Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Descri~ t~ ion
Polymer-Modified Viruses
The present application claims priority from United Kingdom Patent Application
Nos. 9706735.9, 9719625.7 and 9722316.8 filed April 3, September 15 and
October 22,
1997, respectively.
Ba~,~ground of the Invention
Viruses have many potential therapeutic uses, for example in gene therapy,
whereby the viral genome is used as a vector for foreign genes, as well as in
vaccination
and cancer therapy, for example by exploiting the phenomenon of viral
oncolysis, which
exploits cell destruction following selective virus replication in certain
tumors.
However, clinical use of viruses presents certain problems. For example, many
human subjects are pre-immune to common viruses such as adenoviruses, and thus
have
circulating antibodies. In cases in which the circulating antibodies are
neutralizing in
nature, the administered viral particles may have reduced or no infectivity.
Repeated
administration may exacerbate this problem, since most viruses are highly
immunogenic.
Immune responses may also contribute to the toxicity of viral administration,
and in cases
in which cellular immunity is involved, some profound tissue damage may
result.
In addition to problems related to the immune system, virus particles are also
potentially vulnerable to other clearance mechanisms. Particulates tend to be
filtered by
the liver and spleen via a mechanism involving phagocytic/endocytic uptake by
macrophages. Viral aggregates may be cleared by such mechanisms. In addition,
activation of the complement system by viruses may be a factor involved in the
inactivation of some viral vectors. Proteolysis and, where relevant,
lipolysis, may also
potentially damage viral particles.
Viral particles also often have highly specific tissue distribution. This is
not
always desirable in the therapeutic applications envisaged for the virus. For
example, it is
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desirable in some settings to circumvent the natural viral tissue
distribution, possibly
simultaneously 'targeting' the virus to a new site such as a tumor. With
appropriate
modification of viral vectors, both active and passive targeting strategies
should be
feasible with such vectors. However, abrogation of tissue specific
localization systems
may make viral particles more susceptible to non-specific uptake mechanisms.
One form
of passive targeting particularly relevant to viral vectors for use in gene
therapy for cancer
or in viral oncolysis is the so-called enhanced permeability and retention
effect, which
exploits leaky vasculature and poor lymphatic drainage in tumors, which can
achieve
enhanced localization of particulates.
Virus particles also have veterinary and agricultural uses which share some of
the
above problems.
Polymer modification has been shown, in the context of polymer-protein and
polymer-liposome constructs, to have the potential to solve many problems. For
example, polymer cover has been demonstrated to reduce antigenicity and
1 S immunogenicity. In addition, light polymer cover can turn an antigen into
a tolerogen.
Polymer cover can also ameliorate reticuloendothelial system (RES) uptake of
particulates. Further, polymer can serve as a linker to couple targeting
devices to the
surface of other molecules or macromolecular structures to target them to
specific sites.
However, living viruses are very different in their characteristics to
proteins and
liposomes. The surface structures involved in infectivity might well be
compromised by
polymer modification. Virtually all clinical applications of viruses require
infectivity to
be maintained.
It has been surprisingly found in accordance with the present invention that
viral
particles can be polymer modified and yet retain infectivity. It has also been
discovered
that polymer modification of viruses results in the acquisition of beneficial
properties
such as improved capacity to infect in the presence of neutralizing
antibodies.
Summary of the Invention
The present invention provides viruses modified by polymers. In a preferred
embodiment the polymer is polyethylene glycol (PEG). In one embodiment, the
polymer
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is directly covalently attached to the virus. In another embodiment, the
polymer is
indirectly covalently attached to the virus via an intermediate coupling
moiety. In yet
another embodiment, the polymer is indirectly noncovalently attached to the
virus via a
ligand. In a preferred embodiment, the ligand has specificity for a viral
surface
component. For example, the ligand may be an antibody.
The present invention further provides a method of making viruses modified by
polymers, whereby the modified viruses retain infectivity.
Another embodiment of the present invention provides a method for introducing
a
transgene into a target cell comprising contacting the target cell with a
polymer-modified
virus, wherein the virus comprises the transgene.
The present invention further provides a method of delivering a virus to a
tumor,
comprising administering a polymer-modified virus of the invention to a
subject in need
of such treatment under conditions whereby the polymer-modified virus
localizes to a
tumor.
In another embodiment, the present invention provides a composition comprising
a virus modified by a polymer and a carrier.
Brief Description of the Drawings
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.
Fig. 3A-D shows 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.
Fig. SA-E depicts infectivity (CPRG) assay results for stepwise additions of
5%
PEGsooo, PEG~ZOOO~ or PEGzoooo.
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Fig. 6A-C depicts infectivity (chemiluminescence, RLU) assay results for
stepwise additions of 3%, 5% or 8% PEGsooo~
Fig. 7A-C depicts infectivity (chemiluminescence, RLU) assay results for
stepwise additions of 5% PEGSOOO~
S Fig. 8A-C depicts infectivity (chemiluminescence, RLU) assay results for
stepwise additions of S% PEG~ZOOO and PEGZOOOO~
Fig. 9 depicts infectivity {chemiluminescence, RLU) assay results for a single
addition of 3 % PEGsooo~
Fig. l0A and B shows graphs of an antibody neutralization assay for the impact
of
stepwise additions of 5% PEGsooo on neutralization of infectivity
(chemiluminescence,
RLU assay), 10,000:1 antibody molecules to virus particles.
Fig. 1 lA and B shows graphs of antibody neutralization assays for the impact
of
stepwise additions of 5% PEGSOOO on neutralization of infectivity
(chemiluminescence
RLU assay); 5,000:1 antibody molecules to virus particles.
1 S Fig. 12A and B shows graphs of an antibody neutralization assay for the
impact of
stepwise additions of 5% PEG,ZOOO on neutralization of infectivity
(chemiluminescence
RLU assay); 10,000:1 antibody molecules to virus particles.
Fig. 13 shows a graph of a fluorescamine assay of anti-hexon antibody modified
using TMPEG.
Fig. 14 shows a graph of a fluorescamine assay of MAb 8052 modified using
cyanuric chloride-MPEG.
Fig. 15 shows an SDS-PAGE gel showing immunoprecipitation of adenoviral
hexon by PEGylated anti-hexon antibody.
Fig. 16A-E depicts gel permeation chromatography of antibody and PEGylated
antibody on a Superose 12 column.
Fig. 17A-J depicts antibody competition ELISA, showing competition of
biotinylated anti-hexon antibody by binding to virus in the presence of
increasing
concentrations of PEG antibody.
Fig. 18A-C shows the elution profile of control and TMPEG-treated virus from
DEAE ion exchange resin following chromatography.
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Fig. 19 A-C shows the elution profile of untreated (panel 19a), MPEG treated
(panel 19b) and TMPEG treated (panel 19C) Adenovirus ONYX-015 from 1 ml
Resource
Q column (Pharmacia).
Fig. 20 depicts infectivity assay results (ELISA for hexon protein) following
stepwise additions of 5% TMPEGsooo or MPEGsooo to Adenovirus ONYX-015.
Fig. 21 A-F shows a laser copy of photographs demonstrating cytophatic effect
{CPE) for untreated Adenovirus ONYX-015 (panels A-B) and ONYX-O15 incubated
with
5% MPEGsooo (P~els C-D) or TMPEGsooo (P~els E-F).
Fig. 22 shows a laser copy of immunofluorescence photographs (staining with
anti-hexon antibody) demonstrating infectivity and replication of adenovirus
ONYX-015
incubated with TMPEGSOOO~
Fig. 23 shows the infectivity measured by plaque assay of vaccinia virus
following stepwise addition of MPEG500 or TMPEGsooo~
Fig. 24 shows photographs demonstrating infectivity measured by ~3-
galactosidase
expression of vaccinia virus, following step-wise addition of MPEGsooo or
TMPEGsooo~
Fig. 25 shows an autoradiograph of an SDS-PAGE demonstrating the early gene
expression (production of 'y-IFNg receptor) following infection with vaccinia
virus which
had been incubated with MPEGsooo or TMPEGsooo using step-wise addition.
Fig. 26 demonstrates the expression of late genes (IL-1 ~3 receptor) following
infection with vaccinia virus which had been incubated with MPEGsooo or
TMPEGsooo
(step-wise addition).
Fig. 27 demonstrates protection from neutralisation by anti-vaccinia serum for
Vaccinia virus which had been incubated with TMPEGsooo (step-wise addition).
Fig. 28 A-B shows the infectivity measured by plaque assay of Retrovirus
following step-wise addition of MPEGSOOO or TMPEGsooo~
Fig. 29 A-F shows lacZ expression following infection with Retrovirus which
had
been incubated with MPEGsooo or TMPEGSOOO (step-wise addition).
Fig. 30 A-B shows the infectivity measured by plaque assay of Herpesvirus
following step-wise addition of MPEGsooo or TMPEGsooo.
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Fig. 31 A-B shows the elution profile of ONYX-O 1 S incubated with PVP (panel
32a) and activated PVP (panel 32b) from 1 ml Resource Q column (Pharmacia).
Fig. 32 shows immunofluorescent staining of liver (A) and tumor sections (B
and
C) taken from nude mice bearing LS 174T human colon carcinoma injected with
PEGylated virus (A and B) or control virus (C).
Fig. 33 shows transgene expression in mice infected with PEGylated or sham
treated adenoviral vectors.
Detailed Description of the Invention
The present invention provides viruses modified by polymers. Such a viral
particle has one or more polymer molecules covalently or noncovalently bound
thereto.
The polymer-modified viruses of the present invention maintain the biological
property
of infectivity.
