Note: Descriptions are shown in the official language in which they were submitted.
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CATIONIC COMPLEXES OF POLYMER-MODIFIED ADENOVIRUS
BACKGROUND OF THE VENTION
Effective use of transgenes for the treatment of inherited and acquired
5 disorders requires efficient delivery of transgenes. Various vector systems
have been
developed that are capable of delivering a transgene to a target cell. While
newer
generations of vectors having improved characteristics have been developed,
there
still remains a need to improve efficiency of available gene transfer methods.
Improved efficiency is desirable both to increase the ability of the vector to
deliver the
10 transgene to target cells to correct the cellular defect, or provide a gene
encoding a
desirable product and to decrease the required amount of the vector and
thereby
reduce toxicity, including immunogenicity.
Adenoviral vectors, have been designed to take advantage of the
desirable features of adenovirus which render it a suitable vehicle for
nucleic acid
15 transfer. Adenovirus is a non-enveloped, nuclear DNA virus with a genome of
about
36 kb, which has been well-characterized through studies in classical genetics
and
molecular biology (Horwitz, M.S., "Adenoviridae and Their Replication," in Vir
,
2nd edition, Fields et al., eds., Raven Press, New York, 1990). The viral
genes are
classified into early (known as El-E4) and late (known as L1-LS)
transcriptional units,
20 referring to the generation of two temporal classes of viral proteins. The
demarcation
between these events is viral DNA replication. The human adenoviruses are
divided
into numerous serotypes (approximately 47, numbered accordingly and classified
into
6 subgroups: A, B, C, D, E and F), based upon various properties including
hemaglutination of red blood cells, oncogenicity, DNA base and protein amino
acid
25 compositions and homologies, and antigenic relationships.
Recombinant adenoviral vectors have several advantages for use as
gene transfer vectors, including tropism for both dividing and non-dividing
cells,
minimal pathogenic potential, ability to replicate to high titer for
preparation of vector
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2
stocks, and the potential to carry large inserts (Berkner, K.L., Curr. Ton
Micro
Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Theranv 1:51-64, 1994).
The cloning capacity of an adenovirus vector is proportional to the size
of the adenovirus genome present in the vector. For example, a cloning
capacity of
about 8 kb can be created from the deletion of certain regions of the virus
genome
dispensable for virus growth, e.g., E3, and the deletion of a genomic region
such as El
whose function may be restored in trans from 293 cells (Graham, F.L., J. Gen.
Virol.
36:59-72, 1977) or A549 cells (Imler et al., gene Theranv 3:75-84, 1996). Such
E 1-
deleted vectors are rendered replication-defective. The upper limit of vector
DNA
10 capacity for optimal carrying capacity is about 105%-108% of the length of
the
wild-type genome. Further adenovirus genomic modifications are possible in
vector
design using cell lines which supply other viral gene products in trans, e.g.,
complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996),
complementation of E4 (Krougliak et al., Hum. Gene Ther_ 6:1575-1586, 1995;
Wang
et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX
(Caravokyri et
al., J.J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene T er 6:1575-
1586,
1995).
Adenoviral vectors for use in gene transfer to cells and in gene
therapy applications commonly are derived from adenoviruses by deletion of the
20 early region 1 (E1) genes (Berkner, K.L., Curr. Top. Micro Immun~l 158:39-
66,
1992). Deletion of E1 genes renders the vector replication defective and
significantly reduces expression of the remaining viral genes present within
the
vector. However, it is believed that the presence of the remaining viral genes
in
adenovirus vectors can be deleterious to the transfected cell for one or more
of the
following reasons: (1) stimulation of a cellular immune response directed
against
expressed viral proteins, (2) cytotoxicity of expressed viral proteins, and
(3)
replication of the vector genome leading to cell death.
Transgenes that have been expressed to date by adenoviral vectors
include p53 (Wills et al., Human Gene Theranv 5:1079-188, 1994); dystrophin
(Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et
al.,
Human Gene Theranv 5:979-985, 1994; ornithine transcarbamylase
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3
(Stratford-Perncaudet et al., Human Gene Theranv 1:241-256, 1990; We et al.,
J. 'ol.
Chem. 271;3639-3646, 1996;); adenosine deaminase (Mitani et al., Human Gene
Thera~,y 5:941-948, 1994); interleukin-2 (Haddada et al., Human Gene Theranv
4:703-711, 1993); and al-antitrypsin (Jaffe et al., Nature Genetics 1:372-378,
1992);
thrombopoietin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase
(Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).
The particular tropism of adenoviruses for cells of the respiratory tract
has relevance to the use of adenovirus in gene transfer for cystic fibrosis
(CF), which
is the most common autosomal recessive disease in Caucasians. Mutations in the
cystic fibrosis transmembrane conductance regulator (CFTR) gene that disturb
the
CAMP-regulated Cl- channel in airway epithelia result in pulmonary dysfunction
(Zabner et al., Nature Genetics 6:75-83, 1994). Adenovirus vectors engineered
to
carry the CFTR gene have been developed (Rich et al., Human Gene Theranv
4:461-476, 1993) and studies have shown the ability of these vectors to
deliver CFTR
to nasal epithelia of CF patients (Zabner et al., ~j 75:207-216, 1993), the
airway
epithelia of cotton rats and primates (Zabner et al., Nature Genetic 6:75-83,
1994),
and the respiratory epithelium of CF patients {Crystal et al., Nature Genetics
8:42-51,
1994). Recent studies have shown that administering an adenoviral vector
containing
a DNA sequence encoding CFTR to airway epithelial cells of CF patients can
restore a
functioning chloride ion channel in the treated epithelial cells (Zabner et
al., J.J. Clin
v t. 97:1504-1511, 1996; U.S. Patent No. 5,670,488, issued September 23,
1997).
Transfer of the cystic fibrosis transmembrane conductance regulator
(CFTR) cDNA to airway epithelia of patients with cystic fibrosis (CF) thus
provides
an example of successful use of gene transfer to correct a cellular defect,
i.e., the CF
defect in electrolyte transport. Vector systems including adenoviral vectors
(Zabner et
al. (1993) ~1 ~5: 207; Knowles et al. (1995) New Engl J Med ~3: 823; Hay et
al.
(1995) Hum. Gene. They, _6: 1487; Zabner et al. (1996) J. Clin Invest 97: 1504
and
U.S. Patent No. 5,670,488) and cationic lipids (Caplen et al. (1995) N t. ed
1_: 39
and U.S. Patent No. 5,650,096) have been shown to be capable of transferring
the
CFTR cDNA and expressing CFTR in mature ciliated human airway epithelia. The
successful delivery of CFTR in such cells is manifest in the appearance of a
functional
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4
chloride ion channel in the treated cells.
While CFTR cDNA can be delivered to target cells for expression,
current adenoviral vectors are less than optimal in delivering the CFTR cDNA
to
airway epithelia because the binding of the virus to the apical (exposed)
surface of the
S epithelium is limited. Grubb et al. ( 1994) Nature 71: 802. The limited
infection can
be partially overcome by increasing the contact time between the virus and the
apical
surface. Zabner et al. (1996) J. Virol. Z: 6994.
Cationic lipid vector-mediated gene transfer to mature human airway
epithelia is also suboptimal. Caplen et al. (1995) Nat.Nat. Med. 1_: 39. While
it appears
10 that cationic molecules bind to the cell surface and in some cases are
taken up by the
cell, important barriers to transgene expression may be release of DNA from
the
endosome, entry into the nucleus, release of DNA from the cationic molecule,
and
transcription of the DNA. Zabner et al. (1995) J. Biol Chem ~7 : 18997.
Gene transfer systems that combine viral and nonviral components
15 have been reported. Cristiano et al. (1993) Proc. Natl. Acad Sci ~A ~;
11548; Wu
et al. (1994) J. Biol. Chem. ~: 11542; Wagner et al. (1992) Proc. Natl. Acad
Sci
,$~: 6099; Yoshimura et al. (1993) J. Biol. Chem ~$: 2300; Curiel et al.
(1991)
Proc Natl Acad Sci USA $8_: 8850; Kupfer et al. (1994) Hum. Gene Ther. ~:
1437;
and Gottschalk et al. (1994) Gene Ther. ~: 185. In most cases, adenovirus has
been
20 incorporated into the gene delivery systems to take advantage of its
endosomolytic
properties. The reported combinations of viral and nonviral components
generally
involve either covalent attachment of the adenovirus to a gene delivery
complex or co-
internalization of unbound adenovirus with cationic lipid: DNA complexes.
Further,
the transferred gene is contained in plasmid DNA that is exogenous to the
adenovirus.
25 In these formulations, large amounts of adenovirus are required, and the
increases in
gene transfer are often modest.
Accordingly, there is a need in the art for improved vector systems for
the efficient delivery of transgenes to target cells. The present invention
overcomes
certain limitations associated with adenoviral vectors and while retaining the
desirable
30 features of the vector system.
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SUMMARY OF THE INVENTION
The present invention is an adenovirus complex of a cationic molecule
and of an adenovirus having at least one polyalkalene glycol polymer bound
thereto.
Exanples of polyalkalene glycol polymers that can be used include, but are not
limited to, polyethylene glycol, methoxypolyethyleneglycol, polymethyl-
ethyleneglycol, polyhydroxypropyleneglycol, polypropylene glycol, and
polymethylpropylene glycol, in which polyethylene glycol is more preferred.
The
polyethylene glycol polymers have an average molecular weight of from 200
daltons
to 20,000 daltons, with 2000 daltons to 12,000 daltons being preferred, and
about
5000 daltons being even more preferred.
In another embodiment of the present invention, the polyalkalene
glycol poymer is an activated polyalkylene glycol polymer. Examples of
activated
polyalkylene glycol polymer that can be used include, but are not limited to,
methoxypolyethylene glycol-tresylate (TMPEG), methoxypolyethylene glycol-
acetaldehyde, methoxypoiyethylene glycol activated with cyanuric chloride, N-
hydroxysuccinimide polyethylene glycol (NHS-PEG), polyethyleneglycol-N-
succimimide carbonate and mixtures thereof.
The adenovirus component of the complex is preferably a recombinant
adenoviral vector. Adenoviral vectors containing a transgene such as a nucleic
acid
encoding CFTR are particularly preferred.
