 35935). The transfer plasmid
used,
pXCl-E1B55-2A, contains wtAdS sequences from 22-2246. For recombination to
produce adenovirus cFAMA, the viral large fragment and pXCl-E1B53-2A were
cotransfected into 293 cells by calcium phosphate mediated transfection. After
5 hours
the precipitate was rinsed from the cells and normal media replaced. At 15
days after
the initial transfectian, viral "comets" 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.
Exaynle 2. In vitro Evaluation of E1Bd155K-MLP-n53 (FAMA~
Example 2.a. p53 Expression MRC9 Cells
Levels of p53 expression from two different viral constructs (E1Bd155K-MLP-
p53 (cFAMA) and ACN53 (Wills, et al. (1994) Human Gene Therapy, supra)) were
evaluated in MRC9. Six well plates were seeded with 6 x 105 MRC9 cells per
well.
Infection was performed for a one hour pulse in a volume of 250 microliters at
two
different concentrations, 1.8x108 particles/ml and 1.8x109 particles/ml. The
cells were
harvested and assayed using a BioRad Catalog Number 500-0006 Kit in
substantial
accordance with the instructions provided by the manufacturer. The cells were
harvested by scraping at 24; 48, and 72 hours as indicated. The cells were
resuspended
in lysis buffer (50mM Tris, 250 mM NaCI, 0.1% NP40, 50 mM NaF, 5 mM EDTA)
and lysed by the freeze thaw method (repeated three times). The cells were
stained with
the BioRad dye reagent concentrate provided with the kit and the absorbance
was
determined at a wavelength of 595 nm. The primary antibody was a marine anti-
p53
antibody (commercially available from Novacastra as catalog number 1801
diluted
1:1000). The secondary antibody was Goat-anti-Mouse HRP (commercially
available
from Amersham as catalog number NA931). The data is presented in Figure 1 of
the
attached drawings. As can be seen from the data presented, the replication
competent
MLP-p53 constrict resulted in expression later in time than the replication
deficient
CMV-p53 virus (ACN53).
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Example 2.b. p53 Expression SK-HEP1 Cells
This evaluation was performed in substantial accordance with the teachining of
Example 2.a above except that the experiment was conducted in SK-HEP1
hepatocellular carcinoma cells and the six well plate was seeded with 7.5 x
105 cells per
well. The results are presented in Figure 2 of the attached drawings.
Exam; 2.c. p53 Expression NCI H358 Cells
This evaluation was performed in substantial accordance with the teachining of
Example 2.a above except that the experiment was conducted in NCI H358 cells
except
that the six well plate was seeded with 1.5 x 105 cells per well. The cells
were infected
as in Example 2.a. above using 1.8 x 109 particles/ml concentration of the
indicated
viruses for Figure 3 and 1.8 x 108 for Figure 4. The results are presented in
Figure 3
and 4 of the attached drawings.
Example 2.d. Time Course of Viral Replication
SK-BR3 cells were infected with a one hour pulse of the following recombinant
adenoviral ectors at a concentration of 1.8 x 109 particles/ml:
1. Mock: non-infected cells
2. rAdcon: a recombinant adenovirus lacking El and protein IX function
without a p53 coding sequence (Wills, et al.)
3. E1B~155K: A recombinant adenovirus containing the E1B-55K deletion
described in Example 1 above with no exogenous transgene cassette.
4. 55K/N>LPp53: The recombinant adenovirus cFAMA prepared in substantial
accordance with the teaching of Example 1 above.
5. rAd-p53: ACN53 (Wills, et al.)
6. 55K/CMVp53: A recombinant adenovirus cFAIC containing the E1B-55K
deletion described above further comprising an expression cassette encoding
p53 under control of the CMV promoter
7. AdSWT: Wild type adenovirus type 5.
The cells were harvested at approximately 48 hours post infection. The DNA was
applied to a agarose gel and stained with ethidium bromide according to
techniques well
known in the art. The results are presented in Figure 5 of the attached
drawings.
Example 3. Demonstration of Therapeutic Efficacy In vivo
PC-3 cells (prostate carcinoma, p53 null) were injected subQ into flanks of
nude
mice. When tumors were palpable (day 11) , virus was intratumorally injected
for 5
consecutive days at a dose of 1 x 10'° particles per injection on days
11-15 post PC-3
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cell injection. The six different viral constructs cFAMA, cFAIC, FTCB, ZZCB,
cZAZA
and wildtype Ad5 (described in Example 2 above) were compared. V-PBS refers to
phosphate buffered saline containing 3% sucrose and 2mM magnesium chloride.
Tumor volume was evaluated on days 1, 4, 7, 11, 15, 19, 23, and 27. The
results of
the evaluations are presented in Figure 5 of the accompanying drawings and
presented
in the Table 1 above.
