U.S. patent application number 13/005387 was filed with the patent office on 2011-06-30 for treatment using herpes simplex virus.
This patent application is currently assigned to CRUSADE LABORATORIES LIMITED. Invention is credited to Susanne Moira Brown, Guy Michael Gary Hamilton.
Application Number | 20110158948 13/005387 |
Document ID | / |
Family ID | 46331779 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110158948 |
Kind Code |
A1 |
Brown; Susanne Moira ; et
al. |
June 30, 2011 |
Treatment Using Herpes Simplex Virus
Abstract
Herpes Simplex Viruses are disclosed having single-chain
antibodies (scFv) embedded in the viral envelope via fusion with
glycoprotein D and with glycoprotein H and L.
Inventors: |
Brown; Susanne Moira;
(Glasgow, GB) ; Hamilton; Guy Michael Gary;
(Glasgow, GB) |
Assignee: |
CRUSADE LABORATORIES
LIMITED
Glasgow
GB
|
Family ID: |
46331779 |
Appl. No.: |
13/005387 |
Filed: |
January 12, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12065350 |
Apr 18, 2008 |
7897146 |
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PCT/GB2006/003215 |
Aug 30, 2006 |
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13005387 |
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10579606 |
May 16, 2006 |
7498161 |
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PCT/GB04/04851 |
Nov 17, 2004 |
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12065350 |
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Current U.S.
Class: |
424/93.2 |
Current CPC
Class: |
A61P 35/04 20180101;
C12N 7/00 20130101; A61K 31/396 20130101; C12N 2710/16632 20130101;
A61K 38/44 20130101; A61K 38/44 20130101; A61K 35/763 20130101;
A61K 31/396 20130101; C12N 15/86 20130101; A61K 48/0075 20130101;
C12N 2710/16643 20130101; C12Y 105/01034 20130101; A61K 45/06
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 35/763
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.2 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61P 35/04 20060101 A61P035/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2003 |
GB |
0326798.6 |
Aug 30, 2005 |
GB |
0517617.7 |
May 11, 2006 |
GB |
0609381.9 |
Claims
1. A method of treating a tumor in a human comprising the steps of
administering to an individual human in need of treatment a
therapeutically effective amount of a pharmaceutical, wherein the
pharmaceutical is an activatable prodrug, and a therapeutically
effective amount of an herpes simplex virus (HSV-1) wherein the
HSV-1 genome encodes a polypeptide capable of converting the
activatable prodrug to a therapeutically active pharmaceutical,
wherein the HSV-1 genome has a mutation in each ICP34.5 locus such
that the HSV-1 cannot express a functional ICP34.5 gene product,
and wherein the HSV-1 is administered at an extratumoral
location.
2. The method of claim 1 wherein the method involves simultaneous,
separate or sequential administration of the HSV-1 and activatable
prodrug.
3. The method of claim 1 wherein the nucleotide sequence encoding
said polypeptide is located entirely within, or so as to overlap,
the ICP34.5 encoding nucleotide sequence of the HSV-1 genome.
4. The method of claim 1 wherein the extratumoral administration is
into a circulating fluid of the patient.
5. The method of claim 1 wherein the extratumoral administration is
into the blood of the human in need of treatment.
6. The method of claim 1 wherein said activatable prodrug is
administered directly to the tumor.
7. The method of claim 1 wherein said HSV-1 and activatable prodrug
are administered at the same extratumoral location.
8. The method of claim 1 wherein said HSV-1 and activatable prodrug
are administered to the same circulating fluid.
9. The method of claim 1 wherein said HSV-1 and activatable prodrug
are administered at different extratumoral locations.
10. The method of claim 1 wherein the HSV-1 is oncolytic.
11. A kit of parts for use in treating a tumor in a human patient
by combination therapy, said kit comprising a first container
containing an herpes simplex virus (HSV-1) and a second container
comprising a pharmaceutical, wherein the pharmaceutical is an
activatable prodrug and the HSV-1 genome encodes an expressible
polypeptide capable of converting the activatable prodrug to a
therapeutically active pharmaceutical, and wherein the HSV-1 has a
mutation in each ICP34.5 locus such that the HSV-1 cannot express a
functional ICP34.5 gene product, the kit further comprising
instructions for the therapeutically effective administration of
said HSV-1 and/or pharmaceutical to a human patient in need of
treatment at an extratumoral location in order to treat the
tumor.
12. The kit of claim 11 wherein the nucleotide sequence encoding
said polypeptide is located entirely within, or so as to overlap,
the ICP34.5 encoding nucleotide sequence of the HSV-1 genome.
13. The kit of claim 11 wherein the HSV-1 is oncolytic.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the treatment of cancer
using herpes simplex virus.
BACKGROUND TO THE INVENTION
[0002] The herpes simplex virus (HSV) genome comprises two
covalently linked segments, designated long (L) and short (S). Each
segment contains a unique sequence flanked by a pair (terminal and
internal) of inverted repeat sequences. The long repeat (RL or
R.sub.L) and the short repeat (RS or R.sub.S) are distinct.