In accordance with the present invention, the polyalkalene glycol
polymer is directly covalently bound to the virus particle, indirectly
covalently bound
to the virus particle by an intermediate coupling moiety, directly
noncovalently
attached to the virus particle, or indirectly noncovalently attached to the
virus particle
by a ligand. The ligand for indirect noncovalent attachment is preferably a
ligand
having specificity for a viral surface component, such as an antibody. One
particularly
preferred antibody to be used is a non-neutralizing anti-adenovirus antibody,
such as a
non-neutralizing anti-hexon antibody.
The cationic molecule component of the complexes of the present
invention is a cationic polymer, with DEAE-Dextran being preferred.
Alternatively,
the cationic molecule is a cationic lipid.
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In another embodiment, the present invention provides a composition
containing the above-described adenovirus complexes in a carrier.
BRIEF DESCRT_PTION OF THE DRA~NG
Fig. 1 shows capillary electropherographs of adenovirus treated with
3% (w/v) TMPEG and MPEG.
Fig. 2 is a graph of the time course of mobility change on capillary
electropherographs of adenovirus treated with 3 % (w/v) TMPEG.
Figs. 3A-D show photon correlation spectroscopy results
demonstrating the change in viral particle size during PEGylation.
Fig. 4 depicts infectivity (CPRG) assay results for a single addition of 3
TMPEG, 3% MPEG and control virus exposed for 0-6h.
Figs. SA-E depict infectivity (CPRG) assay results for stepwise
additions of 5% PEGS°~, PEG,ZO~, or PEG2~~.
Figs. 6A-C depict infectivity (chemiluminescence, RLU) assay results
I S for stepwise additions of 3%, S% or 8% PEGS.
Figs. 7A-C depict infectivity (chemiluminescence, RLU) assay results
for stepwise additions of 5% PEGS.
Figs. 8A-C depict infectivity (chemiluminescence, RLU) assay results
for stepwise additions of S% PEG,2~ and PEGZOOOO~
Fig. 9 depicts infectivity (chemiluminescence, RLU) assay results for a
single addition of 3 % PEGS.
Figs. l0A - C show graphs of an antibody neutralization assay for the
impact of stepwise additions of 5% PEGS on neutralization of infectivity
(chemiluminescence, RLU assay), 10,000:1 antibody molecules to virus
particles.
Figs. 11 A - C show graphs of antibody neutralization assays for the
impact of stepwise additions of 5 % PEGS on neutralization of infectivity
(chemiluminescence RLU assay); 5,000:1 antibody molecules to virus particles.
Figs. 12A - C show graphs of an antibody neutralization assay for the
impact of stepwise additions of 5% PEG,z~ on neutralization of infectivity
(chemiluminescence RLU assay); 10,000: I antibody molecules to virus
particles.
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Figs. 13A - C show the elution profile of control and TMPEG-treated
virus from DEAE ion exchange resin following chromatography.
Fig. 14 depicts comparative infectivity (chemiluminescence, RLU)
assay results as illustrated by transgene expression in naive and immunized
mice
infected with PEGylated or sham treated adenoviral vectors.
Fig. I S depicts infectivity (chemiluminescence, RLU) assay results in
naive mice for (i) adenovirus alone, (ii) adenovirus with 10%
TMPEGso°o, (iii)
adenovirus/10% TMPEGS°oo complexed with poly-L-lysine , and (iv)
adenovirus/10%
PEGsooo complexed with DEAE-Dextran.
Fig 16 depicts comparative infectivity (chemiluminescence, RLU)
assay results in naive and immunized mice for (i) adenovirus with 10%
TMPEGsooo,
and (ii) adenovirus/10% TMPEGsooo complexed with DEAE-Dextran, (iii) sham-
treated adenovirus ( 10% MPEGsooo) and (iv) sham-treated adenovirus ( 10%
MPEGSOOO) complexed with DEAE-Dextran.
DETAILED DE~C'.R 1PTT N OF THE TNUFNTIt~7~
The present invention provides complexes of cationic molecules and
polymer-modified adenovirus that advantageously exhibit increased infectivity
and
reduced immunogenicity. The cationic complexes of the present invention have
surprisingly been found to exhibit heightened levels of infectivity in cells
previously
immunized with adenovirus, in addition to heightened levels of infectivity in
naive
(i.e., non-immunized) cells.
In accordance with the present invention, adenoviral vector particles
are polymer-modified by covalently or noncovalently binding to the virus a
polyalkalene glycol polymer, which renders the viral vector substantially non-
immunogenic. The polyaklene glycol polymers used in the present invention
preferably have an average molecular weight of from about 200 to about 20,000
daltons. Examples of glycol polymers that can be used include, but are not
limited to,
polyoxymethylene glycols, polyethylene glycols (PEG), methoxypolyethylene
glycols,
and derivatives thereof including for example polymethyl-ethylene glycol,
polyhydroxypropylene glycol, polypropylene glycol, and polymethylpropylene
glycol.
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A preferred glycol polymer used in accordance with the present
invention is PEG. PEG is a water-soluble polymer having the formula
H(OCHzCHz)~OH, wherein n is the number of repeating units and determines the
average molecular weight. PEGS having average molecular weights of from 200 to
20,000 daltons are commercially available from a variety of sources. In
accordance
with the present invention, PEG having an average molecular weight of from 200
(PEGzoo) to 20,000 (PEGzo,ooo)can be used to prepare adenoviruses modified
with PEG.
In a preferred embodiment, PEG has an average molecular weight from about 2000
to
about 12,000, with an average molecular weight from about 4000 to about 6000
(e.g.,
5000) being more preferred.
In accordance with the invention, the adenoviruses are polymer-
modified by direct covalent, indirect covalent, or indirect noncovalent
attachment of
the polyaklalene glycol polymer to the virus particle. A variety of schemes
exist for
covalent and non-covalent attachment: 1 ) the glycol polymer can be attached
via
direct covalent coupling to the surface of the adenovirus; 2) the glycol
polymer can be
attached via indirect covalent coupling (e.g., via an intermediate coupling
moiety that
links the polymer to the adenovirus surface); or 3) the glycol polymer can be
attached
via an indirect non-covalent linkage using, for example, a suitable PEGylated
ligand.
Examples of suitable ligands include, but are not limited to, antibodies to
surface
proteins, lipids or carbohydrates.
Targets for polymer modification include reactive groups on the viral
surface with which the polymer or coupling agent can interact, including for
example
primary and secondary amine groups, thiol groups and aromatic hydroxy groups.
As
will be apparent to one of ordinary skill in the art, the preferred method for
polymer
modification of the adenovirus is dependent upon the available target sites
found on
the viral surface. Examples of available target sites for attachment of the
glycol
polymer to the adenovirus include, but are not limited to, the hexon, penton
cell base,
and fiber proteins. The adenoviral hexon protein is a particularly preferred
site for
attachment of the alkalene glycol polymer. While not wishing to be bound by
theory,
it is believed that modification of these sites masks epitopes from
neutralizing
antibodies thereby providing the adenoviral vector with reduced antigenicity
and/or
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9
immunogenicity.
Methods for the direct or indirect covalent attachment of polymers to
polypeptides that are known in the art may be used to provide the polymer-
modified
adenoviruses of the present invention. Methods are described, for example, in
WO
5 90/04606, and in U.S. Patent Nos. 4,179,337 and 5,612,460, the disclosures
of which
are incorporated herein by reference. Generally, the glycol polymer is
activated by
converting a terminal moiety of the polymer to an activated moiety, or by
attaching an
activated coupling moiety to the polymer. The activated polymer is then
coupled to
the target via the activated moiety. The activated moiety or activated
coupling moiety
can be selected based upon its affinity for the desired target site on the
viral surface.
For example, the terminal hydroxyl groups of PEG can be converted
into reactive functional group or attached to an activated coupling moiety to
provide a
molecule known as "activated" PEG. Various forms of activated PEG are known in
the art and are commercially available. For direct covalent linkage to the
adenovirus a
15 suitable activated PEG is MPEG-tresylate (TMPEG), which is believed to
react with
E-lysine groups, or MPEG-acetaldehyde. For indirect covalent linkage other
forms of
activated PEG are known in the art and commercially available, including for
example
methoxypolyethylene glycol (MPEG) derivatives such as MPEG activated with
cyanuric chloride, PEG N-hydroxysuccinimide PEG (NHS-PEG), which reacts with
20 amine groups, and PEG-N-succimimide carbonate. These and other activated
PEGS
are disclosed in W095/06058, and in U.S. Patent Nos. 4,179,337 and 5,612,460,
which are incorporated herein by reference.
The covalent attachment of PEG to the adenovirus surface
("PEGylation") is accomplished by incubating the virus with the activated PEG
(e.g.,
25 TMPEG). Single addition or multiple addition incubation regimes can be
used. The
optimal ratios of TMPEG to adenoviral particles to achieve reduced
antigenicity,
along with heightened infectivity, may be ascertained by performing the
various
assays described below. Under conditions designed to provide direct TMPEG
modified adenovirus, PEGylation in the amount of about 5-20% w/v is preferred,
with
30 a concentration of about 10% w/v being most preferred.
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Preferably, when high concentrations (e.g., 10% or greater) of glycol
polymer are attached to the virus, the activated polymer is added in a
stepwise fashion.
Stepwise addition is preferred since viral particles tend to aggregate at high
concentrations, which reduce the overall effeciency of PEGylation. Moreover,
S aggregation is exacerbated by the use of certain activated polymers, e.g.,
TMPEG.
Thus, the inital use of low polymer concentrations in a stepwise manner can
reduce
the tendency of the particles to aggregate, thereby facilitating a higher
degree of
PEGylation. For example, activated PEG such as TMPEG may be added in separate
steps to a viral stock solution every thirty minutes to increase the polymer
10 concentration each time by 3%, S% or 8% (wlv) in the reaction mixture to
obtain final
polymer concentrations of 12%, 20% and 32% respectively (approximately w/v,
i.e.,
not correcting for the volume of the polymer). In addition, after the last
addition of
the glycol polymer, a further incubation time might be allowed. These
necessary
adjustments to the reaction parameters (e.g., the number of steps,
concentration of the
polymer, and reaction time) for optimal results can easily be ascertained by
one of
ordinary skill in the art..
The attachment reaction may be quenched by dialysis or by addition of
excess lysine (e.g., a 10 to100-fold excess lysine). Alternatively, the
reaction might
be run to completion (i.e., the point at which the activated PEG, such as
TMPEG, is
either completely consumed in the PEGylation reaction or rendered inactive by
hydrolysis).