In accordance with the present invention, polymers are generally large non-
immunogenic, biologically inert molecules comprising a chain of smaller
molecules
linked by covalent bonds. Polymers useful in accordance with the present
invention are
those polymers which, when covalently or noncovalently bound to a virus,
provide a
polymer-modified virus that retains detectable levels of infectivity and is
substantially
non-immunogenic. The polymers preferably have an average molecular weight of
from
about 200 to about 20,000 daltons. The polymers are biocompatible, and may be
linear or
branched. The polymers may be homopolymers or heteropolymers. Suitable
polymers
for use in the present invention include polyalkalene compounds such as
polyalkalene
oxides and glycols. Polyalkalene compounds include polyoxymethylene,
polyethylene
glycols (PEG) and oxides, and methoxypolyethyleneglycols, and derivatives
thereof
including for example polymethyl-ethyleneglycol, polyhydroxypropyleneglycol,
polypropylene glycol, polymethylpropylene glycol, polyhydroxypropylene oxide
and
polyvinyl pyrrolidone (PVP).
A preferred polymer 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
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molecular weights of from 200 to 20,000 daltons are commercially available. In
accordance with the present invention, PEG having an average molecular weight
of from
200 (PEGZOO) to 20,000 (PEGZO,ooo) may be used to prepare viruses modified by
PEG. In a
preferred embodiment, the PEG has an average molecular weight of from about
2000 to
about 12,000. In a more preferred embodiment, the PEG has an average molecular
weight of about 5000.
It has been discovered in accordance with the present invention that polymer-
modified viruses can exhibit reduced antigenicity while retaining infectivity.
Accordingly, viruses that are useful for the present invention include viruses
for which
I O the properties of infectivity and reduced antigenicity are desired.
Further, the polymer-
modified viruses of the present invention may exhibit increased circulation
time in vivo.
Thus the present polymer-modified viruses have utility for therapeutic and
diagnostic ill
vivo applications.
The polymer-modified viruses have utility in medical therapy and diagnosis in
medical and veterinary practice and in agriculture. They are 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, 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.
More specifically, the present invention is directed to a virus selected from
RNA and
DNA viruses. Preferably the virus used is selected from the following families
and
groups: Adenoviridae; Birnaviridae; Bunyaviridae; Caliciviridae; Capillovirus
group;
Carlavirus group; Carmovirus virus group; Group Caulimovirus; Closterovirus
Group;
Commelina yellow mottle virus group; Comovirus virus group; Coronaviridae; PM2
phage group: Corcicoviridae; Group Cryptic virus; group Cryptovirus;
Cucumovirus
virus group Family ~6 phage group; Cystoviridae; Group Carnation ringspot;
Dianthovirus virus group; Group Broad bean wilt; Fabavirus virus group;
Filoviridae;
Flaviviridae; Furovirus group; Group Geminivirus; Group Giardiavirus;
Hepadnaviridae;
Herpesviridae; Hordeivirus virus group; Ilarvirus virus group; Inoviridae;
Iridoviridae;
Leviviridae; Lipothrixviridae; Luteovirus group; Marafivirus virus group;
Maize chlorotic
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_g_
dwarf virus group; icroviridae; Myoviridae; Necrovirus group; Nepovirus virus
group;
Nodaviridae; Orthomyxoviridae; Papovaviridae including adeno-associated
viruses;
Paramyxoviridae; Parsnip yellow fleck virus group; Partitiviridae;
Parvoviridae; Pea
enation mosaic virus group; Phycodnaviridae; Picornaviridae; Plasmaviridae;
Podoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxviridae;
Reoviridae;
Retroviridae; Rhabdoviridae; Group Rhizidiovirus; Siphoviridae; Sobemovirus
group;
SSV1-Type Phages; Tectiviridae; Tenuivirus; Tetraviridae; Group Tobamovirus;
Gioup
Tobravirus; Togaviridae; Group Tombusvirus; Group Torovirus; Totiviridae;
Group
Tymovirus; Plant virus satellites.
Particularly preferred viruses for the purpose of delivery of transgenes
include, for
example, retrovirus, adenovirus, adenoassociated virus, herpesvirus and
poxvirus.
Adenovirus is particularly preferred.
As used herein, the term virus includes recombinant genetically engineered
viruses. For example, the virus may be a virus that has been engineered such
that it is
incapable of replicating and exhibits minimal gene expression. The recombinant
viruses
may contain transgenes. Transgenes are defined herein as nucleic acids that
are not
native to the virus. For example, a transgene may encode a biologically
functional
protein or peptide, an antisense molecule, or a marker molecule.
The polymer-modified viruses of the present invention may be provided by
direct
covalent, indirect covalent, or indirect noncovalent attachment of the polymer
to the
virus.
A variety of schemes for covalent and non-covalent attachment exist: 1 )
polymer
may be attached via direct covalent coupling to the viral surface; 2) polymer
may be
attached via indirect covalent coupling (e.g. via an intermediate coupling
moiety which
links the polymer to the viral surface); or 3) attached via an indirect non-
covalent linkage
using, for example, a suitable PEGylated ligand. Suitable ligands are not
restricted to
antibodies to surface proteins or lipid and could include hydrophobic ligands
for viral
particles with hydrophobic surface components such as envelope viruses.
The polymer may be attached via direct or indirect covalent coupling to the
viral
surface by methods that are generally known in the art for covalent attachment
of
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polymers to other molecules, such as proteins. 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 amino groups, thiol
groups and
aromatic hydroxy groups. Thus the preferred method for polymer modification of
a virus
depends upon the available target sites on the surface of the particular
virus. The
specificity of particular methods of polymer modification for particular
target groups is
well-known, and thus the ordinarily skilled artisan can select a method
suitable for the
desired target.
Different methods of polymer modification may be selected depending upon
whether the virus is enveloped or non-enveloped. The surface of a non-
enveloped virus is
a protein shell, or capsid, often containing multiple types of polypeptides.
Representative
non-enveloped viruses include adenovirus, parvovirus and picornavirus. In
enveloped
viruses, the protein capsid is enclosed by a lipid bilayer that contains viral-
encoded
polypeptides. Representative enveloped viruses include herpesvirus, poxvirus
and
baculovirus. Both the capsid and the envelope polypeptides provide targets for
polymer
modification. For example, in a nonenveloped virus such as adenovirus, the
hexon,
penton cell base, and fiber proteins are targets for polymer modification.
Viral
polypeptides that provide sites of exposed epitopes for neutralizing
antibodies, for
example the adenoviral hexon protein, are particularly preferred sites for
polymer
modification. Modification of these sites is believed to mask the epitope from
neutralizing antibodies, thus providing a viral vector with reduced
antigenicity.
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 viruses
of the
present invention. Methods are described, for example, in WO 90/04606, U.S.
Patent
4,179,337 and 5,612,460, the disclosures of which are incorporated herein by
reference.
Generally, the 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.
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For example, the hydroxyl end groups of PEG may 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 virus a 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-PEGj, which reacts with amine groups, and PEG-N-
succimimide carbonate. These and other activated PEGS are disclosed in
W095/06058,
U.S. Patents 4,179,337 and 5,612,460 incorporated herein by reference.
For example, the covalent attachment of PEG to the viral surface is
accomplished
by incubating the virus with the activated PEG, for example TMPEG. Several
incubation
regimes may be used. For instance, a single addition of the activated polymer
with or
1 S without gentle mixing can be used. The optimal ratios of TMPEG to viral
particles to
achieve modified virus having reduced antigenicity with maintenance of
infectivity may
be determined by performing the assays described below. For example, virus and
activated TMPEG are combined at molar ratios of activated PEG to E-amino
termini of
lysine residues of from about 1:1 to about 400:1. As the amount of activated
polymer to
be added to the virus increases, it may be alternatively advantageous to add
the activated
polymer in a stepwise fashion. The rationale behind stepwise addition is that
viral
particles tend to aggregate and this is exacerbated by certain activated
polymers, e.g.
TMPEG, especially at high concentrations. Thus initial PEGylation at low
polymer
concentration can serve to reduce the tendency to aggregate at subsequent
higher polymer
concentrations and hence help to achieve 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 concentration each time by 3%, 5%
or 8%
(w/v) 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 polymer, a further incubation time
might be allowed.
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The ordinarily skilled artisan can adjust the number of steps, concentrations
of polymer,
and time intervals to achieve optimal results.
The reaction may be quenched by dialysis or by addition of excess lysine, for
example from 10 to 100-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).
For some applications, for example those requiring repeat dosing of a polymer
modified virus, it may be desirable to separate unreacted polymer from polymer-
modified
virus, which may then be purified by standard methods as necessary for the
intended use.
Separation and purification may be performed by methods known in the art, for
example
ion exchange chromatography, 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.
For some applications, it may be desirable to separate unmodified virus from
modified virus. In cases in which the polymer is a polyalkylene glycol,
separation of
modified from unmodified 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 modification of virus by PEG ("PEGylation") may be 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, for example, DEAE-chromatography, can be
performed by standard methods to evaluate the modified viruses based upon
altered
charge.
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Whole virus CE provides a means to monitor the modification of virus by
polymer as a function of altered surface charge. For example, covalent
attachment of
PEG to the virus surface seems to result in shrouding of the negative surface
charges on
the viral particle and thus this polymer-modified virus displays a more
neutral mobility to
the virus. 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
eiectrophorese the
highly mobile salt ions in which the virus may be formulated for stability,
before trite,
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 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 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 covalentiy 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 a virus
particle. The
ELISA can be performed by standard methods known in the art.
In a preferred embodiment of the present invention, the polymer-modified virus
is
a recombinant virus prepared under conditions believed to provide a virus
covalently
modified by PEG. In a particularly preferred embodiment, the virus is a
recombinant
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adenoviral vector. Suitable recombinant adenoviral vectors include vectors
derived from
adenovirus type 2 (Ad2) and type 5 (Ad5) which have been deleted for the E1
regions.