[0003] The HSV ICP34.5 (also .gamma.34.5) gene, which has been
extensively studied.sup.1,6,7,8, has been sequenced in HSV-1
strains F.sup.9 and syn17+.sup.3 and in HSV-2 strain HG52.sup.4.
One copy of the ICP34.5 gene is located within each of the R.sub.L
repeat regions. Mutants inactivating both copies of the ICP34.5
gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 1716.sup.2
(HSV 1716) or the mutants R3616 or R4009 in strain F.sup.5, are
known to lack neurovirulence, i.e. be avirulent, and have utility
as both gene delivery vectors or in the treatment of tumours by
oncolysis. HSV-1 strain 17 mutant 1716 has a 759 bp deletion in
each copy of the ICP34.5 gene located within the BamHI s
restriction fragment of each RL repeat.
[0004] ICP34.5 null mutants of HSV-1 strain 17 have consistently
shown much better clinical oncolytic efficacy than mutants in other
HSV strains, such as strain F, to the extent that some strain 17
mutants are now in advanced stage clinical trials for the treatment
of tumour. Strain 17 ICP34.5 null mutants are additionally
advantageous over those of other strains in that they achieve
clinical efficacy when administered directly to tumours at dosages
that are one or more logs lower than those required to achieve a
comparable effect using mutants of other strains.
[0005] HSV 1716 is one example of such a mutant and is described in
WO 92/13943.sup.2, specifically incorporated herein by reference.
HSV 1716 has been deposited on 28 Jan. 1992 at the European
Collection of Animal Cell Cultures, Vaccine Research and Production
Laboratories, Public Health Laboratory Services, Porton Down,
Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession
number V92012803 in accordance with the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure (herein
referred to as the `Budapest Treaty`).
[0006] HSV1716/CMV-NTR/GFP (referred to herein as HSV 1790) is
another exemplary ICP34.5 null mutant of HSV-1 strain 17. This
virus is an engineered herpes simplex virus ICP34.5 null mutant
which expresses the nitroreductase (NTR) gene and is described in
WO 2005/049845.sup.14, specifically incorporated herein by
reference. It is modified in each ICP34.5 locus by insertion of the
E. coli nitroreductase (NTR) gene which disrupts the ICP34.5
protein coding sequence such that the virus lacks a functional
ICP34.5 protein. The virus is ICP34.5 deficient, non-neurovirulent
and exhibits good oncolytic properties.
[0007] In HSV 1790 the NTR gene is operably linked to a
transcription control element permitting expression of the NTR
gene. As such the virus may be used in gene therapy techniques
wherein the virus acts as a vector for the expression of NTR in an
HSV infected cell. NTR is capable of converting a range of prodrug
molecules, such as CB1954, into cytotoxic active pharmaceutical
agents. Thus, HSV1790 can be used in targeted combination therapy
in which the oncolytic ability of HSV1790 is combined with
localised prodrug activation in tumour cells.
[0008] HSV 1790 has been deposited (under the name
HSV1716/CMV-NTR/GFP) in the name of Crusade Laboratories Limited
having an address at Department of Neurology Southern General
Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotland on 5 Nov.
2003 at the European Collection of Cell Cultures (ECACC), Health
Protection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG,
United Kingdom under accession number 03110501 in accordance with
the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure (herein referred to as the `Budapest Treaty`).
[0009] To date, the treatment of tumours using ICP34.5 deficient
HSV has relied upon direct introduction of the HSV to the tumour,
usually by intratumoral injection. This has been considered
necessary in order to ensure that the HSV reaches its intended
target, i.e. the tumour that is to be treated. Moreover, this
approach reduces the risks associated with introduction of a viral
vector into healthy tissue in as far as the lytic capacity of the
virus is focused on the tumour.
[0010] It is well known that tumours may occur in virtually any
tissue and at virtually any position in the body. As such it can
often be procedurally difficult, as well as the cause of
considerable discomfort and possible risk to the patient, to
deliver the HSV directly to the tumour. Accordingly, it would be of
significant clinical benefit if the oncolytic effect of these HSV
could be obtained without having to administer the HSV directly to
the tumour. However, the ability of a clinically efficacious
oncolytic HSV that is administered to a patient's healthy tissue to
successfully and selectively target and lyse tumour tissue located
elsewhere in the body, and which does not exhibit serious
disadvantageous side-effects on the patient's healthy tissue,
remains uncertain.
SUMMARY OF THE INVENTION
[0011] The inventors have now shown that oncolytic HSV of strain 17
administered systemically at a site distal from a tumour may be
used to treat the tumour without causing damaging side effects to
the patient being treated.
[0012] In particular, the inventors have shown that successful
tumour treatment may be obtained by intravenous administration of
the HSV, i.e. to the circulating blood. These results demonstrate
that oncolytic HSV of strain 17 introduced to healthy tissue may
circulate in the body to target and treat the cancerous tissue
without pathologically infecting or lysing the intervening healthy
tissue.