In another embodiment of the present invention, the glycol polymer is
indirectly noncovalently attached to the adenovirus via a suitable ligand. In
a
preferred embodiment, the ligand is an antibody or antibody fragment,
including for
example a non-neutralizing anti-virus antibody or fragment therefrom (e.g.,
Fab,
F(ab')2 , Fv). As used herein, the term "antibody" includes monoclonal and
polyclonal
antibodies. In a particularly preferred embodiment, the ligand is a non-
neutralizing .
anti-hexon antibody. Such antibodies are commercially available and include,
for
example, MAb 8052 and MAb 805 available from Chemicon International, Temecula,
CA, USA.
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Indirect non-covalent attachment of glycol polymer to the adenovirus is
accomplished by incubation of the virus with a suitable ligand that has been
modified
by the covalent attachment of polymer. The glycol polymer can be covalently
attached (i.e., bound) to the ligand by standard methods as described herein
above.
S For example, a non-neutralizing anti-virus antibody such as anti-hexon
antibody may
be PEGylated using an activated PEG molecule as described above. In a
preferred
embodiment, anti-hexon antibody is modified using TMPEG. One of ordinarly
skilled
in the art can ascertain the optimal ratios of activated PEG to antibody,
concentrations
of activated PEG and antibody, buffer and time and temperature of incubation
to
10 achieve optimal modification of the antibody. The polymer-modified ligand
is then
incubated with adenovirus to allow non-covalent binding of the polymer-
modified
ligand to the virus surface.
Antibodies modified with PEG at the epitope binding site (e.g.,
complementarity determining regions (CDRs)) can exhibit reduced affinity to
the
15 adenovirus thereby decreasing the e~ciency noncovalentl attachment. In
order to
prevent PEGylation at the epitope binding site an antibody is preferably
immobilized
prior to PEG modification. For example, anti-hexon antibody is bound to
purified
immobilized hexon (e.g., hexon-Sepharose~) prior to PEG modification of
antibody.
The PEGylated antibody is then released from immobilized hexon.
20 Alternately, non-immobilizied anti-hexon antibodies can be PEGylated
creating a population of antibodies PEGylated on the epitope binding site in
addition
to other sites, which are thereafter separated by immunoaffinity
chromatography. For
example, the mixed population of modified antibodies can be incubated with
immobilized hexon, to which antibodies modified only at sites other than the
epitope
25 binding site will bind. These PEGylated antibodies are then released from
the
immobilized hexon for use in accordance with the present invention.
For some applications, for example, those requiring repeat dosing of a
polymer modified virus, it may be desirable to separate the unreacted glycol
polymer
from the polymer-modified adenovirus, which may then be purified by standard
30 methods as necessary for the intended use. Separation and purification may
be
performed by methods known in the art, for example ion exchange
chromatography,
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12
gel filtration chromatography, or cesium chloride gradient purification. In
situations
in which there is indirect PEGylation of an antibody, hexon affinity resin may
be
useful to separate the PEGylated antibody from unreacted PEG.
In addition, it may be desirable to separate unmodified adenovirus from
polymer-modified adenovirus. Separation of the unmodified from polymer-
modified
virus may be performed by partitioning in an aqueous biphasic polyalkylene
glycol
solution. For example, phase partitioning in an aqueous biphasic system of PEG
and
dextran may allow the separation of PEG-modified virus from unmodified virus.
Partitioning may be performed by counter-current distribution. Generally, the
phase
system is prepared by mixing solutions of dextran and PEG. PEG and PEG-
modified
virus are incorporated into the phase system, mixed by inversion or rotation,
and
allowed to separate. PEG modified virus partitions into the PEG phase, and
unmodified virus partitions into the dextran phase.
The efficiency of adenovirus polymer modification (e.g., PEGylation)
is evaluated by methods known in the art, including ion exchange
chromatography,
capillary electrophoresis (CE), photon correlation spectroscopy (PCS), and
through
the use of a labeled (e.g., biotinylated) PEG in a quantitative ELISA. Ion
exchange
chromatography (e.g., DEAE-chromatography) can be performed by standard
methods
to evaluate the modified viruses based upon altered charge.
Whole virus CE provides a means to monitor the modification of
adenovirus by the glycol polymer as a function of altered surface charge. For
example, covalent attachment of PEG to the adenovirus surface seems to result
in
shrouding of the negative surface charges on the viral particle thereby
causing virus to
exhibit a more neutral mobility. CE may be performed by methods known to those
of
ordinary skill in the art. For instance, a ramped low-high voltage pre-
treatment is used
to electrophorese the highly mobile salt ions in which the virus may be
formulated for
stability, before true, high voltage separation begins. In plots derived from
CE, virus
particles with PEG covalently attached run at a position closer to the neutral
point
than virus without covalently attached PEG. CE may be conveniently used to
assess
the influence of various conditions, including molar ratios, concentrations
and
incubation times, on the covalent attachment of PEG to the virus particles.
Increasing
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13
neutrality reflects increasing PEG-chain density on the virus surface.
PCS uses the relationship between particle size and movement in
suspension (via Brownian motion) to gain accurate measurements on the size of
the
particles. This method is widely applied to monitor polymer attachment to
particles
S including liposomes, microspheres and nanoparticles by measuring their
increase in
size. These data suggest that covalently attached PEG at relatively low
density forms
globular "mushroom" shapes and thus the increase in size is relatively small.
Altering
the conditions under which one would expect to increase the density of
covalently
attached PEG chains results in a more extended conformation of the polymer or
"brush" shapes which is reflected by a relatively larger increase in particle
size. Thus
PCS may be used using methods known to those of ordinary skill in the art to
monitor
the size changes of the virus particle under different reaction conditions.
The ELISA analysis of a biotinylated PEG can provide the most
quantitative assessment of the number of molecules of PEG covalently bound to
the
adenovirus virus particle. The ELISA can be performed by standard methods
known
in the art.
The indirect noncovalent attachment of glycol polymer via a polymer-
modified ligand can also be monitored by displacement of labeled .ligand from
the
adenovirus in a competition enzyme-linked immunosorbent assay (ELISA). For
example, the ability of a PEGylated anti-hexon antibody to bind to the
adenovirus
surface is measured in a standard competition ELISA using a biotinylated anti-
hexon
antibody.
As used herein, the term "adenovirus" includes genetically engineered
adenoviruses (i.e., recombinant adenoviral vectors). Preferably, the
adenovirus of the
present invention is a recombinant adenovirus engineered to be incapable of
replicating and exhibits minimal expression of adenoviral genes. Suitable
recombinant adenovirus include adenoviral vectors derived from adenovirus type
2
(Ad2), type S (Ad5) and type 6 (Ad6) which have been deleted for the El
regions.
Representative adenoviral vectors that are useful for delivery of a transgene
are
disclosed by Zabner et al. (1996) ~. Clin. Invest. ~ : 1504, Zabner et al.
(1993);,~gll 75
207, U.S. Patent Nos. 5,707,618 and 5,670,488, the disclosures of which are
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14
incorporated herein by reference.
The recombinant adenoviruses also preferably contain transgenes
operably linked to suitable promoter and other regulatory sequences.
"Transgenes"
are defined herein as nucleic acids that are not native to the adenovirus.
Examples of
5 transgenes to be utilized are nucleic acids encoding a biologically
functional protein or
peptide, an antisense molecule, or a marker molecule. The promoter may be an
endogenous adenovirus promoter, for example the E 1 a promoter or the Ad2
major late
promoter (MLP) or a heterologous eucaryotic promoter, for example a
phosphoglycerate kinase (PGK) promoter or a cytomegalovirus (CMV) promoter.
Similarly, those of ordinary skill in the art can construct adenoviral vectors
utilizing
endogenous or heterologous poly A addition signals.
In a preferred embodiment, the recombinant adenoviral vector contains
a transgene such as the nucleic acid encoding cystic fibrosis transmembrane
conductance regulator (CFTR). CFTR is a phosphorylation and nucleoside
15 triphosphate-regulated Cl- channel located in the apical membrane of
epithelial cells in
the lung, intestine, pancreas and sweat glands. For a review, see Welsh et
al., (1992)
Ne~.uyon 8: 821, incorporated herein by reference. Cystic fibrosis (CF)
results from a
non-functional Cf channel in an individual's epithelial cells caused by
mutations in
the gene encoding CFTR. Such mutations result in loss of function of the
chloride
20 channel and thus defective electrolyte transport in affected epithelial
cells. DNA
encoding wild-type CFTR is known in the art; the sequence is disclosed, for
example,
in U.S. Patent No. 5,670,488, incorporated herein by reference. A deletion
mutant of
CFTR that encodes a regulated Cf channel is disclosed by Sheppard et al.
(1994) ~Il
7ø: 1091, and in , U.S. Patent Nos. 5,670,488 and 5,639,661, the disclosures
of which
25 are incorporated herein by reference. Other examples of recombinant
adenoviral
vectors containing transgenes encoding CFTR are Ad2/CFTR-2, Ad2/CFTR-8, and
Ad2/CFTR-16, which are respectively found in Zabner et al. (1996) ~. Clin
Invest
7:1504-151 I, U.S. Patent No. 5,707,618 and U.S. Serial No.: 08/839,552 filed
April 14, 1997, all incorporated herein by reference.
30 In accordance with the present invention, DNA encoding a CFTR
protein includes the foregoing published sequences as well as other DNAs
encoding
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15
CFTR known to those of skill in the art. Further included are modifications of
the
known DNA molecules, for example mutations, substitutions, deletions,
insertions
and homologs, that encode a functional CFTR protein, i.e., a chloride channel.
In another embodiment of the invention, the polymer-modified
5 recombinant adenovirus is an adenovirus that can induce tumor-specific
cytolysis also
known as viral oncolysis. Representative adenovirus that are useful for viral
oncolysis
are disclosed by Bischoff et al. ( 1996) Science ~7 :373; Heise et al. ( 1997)
Nature
Medicine x:630; and EP689447A, the disclosures of which are incorporated
herein by
reference.