Example 4. Construction of E1Bd155K-MLP-C~rtosine Deaminase
The E1Bd155K-MLP-Cytosine Deaminase vector is prepared in substantial
accordance with the teaching of Example 1 herein. However, the transfer
plasmid
containing the p53 coding sequence is replaced with a DNA sequence encoding
the
cytosine deaminase gene.
Example 5. construction of AFP-E4-E1Bd155K-MLP-X53
The AFP-E4-E1Bd155K-MLP-p53 is a recombinant adenovirus which is similar
to the E1Bd155K-MLP-p53 vector is prepared in substantial accordance with the
teaching of Example 1 herein. However, in this construction the E4 gene is
operably
linked to the alpha-fetoprotein tumor specific promoter sequence (AFP)
facilitating the
replication of this virus in hepatocellular carcinoma cells. Other factors
such as NF-IL6
can substitute for Ela in regulating Ela responsive promoters in the
adenovirus in the
absence of Ela function (Spergel, et al. (1992) J. Viroll 66:1021-1030) and
this can be
avoided by substitution of the E4 promoter with a tumor specific promoter.
Example 6. Construction of AFP-E4-E1Bd155K-MLP-IFN2a
The AFP-E4-E1Bd155K-MLP-iFN2a describes a recombinant adenovirus
which is similar to the AFP-E4-E1Bø~55K-MLP-p53vector is prepared in
substantial
accordance with the teaching of Example 5 herein. However, in this
construction the
p53 gene is replaced with the DNA sequence encoding interferon alpha-2b.
Example 7. Construction of ElAd101/07-E1Bd155K-MLP-p53
The E1A~101/07-ElBd155K-MLP-p53 is a recombinant adenovirus which is
substantially similar to the E1Bd155K-MLP-p53 vector is prepared in
substantial
accordance with the teaching of Example 1 herein. However, in this virus the
Ela gene
is modified to contain the in-frame deletion mutations X11101 and X11107, as
described
in Jelsma et al., (1998) Virology 1 'i, 494-502 and are constructed using the
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oligonucleotide site directed technique of Zoeller and Smith (1984) DNA 3_,
479-488, as
modifed by Kunkel (1985) PNAS $_2, 488-492. All of the reagents, bacterial
strains,
and Ml3 vectors used for mutagenesis are provided in the Muta-Gene in vitro
mutagenesis kit (commercially availble from Bio Rad, Hercules, CA}. The M13
template DNA, useful for mutagenesis of the ElA region, contains Ad5 sequences
from
nucleotide positions 22-1339 inserted between the BamHl and Xbal restriction
enzyme
sites in the multiple cloning sequence of Ml3mpl9. The resulting bacteriophage
construct, Ml3mpI9ElA, is 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, are
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 are first
phosphorylated at the 5' end, and then annealed to uracil containing
M13mp11ElA
single-stranded template DNA. The annealed primer/template reactions are
incubated
with T4 DNA polymerise, 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 are then transfomed into
the
ung+ wild type host bacterial strain, MV 1190. 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 is first screened by restriction enzyme analysis and then by DNA-
sequencing,
in both strands, to confirm the desired ElA-mutations.
Construction of an adenovirus comprising the E1A~01/deledons is carried out
by using homologous recombination in the adenovirus El-region containing 293
cell
line by the method of McGory, et al., (1988) Virology 163, 6I4-6I7. This
method
requires two plasmids, one a viral plasmid containing the entire wtAdS genome
modified as in Example 1 to contain the E1bd155K deletion and the MLP promoter
(pXCI-E1B55-2A), and the other a transfer plasmid containing an ElA gene with
the
x,01/07 double ElA-mutant . The transfer plasmid, pLE2 contains wtAdS
sequences
from 22-1774 cloned in the tetracycline gene of pBR322 Jelsma et al.,
(1988)Virology
,~63, 494-502. For transfer of the ElA d~1101 and X1107 Ela-mutants from the
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M13mp19 background in which they were constructed, wild ElA restriction enzyme
fragments in pLE2 are replaced with cognate mutated ElA fragments from
M13mp19E1d11101 and M13mp19ElAd11107 to create pLE2ElA~101/07. For
recombination to produce adenovirus ElAd101/07 the viral plasmid, pXCI-E1B55-
2A
and pLE2ElAd101/07 are cotransfected into 293 cells by calcium phosphate
mediated
transfection. After 5 hours the precipitate are rinsed and the cells are
overlayed with
growth medium containing agarose to isolate viral plaques. At 7-10 days after
the initial
transfection viral plaques are isolated, plaque purified two times, and
subsequently viral
DNA is screened using restriction enzyme analysis and DNA sequencing. Viral
stocks
are purified by double cesium chloride gradients and quantitated by column
chromatography as described in Huyghe, et al. (1995) Human Gene Therapy 6:1403-
1416.
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