[0013] This finding represents a significant advance in clinical
HSV oncolytic therapy and provides the basis for considerable
improvements in clinical efficacy, procedure and risk management,
as well as in patient comfort.
[0014] At its most general the present invention relates to the use
of an HSV to treat a cancerous condition in a patient in need of
treatment, wherein the HSV is administered to the patient at a
location that is outside of the cancerous condition that is to be
treated.
[0015] The administration may therefore be extratumoral or
extraneoplastic, i.e. where the HSV is not administered directly to
the tumour or neoplastic tissue that is to be treated. Such
administration may involve administration of the HSV to a tissue in
which a tumour or neoplasia to be treated is not present.
[0016] Administration may be systemic, i.e. wherein substantially
the entire body is exposed to the HSV, rather than only the
cancerous tissue that requires treatment. This may be achieved by a
route of administration that permits the HSV to circulate in one of
the body's circulating fluids, e.g. the blood, lymph or spinal
fluid.
[0017] Preferably, administration of the HSV is such that the HSV
can be transported within the body to a cancerous condition that
requires treatment. This may involve transport to more than one
site of the cancerous condition, e.g. to two or more tumours, for
the treatment of one or more of those tumours. In that respect, the
HSV may be considered to be indirectly administered to the
cancerous condition. Administration to the blood may be
particularly preferred (e.g. by intravenous and/or intra-arterial
administration). This route of administration may be preferred for
the treatment of any cancerous condition, but optionally it may
exclude blood cancers, e.g. leukaemia.
[0018] Administration to other circulating fluids such as to the
lymph fluid, through administration to the lymphatic system, or to
the spinal fluid may also be preferred for the treatment of any
cancerous condition, but optionally excluding cancers of the lymph
fluid and/or of the lymphatic system (e.g. lymphoma) or of the
spinal fluid and/or spinal column respectively.
[0019] According to one aspect of the present invention an herpes
simplex virus (HSV) is provided for use in the treatment of a
tumour by extratumoural administration of said HSV.
[0020] According to another aspect of the present invention the use
of an herpes simplex virus (HSV) in the manufacture of a medicament
for use in the treatment of a tumour by extratumoural
administration of said HSV is provided.
[0021] According to a further aspect of the present invention an
herpes simplex virus (HSV) is provided for use in the treatment of
a cancerous condition in a patient by administration of said HSV to
the patient's blood.
[0022] According to another aspect of the present invention the use
of an herpes simplex virus (HSV) in the manufacture of a medicament
for use in the treatment of a cancerous condition in a patient by
administration of said HSV to the patient's blood is provided.
[0023] In another aspect of the present invention there is provided
a method of treating a tumour in a patient in need of treatment
thereof, said method comprising the step of administering an herpes
simplex virus (HSV) to said patient at an extratumoural location on
or in the patient's body.
[0024] In a further aspect of the present invention there is
provided a method of treating a cancerous condition in a patient in
need of treatment thereof, said method comprising the step of
administering an herpes simplex virus (HSV) to the patient's
blood.
[0025] In another aspect of the present invention there is provided
a kit of parts for use in treating a tumour in a patient, said kit
comprising a container containing an herpes simplex virus (HSV) and
instructions for the administration of said HSV to a patient in
need of treatment at an extratumoural location in order to treat
the tumour.
[0026] In yet another aspect of the present invention there is
provided a kit of parts for use in treating a cancerous condition
in a patient comprising a container containing an herpes simplex
virus (HSV) and instructions for the administration of said HSV to
a patient's blood in order to treat a cancerous condition of the
patient.
[0027] The instructions for administration may include information
on suitable HSV dosages.
[0028] The inventors have also found that that the ability of
systemically (extratumourally) administered HSV to preferentially
target and infect cancerous cells may be used in combination with a
pharmaceutical or medicament to treat a cancerous condition. As the
pharmaceutical may also be administered systemically this provides
a significant advance in the available treatments. Such combination
therapy may also provide for therapeutic improvements owing to the
potential synergistic effect provided by the combination.
[0029] Therefore, further aspects of the present invention concern
the use of HSV according to the invention in combination with a
pharmaceutical or medicament, preferably a chemotherapeutic drug or
activatable prodrug, in the treatment of a cancerous condition.
[0030] Accordingly, in a further aspect of the present invention an
herpes simplex virus (HSV) is provided for use in the treatment of
a tumour by combination therapy with a pharmaceutical, wherein the
HSV and/or pharmaceutical is administered at an extratumoural
location.
[0031] In another aspect of the present invention a pharmaceutical
is provided for use in the treatment of a tumour by combination
therapy with an herpes simplex virus (HSV), wherein the HSV and/or
pharmaceutical is administered at an extratumoural location.
[0032] According to a further aspect of the present invention an
herpes simplex virus (HSV) is provided for use in the treatment of
a cancerous condition by combination therapy with a pharmaceutical,
wherein the HSV and/or pharmaceutical is administered to the
patient's blood.