10 In accordance with the present invention, the polymer-modified
adenovirus is complexed with a cationic molecule. The cationic molecule can be
any
cationic compound that exhibits minimal toxicity to mammals and does not
decrease
the infectivity of the polymer-modified virus. Preferably, the cationic
molecule
provides the polymer-modified virus with infectivity levels comparable to, if
not
15 greater than, the infectivity levels exhibited by the corresponding
unmodified
adenovirus. Examples of cationic molecule that can be used include, but are
not
limited to, cationic polymers, cationic lipids, cationic sugars, cationic
proteins, or
cationic dendrimers. The cationic molecules can also be combined with non-
cationic
molecules. Examples of cationic polymers include, but are not limited to,
20 polyethyleneimine (PEI), DEAE-dextran, and histone (fraction V-S),
protamine,
polybrene (Hexadimethrine Bromide) and cationic dendrimers, in which DEAE-
dextran is preferred. Alternatively, the polymer-modified adenovirus can be
dispersed
in a metal salt precipitate such as calcium phosphate, as set forth in pending
application U.S. Serial No. 09/082,510, filed May 21, 1998, which is
incorporated
25 herein by reference. Those of ordinary skill in the art can determine the
molecular
weight of the cationic polymer that provides optimal gene transfer in
accordance with
the methods described herein. PEI is preferably used at an average molecular
weight
of 25 kDa.
Cationic lipids are known to those of ordinary skill in the art.
30 Representative cationic lipids include those disclosed e.g., by U.S. Patent
No.
5,283,185, PCT/LJS95/16174 (W096/18372) and U.S. Patent No. 5,650,096, the
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16
disclosures of which are incorporated herein by reference. In a preferred
embodiment
the cationic lipid is (N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol
(DC-
Chol) disclosed in U.S. Patent No. 5,283,165. In another preferred embodiment,
the
cationic lipid is N4-spermine cholesterol carbamate (GL-67) or N4-spermidine
5 cholesterol carbamate (GL-53) disclosed in W096/18372 and U.S. Patent No.
5,650,096. Other representative cationic lipids include (2, 3-dioleyloxy-N-
[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate
(DOSPA), dioctadecylamidoglycyl spermine (DOGS), commercially available as
TRANSFECTAM~ from Promega, Madison, WI; 1,3-dioleoyloxy-2-(6-
10 carboxyspermyl)-propyl amide (DOSPER); N-[1-(2,3-Dioleoyloxy)propyl] -N,N,N-
trimethyl-ammoniummethylsulfate (DOTAP); N-[1-2(2,3-dioleyloxy)propyl]-N,N,N-
trimethylammonium chloride (DOTMA); (t)-N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-
bis(tetradecyloxy)-1-propanaminium bromide (DMRIE); (~)-N-(2-Aminoethyl)-N,N-
dimethyl-2,3-bis (tetradecyloxy)-1- propanaminium bromide ((3AE-DMRIE);
1 S dimethyldioctadecylammonium bromide (DDAB); LIPOFECTIN~, a 1:1 (w/w)
formulation of DOTMA and dioleoyl phosphotidylethanolamine (DOPE)
commercially available from Life Technolgies, Gaithersburg, MD;
LIPOFECTAMINE~, a 3:1 (w/w) formulation of DOSPA and DOPE commercially
available from Life Technologies, Gaithersburg, MD; LIPOFECTACETM, a 1:2.5
20 (w/w) formulation of DDAB and DOPE, commercially available from Life
Technologies, Gaithersburg, MD; TfxTM -S0, a reagent consisting of N,N,N', N'-
tetramethyl-N-N'-bis(2-hydroxyethyl)-2,3,-dioleoyloxy-1, 4-butanediammonium
iodide and DOPE, commercially available from Promega, Madison, WI; and DMRIE-
CTM, a 1:1 (molar ratio) formulation of DMRIE and cholesterol commercially
25 available from Life Technologies, Gaithersburg, MD. In a preferred
embodiment the
cationic lipid is GL-53 or GL-67. In accordance with the present invention,
the
cationic lipid may be combined with a colipid such as DOPE or cholesterol.
The ratio of cationic molecule to polymer-modified adenovirus to be
used in complex formation is variable. As will be apparent to one skilled in
the art,
30 factors that may affect the cationic molecule : virus ratios include the
cationic
molecule selected, polyalkylene glycol polymer selected, and cell type
targeted for
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infection. However, optimal cationic molecule : virus ratios can easily be
determined
by one skilled in the art utilizing the infectivity assays described herein.
Some
illustrative cationic molecule : virus ratios for the complexes of the present
invention
are set forth below. DEAE-Dextran is complexed with the polymer-modified
adenovirus at a ratio of 100-3000 molecules per virus particle, with 400-600
molecules per virus particle being preferred. PEI is compiexed at a ratio of
200-1200
molecules per virus particle, with 400-600 molecules per virus particle being
preferred. Protamine is complexed with 400-40,000 molecules per virus
particle, with
3000-5000 molecules per virus particle being preferred. Polybrene is complexed
with
4 x 103 - 4 x 105 molecules per virus particle, with 3.5 x 105 - 4.5 x 105
molecules per
virus particle being preferred. The cationic lipid, GL-67, is complexed with 9
x 105 -
9 x 106 molecules per virus particle, with 8.5 x 106 - 9.5 x 106 molecules per
virus
particle being preferred. Alternatively, calcium phosphate can be co-
precipitated in
the presence of the polymer-modified virus by admixing a molar excess of
calcium
(Ca2+) to phosphate (P04 ), (Ca2+ : P04 ), ranging from 6:1 to 42:1, with a
ratio 13:1 to
15:1 being preferred.
The complexes of the present invention can be simply prepared by
admixing the components under suitable conditions. For example, suspensions of
viral particles and cationic molecules are prepared with phosphate-buffered
saline
(PBS) at pH of 7. Prior to admixing the two suspension, the suspensions are
warmed
to 30°C to facilitate complex formation. The two suspensions are mixed
and
incubated at 30°C for approximately 15 minutes to allow sufficient
complex
formation. However, if desired, complex formation can be conducted at room
temperature with additional incubation times. The complexed polymer-modified
adenovirus is then resuspended in PBS and is ready for administration to a
host.
As previously described, the cationic complexes of polymer-modified
(e.g., PEGylated) adenovirus exhibit heightened levels of infectivity in both
immune
subjects and naive (i.e., non-immune) subjects. However, PEGylation of the
virus
exceeding 15% can cause a decrease or ablation of viral infectivity, thereby
providing
a disincentive for further PEGylation. Normally, decreases in viral
infectivity do not
occur at PEGylation levels of 10% or less depending on the glycol polymer
selected
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18
for attachment (e.g., TMPEG vs. MPEG). Accordingly, the cationic complexes of
the
present invention provide a solution to this problem by allowing significantly
greater
levels of polymer modification to be used (e.g., 20% or greater) while
maintaining
viral infectivity levels comparable to unmodified (i.e., non-polymer-modified)
5 adenovirus. In fact, as demonstrated in the examples set forth below, the
complexes
of the present invention provide infectivity levels significantly greater that
either
unmodified adenovirus and polymer-modified adenovirus.
Infectivity of the adenoviral complexes of present invention are
assessed by standard infection assays. For example, the ability of adenovirus
to infect
10 a cell is assessed by monitoring the expression of a transgene (e.g., a
reporter gene
such as lacZ) contained within the adenovirus. Genetic reporter systems are
well-
known in the art, and are disclosed for example in Short Protocols in Molec
~lar
Bio- lo~v, 1995, Ausubel et al., eds., 3'd edition, Wiley and Sons, Inc. The
adenoviral
vector is engineered by standard methods to contain a transgene, and the
complexed
15 adenoviral vector is used to infect cells that are permissive for the
virus. After
infection under standard conditions, cell lysates are analyzed for the
presence of the
product of the transgene, e.g., (3-galactosidase. For example, the product of
the
transgene can be assessed by colorimetric, chemiluminescence or fluorescence
assays,
or immunoassays. In this way, those of ordinary skill in the art can compare
20 complexed and uncomplexed adenoviral vector, and can determine the optimal
percentages and conditions for glycolization and cationic complexing that
result in
optimum retention of infectivity.
Alternatively, if the transgene encodes CFTR, infectivity can be
measured by acsertaining the ability of the CFTR protein expressed in cultured
CF
25 airway epithelia to correct the Cl- channel defect following the methods
described by
Rich et al. (1990) Nature 342: 358, incorporated herein by reference. Briefly,
cultured
CF airway epithelial cells are infected with adenoviral vectors containing DNA
encoding a CFTR protein. Virus-mediated expression of functional CFTR protein
is
assessed using an SPQ [6-methoxy-N-(3-sulfopropyl)-quinolinium, Molecular
Probes,
30 Eugene, OR] halide efflux assay. SPQ is a halide-sensitive fluorophore, the
fluorescence of which is quenched by halides. In this assay, cells are loaded
with
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19
SPQ, CFTR is activated by cAMP agonists, the CFTR Cf channel opens, halides
exit
the cell, and SPQ fluorescence in the cell increases rapidly. Thus increases
in
intracellular fluorescence in response to cAMP provide a measure of a
functional CI'
channel.
In another assay suitable for measuring viral infectivity, CF epithelial
cells are infected with adenoviral vectors containing DNA encoding a CFTR
protein,
and secretion of Cl' from infected cells is measured in response to cAMP
stimulation.
The secretion of Cf can be measured as an increase in transepithelial short-
circuit
current with addition of cAMP agonists, as described for example by Rich et
al.
10 {1993) Human Gene Theraw 4_: 461, the disclosure of which is incorporated
herein by
reference. Expression of a functional CFTR protein can also be assessed by
patch
clamp techniques that detect reversibly activated whole-cell currents in
response to
addition of CAMP agonists, or single-channel currents in excised, cell-free
patches of
membrane in response to cAMP-dependent protein kinase and ATP. Patch clamp
15 techniques are described for example by Sheppard et al. (1994) ~gl~ ~ø:
1091, and
U.S. Patent No. 5,639,661, the disclosures of which are incorporated herein by
reference.
Retention of infectivity is defined herein as an infectivity level
sufficient to have therapeutic value, for example at least about 20% infective
relative
20 to unmodified virus (non-complexed, non-polymer-modified adenovirus). For
some
embodiments, the virus complex maintains at least 60% infectivity. In other
therapeutic embodiments, the complexed modified virus is preferred to maintain
at
least 80% infectivity. Lower percent infectivity of at least 5% may be useful
for
applications such as viral oncolysis.
25 In a particular example of an infectivity assay, an adenoviral vector
containing the (3-galactosidase (~3-gal) reporter gene (IacZ) is covalently
modified by
exposure to various concentrations of TMPEG and subsequently complexed with a
cationic molecule, other than poly-L-lysine (e.g., DEAE-dextran). A cell line
that
supports adenoviral vector propagation, for example 293 human embryonic kidney
30 cells (ATCC CRC 1573), is exposed to unmodified and modified/complexed
adenoviral vector containing the ~i-gal gene. Cells are then incubated under
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conditions appropriate for ~i-gal expression. The presence of (3-gal in cell
lysates is
measured by standard colorimetric, fluorescence, or chemiluminescence assays,
e.g.,
by using X-gal. The quantity of (3-gal in 293 cell lysates provides a
measurement of
the ability of the complexed, PEGylated adenovirus to infect 293 cells. The
complexed, PEGylated virus that maintains SO% infectivity relative to
unmodified
virus is considered to retain infectivity.