Representative adenoviral vectors that are useful for delivery of a transgene
are disclosed
by Zabner et al. (1996) J. Clin. Invest. _6 : i 504, Zabner et al. (1993);
Cell 75 : 207, U.S.
Patent Nos. 5,707,618 and 5,670,488, the disclosures of which are incorporated
herein by
reference. In a preferred embodiment, the recombinant adenoviral vector
contains a
transgene, including for example the cystic fibrosis transmembrane conductance
regulator
(CFTR) gene.
In another embodiment of the invention, the polymer modified virus is a
recombinant 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) S ' n a 74:373; Heise et al. (1997) Nature Medicine
3_:630; and
EP689447A, the disclosures of which are incorporated herein by reference.
In another embodiment of the present invention, the polymer is indirectly
noncovalently attached to the virus via a suitable polymer-modified ligand.
Suitable
ligands are not restricted to those having specificity for a viral surface
component such as
a viral surface protein or lipid, and may include hydrophobic ligands for
viral particles
with hydrophobic surface components such as envelope viruses and also ionic
ligands. In
a preferred embodiment, the ligand is an antibody or antibody fragment,
including for
example a non-neutralizing anti-virus antibody or fragment therefrom. 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.
Indirect non-covalent attachment of polymer to the virus is accomplished by
incubation of the virus with a suitable ligand that has been modified by the
covalent
attachment of polymer. The polymer may be covalently attached to the ligand by
standard methods as described herein above. 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
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modified using TMPEG. The ordinarily skilled artisan can determine the optimal
ratios
of activated PEG to antibody, concentrations of activated PEG and antibody,
buffer and
time and temperature of incubation to achieve optimal modification of the
antibody. The
polymer modified ligand is then incubated with the virus particles to allow
non-covalent
binding of the polymer modified ligand to the virus surface.
Antibodies modified with PEG at the epitope binding site may not efficiently
noncovalently attach to a virus. In order to prevent PEGylation of the
antibody at the
epitope binding site, the PEG modification may be performed on immobilized
antibody.
For example, anti-hexon antibody is bound to purified immobilized hexon (eg.
hexon-
Sepharose) prior to PEG modification of antibody. The PEGylated antibody is
then
released from immobilized hexon. Alternately, anti-hexon antibody is modified
by PEG,
creating a population of antibodies PEGylated on the epitope binding site and
other sites.
The modified antibodies are then incubated with immobilized hexon, to which
only
antibodies modified at sites other than the epitope binding site will bind.
These
PEGylated antibodies are then released from the immobilized hexon for use in
accordance with the present invention.
The indirect noncovalent attachment of polymer via a polymer-modified Iigand
may be monitored by displacement of labeled ligand from virus in a competition
enzyme-
linked immunosorbent assay (ELISA). For example, the ability of PEGylated anti-
hexon
antibody to bind to the virus surface is measured in a standard competition
ELISA using,
for example, biotinylated anti-hexon antibody.
The polymer-modified viruses of the present invention maintain infectivity and
exhibit reduced antigenicity. It has been discovered in accordance with the
present
invention that viral infectivity eventually decreases upon additional polymer
modification. By utilizing standard assays, including the following assays, to
assess
infectivity and antigenicity, those of ordinary skill in the art can determine
the method
and conditions of polymer modification that allow retention of infectivity and
reduction
in antigenicity. Under conditions designed to provide direct TMPEG polymer
modified
adenovirus, the methods correlating with PEGylation due to exposure to TMPEG
of
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about 5-20% w/v are preferred, with a concentration of about 10% w/v being
most
preferred.
The ability of the polymer-modified viruses of the present invention to
maintain
infectivity may be assessed by standard infection assays. For example, the
ability of the
S virus to infect a cell may be assessed by monitoring the expression of a
transgene
contained within the virus, such as a reporter gene. Genetic reporter systems
are well-
known in the art, and are disclosed for example in Short Protocols in
Molecular Biolo ~,
1995, Ausubel et al., eds., 3'd edition, Wiley and Sons, Inc. The virus is
engineered by
standard methods to contain a transgene, and the polymer-modified virus 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. 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 unmodified and modified virus, and can determine the optimal
percentages and
I S conditions for polymer modification that result in retention of
infectivity by the polymer-
modified virus. Retention of infectivity is defined herein as an infectivity
level sufficient
to have therapeutic value, for example at least about 20% infective relative
to unmodified
virus. For some therapeutic embodiments, the polymer-modified virus maintains
at least
60% infectivity. In other therapeutic embodiments, the polymer-modified virus
is
preferred to maintain at least 80% infectivity. Lower percent infectivity of
at least 5%
may be therapeutically useful for applications such as viral oncolysis.
In a particular example of an infectivity assay, adenovirus genetically
modified to
contain the ~i-galactosidase (~3-gal) reporter gene (lacZ) is covalently
modified by
exposure to various concentrations of TMPEG. A cell line permissive for
adenoviral
infection, for example 293 human embryonic kidney cells (ATCC CRC 1573), is
exposed
to unmodified and modified adenovirus containing the ~i-gal gene. Cells are
then
incubated under conditions appropriate for ~3-gal expression. The presence of
(3-gal in
cell lysates is measured by standard colorimetric, fluorescence, or
chemiluminescence
assays. The quantity of ~i-gal in 293 cell lysates provides a measurement of
the ability of
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the unmodified and PEG-modified virus to infect 293 cells. PEG-modified virus
that
maintains 50% infectivity relative to unmodified virus is considered to retain
infectivity.
The polymer-modified viruses of the present invention may exhibit reduced
antigenicity relative to unmodified virus. Reduced antigenicity is defined as
a
statistically significant (p>0.05) reduction in binding of the polymer-
modified virus to
neutralizing antibodies against the virus. Reduced antigenicity can be
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 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 polymer-modified
viruses of
the present invention are protected from neutralization by the polymer
coating, and thus
provide increased 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 PEG modification necessary to provide a modified virus that
maintains
infectivity and exhibits reduced antigenicity.
Another embodiment of the present invention provides a method for introducing
a
transgene into a target cell. The method comprises introducing into the target
cell a
polymer-modified virus of the present invention, wherein the virus is a
recombinant viral
vector comprising the transgene. Use of the present polymer-modified viruses
to deliver
a transgene to a target cell is useful for the treatment of various disorders,
for example in
which 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.
The polymer-modified virus is introduced into the host cell by methods known
in
the art, including for example infection. Infection of a target cell in vivo
is accomplished
by contacting the target cell with the polymer-modified virus. The polymer-
modified
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virus is delivered as a composition in combination with a physiologically
acceptable
carrier. As used herein, the term "physiologically acceptable carrier"
includes any and all
solvents, diluents, isotonic agents, and the like. The use of such media and
agents for
compositions is well known in the art. The polymer-modified viruses 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. In a preferred
embodiment, the
transgene is a DNA molecule encoding CFTR or an analog or variant thereof
which
provides functional regulated chloride channel activity in target cells, and
the complex is
delivered to the airway epithelium by inhalation. DNA molecules encoding CFTR
are
well known in the art and disclosed for example in W094/12649 and W095/25796,
the
disclosures of which are incorporated herein by reference.
The present invention further provides a method for delivering a virus to a
tumor,
comprising administering a polymer-modified virus of the invention to a
subject in need
of such treatment under conditions whereby the polymer-modified virus
localizes to a
tumor. The ability of the polymer-modified viruses of the 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 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 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
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and achieve both good tumor localization and high tumor to blood ratios as
well as high
tumor to 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
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 3:369-645; and EP689447A, incorporated herein by reference.
In accordance with the present method, the polymer-modified virus is
administered to a subject as a composition of polymer-modified virus in
combination
with a physiologically acceptable carrier as described hereinabove. The
composition may
be administered by methods appropriate in view of the location of the tumor,
including
for example ingestion, injection, aerosol, inhalation, and the like. In a
preferred
embodiment, the compositions are delivered intravenously.
The present invention further provides compositions comprising the polymer-
modified viruses and further comprising a physiologically acceptable carrier.
In a
preferred embodiment the polymer-modified virus is a recombinant viral vector
modified
by covalent attachment of PEG.
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 polymer-modified
viruses are
compounded for convenient and effective administration in effective amounts
with a
suitable physiologically acceptable carrier and/or diluent.
The precise effective amount of polymer-modified virus to be used in the
methods
of this invention applied to humans can be determined by the ordinary skilled
artisan with
consideration of individual 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
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herein refers to physically discrete units suited as unitary dosages for the
mammalian
subjects to be treated, each unit containing a predetermined quantity of
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 to
the usual
dose and manner of administration of the ingredients.
The invention is further illustrated by the following specific examples which
are
not intended in any way to limit the scope of the invention.
x a
When performing these assays, the skilled artisan should be aware that
exposure
of the virus to parent polymer that cannot covalently attach to the virus
surface, and
handling of the virus in buffer not containing any polymer, may influence the
infectivity
of the virus. These effects may be exacerbated by the type of activated
polymer used and
its length. Therefore, care should be taken not to associate non-specific
reductions in
infectivity with polymer modification of the virus surface. Suitable controls
include virus
sham treated with parent polymer that cannot attach to the virus surface, and
a handling
control in which virus is exposed to the same incubation but substituting
buffer for
polymer solution (e.g., no-polymer control).
Example 1
Covalent Attachment of Polyethylene lvcol to Adenovirus
Tresyl-monomethoxypolyethylene glycol (TMPEG) was prepared 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.