[0033] According to a further aspect of the present invention a
pharmaceutical is provided for use in the treatment of a cancerous
condition by combination therapy with an herpes simplex virus
(HSV), wherein the HSV and/or pharmaceutical is administered to the
patient's blood.
[0034] In another aspect of the present invention products are
provided containing an herpes simplex virus (HSV) and a
pharmaceutical as a combined preparation for simultaneous,
separate, or sequential use in the treatment of a tumour wherein
the HSV and/or pharmaceutical is administered at an extratumoural
location.
[0035] According to a further aspect of the present invention
products are provided containing an herpes simplex virus (HSV) and
a pharmaceutical as a combined preparation for simultaneous,
separate, or sequential use in the treatment of a cancerous
condition wherein the HSV and/or pharmaceutical is administered to
the patient's blood.
[0036] In another aspect of the present invention the use of an
herpes simplex virus (HSV) in the manufacture of a medicament for
treatment of a tumour by combination therapy of said medicament
with a pharmaceutical, wherein the HSV and/or pharmaceutical is
administered at an extratumoural location, is provided.
[0037] In another aspect of the present invention the use of a
pharmaceutical in the manufacture of a medicament for treatment of
a tumour by combination therapy of said medicament with an herpes
simplex virus (HSV), wherein the HSV and/or medicament is
administered at an extratumoural location, is provided.
[0038] According to a further aspect of the present invention the
use of an herpes simplex virus (HSV) in the manufacture of a
medicament for treatment of a cancerous condition by combination
therapy of said medicament with a pharmaceutical, wherein the HSV
and/or pharmaceutical is administered to the patient's blood, is
provided.
[0039] According to a further aspect of the present invention the
use of a pharmaceutical in the manufacture of a medicament for
treatment of a cancerous condition by combination therapy of said
medicament with an herpes simplex virus (HSV), wherein the HSV
and/or medicament is administered to the patient's blood, is
provided.
[0040] In another aspect of the present invention a method of
treating a tumour is provided comprising the steps of administering
to an individual in need of treatment a therapeutically effective
amount of an herpes simplex virus (HSV) and a pharmaceutical,
wherein the HSV and/or pharmaceutical is administered at an
extratumoural location.
[0041] According to a further aspect of the present invention a
method of treating a cancerous condition is provided comprising the
steps of administering to an individual in need of treatment a
therapeutically effective amount of an herpes simplex virus (HSV)
and a pharmaceutical, wherein the HSV and/or pharmaceutical is
administered to the patient's blood.
[0042] In another aspect of the present invention a kit of parts is
provided for use in the treatment of a tumour by combination
therapy, the kit comprising a first container comprising an herpes
simplex virus (HSV) and a second container comprising a
pharmaceutical, together with instructions for the extratumoural
administration of the HSV and/or pharmaceutical.
[0043] According to a further aspect of the present invention a kit
of parts is provided for use in the treatment of a cancerous
condition by combination therapy, the kit comprising a first
container comprising an herpes simplex virus (HSV) and a second
container comprising a pharmaceutical, together with instructions
for administration of the HSV and/or pharmaceutical to the
patient's blood.
[0044] In preferred embodiments the HSV genome encodes an
exogenous/heterologous (non-HSV originating) polypeptide/protein
such as NTR that may be expressed by the HSV following infection of
cells in vitro and/or in vivo, particularly tumour or other
cancerous cells in vivo (e.g. in a patient requiring treatment).
The expressed polypeptide and pharmaceutical are preferably capable
of interacting, directly or indirectly, to produce a therapeutic
effect, which may be enhanced as compared to the therapeutic effect
of the HSV or pharmaceutical alone.
[0045] In particularly preferred embodiments, the pharmaceutical is
an activatable prodrug and the exogenous polypeptide/protein is
capable of converting the prodrug to a therapeutically active
pharmaceutical. In one particularly preferred embodiment the
expressed polypeptide/protein is an NTR and the prodrug is an NTR
prodrug, such as CB1954. Accordingly, the HSV may be HSV1790. This
therapeutic approach may be referred to as gene directed
enzyme-prodrug therapy (GDEPT).
[0046] Accordingly, the following further aspects of the invention
are also provided.
[0047] According to one further aspect of the present invention an
herpes simplex virus (HSV) is provided for use in the treatment of
a tumour by combination therapy with an activatable prodrug,
wherein the HSV and/or activatable prodrug is administered at an
extratumoural location, and wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical.
[0048] In another aspect of the present invention an activatable
prodrug is provided for use in the treatment of a tumour by
combination therapy with an herpes simplex virus (HSV), wherein the
HSV and/or activatable prodrug is administered at an extratumoural
location, and wherein the HSV genome encodes a polypeptide/protein
capable of converting the activatable prodrug to a therapeutically
active pharmaceutical.