The complexed, polymer-modified adenoviruses of the present
invention exhibit reduced antigenicity relative to unmodified virus. Reduced
antigenicity is defined as a statistically significant (p>0.05) reduction in
binding of the
10 polymer-modified virus to neutralizing antibodies against the virus.
Reduced
antigenicity is assessed by methods known in the art, including in vitro and
in vivo
assays. For example, both modified and unmodified viruses containing reporter
genes
are incubated in the presence or absence of neutralizing antibodies or serum.
The
antibody-treated viruses and non-antibody treated control viruses are then
used to
15 infect cells as described above, and reporter gene expression in infected
cells is
performed as described above. With unmodified viruses, treatment with
neutralizing
antibodies results in lower levels of infection and thus lower levels of
transgene
expression. The complexed, polymer-modified adenoviruses of the present
invention
are protected from neutralization by the polymer coating, and thus provide
increased
20 infectivity and increased transgene expression in the present assays
relative to
unmodified viruses that have been exposed to neutralizing antibodies.
By utilizing the foregoing assays, those of ordinary skill in the art can
determine the conditions for glycolization and subsequent complexing necessary
to
provide a complexed, polymer-modified adenovirus that maintains infectivity
and
exhibits reduced antigenicity.
Because of their unique properties, the cationic complexes of polymer-
modified adenoviruses are particularly useful for therapeutic and diagnostic
'n vivo
applications. The cationic complexes of the present invention have utility in
medical
therapy and diagnosis in medical and veterinary practice and in agriculture.
They are
30 of particular use in gene therapy (for example the delivery of genes for
the localized
expression of a desired gene product) and for non-gene therapy applications
such as,
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21
but without limitation, viral oncolysis. The viruses are useful, for example,
to deliver
genes, toxins and/or diagnostic markers. An additional application is in the
creation
of tolerogens for viral antigens.
In one embodiment of the present invention, a method is provided for
introducing a transgene into a target cell. The method comprises introducing
into the
target cell the complexed, polymer-modified adenovirus of the present
invention,
wherein the adenovirus is a recombinant adenoviral vector comprising the
transgene.
The complexed, polymer-modified adenoviruses are particularly useful for
delivering
a transgene to a target cell for the treatment of various disorders, for
example in which
10 the transgene product is absent, insufficient, or nonfunctional.
Alternatively, the
expression of the transgene may serve to block the expression or function of
an
undesired gene or gene product in the target cell.
Target cells for adenovirus complexes of the present invention are any
cell in which expression of a transgene is desired. Target cells include cell
types
15 permissive to adenovirus infection (e.g., 293 cells and A549 cells) and
cell types
resistant to adenovirus infection (e.g., human epithelial cells, NIH 3TC
cells, and 9L
gliosarcoma cells). In fact, the complexed, polymer-modified adenoviral
vectors are
particularly suitable for infecting adenovirus resistant cells for transgene
expression.
While not wishing to be bound by theory, the complexes of the present
invention do
20 not require binding to the Coxsackie Adenovirus Receptor (CAR) for
internalization.
As will apparent to the skilled artisan, internalization of adenovirus in
permissive cell
types is generally dependent on the CAR pathway. However, the complexes of the
present are internalized by pathways other than CAR, which renders them
particularly
suitable for transgene expression in adenovirus resistant cell types
(Fasbender et al.,
25 (1997) ~. Biol. Chem. 2:6479-6489; Kaplan et al., (1998) Hum. Gen. Ther
~:1469-
1479).
The complexed, polymer-modified adenovirus is introduced into the
host cell by methods known in the art, including for example infection.
Infection of a
target cell i~r vivo is accomplished by contacting the target cell with the
adenovirus.
30 The adenovirus is delivered as a composition in combination with a
physiologically
acceptable carrier. As used herein, the term "physiologically acceptable
carrier"
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22
includes any and all solvents, diluents, isotonic agents, and the like. In a
preferred
embodiment the complexed, polymer-modified adenovirus is a recombinant
adenoviral vector polymer-modified by covalent attachment of PEG and
subsequently
complex with DEAE-dextran. The use of such media and agents for compositions
is
5 well known in the art. The adenoviruses of the invention may be delivered to
the
target cell by methods appropriate for the target cell, including for example
by
ingestion, injection, aerosol, inhalation, and the like. The compositions may
be
delivered intravenously, by injection into tissue, such a brain or tumor, or
by injection
into a body cavity such as pleura or peritoneum.
10 The formulation of compositions is generally known in the art and
reference can conveniently be made to Remington's Pharmaceutical Sciences,
17'" ed.,
Mack Publishing Co., Easton, PA. The forms of the present complexes suitable
for
administration include sterile aqueous solutions and dispersions. The subject
complexed, polymer-modified adenoviruses are compounded for convenient and
15 effective administration in effective amounts with a suitable
physiologically
acceptable carrier and/or diluent.
The effective amounts of the complexed, polymer-modified adenovirus
to be used in accordance with the present invention for humans, or any other
mammal,
can be determined by the ordinary skilled artisan with consideration of
individual
20 differences in age, weight and condition of the subject.
It is especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit
form as used herein refers to physically discrete units suited as unitary
dosages for the
mammalian subjects to be treated, each unit containing a predetermined
quantity of
25 active material calculated to produce the desired effect in association
with the required
carrier. The specification for the novel dosage unit forms of the invention
are dictated
by and directly depend on the unique characteristics of the polymer-modified
viruses
and the limitations inherent in the art of compounding. In the case of
compositions
containing supplementary active ingredients, the dosages are determined by
reference
30 to the usual dose and manner of administration of the ingredients.
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23
The present invention further provides a method for delivering a virus
to a tumor, comprising administering a complexed, polymer-modified adenovirus
of
the invention to a subject in need of such treatment under conditions whereby
the
adenovirus localizes to a tumor. The ability of the complexed adenoviruses of
the
5 present invention to provide retention of infectivity and reduced impact of
neutralizing
antibodies open up this additional method of use for polymer-modified virus.
Particulates of the size range 100-200nm undergo passive tumor targeting in
relation
to the so-called EPR effect (Enhanced Permeability and Retention). Tumors have
leaky vasculature and thus long circulating particles have the opportunity to
leave the
10 circulation and enter the tumor parenchyma via the holes in tumor blood
vessels.
Tumors lack lymphatics which is the main system for removal of macromolecules
and
particles from the tissues (the basis for the Retention element in EPR). PEG
has been
used to enhance the passive targeting of liposomes to tumors via increased
circulation
time. However, data in the scientific literature shows that this approach
leads to
15 unfavorable properties such as unacceptable low tumor to blood ratios (i.
e. less than 1 )
for much of the lifetime of the product. Using different optimization
principles it has
been shown (WO 96/34598) that additional effects of PEGylation, other than
improved circulation time, can be exploited to solve this problem and achieve
both
good tumor localization and high tumor to blood ratios as well as high tumor
to
20 normal tissue ratios. Thus the present invention provides a means of
improving the
tumor localization of virus particles. This is relevant to both gene therapy
applications where viral vectors are used to deliver genes and for non-gene
therapy
applications. The latter include the recently discovered system selective for
the
infection of p53 deficient tumor cells which has the capacity to kill tumor
cells via
25 viral oncolysis Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M,
Ng L,
Nye JA, Sampson-Johannes A, Fattaey, McCormick F (1996) Science 274:373-376;
Heise et al. (1997) Nature Medicine ,x:369-645; and EP689447A, incorporated
herein
by reference.
The invention is further illustrated by the following specific examples
30 which are not intended in any way to limit the scope of the invention.
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24
EXAMPLES
x a
Covalent Attachment of Polveth lene ~ycol to Adenovirus
Tresyl-monomethoxypolyethylene glycol (TMPEG) was prepared
5 using MPEGS~°. In this example and in subsequent examples, except
where
otherwise indicated, TMPEG was prepared as set out in WO 95/06058, which
corresponds to U.S. Application Serial Nos. 08/471,348 and 08/601,040, filed
June 6,
1995 and February 23, 1996, respectively, the disclosures of which are
incorporated
herein by reference.
10 Type 2 adenovirus (genetically modified to carry the ~i-gal reporter
gene), as disclosed in U.S. Patent No. 5,670,488, was prepared by banding in
isopycnic CsCI density centrifugation (three rounds), then extensively
dialysed against
phosphate buffered saline (PBS, pH 7.2) containing 5% sucrose. The stock
solution
used contained 6.4x10'° infectious units per ml (4.8x10" particles/ml).
The virus
15 stock was made 3%w/v by the addition of dry TMPEG, typically 3.Omg to
100,u1 of
stock. The samples were incubated at 25°C with rotary mixing for 24h.
The polymer-treated virus was monitored via capillary electrophoresis
(CE) using a Beckman PACE 5010 system with a 57cm silica capillary of 50,um
Internal diameter (inlet=anode). A preliminary 1.5min wash in 1 M NaOH and
second
20 wash in running buffer (20mM phosphate buffer pH 7.0, S.OmM NaCI) were
performed. After incubation, the samples were transferred to the CE machine
where
the auto sampler removed a few nanolitres by a pressure injection setting of l
Os and
separation was achieved using 2 minute voltage ramping to a final of l7Kv.
Whole virus CE monitors the changes in surface charge of the virus
25 upon treatment with PEG. Incubation with PEG correlates with a
progressively
increased more neutral mobility to the virus. Increasing neutrality is
consistent with
an increased PEG-chain density on the virus surface.
Figure 1 (upper panel) shows superimposed capillary
electropherographs for adenovirus exposed to 3%(w/v) TMPEG and MPEG. The
30 hiatus in each plot marks the trough at the point of neutrality. The TMPEG
treated
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virus ran at a location significantly nearer the neutral point than the sham-
treated
MPEG. Under these PEGylation conditions there is no evidence of residual
unPEGylated virus (i. e. no peak or shoulder on the TMPEG trace corresponding
to the
control virus).