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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 with 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
stock was made 3%w/v
by the addition of dry TMPEG, typically 3.Omg to 1 OO,uI 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 SO,um Internal
diameter
(inlet=anode). A preliminary l .5min wash in 1 M NaOH and second 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
nanoiitres 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 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 hiatus in each plot marks
the
trough at the point of neutrality. The TMPEG treated 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 as
described, above
using 300/cl of virus stock and 3%(w/v) TMPEG. The % mobility was calculated
as
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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: 1
OO,uI of reaction
mixture was analyzed up to this point (using the 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.
Example 2
Covalent Attachment of Polyethylene Glycol to Adenovirus
Type 2 adenovirus stock solution prepared as in Example 1 (1.35x10'°
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.
i 5 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. 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.
am 3
Infectivitv Assays for PEGvlated 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
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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 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). 1n some experiments the 4th
addition was
sampled after 30 rains and a further incubation time (giving 5 reaction
conditions).
Infectivity was measured in two ways (see also Example 4). (3-gal expression
was
monitored in human 293 cells (Graham et al., J. Gen. Virol. 36:59-72, 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 400;u1 per well in a 24 well
microliter plate
using a 1x106/ml cell suspension. Having established a monolayer by 24h, 10,u1
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 5% COz in air at
37°C to
express ~i-gal.
The cell monolayer was depleted of medium and then washed with PBS. Then
601 of lysis buffer (15 % triton X-100, 25OmM 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 50~c1 of each sample was transferred to a fresh
microliter plate.
A set of ~i-gal standards (5.5 units in lysis buffer and doubling dilutions in
lysis buffer)
was added to the same microliter plate. 150 ~cl of CPRG substrate buffer
(1.6mM CPRG,
60mM phosphate buffer: 1mM MgS04; lOmM KC1; 50mM ~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).
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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 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 (P~els C and D, same
symbols), in one experiment TMPEG decreased the 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 (Panel E same symbols).
Example 4
Infectivitv Assavs for PEG lv ated and SHAM Treated Virus
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.
Figure 6a-c compares the effects of 3%, 5% 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.
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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
(Figure 7a
and b, filled circles TMPEG- open circles MPEG). A subsequent 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 (P~els 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 5
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 PEGZOOOO~ TMPEG
treatment produced lower infectivity than MPEG for all additions including the
first, but
approximately one third the initial infectivity value remained even after the
4th addition
of TMPEG.
With a single addition of 3% PEGsooo~ 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).
Example 5
The Impact of PEGvlation on the Reduction of Infectivitv by Neutralizing
Antibodies
Using the infectivity assay given in Example 4, exposure of the TMPEG and
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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 {~40-50%) (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) on antibody neutralisation.
Antibody
treatment is shown by the filled symbols and MPEG treatment by circles and
TMPEG
treatment by squares. In the lower panels, hatched bars indicate TMPEG
treatment.
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 upper panels in each figure show the raw data and the
lower
panels the transgene expression as a percent of the equivalent non-antibody
treated
control. In Figure 10 the amount of 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.
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.
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.
E a 6
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Indirect PEGvlation of Adenovirus Usine a Non-neutralizing Anti-hexon AntibodX
The present invention relates to polymer-modified viruses, processes for
obtaining
them and their use. The invention also provides means of attaching polymer
molecules to
viral particles whilst retaining infectivity of the modified virus.
Initial experiments on the PEGylation of an anti-hexon antibody were performed
using commercially available anti-hexon antibodies from Chemicon (Mab 8052).
Two
types of activated PEGs were tested for their ability to PEGylate the antibody
namejy
cyanuric chloride activated PEG and PEG-tresylate (TMPEG). TMPEGSOOO was
obtained
from Shearwater Polymers, Huntsville, AL.
PEGylation of an anti-hexon antibody using TMPEG was accomplished as
follows. MAb 8052 50 g.g was incubated with TMPEG at the following PEG:lysine
molar ratios, 0.2:1, 0.5:1, 1:1, 2:1, 5:1. The TMPEG and antibody were
incubated for one
hour at room temperature with gentle rocking on a "Vari-Mix" after which time
the
reaction mixture was stored at 4°C or -80°C until further use.
(In some experiments the
1 S treatment with PEG was stopped using excess lysine. However, for samples
analyzed by
the fluorescamine assay, the reaction was stopped by lowering the
temperature.)
Calculation of molar ratios assumed 90 lysine residues per IgG. A
fluorescamine assay of
the IgG treated with TMPEG was performed according to the method of Laurel et
al.
(1994) Methods in Enzvmology, 2?$ incorporated herein by reference, to assess
the
amount of lysine substitution of the anti-hexon antibody treated with TMPEG.
In this
assay lysine residues modified with PEG are not available for reaction with
the
fluorescamine leading to a corresponding decrease in antibody associated
fluorescence.
Results of the fluorescamine assay are provided in Fig. 13. The percent
modification of
IgG lysines was calculated as 1 - {slope modified 1gG/slope unmodified 1gG)
using the
method of Laurel et al. Results are presented in Table 1.
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Table 1
PEG:Lysine % Modification lysines
Control p
0.2:1 25
0.5:1 37
1:1 46
5:1 70
It is concluded that increasing the ratio of PEG:lysine leads to a
corresponding
increase in the number of lysine residues that are substituted with PEG or
alternatively
that treatment with PEG decreases the number of available free lysine residues
on the
lgG.
PEGylation of an anti-hexon antibody using cyanuric chloride activated PEG was
accomplished as follows. Anti-hexon antibody MAb 8052 was dialyzed into 0.1 M
sodium bicarbonate pH 9. Following dialysis 25 pg of the MAb 8052 was
incubated with
the cyanuric chloride activated mPEG at increasing PEG:lysine residues of 2:1,
10:1,
100:1. The PEG and antibody were incubated for one hour at room temperature
with
gentle rocking on a "Vari-Mix" after which time the reaction mixture was
stored at 4 ° C
or -80°C until further use. Calculation of molar ratios assumed 90
lysine residues per
lgG. To assess the amount of lysine substitution of the PEGylated anti-hexon
antibody, a
fluorescamine assay of the PEG-treated IgG was performed according to the
method of
Laurel et al. (1994), Methods in Enz,~lo~v 228. Results of the fluorescamine
assay are
provided in Fig. 14.
The percent modification of IgG lysines was calculated as 1 - (slope modified
IgG/slope unmodified IgG) using the method of Laurel et al. Results are
presented in
_r.
Table 2.
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Table 2
PEG:IgG Lysine % modification of lysines
Control 0
2:1 30
10:1 47
I 00:1 (0
It is concluded that the anti-hexon antibody Mab 8502 was successfully
PEGylated using cyanuric chloride activated mPEG. Using the fluorescamine
assay it
was shown that increasing the ratio of PEG:lysine during the PEGylation
reaction
resulted in a corresponding increase in the modification of lysine residues on
the
antibody.
Example 7
Demonstration that a PEG~ated Anti-hexon Antibody Still Recognizes Viral Hexon
TMPEG modified Mab 8052 (modified at a ratio of 100:1 PEG:lysine as prepared
in Example 6) and unmodified antibody were incubated with a detergent
solubilized
fraction of adenovirus for 2 hrs at 4°C. Antibody antigen complexes
were captured with
Staph A membranes and analyzed on a SDS-PAGE gel. Figure 15 demonstrates that
the
PEGylated antibody was equally effective as the non-PEGylated antibody at
immunoprecipitating viral hexon. Thus PEGylation did not grossly affect the
antigen
recognition site of the antibody.
Example 8
Indirect Adenovirus . ISA Using PEGYlated Anti-hexon Antibodies
An indirect adenovirus ELISA was also performed to demonstrate that the
PEGylated anti-hexon antibody still recognized adenovirus. The ELISA procedure
is as
follows: the 9b wells of a microtiter plate received 0.1 ~g of inactivated
adenovirus in
coating buffer (100 mM carbonate pH 9.2 (Pierce)) and was incubated overnight
at 4°C.
After overnight incubation the plates received 150 ~l of blocking buffer per
well and
were incubated for lh at 37°C. The plates were washed 3 times with wash
buffer (PBS
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containing 0.05% Tween 20, 0.5% BSA (Pierce)). The wells then received 100 ~1
of a
solution containing a 1:250 dilution of antibody (2 mg/ml) (control, TMPEG-Ab
as
prepared in Example 6 and antibody PEGylated with cyanuric chloride PEG as
prepared
in Example 6). A series of twofold dilutions of the antibody were performed
across the
plate. The plates were incubated overnight with antibody and the wells were
subsequently washed 3 times with wash buffer. The antibody bound to the virus
was
quantified using a standard streptavidin - HRP assay kit (Pierce Chemical,
Rockford, IL).
Results are shown in Table 3.
Table 3
Antibody Titre
Control 8000
tmPEG-Antibody 8000
(10:1)
tmPEG-Antibody 4000
(100:1)
tmPEG-Antibody 4000
(200:1 )
CC-PEG Antibody 1000
(25:1)
CC-PEG Antibody >500
(50:1)
CC-PEG Antibody > 500
(75:1)
The results in Table 2 demonstrate that the anti-hexon antibody PEGylated with
cyanuric chloride PEG had a lower titre for adenovirus compared to control or
antibody
PEGylated with TMPEG. This suggests that PEGylation of the antibody using
TMPEG
preserves the antigen recognition site of the antibody to a greater extent
than PEGylation
using cyanuric chloride activated PEG.
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A competition ELISA was designed to determine if PEGylation of the antibody
resulted in large changes in the affinity of the antibody for viral antigen.
Anti-hexon
antibody was PEGylated with either TMPEG or cyanuric chloride activated PEG.