[0049] According to a further aspect of the present invention an
herpes simplex virus (HSV) is provided for use in the treatment of
a cancerous condition by combination therapy with an activatable
prodrug, wherein the HSV and/or activatable prodrug is administered
to the patient's blood, and wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical.
[0050] According to a further aspect of the present invention an
activatable prodrug is provided for use in the treatment of a
cancerous condition by combination therapy with an herpes simplex
virus (HSV), wherein the HSV and/or activatable prodrug is
administered to the patient's blood, and wherein the HSV genome
encodes a polypeptide/protein capable of converting the activatable
prodrug to a therapeutically active pharmaceutical.
[0051] In another aspect of the present invention products are
provided containing an herpes simplex virus (HSV) and an
activatable prodrug as a combined preparation for simultaneous,
separate, or sequential use in the treatment of a tumour wherein
the HSV and/or activatable prodrug is administered at an
extratumoural location, and wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical.
[0052] According to a further aspect of the present invention
products are provided containing an herpes simplex virus (HSV) and
an activatable prodrug as a combined preparation for simultaneous,
separate, or sequential use in the treatment of a cancerous
condition wherein the HSV and/or activatable prodrug is
administered to the patient's blood, and wherein the HSV genome
encodes a polypeptide/protein capable of converting the activatable
prodrug to a therapeutically active pharmaceutical.
[0053] In another aspect of the present invention the use of an
herpes simplex virus (HSV) in the manufacture of a medicament for
treatment of a tumour by combination therapy of said medicament
with an activatable prodrug is provided, wherein the HSV and/or
activatable prodrug is administered at an extratumoural location,
and wherein the HSV genome encodes a polypeptide/protein capable of
converting the activatable prodrug to a therapeutically active
pharmaceutical.
[0054] In another aspect of the present invention the use of an
activatable prodrug in the manufacture of a medicament for
treatment of a tumour by combination therapy of said medicament
with an herpes simplex virus (HSV) is provided, wherein the HSV
and/or activatable prodrug is administered at an extratumoural
location, and wherein the HSV genome encodes a polypeptide/protein
capable of converting the activatable prodrug to a therapeutically
active pharmaceutical.
[0055] According to a further aspect of the present invention the
use of an herpes simplex virus (HSV) in the manufacture of a
medicament for treatment of a cancerous condition by combination
therapy of said medicament with an activatable prodrug is provided,
wherein the HSV and/or activatable prodrug is administered to the
patient's blood, and wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical.
[0056] According to a further aspect of the present invention the
use of an activatable prodrug in the manufacture of a medicament
for treatment of a cancerous condition by combination therapy of
said medicament with an herpes simplex virus (HSV) is provided,
wherein the HSV and/or activatable prodrug is administered to the
patient's blood, and wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical.
[0057] In another aspect of the present invention a method of
treating a tumour is provided comprising the steps of administering
to an individual in need of treatment a therapeutically effective
amount of an herpes simplex virus (HSV) and an activatable prodrug,
wherein the HSV and/or activatable prodrug is administered at an
extratumoural location, and wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical.
[0058] According to a further aspect of the present invention a
method of treating a cancerous condition is provided comprising the
steps of administering to an individual in need of treatment a
therapeutically effective amount of an herpes simplex virus (HSV)
and an activatable prodrug, wherein the HSV and/or activatable
prodrug is administered to the patient's blood, and wherein the HSV
genome encodes a polypeptide/protein capable of converting the
activatable prodrug to a therapeutically active pharmaceutical.
[0059] In another aspect of the present invention a kit of parts is
provided for use in the treatment of a tumour by combination
therapy, the kit comprising a first container comprising an
activatable prodrug and a second container comprising an herpes
simplex virus (HSV) wherein the HSV genome encodes a
polypeptide/protein capable of converting the activatable prodrug
to a therapeutically active pharmaceutical, together with
instructions for the extratumoural administration of the HSV and/or
activatable prodrug.
[0060] According to a further aspect of the present invention a kit
of parts is provided for use in the treatment of a cancerous
condition by combination therapy, the kit comprising a first
container comprising an activatable prodrug and a second container
comprising an herpes simplex virus (HSV) wherein the HSV genome
encodes a polypeptide/protein capable of converting the activatable
prodrug to a therapeutically active pharmaceutical, together with
instructions for administration of the HSV and/or pharmaceutical to
the patient's blood.
[0061] In preferred embodiments, both the HSV and pharmaceutical
are administered to the patient at a location that is outside of
the cancerous condition that is to be treated, i.e. extratumoural.
For example, administration may be systemic into any circulating
fluid, e.g. to the blood by intravenous administration. In another
embodiment, one of the HSV or pharmaceutical may be administered
extratumourally, with the other being administered directly to the
tumour, e.g. by injection.
[0062] In terms of route of administration, the HSV and
pharmaceutical may be administered either by the same route of
administration, e.g. both intravenously, or by separate routes of
administration, e.g. one intravenously and the other
intraperitoneal.