In order to confirm that the mobility shift was not an artifact, a mixture
of equal volumes of the two samples was loaded (Figure 1, lower panel). Two
well
separated peaks were evident, corresponding to those shown in the upper panel.
Figure 2 shows the time course of the change in electrophoretic
mobility of virus with duration of exposure to TMPEG 3% (w/v), prepared
essentially
10 as described, above using 300,u1 of virus stock and 3%(w/v) TMPEG. The %
mobility
was calculated as follows: (mobility of modified virus peak- mobility of
neutral
position)/(mobility of unmodified virus peak-mobility of neutral position) X
100.
Since the reaction co-product can influence the running buffer, this was
renewed at the
point arrowed: 1001 of reaction mixture was analyzed up to this point (using
the
1 S repeat sampling function of the CE machine, i. e. without mixing) and a
fresh 1 OO,uI
aliquot of the reaction mixture was used thereafter.
Eacample 22
Covalent Attachment of Pol et y ne Glycol to Adenovir~
Type 2 adenovirus stock solution prepared as in Example 1
(1.35x10°
20 infectious units per ml; 9.3x10 ~ particles per ml) was PEGylated using
3%(w/v)
TMPEG except that rotary mixing was not used so that repeated size analyses
could
be made.
Viral particle size was monitored using photon correlation
spectroscopy (PCS) in a Malvern Instrument's ZetaMaster 5.
25 Figures 3A and 3B show the diameter versus time for TMPEG treated
and untreated virus respectively. Results are expressed as % time 0 values.
Figures
3C and 3D show measurements taken during a PEGylation reaction over a longer
time
period. Reaction with TMPEG is shown in Figure 3D and sham treatment with
MPEG is shown in Figure 3C. Treatment with TMPEG results in an increase in
particle size (Figs. 3B and 3D) which is not seen in the control untreated
virus (Fig.
CA 02341516 2001-02-22
WO 00/11202 PCTNS99/19162
26
3A) or in the MPEG treated virus (Fig. 3C). Increases in size are shown in
Figs. 3B
and 3D. PCS has the advantage of giving numeric data and thus the method gives
an
ability to rank samples.
1;~ a~
Infectivitv Assavs for PEG3rlate~ and Sham Treated Virus
Several regimes of PEG treatment were evaluated with respect to
retention of infectivity (see also Example 4). In addition to exposure to
3%(w/v)
TMPEG, stepwise addition was also used (the objective being to achieve higher
ultimate PEGylation). The rationale behind step wise addition is that viral
particles
tend to aggregate and this is exacerbated by PEG, especially at high
concentrations.
However, PEGylation has been shown, in the context of other particles (e.g.
liposomes), to prevent aggregation. Thus initial PEGylation at low polymer
concentration can serve to reduce the tendency to aggregate at subsequent
higher
polymer concentrations and hence achieve a higher degree of PEGylation. Three
step
1 S wise addition regimes were used: TMPEG or MPEG were added every thirty min
to
viral stock solution (prepared as in Example 1 ) to increase the polymer
concentration
by 3%, 5% or 8% in the reaction mixture. Viral stocks used for these
experiments
ranged from 1.35-7.6x10 infectious units per ml and 9.3-20x10" particles per
ml. In
each experiment a maximum of four additions of dry polymer were made, equating
to
final polymer concentrations of 12 %, 20 % and 32% (~w/v, i. e. not correcting
for the
volume of the polymer). In some experiments the 4th addition was sampled after
30
mins and a further incubation time (giving 5 reaction conditions).
Infectivity was measured in two ways (see also Example 4). ~i-gal
expression was monitored in human 293 cells (Graham et al., ~. Gen. Virol x:59-
72,
1977) exposed to virus in culture (this cell line is permissive for adenoviral
replication). Cells were trypsinised 1 day prior to assay and seeded at 4001
per well
in a 24 well microliter plate using a 1x106/ml cell suspension. Having
established a
monolayer by 24h, 101 of reaction mixture was added to each of 4 replicate
wells
containing 293 cells. The cells were incubated overnight in a fully humidified
atmosphere of S% CO, in air at 37°C to express ~3-gal.
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The cell monolayer was depleted of medium and then washed with
PBS. Then 60,u1 of lysis buffer (15 % triton X-100. 250mM Tris-HCI, pH 7.0)
was
added and the microliter plate incubated at room temperature for 30 min in an
orbital
shaker. After the cells had lysed for 30 min SOuI of each sample was
transferred to a
5 fresh microliter plate. A set of (3-gal standards (5.5 units in lysis buffer
and doubling
dilutions in lysis buffer) was added to the same microliter plate. 1 SO ,ul of
CPRG
substrate buffer (l.6mM CPRG, 60mM phosphate buffer: 1mM MgS04; IOmM KC1;
SOmM (3-mercaptoethanol; 250 ml distilled water) was added to each well. After
brief
mixing (4 min) the plate was read at 555 nm on a microliter plate reader
(Titertek
Multiskan Plus MKII, ICN, flow Laboratories, Switzerland).
The single addition of 3 % (w/v) TMPEG was examined using the
CPRG assay. Figure 4 shows the results of CPRG assays on TMPEG treated virus
(open circles) and MPEG sham-treated virus (triangles) and control virus
(filled
circles). None of the treatments produced a trend of falling infectivity over
the time
1 S period studied (six hours). A second independent experiment confirmed this
result,
showing no significant decline in OD over 6 hours for either control virus,
TMPEG
treated virus or virus sham-treated with MPEG (data not shown). Thus the PEG
treatment of virus in Examples 1 and 2 demonstrated no reduction in
infectivity.
The stepwise addition of 5% of PEGsooo, PEG,ZOOO or PEGZOOOO produced
a variable impact on infectivity (Figure 5). Panels A and B show the impact of
stepwise addition of 5% of PEGsooo (mean of 2 and mean LSD of 4 replicates
respectively, some error bars are hidden by the symbols). The TMPEG (filled
circles)
produced a reduction in infectivity as compared to the MPEG (open circles).
With
PEG,ZOOO (panels C and D, same symbols), in one experiment TMPEG decreased the
25 infectivity of the virus as compared with the MPEG treated virus, but in
the other,
MPEG and TMPEG were not significantly different (i. e. MPEG and TMPEG had a
similar effect on infectivity). Treatment with TMPEGzoooo also did not show
any
significantly greater effect than the equivalent amount of MPEGZOOOO (P~el E
same
symbols).
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1 4
Infectivitv Acsayc fnr pE~vlatPd and SHAM Treated Vinus
Single and stepwise additions of TMPEG and MPEG were prepared as
in Example 3 and analyzed with respect to infectivity using a chemiluminesent
reporter assay system for the detection of the virally encoded ~3-
galactosidase
(Galacto-LightTM). This assay system uses a chemiluminescent substrate and was
performed in accordance with the manufacturer's instructions.
Figures 6A - C compares the effects of 3%, S% and 8% incremental
additions of TMPEGsooo (filled circles) or MPEGsooo (open circles) on viral
infectivity.
Note that in Figure 6A and B the MPEG and TMPEG treated viral samples show
similar infectivity. A modest decline in infectivity with treatment with
either MPEG
or TMPEG was observed. In subsequent experiments with no-PEG controls these
showed a similar decline in infectivity, suggesting that this was a handling
effect and
not due to PEG. In Figure 6C the MPEG and the TMPEG treated virus performed
similarly. Thus, this experiment shows that treatment with TMPEG or MPEG does
not result in loss of infectivity.
Apparent loss of infectivity due to the addition of PEG chains was seen
twice with this assay in experiments using PEGsooo in the 5% incremental
addition
scheme (Figures 7A and B, filled circles TMPEG- open circles MPEG). A
subsequent
20 assay of the same sample as shown in Figure 7B showed no significant
difference
between the MPEG and TMPEG treatments, indicating that no significant loss of
infectivity had in fact occurred (Figure 7C, same symbols).
Figure 8 shows comparable results for PEG,ZOOO (panels A and B, filled
circles TMPEG; open circles MPEG) and PEGZOOOO (panel C, same symbols). As
above, condition 0 is an untreated virus control and conditions 1-4 are
stepwise
additions of S % TMPEG or MPEG. With the PEG,ZOOO there was a modest
additional
loss of infectivity with TMPEG in one of the two experiments after the 3rd and
4th
addition of TMPEG (panel B). In the other experiment (Panel A) using PEG,ZOOO
no
significant reduction in infectivity was observed with either TMPEG or MPEG.
With
30 PEGZOOOO, TMPEG treatment produced lower infectivity than MPEG for all
additions
including the first, but approximately one third the initial infectivity value
remained
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even after the 4th addition of TMPEG.
With a single addition of 3% PEGS, i.e. prepared as in Example 3,
with the chemiluminescence assay there was a modest decline in infectivity
(Figure 9).
It should be noted that the decline in infectivity observed over time was seen
both in
the case of the TMPEG (filled circles) and MPEG treated virus (open circles)
as well
as the untreated "handling" control (triangles).
Th PE a ' a iv' N ut 'e
Using the infectivity assay given in Example 4, exposure of the
TMPEG and MPEG treated virus to neutralizing antibodies was used to seek
evidence
of the protection from neutralization afford by the polymer treatment.
Transgene expression was monitored in the presence and absence of a
polyclonal neutralizing antibody purified from rabbit anti-hexon serum using a
hexon
affinity resin. The polyclonal antibody was titered with untreated virus and
the ratio
was established where 30 to 50% infectivity was retained in the presence of
the
neutralizing antibody. Two antibody titers were used 10,000:1 (~30%) or
5,000:1
(~40-SO%) (antibody molecules to virus particles) where indicated.
Figures 10-12 show the impact of incremental additions of 5%
TMPEGsooo (Figures 10 and 11) and TMPEG,ZOOO (Figure 12) to the adenovirus on
antibody neutralisation (B Panels) compared to incremental addition of MPEG (A
Panels) to the virus. The open circles in the A and B panels of Figures 10 -
12 are
infectivity in the absence of antibody; the closed circles are infectivity in
the presence
of antibody.
In all three cases there is evidence of significant protection from
neutralization and a trend of improving protection with the highest/longest
TMPEG
exposure giving maximum protection. The A and B panels in each figure show the
raw data, while Figures 1 OC, 11 C, and 12C show the transgene expression as a
percent of the equivalent non-antibody treated control. In each of the C
panels the
open bars indicate transgene expression from MPEG treated virus while the
hatched
bars indicate transgene expression from TMPEG virus. In Figure 10 the amount
of
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virus added to the assay was adjusted to compensate for differences in the
number of
infectious units of the non-antibody treated controls. In Figures 11 and 12
the same
number of viral particles was assayed for each condition. The antibody titers
were
10,000:1, 5,000:1 and 10,000:1 respectively.