Antibody PEGylated with TMPEG was more capable of binding to the virus than
antibody PEGylated with cyanuric chloride activated PEG as shown by
competition of
biotinylated anti-hexon antibody in a competition ELISA. The ELISA plate was
coated
with adenovirus as described in Example 9. After coating the wells received
biotinylated
antibody alone or biotinylated antibody and test antibody which included TMPEG
antibody or cyanuric chloride - PEG antibody. The biotinylated antibody bound
to the
virus was then quantified using a standard strepavidin - HRP assay. If
PEGylated
antibody can compete effectively with the biotinylated parental antibody for
sites on the
virus there will be less biotinylated antibody bound the surface of the virus
resulting in a
lower titre value. Results are shown in Table 4.
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Table 4
Antibody Titre
Biotinylated Parental 4000
Biotinylated parental 1000
+ tmPEG-antibody
(10:1)
Biotinylated parental 1000
+ tmPEG-antibody
(100:1)
Biotinylated parental 4000
+ tmPEG-antibody
(200:1 )
Biotinylated parental 4000
+ CC PEG-antibody
(25:1)
Biotinylated parental 2000
+ CC PEG-antibody
(50:1 )
Biotinylated parental 2000
+ CC PEG-antibody
(75:1 )
Table 4 shows that antibody PEGylated with TMPEG at the ratios of PEG:lysine
of 10:1 and 100:1 could still effectively compete with the biotinylated
parental antibody
for virus. This resulted in less biotinylated antibody bound to the virus and
hence a lower
titre value. Antibody PEGylated with TMPEG at a ratio of 200:1 PEG:lysine was
ineffective at competing with the biotinylated parental antibody suggesting
that at this
high ratio of PEG the antigen binding site of the antibody is compromised.
Antibody PEGylated with cyanuric chloride activated PEG was not effective at
competing with the biotinylated parental antibody for binding to virus
suggesting that
PEGylation with cyanuric chloride PEG had compromised the antigen binding site
of the
antibody.
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Example 9
Indirect PEGylation Via PEGylated A_r~tibo~
Further experiments were performed in which non-neutralizing anti-hexon
antibody purified from hybridoma cell line HB8117, American Type Culture
Collection,
Rockville, MD was used as a ligand with which to attach PEG to the virus. The
antibody
was incubated with TMPEG (as described in Example 1 ) in PBS at room
temperature for
2h using a rotary mixer. The final concentration of antibody was 1 OOp.g/ml
and the
TMPEG was 10.6mg/ml added to provide an excess of TNIPEG:NH,. The excess
TMPEG was neutralized by addition of glycine and a further 2h incubation.
PEGylation of the antibody was confirmed by the increase in size shown by gel
permeation chromatography (Figure 16). The antibody preparation did not
contain any
significant proportion of residual unmodified antibody (note the lack of a
subsidiary peak
in the unmodified position). Incubation of the antibody with increasing
concentrations of
TMPEG-5K lead to a progressive displacement of the protein elution peak from
circa
11.1 ml to circa 9.5m1, 9.1 ml and 8.95m1, indicative of increasing degree of
modification
(Figure 16, left panels). Reactions prepared with 10.6 mg/ml and 22.5 mg/ml
did not
contain any significant proportion of residual unmodified antibody (note the
lack of a
subsidiary peak in the unmodified position). (Figure 16, two top left
p~.nels). However,
when the TMPEG concentration was increased to 45 mg/ml (Fig. 16, bottom left
panel),
the reaction mixture contained a small proportion of unmodified antibody. This
might be
due to partial precipitation of the protein induced by the high concentration
of polymer,
thus making the protein unavailable for PEGylation. Incubation of the antibody
with
TMPEG 12K lead to a displacement of the protein peak to circa 7.98m1 and
7.52m1
(Figure 16, right panels). None of the reactions contained any significant
proportion of
unmodified antibody. The displacement of the protein elution peak by
PEGylation was
more marked for the conjugates obtained with TMPEG-12K than that observed for
conjugates prepared with TMPEG-5K. Thus the conjugates obtained with TMPEG-12K
have an overall hydrodynamic radius grater than that of the conjugates
obtained with
TMPEG-5K. A greater hydrodynamic radius could indicate: either a) greater
impact per
PEG chain for the TMPEG-12K than for the TMPEG-5K, or b) greater number of PEG
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chains attached with TMPEG-12K than with TMPEG-SK. However, the chromatograms
do not allow to discriminate between these two possibilities.
Five preparations of PEGylated antibody covering a range of degrees of
modification, three MPEG-SK-antibody conjugates (Preps 1 to 3 with elution
volumes on
the Superose 12 column at 9.31 ml, 9.08m1 and 8.96m1, respectively) and two
MPEG-12K
antibody conjugates (Preps 4 and 5 with elution volumes on the Superose 12
column at
7.98m1 and 7.72m1, respectively) were tested for binding to the viral surface.
PEGylated antibody was capable of binding to the virus using a biotinylated
anti-
hexon antibody in a competition ELISA (Figure 17). The wells of a microtiter
plate (96
wells) received 100 ~cl of a 1 ~g/ml stock inactivated adenovirus in coating
buffer and
were incubated overnight at 4 °C. After the overnight incubation, the
plates received 150
,ul of blocking buffer per well and were incubated for 1 h at 37 °C.
The plates were then
washed 3 times with 400 ,ul of wash buffer per well. The wells then received
100 ,uI of a
solution containing biotinylated antibody at 21.6 nM and test antibody
{control, MPEG
treated or TMPEG treated) at increasing concentrations ranging from 1.1 nM to
540 nM.
The plates were incubated for 1 h at 37°C and then the wells were
washed 3 times with
400 /,cl of wash buffer. The biotinylated antibody bound to the virus was then
quantified
using a standard streptavidin-HRP assay. The stock inactivated adenovirus type
2 was
obtained in lyophilized form, 200 ~g/vial, from Lee Biomolecular Research, San
Diego
CA, Cat No.405001. To produce the 1 ug/ml stock, the lyophilized powder was
dissolved in 1 ml of distilled water and 50 ,ul were then diluted up to 10 ml
with coating
buffer. The coating buffer was 100 mM carbonate pH 9.2 (Pierce). Blocking
buffer was
PBS containing 0.05% Tween 20, 0.5 % BSA (Pierce 10X). Wash buffer was PBS
containing 0.05% Tween 20. The biotinylated antibody was at a concentration of
10.8
,uM.
Figure 17 shows the binding of biotinylated anti-hexon antibody to the
viral surface in the presence of increasing concentrations of untreated
monoclonal anti-
hexon antibody (dotted lines), monoclonal anti-hexon antibody incubated with
MPEG
(open circles) and PEG-antibody (filled circles). The latter was obtained by
incubation of
the monoclonal anti-hexon antibody with TMPEG (see chromatogram in Figure 16).
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Thus PEGylated antibody serves as an alternate approach for binding PEG to the
viral
surface.
Example 10
Quantitative Analysis OFPEGylated Adenoviral Vector
An Ad2/(3-gal 2 vector (U.S. Patent No. 5,670,488 and described by Zabner et
al.
( 1996) J. V irol. 70 : 6994) was covalently modified by PEG with 0.01 %, 0.1
%, 1.0% or
5.0% biotinylated NHS-PEGsooo (Shearwater Polymers). PEGylated 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-(3-gal 2
vector
was treated with increasing amounts of TMPEG-biotin 5%, 10%, or NHS-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
assay with
an avidin HRP conjugate as reporter. A standard curve of PEG-biotin (0-250
ng/ml) was
generated to determine the number of molecules of PEG-biotin attached per
virus particle.
Results are shown in Table 5.
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Table S
Sample Molecules PEG-biotin:virus particle
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
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 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.
Covalent Attachment of Polvg~ rP Glvcol to 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) cyanuric chloride
activated
mPEGsooo b) TMPEGsooo and c) amino-PEGsooo~ The mPEGs were obtained from
Shearwater Polymers. Activation of mPEG with cyanuric chloride couples one
triazine
ring per mPEG molecule. This activated mPEG can react with amino groups on
proteins.
Alternatively mPEG can be activated with tresyl chloride (2,2,2,-
trifluoroethanesulphonyl
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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 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 sulfliydryl 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-~3-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 O.1M sodium carbonate buffer pH 8.5 containing O.15M NaCI before
treatment with cyanuric chloride activated mPEG or 0.2M sodium phosphate
buffer pH
7.5 containing O.15M NaCI before treatment with TMPEG. Ali 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 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 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 6 and demonstrate that infectivity of the virus
is
retained following PEGylation with TMPEG. (Error in the assay is t0.5 log.)
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Table 6
PEG:Lysine Infectivity
5:1 3.8e8 iu/ml
2.5:1 l.Se8 iu/ml
I :l 2.2e8 iu/ml
Control Se8 iu/ml
~xam~le 12
Reduced Binding of Neutralizing Antibodies to PEGy~ated Vector
Ad2-~i-gal 2 virus was PEGylated with TMPEG as described in Example I 1.
Virus was incubated with serial two-fold dilutions of neutralizing human serum
for I
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 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 7.
Table 7
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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Example 13
Ion-exchange Chromatography of PEGvlated Virus Particles
Ad 2-~i-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 DEAE
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:Iysine and virus treated with PEG at a ratio of
10:1
PEG:lysine. All samples had equivalent protein values before chromatography.
Figure 18, panel A shows the elution profile from the DEAF-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 18, panel B shows the elution profile from the DEAE-
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 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 longer bind to the resin and are recovered in the flow
through peak.
The elution profile from the DEAF-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 greater
fraction of particles eluting in the flow through peak, the virus particles
had increased
levels of PEGylation. Table 8 expresses the size of the two peaks (expressed
as area
under peak) in relation to the PEG:lysine ratios used during PEGylation. In
conclusion,
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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 charge differences.