[0063] In order to provide a `combination therapy`, therapeutically
effective amounts of the HSV and pharmaceutical should be present
in the body. This may be achieved by simultaneous, separate or
sequential administration of the HSV and pharmaceutical. Where the
HSV and pharmaceutical are not administered simultaneously the time
period between administration of the first and second compounds may
be predetermined such that the HSV, or a polypeptide/protein
encoded by the HSV genome, and pharmaceutical are present in active
form in the patient's body at the same time in order that they may
directly or indirectly interact and provide a therapeutic benefit,
which may optionally involve a synergistic effect of the HSV
(and/or the polypeptide/protein encoded by the HSV genome) and
pharmaceutical. Preferred time periods may be less than 15 minutes,
less than one hour, two hours, three hours, four hours, five hours
or six hours, twelve hours, twenty four hours, forty eight hours,
one week or two weeks. Either the herpes simplex virus or
pharmaceutical may be administered first.
[0064] The administered pharmaceutical may comprise a
therapeutically active compound, or a pharmaceutically acceptable
salt or ester thereof. In some preferred embodiments of the
invention the pharmaceutical is administered in the form of an
activatable prodrug, e.g. an NTR prodrug. An activatable prodrug
may not be therapeutically active, e.g. as a chemotherapeutic, or
may be only partially active. Preferably, the prodrug may be
converted to a therapeutically active pharmaceutical form in the
body, e.g. to a chemotherapeutic form. In preferred embodiments,
the prodrug requires a factor, preferably an enzyme or protein, to
be present for conversion to occur. This factor is preferably
encoded by the HSV and expressed in cells infected by the HSV. This
system provides a means of highly effective targeting of the active
pharmaceutical form to cells infected by the HSV.
[0065] HSV modified so as to be capable of targeting specific cells
and tissues are described in PCT/GB2003/000603 (WO 03/068809),
hereby incorporated in its entirety by reference.
[0066] Preferably the administered HSV is non-neurovirulent. The
HSV is also preferably oncolytic. More preferably the HSV is
modified in at least one of the long repeat regions (R.sub.L) of
the HSV genome, relative to the genome of the corresponding
wild-type strain, such that the HSV lacks neurovirulence. The
modification may be within the BamHI s restriction fragment of one
or each R.sub.L repeat. As such, the HSV genome may be modified
within the Bam HI s region of the internal repeat R.sub.L
(0.81-0.83 mu) and within the counterpart region of the terminal
R.sub.L (0-0.02 mu) such that the variant lacks neurovirulence.
[0067] Such modification may take the form of at least one
addition, deletion, substitution or insertion of one or more
nucleotides.
[0068] In one arrangement the genome is modified in each said
region by a deletion of one or more nucleotides. The deletion may
be of at least 50 or at least 100 nucleotides or from 0.5 Kb to 3
Kb or from 0.7 Kb to 2.5 Kb. In one arrangement the deletion is 759
bp in length and is located between nucleotide positions 125213 and
125972 of the internal long repeat (IR.sub.L) and in the
counterpart region of the terminal long repeat (TR.sub.L) of HSV-1
strain 17.
[0069] Suitable modifications may also include the insertion of an
exogenous nucleic acid sequence or exogenous/heterologous cassette
comprising said sequence into the herpes simplex virus genomic DNA.
The insertion may be performed by homologous recombination, or by
site-specific recombination using an HSV genome with appropriate
recombination sites, of the exogenous nucleic acid sequence into
the genome of the selected herpes simplex virus. For example, the
modification may take the form of insertion of a sequence of
nucleotides encoding a gene product, such as NTR, which may be
operably linked to one or more control sequences enabling
expression of the gene product from the HSV vector.
[0070] Where a plurality of nucleotides are inserted, e.g. in the
case of insertion of a gene sequence, the inserted nucleotides may
be located entirely within, or may overlap, at least one of the
ICP34.5 protein coding sequences of the HSV genome. The inserted
nucleic acid may be located in both (this will usually be all)
copies of the RL1 locus or ICP34.5 protein coding sequence.
[0071] The HSV may, therefore, have an inactivating mutation in the
RL1 locus of the HSV genome, more specifically a mutation which
inactivates the function of the ICP34.5 gene product, such that the
herpes simplex virus does not produce a functional ICP34.5 gene
product and is non-neurovirulent.
[0072] Accordingly, an inactivating mutation may be present in one
or each ICP34.5 locus, disrupting the ICP34.5 protein coding
sequence such that the ICP34.5 gene is non-functional and cannot
express a functional ICP34.5 gene product.
[0073] Preferably, both copies of the ICP34.5 gene sequence contain
inactivating mutations, which may be the result of one or more
modifications of the HSV genome, as described above.
[0074] Where all copies of the ICP34.5 gene present in the herpes
simplex virus genome (two copies are normally present) are
disrupted such that the herpes simplex virus is incapable of
producing a functional ICP34.5 gene product, the virus is
considered to be an ICP34.5 null mutant.
[0075] The HSV may lack at least one expressible ICP34.5 gene, but
is preferably an ICP34.5 null mutant.