5 These data show protection from immune recognition. For the
purposes of clarification, protection is defined as there being a
statistically significant
difference in transgene expression in the presence of the immune agent under
test (e.g.
antibody or cell suspension) as compared with the expression observed in
untreated
control.
10 The single addition of 3% TMPEGsooo showed some protection after 4h
and 6h incubation in two independent assays. Taken in conjunction the above
examples indicate the presence of a PEGylation "window" where treatment with
PEG
does not abrogate all infectivity but conveys statistically significant
protection from
neutralisation by antibody.
15 x 1~
Quantitative Analv i OFP .('YlatP~ Adenoviral Vector
An Ad2/(3-gal 2 vector (U.S. Patent No. 5,670,488 and described by
Zabner et al. ( 1996) . Vir . Z : 6994) was covalently modified by PEG with
0.01 %,
0.1%, 1.0% or 5.0% biotinylated NHS-PEGsooo (Shearwater Polymers). PEGylated
20 vector proteins were analyzed by SDS-PAGE. SDS-PAGE demonstrated that the
hexon, penton base and fiber were the primary targets for covalent
modification by
PEG, and increasing concentration of PEG led to modification of additional
proteins.
PEGylation of adenovirus was also assessed quantitatively. Ad 2-~i-gal
2 vector was treated with increasing amounts of TMPEG-biotin S%, 10%, or NHS-
25 PEG-biotin 0.01 %, 0.1 %, 1 %, 5%. Both PEGsooo's were obtained from
Shearwater
Polymers. Stepwise additions of PEG were made every 30 minutes up to a period
of 1
hour for TMPEG-biotin and 2 hours for NHS-PEG-biotin. Following PEG treatment
the unreacted PEG was separated from the PEG-virus by CsCI gradient
purification
and the amount of PEG-biotin attached to the virus was quantitated using an
ELISA
30 assay with an avidin HRP conjugate as reporter. A standard curve of PEG-
biotin (0-
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31
250 ng/ml) was generated to determine the number of molecules of PEG-biotin
attached per virus particle. Results are shown in Table 1.
Table 1
Sample Molecules PEG-biotin:virus particle
S 0.1% NHS-PEG-Biotin 600:1
1 % NHS-PEG-Biotin 3077:1
5% NHS-PEG-Biotin 3191:1
5% TMPEG-Biotin 1500:1
10% TMPEG-Biotin 1000:1
10 Treatment of adenovirus with either TMPEG-biotin or NHS-MPEG-
biotin led to the covalent attachment of PEG-biotin to the surface of the
virus. The
data indicates that at comparable concentrations of tresyl and NHS PEG-biotin,
more
PEG-biotin was attached to the virus particle after treatment with the NHS-PEG
biotin, which is consistent with reports that the reaction of NHS-PEG with
lysine
15 residues occurs more quickly (30-45 minutes) compared to the reaction of
tresyl
MPEG with lysine residues which occurs over an extended period of time (2-3
hours).
This data provides quantitative results regarding the extent of
covalently bound PEG.
cm 7
20 Covalent Attachment of Polvethylen~ lvcol t~ Adenoviru~
Type 2 adenovirus (genetically modified to carry the ~i-gal reporter
gene) was prepared by banding with isopycnic CsCI density centrifugation then
extensively dialysed against phosphate buffered saline (PBS pH 7.2). Three
different
types of MPEGs were tested for their ability to PEGylate adenovirus namely a)
25 cyanuric chloride activated MPEGS~ b) TMPEGS~ and c) amino-PEGS~o. The
MPEGs were obtained from Shearwater Polymers. Activation of MPEG with
cyanuric chloride couples one triazine ring per MPEG molecule. This activated
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MPEG can react with amino groups on proteins. Alternatively MPEG can be
activated with tresyl chloride (2,2,2,-trifluoroethanesulphonyl chloride) to
form
tresylated MPEG which can react with epsilon amino groups on proteins to form
a
highly stable amine linkage. SPDP-amino MPEG couples to proteins via cysteine
S residues. The activated NHS ester end of SPDP reacts with the amine groups
on the
amino PEG to form an amide linkage. The 2-pyridyldithiol group at the other
end is
free to react with sulfhydryl groups to form a disulfide linkage. SPDP -
aminoPEG
was synthesized by the addition of SPDP (N-succinimidyl 3-(2-pyridylditthio)
propionate) to amino PEG in the presence of methanol. Following an overnight
incubation at room temperature the SPDP-aminoPEG was collected by
precipitation
with ether.
Ad2-~i-gal 2 virus was incubated with either a) cyanuric chloride
activated MPEG b) TMPEG or c) amino PEG at increasing ratios of PEG:lysine.
Ad2-(3-gal 2 virus was dialysed into 0.1 M sodium carbonate buffer pH 8.5
containing
15 O.15M NaCI before treatment with cyanuric chloride activated MPEG or 0.2M
sodium
phosphate buffer pH 7.5 containing 0.1 SM NaCI before treatment with TMPEG.
All
PEGylation reactions were performed at room temperature. Samples were mixed on
a
rotary platform, the PEGylation reaction was terminated by the addition of
excess
lysine or alternatively by lowering the temperature. Infectivity of the
PEGylated
20 viruses was initially assessed qualitatively by infecting 293 cells with
PEGylated virus
followed by measurement of transgene expression ((3-galactosidase) using X-gal
staining. Using this assay the TMPEG treated virus had greater infectivity
than the
virus that had been treated with cyanuric chloride activated PEG or SPDP-PEG.
The
TMPEG treated virus was further measured for infectivity using the more
quantitative
25 assay of end-point dilution in 293 cells using fluorescence isothiocyanate
(FITC)-
conjugated anti-hexon antibody as described by Rich, DP, Couture LA, Cardoza
LM,
Guiggio, VM, Armentano, D., Espino, PC, Hehir, K., Welsh, MJ, Smith, AE and
Gregory, RJ, 1993, Hum. Gen. Ther. 4:461-476.
The results are shown in Table 2 and demonstrate that infectivity of the
30 virus is retained following PEGylation with TMPEG. (Error in the assay is
t0.5 log.)
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Table 2
PEG:Lysine Infectivity
5:1 3.8e8 iu/mI
2.5:1 1.5e8 iu/ml
1 ~ 1 2.2e8 iu/ml
Control Se8 iu/ml
Exa~mnle 8
Reduced Bindin of Neutral-izin Ant~hnrt;PC r
O P ~YlBttP~ VPrtnr
Ad2-(3-gal 2 virus was PEGylated with TMPEG as described in
Example 11. Virus was incubated with serial two-fold dilutions of neutralizing
human serum for 1 h/37°C and 293 cells were added. The assay was read
when 293
cells incubated alone reached confluency. The neutralizing titer was defined
as the
reciprocal of the highest dilution of serum that showed detectable protection
of 293
cells from cytopathic effect when compared to cells incubated with virus not
exposed
1 S to serum. Prior to the assay, the different virus preparations to be
tested were titrated
to ascertain the lowest dilution that caused 100% cytopathic effect. Results
are shown
in Table 3.
Table 3
Virus Neutralizing titre
PEG:lysine ratios
5:1 800
2.5:1 3200
Control 6400
According to the results, more serum is required to neutralize the
PEGylated virus compared to the untreated virus suggesting that PEGylation
covers
sites recognized by neutralizing antibodies.
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19
Ion-exchange Chromato ~r ~y odd Virus Pa_rtirles
Ad 2-(3-gal virus was PEGylated as described in Example 11 with
TMPEG at ratios of 50 moles and 10 moles PEG:lysine. The virus was applied to
a
S DEAF ion-exchange resin (Millipore, Bedford, MA) in phosphate buffer
containing
NaCI. Bound virus was eluted from the resin using an increasing salt gradient
and the
flow through peaks and eluted protein peaks were analyzed for control virus,
virus
treated with TMPEG at a ratio of 50:1 PEG:lysine and virus treated with PEG at
a
ratio of 10:1 PEG:lysine. All samples had equivalent protein values before
chromatography.
Figure 13, panel A shows the elution profile from the DEAE-ion
exchange resin (Millipore, Bedford, MA) following chromatography of control
virus.
One main protein peak was eluted from the resin and this was shown to contain
infectious virus particles (data not shown). Figure 13, panel B shows the
elution
15 profile from the DEAF-ion exchange resin following chromatography of virus
that
had been treated with TMPEG (10:1 ratio). In contrast to the profile for the
control
virus there is the appearance of a flow through peak in addition to the eluted
protein
peak, which has diminished in size. The appearance of the flow through peak
suggests that PEGylation has generated viral particles which no longer can
bind to the
20 DEAE-resin under these conditions and as a result are now present in the
flow through
peak along with unreacted PEG. Since ion-exchange chromatography is based on
charge interactions between the protein and the ion-exchange resin, apparently
PEGylation has produced a heterogenous population of virus particles which
have
altered surface charges. Those with significant surface charge differences can
no
25 longer bind to the resin and are recovered in the flow through peak. The
elution
profile from the DEAE-ion exchange resin following chromatography of virus
PEGylated with TMPEG at a ratio of 50:1 showed a similar profile. The flow
through
peak in this sample was significantly larger while the eluted protein peak was
in
contrast reduced. At the increased ratio of PEG:lysine of 50:1 which resulted
in a
30 greater fraction of particles eluting in the flow through peak, the virus
particles had
increased levels of PEGylation. Table 4 expresses the size of the two peaks
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(expressed as area under peak) in relation to the PEG:lysine ratios used
during
PEGylation. In conclusion, ion exchange chromatography may be used to resolve
heterogeneous populations of PEGylated virus particles and may be used to
separate
highly PEGylated virus particles from lightly PEGylated particles on the basis
of
5 charge differences.
Table 4
Flow Through Peak Area Eluted Peak Area
Control NA 0.272
PEG-Virus 0.022 0.132
10:1
10 PEG-Virus 0.063 0.031
50:1
Example 10
Transgene nres~ion b~E~vlated Ad2/~i Gal2 in Immune M~rP
Two batches of Type 2 adenovirus stock solution prepared as in
15 Example 1 were mixed (2m1 of a batch at 5.38x10'° infectious units
per ml,
2.055x10''- particles per ml and 4 ml of a batch at 1.35x10'°
infectious units per ml,
9.3x10" particles per ml) and subjected to treatment with PEG using a stepwise
addition regime of 5% TMPEG as in Example 3. Samples obtained following two
and three additions of TMPEG (i.e., total 10% and 15% TMPEG, respectively)
were
20 purified from unreacted TMPEG by a standard CsCI (Sigma Chemical, St.