Table 8
Flow Through Peak Area Eluted Peak Area
Control NA 0.272
PEG-Virus 0.022 0.132
10:1
PEG-Virus 0.063 0.031
50:1
Example 14
Trans ene Ex»ression by PEGylated Ad2/~3-Gal2 in Immune Mice
Two batches of Type 2 adenovirus stock solution prepared as in Example 1 were
mixed (2ml of a batch at 5.38x10'° infectious units per ml, 2.055x10'2
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 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 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
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).
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The two PEGylated viral suspensions were compared to untreated Type 2
adenovirus (3.19x10'° infectious units per ml) for ability to effect
gene transfer in 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 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 ~l to each of four mice in the naive group and
four mice in
the pre-immunized group, b) "PEGylated virus 10%", 3x108 infectious units
(2.7x10'°
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 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 a-gal in an AMPGD
assay
(Galacto-LightTM Kit, 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 9 shows the beta-galactosidase expression per microgram of protein
(relative light units, RLU per microgram of protein) for untreated virus,
"PEGylated virus
10%" and "PEGylated virus 15%" in both naive and pre-immunized mice. Beta-
galactosidase expression in the naive mice was observed for all three viral
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
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animals in 4/4 and 3/4 animals for the "PEGylated virus 10%" and "PEGylated
virus
15%" preparations, respectively (see Table 5). Thus PEGylation of the virus
conveys
protection from neutralization ~g vivo resulting in substantial expression of
the vector in
the target tissue i~ vivo.
Table 9. Beta-Galactosidase expression in lung tissue expressed as relative
light
units per microgram of protein (RLU/,ug protein).
Preparation Mouse Number RLU/,ug proteinRLU/~g protein
(infectious Native Immunized
units)
Control virus 1 955 25
(2x10$ iu)
2 1457 90
3 649 28
4 1388 38
PEGylated IO% 1 2341 218
(3x108 iu)
2 2108 1296
3 3694 164
4 1730 1964
PEGylated 15% 1 705 34
{6.4x 10' iu)
2 172 305
3 715 198
4 1128 108
Exam In a 15
Pe~vlation of Adenovirus ONYX-O 15
5 Genetic modification of viruses to produce replication competent viruses
with
restricted permissiveness has been demonstrated in a number of cases (e.g. for
tumour
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cells, hypoxic tissues and tissues having specific promoters). Adenovirus ONYX-
O15
(ONYX Pharmaceuticals) is an example of such viruses, which has been designed
to
propagate selectively in tumours. The virus is a chimera of adenovirus types 2
and 5,
which replicates more efficiently in cells lacking the regulatory protein p53.
Such cells
S include a number of tumour cell lines. The covalent attachment of polymer to
the virus
would be expected to enhance the tumour targeting ability of the virus, adding
further
advantages to those achieved with PEGylation, ideally whilst maintaining
infectivity and
protecting the virus from the effects of neutralising antibodies.
TMPEG was prepared as disclosed in Example 1. Adenovirus ONYX-015 was
prepared following infection of human 293 cells, by ion exchange
chromatography (IEC)
using Resource Q media on a PerSeptive BioSystems chromatograpy workstation.
The
running buffers used were as follows; Buffer A: 1 SOmM HEPES; 20 mg/ml
sucrose;
2mM MgCl2, pH 7.5 (adjusted with NaOH), Buffer B: 1.SM NaCI in buffer A.
Virus purification was effectuated using a gradient of 0-5 minutes, 20% B; 5-
15
minutes 20-50% B; 15-20 minutes, 100% B; 25-30 minutes, 20% B. Concentrated
stocks of virus (9x10" pfu/ml) were diluted in virus storage buffer (VSBIOmM
Tris
base,pH 7.4, 1mM MgCl2, 150mM NaCI, 10% glycerol) to give a working
concentration
of 1 x 10"pfu/mi. Aliquots of virus were stored at -70°C. It should
noted that TRIS is
undesirable in PEGylation reactions since it is a nucleophile and must either
be diluted
sufficiently or the buffer must be exchanged.
Adenovirus ONYX-015 (1x10" pfu/ml and circa 1x10'z particles/ml) was reacted
with PEGs~o using an addition of PEG in 5%(w/v) steps (as described in Example
3).
Each activated polymer addition was incubated for 30 min at 25°C, on a
rotary wheel.
Final concentrations of TMPEG or MPEG at S, 10, 15 and 20% (w/v), were
obtained.
The polymer modified virus was assessed by IEC. The IEC method was run with a
1 ml
Resource Q column (Pharmacia), using a HP 1100 HPLC system. The running
buffers
used were as follows; Buffer A: 150mM HEPES; 20 mg/ml sucrose; 2mM MgCl2, pH
7.5 (adjusted with NaOH), Buffer B: 1.5M NaCI in buffer A.
They were run in a gradient of, 0-5 minutes, 20% B; S-15 minutes 20-50% B; 15-
20
minutes, 100% B; 25-30 minutes, 20% B.
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The IEC method used demonstrates, that in virus samples treated with TMPEG,
the shrouding effect of the PEG chains have resulted in sufficient
neutralisation of
surface charge of the virus particles to inhibit interaction with the column.
The
chromatogram in Figure 19, demonstrates this effect. Untreated virus particles
(Figure
19a) were effectively eluted at 10.90 minutes (570mM NaCI), whereas MPEG
treated
virus (Figure 19b), samples were eluted at 10.91 minutes (570mM NaCI). In the
TMPEG
treated virus samples, no peak was present at this location, with a peak at
the column void
volume being observed (Figure 19c).
A peak also appeared at 0.75min in the TMPEG treated sample and examination
of the spectrum at that location (not shown) was consistent with the new peak
being due
to PEGylated virus. In some examples, an increase in peak height was observed
rather
than a new peak at this location indicating that not only PEGylated virus but
also other
material can elute at this location.
E~ple 16 and Comparative Example 16
Infectivitv Assavs for PEG-treated ~'MPEG~ and Sham-treated (MPEC~I Adenovinzs
ONYX-O 15
PEGylated adenovirus ONYX-015 was prepared as in Example 15 and assessed in
infectivity assays. Infectivity was assessed in an ELISA assay, using antibody
detection
of the major structural hexon protein.
Human 293 cells were seeded at Sx 1 OS cells/ml, in 96-well plates ( 100
ml/well),
and allowed to adhere preferably overnight, or for at least 2 hrs at
37°C. The PEG-
reacted virus samples were diluted in Dulbecco's Minimal Essential Media
(DMEM),
containing 2% fetal calf serum, to give virus concentrations of 1x106, 5x105
and then four
half log dilutions. Semi-confluent cell monolayers were infected with
100 ul/well of diluted virus (6 replicates for each), for 48 hrs at
37°C and 5% CO2.
After 48 hrs, the cells were examined for cytopathic effect (CPE) using phase
contrast microscopy, and results were recorded by photography.
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The cells were then pelleted at 1000 rpm for 2 min, washed twice in phosphate
buffered saline (PBS), fixed in iced ethanol containing S% acetic acid for 10
min at -
20°C, washed in PBS and blocked in Superblock (Pierce Chemical Co., :
Cat. No.37535),
for 1 hr at room temperature or overnight at 4°C. Cells were incubated
with primary anti-
hexon antibody, Access Biomedic Inc. (diluted 1:1000 in PBS comprising 3%
Bovine
serum albumin - BSA - PBSB) for 1 hr at room temperature. This was followed by
incubation in a secondary antibody (rabbit alkaline phosphatase, diluted
1:1000 in PBSB
with 0.1 % Triton X 100, Pierce Cat No:121 ) for 1 hr at room temperature. The
cells were
washed in Tris Buffered saline (TBS), and incubated in PNPP (p-Nitrophenyl
phosphate,
disodium salt) substrate, prepared according to manufacturers instructions,
(Pierce Cat
No:37620), for 20 min. The reaction was stopped with 100 ~cl/well 2N NaOH, and
the
results read at 405nm (Molecular Devices Emax Microplate Reader).
Single and stepwise additions of TMPEGSOOO ~d MPEGSOOO were prepared as in
Example 15 and the preparations were monitored by IEC for PEGylation.
Figure 20a-d shows the effect of 5% additions of TMPEGSOOO ~d MPEGsooo on
adenovirus ONYX-015 infectivity. The infectivity of virus treated with 5 or
10% PEG is
similar for each treated virus sample (open circles MPEG; closed circles
TMPEG) and the
untreated sample (triangles), whereas at 15 and 20% PEG the infectivi~.-y of
the TMPEG
treated virus is reduced with respect to the other two samples, but is still
maintained at a
significant level. Figure 21 a-f, shows that the CPE exhibited by cells
infected with
untreated virus (a & b), TMPEG-treated virus (c & d) and MPEG-treated virus (e
& f) are
similar, suggesting that treatment with TMPEG does not result in substantial
loss of
infectivity or replication ability of this virus.
The effect of PEG treatment on virus infectivity was also assessed using
plaque
assays. Virus samples were prepared as in Example 1 and 15. The PEG treated
and
untreated virus samples were serially diluted in DMEM (with 2% FCS) to give
dilutions
of 10'~ to 10'9 of the original innoculum ( 1 x 10" pfu/ml).