[0076] In another arrangement the herpes simplex virus may lack
only one expressible ICP34.5 gene.
[0077] The HSV may be a mutant of HSV-1 or HSV-2, more preferably
of one of HSV-1 strains 17, F or HSV-2 strain HG52 and most
preferably of HSV-1 strain 17. Non-neurovirulent ICP34.5 null
mutants of HSV-1 strain 17 are particularly preferred and suitable
examples include: [0078] (a) HSV 1716 (ECACC accession number
V92012803); and [0079] (b) HSV 1790 (ECACC accession number
03110501).
[0080] The HSV may be a further mutant of HSV 1716 or HSV 1790.
[0081] Suitable HSV may therefore be described as mutants or
variants of the parent HSV strain from which they are derived or to
which they correspond. For example, HSV 1716 and HSV 1790 are
mutants of HSV-1 strain 17 and may be obtained by modification of
the strain 17 genomic DNA. Suitable mutant HSV may be non-wild type
and may be recombinant. Mutant herpes simplex viruses may comprise
a genome containing modifications relative to the wild type, as
described above.
[0082] The present invention provides HSV for use in a method of
medical treatment. Preferably they are provided for use in the
treatment of cancer, i.e. in oncotherapy. This treatment may
comprise the oncolytic treatment of the cancer, which may take the
form of a tumour. Accordingly, the method of treatment may involve
the killing of tumour cells by the HSV. Treatment may involve the
selective infection and/or lysis of dividing cells. The use of HSV
in the manufacture of a medicament, pharmaceutical composition or
vaccine for the treatment of cancer is also provided. Such
medicaments, pharmaceutical compositions or vaccines may comprise
suitable HSV together with a pharmaceutically acceptable carrier,
adjuvant or diluent.
[0083] Medicaments and pharmaceutical compositions according to
aspects of the present invention may be formulated for
administration by a number of routes, including but not limited to,
systemic, parenteral, intravenous, intra-arterial, intramuscular,
intraperitoneal, oral and nasal. The medicaments and compositions
may be formulated in fluid or solid form. Fluid formulations may be
formulated for administration by injection to a selected region of
the human or animal body.
[0084] The HSV may be administered systemically, topically,
parenterally, intravenously, intra-arterially, intramuscularly,
intrathecally, intraocularly, subcutaneously, orally or
transdermally. Any one of these routes of administration may
involve injection of the HSV. Injectable formulations may comprise
the HSV in a sterile and/or isotonic medium.
[0085] The route of administration may be selected by the ability
of that route to expose substantially the entire body to the HSV.
This may be determined by the ability of the HSV to circulate
throughout substantially all parts of the body via the selected
route. Circulation throughout substantially all of the body may
exclude exposure of the HSV to one or a small number of tissues.
For example, where the HSV is circulated in the blood it may be
excluded from the brain by the blood brain barrier.
[0086] In preferred embodiments of the invention the HSV is
administered by injection to the circulating blood, e.g. by
intravenous or intra-arterial injection.
[0087] The delivery of suitable HSV to cancerous cells that are to
be treated may be performed using naked virus or by encapsulation
of the virus in a carrier, e.g. in nanoparticles, liposomes or
other vesicles.
[0088] Administration is preferably in a "therapeutically effective
amount", this being sufficient to show benefit to the individual.
The actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of the
tumour being treated. Prescription of treatment, e.g. decisions on
dosage etc, is within the responsibility of general practitioners
and other medical doctors, and typically takes account of the
disorder to be treated, the condition of the individual patient,
the site of delivery, the method of administration and other
factors known to practitioners. Examples of the techniques and
protocols mentioned above can be found in Remington's
Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott,
Williams & Wilkins
[0089] The HSV may be administered at any therapeutically effective
dosage amount. Therapeutically effective dosages may comprise less
than or equal to one of 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8 or 10.sup.9 plaque forming units (pfu).
[0090] The patient to be treated may be any animal or human. The
patient may be a non-human mammal, but is more preferably a human
patient. The patient may be male or female.
[0091] In this specification a cancerous condition may be any
unwanted cell proliferation (or any disease manifesting itself by
unwanted cell proliferation), neoplasm or tumour. The cancerous
condition may be a cancer and may be a benign or malignant cancer
and may be primary or secondary (metastatic). A neoplasm or tumour
may be any abnormal growth or proliferation of cells and may be
located in any tissue. Examples of tissues include the colon,
pancreas, lung, breast, uterus, stomach, kidney, testis, central
nervous system (including the brain), peripheral nervous system,
skin, blood or lymph.
[0092] Tumours to be treated may be nervous system tumours
originating in the central or peripheral nervous system, e.g.
glioma, medulloblastoma, meningioma, neurofibroma, ependymoma,
Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma,
or may be non-nervous system tumours originating in non-nervous
system tissue e.g. melanoma, mesothelioma, lymphoma, hepatoma,
epidermoid carcinoma, prostate carcinoma, breast cancer cells, lung
cancer cells or colon cancer cells. HSV mutants of the present
invention may be used to treat metastatic tumours occurring in the
central or peripheral nervous system which originated in a
non-nervous system tissue or metastatic tumours occurring outside
the central or peripheral nervous system which originated in a
central or peripheral nervous system tissue.