Louis,
MO) centrifugation procedure involving a step gradient and two sequential
equilibrium gradients. The purified PEG treated vectors were then dialyzed
against
phosphate buffered saline containing 5% sucrose and frozen at -80°C in
small
aliquots. The titers were determined by end point dilution on 293 cells using
'
25 fluorescence isothiocyanate (FITC)-conjugated anti-hexon antibody as
described by
Rich, DP, Couture LA, Cardoza LM, Giuggio VM, Armentano D, Espino PC, Hehir
K, Welsh MJ, Amith AE and Gregory RJ, 1993, Hum. Gen Ther 4_:461-476. The
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36
purified PEG treated viral suspension prepared with total 10% TMPEG contained
2.7x10" particles/ml (3x109 infectious units/ml) and the purified PEG treated
viral
suspension prepared with total 15% TMPEG contained 2.4x10" particles/ml
(6.4x108
infectious units/ml).
5 The two PEGylated viral suspensions were compared to untreated Type
2 adenovirus (3.19x10'° infectious units per ml) for ability to effect
gene transfer jg
vivo in naive and pre-immunized BALB/c mice. Mice were pre-immunized by the
intra-nasal administration of 109 infectious units of a replication defective
Type 2
adenovirus encoding human CFTR (Ad2/CFTR). The animals chosen for the study
10 had serum anti-adenovirus antibody titers of circa 1/25,000 to 1/50,000.
Naive
BALB/c mice were simply mice that had not been exposed to adenovirus vector.
On
day 0, the viral preparations were administered as follows: a) untreated
virus, 2x108
infectious units were instilled in a volume of 100 ,ul to each of four mice in
the naive
group and four mice in the pre-immunized group, b) "PEGylated virus 10%",
3x108
1 S infectious units (2.7x 10'° particles) were instilled in a volume
of 100 ,ul to each of
four mice in the naive group and four mice in the pre-immunized group, c)
"PEGylated virus 15%", 6.4x10' infectious units (2.4x10'° particles)
were instilled in
a volume of 100 ,ul to each of four mice in the naive group and four mice in
the pre-
immunized group. All animals in the pre-immunized group were subjected to
20 eyebleed on the day of instillation and the blood was analyzed for antibody
titers. All
mice were sacrificed three days after instillation and lung tissue, right
caudal lobe and
left lobe, was excised. The right caudal lobe from all four naive and four
immunized
animals per condition (untreated, "PEGylated virus 10%" and "PEGylated virus
15%") was used for quantification of 13-gal in an AMPGD assay (Galacto-LightTM
Kit,
25 Tropix, Bedford, MA). The protein concentration of lung homogenates was
determined using the BioRad DC reagent (BioRad, Hercules, CA). The left lobe
from
two naive and two immunized animals per condition was used for x-gal staining.
Table 5 shows the beta-galactosidase expression per microgram of
protein (relative light units, RLU per microgram of protein) for untreated
virus,
30 "PEGylated virus 10%" and "PEGylated virus 1 S%" in both naive and pre-
immunized
mice. Beta-galactosidase expression in the naive mice was observed for all
three viral
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37
preparations in all four mice per condition. In the pre-immunized mice, the
untreated
vector gives only background levels of beta-galactosidase expression in all
four mice.
In contrast, the two PEGylated viral preparations gave levels of beta-
galactosidase
above those for the control animals in 4/4 and 3/4 animals for the "PEGylated
virus
10%" and "PEGylated virus I S%" preparations, respectively (see Table 1 ).
Thus
PEGylation of the virus conveys protection from neutralization ~ vivo
resulting in
substantial expression of the vector in the target tissue ~ vivo.
Table 5. Beta-Galactosidase expression in lung tissue expressed as
relative light units per microgram of protein (RLU/~g protein).
Preparation Mouse RLU/~g proteinRLU/,ug protein
(infectious units) Number Native Immunized
Control virus (2x108 1 955 25
iu)
2 1457 90
3 649 28
4 1388 3g
PEGylated 10% (3x108 1 2341 218
iu)
2 2108 1296
3 3694 164
4 1730 1964
PEGylated IS% (6.4x10'1 705 34
iu)
2 172 305
3 715 198
4 1128 108
Exam lp a 11
Transeene Expression of PEGylated Ad2/j~gal 4 virus in Immune Mi~g
Ad2/~3-gal 4 virus (LJ.S. Patent No. 5,670,488) was PEGylated with
10% tresyl MPEG (TMPEG) as already described. PEGylated virus was purified
from
unreacted TMPEG by banding on cesium chloride gradients (Rich et al., ~ Gene
Theranv 4:461-476, 1993). The purified PEGylated virus was dialysed into
phosphate
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38
buffered saline (PBS), S% sucrose and the titre was determined by end point
dilution
on HEK293 cells using fluorescent isothiocyanate (FITC)-conjugated anti-hexon
antibody (Rich et al., 1993). Control or sham treated vector was treated with
non-
reactive MPEG and was purified and titred as described for TMPEG virus.
PEGylated
and sham treated virus were instilled into immune and naive mice. The dose for
each
vector was 2 x 108 iu/mouse (equivalent to ~2 x 10'° particles), the
dose volume per
mouse was 100 ~l. Immune mice had previously been instilled with Ad2 - CFTR-8
vector (U.S. Patent No. 5,707,618) and had titres to adenovirus in the range
25,000 -
S 1,200.
Three days after instillation the animals were sacrificed and lung tissue
from individual animals were homogenised and (3-galactosidase activity in the
homogenate was assessed using a commercially available assay kit according to
manufacturer's instructions (GalactolightTM Kit, Tropix, Bedfor, MA.). The
protein
concentration of lung homogenates was determined using the BioRad DC reagent
(BioRad, Hercules, CA) and the results expressed as relative light units
(RLU)/ug
protein.
Figure 14 shows the (3-galactosidase expression for PEGylated virus
(Ad TMPEG) and sham treated virus (Ad MPEG). Results shown are the mean t
standard deviation of the values obtained with individual animals. ~i-
Galactosidase
expression was measured in the lungs of naive mice for both the MPEG and the
TMPEG (N=2) viral preparations. In the pre-immunised mice (N=4) the sham
treated
virus (Ad MPEG) had reduced levels of ~i-galactosidase expression (~47% of the
~i-
galactosidase expression measured in naive animals), presumably due to
neutralisation
by adenovirus specific antibodies. In contrast in the pre-immunised mice (N=3)
the
PEGylated virus gave levels of ~i-galactosidase expression equivalent to those
measured in naive animals (~89% of the expression measured in naive animals).
Thus
PEGylation of the adenovirus protects the virus from neutralisation, allowing
full
expression of the vector in the target tissue in the presence of an immune
response.
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39
Transgene Expression of Catinn;r tom 1 vP
of PEGvlated Ad2/(3-gal 2 virus in Naive and Imm mized Mice
Following the procedure of Example 1, Ad2-~i-gai 2 virus was
PEGylated with 10% TMPEG (Shearwater Polymers, Huntsville, AL). The
PEGylated virus was purified from unreacted TMPEG by banding on CsCI gradients
and purified by dialysis against phosphate buffered saline (PBS) as previously
described. The titre was determined by end point dilution on 293 cells using
fluorescence isothiocyanate (FITC)-conjugated anti-hexon antibody as set forth
in
Rich et al., (1993) Hum.Gen.Ther. 4_:461-476. The PEGylated virus had a titre
of 7.6
x 108 iu/ml and a particle:iu ratio of 800. Interestingly, the titre of the
virus before
PEGylation was 9.5 x 109 iu/ml with a particle:iu ratio of 80. Thus, viral
infectivity
was compromised during PEGylation.
Naive mice were instilled with samples of unmodified virus (control),
the PEGylated virus and the PEGylated virus complexed with either DEAF-dextran
or
Poly-L-lysine (PLL). The dose for the control was 2 x 108 iu/animal, which was
equivalent to 1.0 x 10'° particles/mouse ratio. The dose for PEGylated
virus
(complexed and non-complexed) was 7.6 x 10' iu/animal, which was equivalent to
6.4
x 10'° particles/mouse. PEGylated virus was complexed with DEAE-dextran
at a ratio
of 3000 molecules DEAE-dextran per virus particle while the virus was
complexed
with PLL at a ratio of 500 molecules per virus particle. The dose volume per
mouse
was 100 ul.
Three days after instillation the animals were sacrificed and lung tissue
from individual animals were homogenised and (3-gal activity in the homogenate
was
assessed using the GalactolightTM Kit (Tropix, Bedford, MA) according to
manufacturer's instructions. The protein concentration of lung homogenates was
determined using the BioRad DC reagent (BioRad, Hercules, CA) and the results
expressed as relative light units (RLU)/ug protein. The results are shown in
Figure 15.
Figure 15 shows that the PEGylated virus complexed with DEAE-
dextran had increased infectivity as compared to the non-complexed PEGylated
virus,
the unmodified control virus and the PEGylated virus complexed with PLL.
Results
CA 02341516 2001-02-22
WO 00/11202 PCTNS99/I9162
shown are the mean ~ standard deviation of the values obtained with individual
animals.
The infectivity of the above-described adenovirus PEGylated with
either TMPEG or MPEG and complexed with DEAF-dextran was also ascertained in
naive and immune mice following the procedure set forth above, but with TMPEG
from a different supplier being used (Sigma Chemical, St. Louis, MO). The
immune
mice had previously been instilled with Ad2-CFTR-8 vector and had titres of
adenovirus in the range 25000 - S 1200. The results are shown in Figure 16.
Figure 16 shows that the PEGylated virus when complexed with
10 DEAE-dextran and administered to naive and immune mice exhibited increased
infectivity. Moreover, transgene expression in the immune mice that received
virus
PEGylated with TMPEG and complexed with DEAF-dextran was equal to that
measured for naive mice which demonstrates the reduced antigenicity of the
PEGylated virus complexed with DEAF-dextran. In comparison, the transgene
15 expression of virus PEGylated with MPEG and complexed with DEAE-dextran was
increased only in naive mice. Thus, virus PEGylated with TMPEG and complexed
with DEAF-dextran exhibited both increased infectivity and reduced
antigenicity.