Semi-confluent monolayers of HEK 293 cells were set up in 6-well plates, and
allowed to establish overnight at 37°C. The medium was removed and the
cells infected
with 200 ,ul/well of diluted virus inoculum. Cells were infected for 1 hr at
37°C, the
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innoculum removed and the cells overlayed with 2 x DMEM (with 10% FCS) and 3%
Seaplaque agarose (Flow laboratories) ( 1:1 v/v). The overlay was allowed to
solidify and
then overlayed with liquid DMEM (with 10% FCS). The assays were set up in
duplicate,
and incubated at 37°C. Assays were examined for plaque formation at 5-6
days post
infection (dpi). Once plaques were observed, the assays were stained with
neutral red
stain (0.1 % in PBS) and the numbers of plaques recorded.
Virus samples treated with 3% TMPEGsooo and MPEGSOOO Produced plaque
titration of 3.0x109 pfu/ml and 4.5x109 pfu/ml respectively, whereas the
untreated
control virus produced a titre of 6x109 pfu/ml. This suggested that both
sample
handling and attachment of PEG chains have a modest and independent impact on
infectivity.
In samples treated with 5% additions of TMPEGSOOO ~d MPEGSOOO ~ resulting in
20% PEG treatment, titres of 6.8x109 pfu/ml and 7xI09 pfu/ml respectively were
attained. In comparison, untreated virus produced titres of 5x109 pfu/ml.
Thus, in this
experiment neither handling nor PEG chain attachment appear to have reduced
infectivity
or replication ability.
Further observations on cells infected using TMPEGsooo-treated virus, were
made
using antibody staining and immunofluorescence microscopy.
Virus samples were treated with TMPEGSOOO ~d MPEGsooo as described in
Example 15.
Semi-confluent monolayers of I-iEK 293 cells were set up in 8 well slide
chambers (Nunc), and allowed to adhere overnight at 37°C. The medium
was removed
and the cells were infected with TMPEG-treated, MPEG-treated or untreated
virus
innoculum diluted in DMEM (with 2% FCS) to 1x106 pfu/ml (50 ~cl/well) for 1 hr
at
37°C, after which the inoculum was replaced with DMEM, containing 5%
FCS. Cells
were incubated at 37°C and prepared for microscopy at 48 and 72 hours
post infection
(hpi), as follows.
Cells were washed in PBS (5 min), blocked in PBSB for 1 hr at room
temperature,
washed in PBS and then incubated in primary anti-hexon antibody (diluted
1:1000 in
PBSB, Access Biomedic Inc.) for lhr at 37°C. The cells were washed in
PBS, and
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incubated in secondary goat anti-rabbit FITC conjugate (diluted 1:80 in PBS,
Sigma
Chem. Co.) for 1 hr at 37°C. The cells were washed extensively in PBS,
three times in
sterile distilled water, and mounted in Citifluor anti-fade mountant (Agar
Accessories
Ltd.). Slides were viewed using a Olympus Epifluoresence Microscope.
Immunofluoresence micrographs in Figure 22 show staining with anti-hexon
antibody in cells infected with TMPEGsooo treated ONYX-O 15 virus, at 48 h
post
incubation, suggesting that treatment with TMPEG produces no inhibitory effect
on virus
replication.
Example 17
Covalent Attachment of Pohrethylene Glycol to Poxvirus
As a representitive virus vector from the Poxvirus family Vaccinia virus
strain MJ
was selected. Strain MJ of Vaccinia virus containing a IacZ gene which encodes
(3-
galactosidase (VVMJ.IacZ), was used to demonstrate the covalent attachment of
TMPEG
to a Poxvirus vector. Vaccinia virus strain MJ.IacZ was prepared from infected
BS-C-1
cells, grown in minimal essential medium (MEM), supplemented with 10% FCS.
Vaccinia virus MJ.IacZ and BS-C-1 cells were obtained from Dr. A. Alcami,
Division of
Virology, Department of Pathology, University of Cambridge, Tennis Court Road,
Cambridge, U.K. Purified virus stocks were prepared by sedimentation through a
sucrose
cushion, dialysed against PBS overnight at 4°C, and titrated by plaque
assay in TK-143B
cells (provided by Dr. Alcami). Titres of 6x109pfu/ml were obtained.
Aliquots of virus were reacted with TMPEGsooo ~d MPEGsooo in 5% (w/v)steps as
described in Example 15. Samples from the 5% and 20% reactions were diluted in
minimal essential medium (MEM) supplemented with 2% FCS, to give serial
dilutions
of 10-5 to 10'9.
Plaque assays were carried out to assess the effect of 5% and 20% (w/v)
treatment
with TMPEG on virus infectivity. TK'143B cells, grown in MEM, supplemented
with
10% FCS, were seeded in 6-well plates and allowed to adhere overnight at
37°C. Cell
monolayers were infected with dilutions, 10-' to 10-8 of TMPEG treated and
MPEG
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treated virus (500 ,ul/well), for I hr at 37°C. After washing with PBS
containing 2%
FCS, the cells were overlayed with MEM containing 2.5% FCS and I .5%
carboxymethyl
cellulose (CMC). After 2 days, the cell monolayers were stained with 0.1 %
crystal violet
in 1 S% ethanol and the number of plaques recorded. The reduction in the
numbers of
plaques is shown in Figure 23 (results of two independent experiments in upper
and lower
panels). Note that the number of pfu/ml does not have a linear relationship to
the virus
dose, thus % retention of infectivity cannot be precisely ascertained, but
that TMPEG
treatment does not abrogate all infectivity.
Infectivity assays were carried out by plaque assay in TK-134B cells which
were
stained after two days, by the addition of 300ug/mt X-gal of (3-galactosidase
in the cells
was ascertained following overnight incubation. The results, which are in
broad
agreement with the findings above, are shown in Figure 24.
The effect of TMPEG treatment on vaccinia virus replication was assessed using
assays for expression of early and late virus proteins with immunomodulatory
activity.
I 5 The soluble interferon-g receptor expressed from an early promoter was
assayed as
follows: Tk-143B cell monolayers were infected with vaccinia virus at a
multiplicity of
infection (moi) of 1 pfu/cell. Culture supernatants were harvested at 24 hours
post
infection hpi and tested for expression of INF-y receptors using a cross-
linking assay.
Media from uninfected or infected cultures (24hpi) were incubated with 1.7nM
'z5I-INF-
y, in the absence or presence of i 00-fold excess IL-1 ~i or IFN-y. IFN-g
receptor
complexes were cross-linked by the addition of EDC and samples were analysed
by SDS-
PAGE in I2% polyacrylamide gels and autoradiography (Figure 25). The effect of
TMPEG and MPEG treatment on expression of the INF-y receptor is shown in
Figure 25,
using the doses of medium (,ul) indicated, from uninfected or infected
cultures at 24 hpi.
No variation in expression was detected. No IFN-y binding activity was
detected in
medium harvested after the absorption period (data not shown). Recombinant
baculovirus-infected cells expressing the vaccinia IL-1 ~i receptor or the
vaccinia INF-y
receptor were used as negative and positive controls, respectively. The
specificity of the
'ZSI-IFN-y binding was confirmed by competition with unlabelled IFN-y, but not
unlabelled IL-1 Vii.
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Expression of the soluble interleukin-1 (3 receptor, expressed from a late
promoter,
was assayed as follows. TK-1438 cell monolayers were infected with vaccinia
virus at an
moi of 1 pfu/cell. Culture supernatants were harvested at 24 hpi and tested
for expression
of IL-1 ~i receptors in a soluble binding assay. Media from uninfected and
infected
cultures (24 hpi) were incubated with 140pM'ZSI-IL-lei, in the presence or
absence of 100
fold excess IL-1(3 or IFN-y. Bound IL-(3 was determined by precipitation with
polyethylene glycol and the precipitate collected on Whatman GF/C filters.
Background
radioactivity precipitated in the presence of binding medium was subtracted.
One ~cl of
medium was equivalent to 1500 cells. Specific bound radioactivity (~ standard
deviation)
is shown in Figure 26. At the indicated doses (,ul), media assayed from cells
infected
with 20%MPEG and 20% TMPEG treated virus, showed little or no difference in
activity. Binding activity of medium harvested after the absorption period is
given as t =
0. Supernatants from recombinant baculovirus infected cells, expressing
vaccinia IL-lb
or vaccinia IFN-g were used as positive and negative controls respectively.
Figure 27 shows the impact of incubation with TMPEGSOOO or MPEGsooo on
neutralisation of the virus by anti vaccinia serum. Wtih the MPEG treated
virus, all
dilutions of serum produced a similar reduction in pfu/ml, indicative of
neutralisation. A
protective effect was evident in the TMPEG treated samples at 1/1000, 1/500
and
possibly 1/250 dilutions of serum.
Ex~ple 18
Covalent attachment of Pol~vlene Glycol to Retrovirus
As a representitive example of retrovirus vectors, the mammalian C-type
retrovirus, Molony marine leukaemia virus (MMLV) was used to demonstrate
retention
of infectivity in TMPEG treated virus samples.
MMLV containing a lacZ gene, encoding ~i-galactosidase sequence (AM-
l2.lacZ), obtained from Dr. Massimo Pizzato, Cancer Research Institute, Inlham
Road,
London, U.K., was produced in 3T3 fibroblasts grown in serum free DMEM.
Monolayers of cells were infected at low multiplicity of infection (moi) for
24 hours at
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37°C. Virus stocks were harvested from culture supernatant, and
filtered through 0.45um
filters prior to use. Virus stocks titrated by infectivity assays in 3T3
cells, were found to
be at 1 x 1 O6 pfu/ml. The filtered culture supernatants, containing 1 x 1 O6
pfu/ml were
treated with TMPEGsooo or MPEGsooo in 5% steps as described in Example 16. The
reactions were carried out at 25°C, allowing 30 min for each addition.
The pH of the
reactions was monitored, and a drop from pH 7.0 to pH 6.8 was recorded in the
TMPEG
15% and 20% reactions.
Monolayers of 3T3 and CE cells were set up in 24 well plates and allowed to
adhere overnight at 37°C. TMPEG and MPEG treated MMLV was diluted in
serum free
DMEM media (10-' to 10-Sdilutions), and cells were infected with each dilution
(O.SmI/well) for 4 hrs at 37°C. Following infection, the virus inoculum
was removed, and
replaced with lml of DMEM containing 10% FCS. At 48 hours post infection
(hpi), the
cells were stained overnight at 37°C with X-gal (300mg/ml). Cells
expressing ~3-
galactocidase activity were counted, and representative dilutions recorded by
photograph.
The infectivity of TMPEG and MPEG treated MMLV is shown in Figure 28 (two
independent experiments are shown in 28a and b). In both experiments TMPEG
treated
samples showed somewhat lower infectivity than the equivalent MPEG treated
controls.
The impact of sham treatment with MPEG differed between the two experiments,
showing modest reduction in infectivity in Figure 28a and an apparent increase
versus
untreated control at 5% MPEG and no reduction versus untreated control at 10,
15 and
20% MPEG respectively in Figure 28b.
Cells showing ~3-galactocidase activity are represented in Figure 29. Panels A
and
B show untreated virus at dilutions 10-' and 10-4 respectively. In the
remaining panels the
dilutions giving the most comparable levels of infectivity in the treated
samples are
shown. Panels C and D show 5% and 20% TMPEG treated virus both at 10'4
dilution and
panels E and F show 5% and 20% MPEG treated virus at 10--' and 10-3 dilutions
respectively.
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E 19
Covalent attachment of Polvethylene Glycol to Herpesvirus
As an example of Herpesvirus based vectors, Herpes simplex I strain 17 (HSV-I
strain 17), obtained from Dr. S. Efstathiou, Division of Virology, Department
of
Pathology, University of Cambridge, Tennis Court Road, Cambridge, U.K., was
used to
demonstrate the covalent attachment of TMPEG to a Herpesvirus vector, and to
assess the
effect of the polymer on virus infectivity.
Virus stocks of HSV-I strain 17, was prepared by infecting roller bottles of
BHK
cells, at an moi of 0.01 (0.01 pfu/cell). The infected cells were incubated in
Glasgow
MEM (GMEM Life Technologies, Inc.) containing 10% FCS (plus
penecillin/streptomycin 1000u/m1), at 37°C until complete cytopathic
effect (CPE) was
observed. The virus from both the infected cells, and the culture supernatant
was
harvested, and purified on a 1 S% Ficoll gradient in endotoxin-free PBS. The
virus was
then separated by ultracentrifugation, and resuspended in PBS. Aliquots of the
purified
virus stock were stored at -70°C. The stock innoculum was titrated by
plaque assay and
was determined to be at 1.1 x 109 pfu/ml.
Aliquots of the purified virus were reacted with either TMPEG or MPEG, in 5%
(w/v) steps as described in Example 16. The reactions were carried out on a
rotary wheel,
at 25°C, allowing 30 min for each addition of PEG. The pH of the
reactions were
monitored at each step, and was found to remain stable at pH 7Ø Following
treatment
with PEG, the reacted virus samples were stored at -70°C.
The retention of HSV-I Infectivity was assessed following treatment with TMPEG
as follows. Vero cells and BHK cells were trypsinised using standard
procedures, and
maintained on ice. Serial 10 fold dilutions of the untreated, MPEG treated and
TMPEG
treated virus samples were prepared in GMEM, containing 2% FCS. 2 x 106 Vero
cells
and 3 x 10' BHK cells were added to each virus dilution ( 10-' to 10~g), and
the cells were
infected by shaking gently at 37°C. The infected cells were seeded in 6
cm dishes, with
the addition of GMEM, containing 10% FCS (plus penecillin/streptomycin
1000units/ml)
and 1% carboxymethyl cellulose (CMC). The cells were incubated at 37°C
for 48 hrs. At
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48 hpi, the assays were fixed in 10% formalin, and stained with toluidine
blue. The
number of plaques was recorded.
Figure 30 shows the HSV-I infectivity assays carned out in Vero cells (Panel
A)
and BHK cells (Panel B), respectively. Reaction with 5% MPEG produced a
reduction of
infectivity in Vero cells, but infectivity was not affected by this level of
exposure to
MPEG in BHK cells. Although treatment with TMPEG resulted in some loss of HSV-
I
infectivity, in both cell lines some retention of infectivity was observed in
both cell lines.
Exam lp a 20
Covalent Attachment of Polvvin~Pvrrolidone IPVP) to Adenovirus ONYX 015
PVP is a linear water soluble polymer which can be activated in a similar
fashion
to polyethylene glycol. In this example PVP carboxylic acid was activated by
the
succinimidyl active ester method (Delgado et al., Crit Rev Therap Drug Carrier
S,
_9:249-304, 1992) to provide activated PVP which can form PVP-modified virus
(kindly
supplied by Prof F. Veronese, University of Padua, Padua, Italy). PVP
carboxylic acid
was used as a control polymer with which to sham treat the virus, since this
is unable to
attach covalently to the virus. Activated and unactivated polyvinyl
pyrrolidine (PVP)
were added at a concentration of 5% (w/v) to adenovirus ONYX-015 (1 x 10"
pfu/ml)
and incubated for 30 min at 25°C. The samples were then assessed for
polymer
attachment using IEC essentially as described in Example 15, using the buffers
A and B
detailed above, but with the following gradient conditions: 0-5 minutes 0%
buffer-B; 5-
22 minutes 0-50% buffer-B; 22-27 minutes 100% buffer-B.
The IEC data are shown in Figure 31 a and b. In the virus sample treated with
unactivated PVP, a peak for unmodified virus is detected at 17.3 min, whereas
in the
sample treated with 5% activated PVP, no peak is detected at this position,
suggesting
that complete modification of the virus had occurred with 5% activated PVP.
The peak at
circa 11 min in Figure 31 a, which is much smaller in Figure 31 b, is a
variable artefact. In
the sample treated with activated PVP a large peak is evident at 2.2min
(truncated in the
figure). This relates to the PVP in the sample, but may also obscure the PVP-
virus.
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Shrouding of surface charge of the virus particle by PVP is anticipated thus
the PVP
modified virus would be expected to eluted much earlier than the virus itself.
Example 2I
Tumor Localization of PEGylated Virus
PEGylated Adenovirus ONYX-015 was prepared by incubation with
TMPEG5000 as described in Example 16, to give final concentrations of 20%
polymer.
Control Adenovirus ONYX-015 was prepared by incubation with MPEG5000. The
PEGylated and control virus samples were analyzed by IEC as described in
Example 15,
and the 20% TMPEG sample was found to contain no unmodified virus.
A human LS i 74T colon carcinoma (obtained from the Clinical Oncology
Department, Royal Free Hospital School of Medicine, London, NW3, U.K.) was
implanted on the flank of nude mice (MF 1 ) (obtained from the Comparative
Biology
Unit, Royal Free Hospital School of Medicine, London, NW3, U.K.) by placing a
small
piece of tumor under the skin. Once the tumor was established (typically 3
weeks after
implantation), the animals were injected into the tail vein, with a dose of
equivalent 1 X
108 pfu/animal (100~1/animal), of PEGylated or control virus. At 24 hours post
injection,
the animals were sacrificed and tumor and liver were taken. The tissues were
prepared
for microscopy as follows: the tissues were cut into small pieces and washed
once in
PBS, fixed in 3% paraformaldehyde/0.3% glutaraldehyde for 1 hr. at 4°C
and then
infiltrated with 2.3M sucrose for 24-48 hours at 4°C. The samples were
frozen at -20°C,
and cryosectioned onto slides. Semi-thin section were stained for 1 hour at
room
temperature in primary anti-hexon antibody (Access Biomedic, Inc., diluted
1:1000 in
PBSB), washed in PBS, and then incubated for 1 hour in secondary goat anti
rabbit FITC
conjugate {Sigma, diluted 1:40 in PBS). The sections were washed in PBS and
distilled
water, and mounted in Citifluor anti-fade mountant. Sections were examined by
confocal
microscopy.
Sections taken from tumor tissues showed distribution of PEGylated and control
virus within the tissue (Figures 32B and C). Sections taken from the liver
tissue showed
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no localization of virus in either PEGylated virus (Figure 32A) or control
virus (data not
shown). The localization of the PEGylated virus in the tumor is shown in
Figure 32B.
Some tumor localization was also seen for the sham PEGylated virus (Figure
32C).
(Figures 32B and C are at the same magnification).
Exam In a 22
Transgene Expression of PEC~vlated Ad2/(3-gal 4 virus in Immu_r~e Mice
Ad2/~i-gal 4 virus (U.S. Patent No. 5,670,488) was PEGylated with 10% tresyl
mPEG (TMPEG - Sigma Chemicals, St. Louis, MO) as already described. PEGylated
virus was purified from unreacted TMPEG by banding on cesium chloride
gradients
(Rich et al., Human Gene Thera~v_ 4:461-476, 1993). The purified PEGylated
virus was
dialysed into phosphate buffered saline (PBS), 5% 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 ,ul. 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 - 51,200.
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 (Galactolight Kit, Tropix, Bedfor, MA.). The protein
concentration of lung
homogenates was determined using the BiolZad DC reagent {BioRad, Hercules, CA)
and
the results expressed as relative light units (RLU)/ug protein.
Figure 33 shows the (3-galactosidase expression for PEGylated virus (Ad tmPEG)
and sham treated virus (Ad mPEG). Results shown are the mean ~ standard
deviation of
the values obtained with individual animals. (3-Galactosidase expression was
measured in
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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
(3-galactosidase expression (~47% of the (3-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 (3-
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.