[0093] In this specification, non-neurovirulence is defined by the
ability to introduce a high titre of virus (approx 10.sup.6 plaque
forming units (pfu)) to an animal or patient.sup.12, 13 without
causing a lethal encephalitis such that the LD.sub.50 in animals,
e.g. mice, or human patients is in the approximate range of
.gtoreq.10.sup.6 pfu.sup.11.
[0094] In this specification the term "operably linked" may include
the situation where a selected nucleotide sequence and regulatory
or control nucleotide sequence are covalently linked in such a way
as to place the expression of a nucleotide coding sequence under
the influence or control of the regulatory sequence. Thus a
regulatory sequence is operably linked to a selected nucleotide
sequence if the regulatory sequence is capable of effecting
transcription of a nucleotide coding sequence which forms part or
all of the selected nucleotide sequence. Where appropriate, the
resulting transcript may then be translated into a desired protein
or polypeptide.
[0095] NTR Prodrug
[0096] In this specification, "NTR prodrug" means any chemical
compound or agent which is not toxic, or exhibits low toxicity, to
a selected human or animal body, or to selected cells or tissues of
the human or animal body, and which may be activated by a
nitroreducase enzyme to a chemical compound or agent which is
cytotoxic to the human or animal body or to those selected
cells.
[0097] "Activation" may involve conversion of a non-toxic (or low
toxicity) prodrug to an active cytotoxic form. That conversion may
involve enzymatic reduction of the prodrug by NTR. The enzymatic
reduction reaction may involve the prodrug as a substrate for NTR
and may involve other co-factors.
[0098] Examples of NTR prodrugs may include compounds from the
following classes of molecules: [0099] 1. dinitirobenzamides;
[0100] 2. dinitroaziridinylbenzamides (e.g. CB1954); [0101] 3.
dinitrobenzamide mustard derivatives (e.g. SN23862); [0102] 4.
4-nitrobenzylcarbamates; [0103] 5. nitroindolines; [0104] 6.
nitroaromatics that are substrates of NTR and are activated to
release a cytotoxic phosphoramide mustard or like-reactive species
upon NTR-reduction (also called nitroaryl phosphoramides); [0105]
7. nitroaromatic prodrugs of the dinitrobenzamide class.
[0106] Examples of NTR prodrugs are disclosed in:
[0107] Johansson E, Parkinson G N, Denny, W A and Neidle S. Studies
on the Nitroreductase Prodrug-Activating System. Crystal Structures
of Complexes with the Inhibitor Dicoumarol and Dinitrobenzamide
prodrugs and of the Enzyme Active Form. J. Med. Chem. 2003, 46,
4009-4020; and
[0108] Hu L, Yu C, Jiang Y et al. Nitroaryl Phosphoramides as Novel
Prodrugs for E. coli Nitroreductase Activation in Enzyme Prodrug
Therapy. J. Med. Chem. 2003 46, 4818-4821.
[0109] both of which are incorporated herein in their entirety by
reference.
[0110] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0111] Aspects and embodiments of the present invention will now be
illustrated, by way of example, with reference to the accompanying
figures. Further aspects and embodiments will be apparent to those
skilled in the art. All documents mentioned in this text are
incorporated herein by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0112] Embodiments and experiments illustrating the principles of
the invention will now be discussed with reference to the
accompanying figures.
[0113] FIGS. 1 to 31 show light micrographs of tissue sections
following tail vein administration of HSV 1790. The tissue sections
illustrated, time after administration and other relevant
information are set out below:
[0114] FIG. 1. Tumour tissue at day 1 following treatment; .times.5
magnification. Areas of positive staining are highlighted by
arrows.
[0115] FIG. 2. Tumour tissue at day 1 following administration;
.times.20 magnification showing view of the stained area marked A
in FIG. 1.
[0116] FIG. 3. Tumour tissue at day 1 following administration;
.times.20 magnification showing view of the stained area marked B
in FIG. 1. An island of positive HSV staining is surrounded by HSV
negative cells. The positively stained cells show the classic
appearance of HSV infected cells--they are necrotic and form large
multi-nucleated cells.
[0117] FIG. 4. Tumour tissue at day 7 following administration;
.times.5 magnification. Large and more widespread areas of HSV
staining are present, as compared to day 1 (FIG. 1).
[0118] FIG. 5. Tumour tissue at day 7 following administration. The
area marked C in FIG. 4 is shown at higher magnification.
[0119] FIG. 6. Tumour tissue at day 7 following administration;
.times.20 magnification. Cells stained positively for HSV are shown
in the area marked D in FIG. 4.
[0120] FIG. 7. Tumour tissue at day 7 following administration.
Highly HSV positive cells are shown in the area marked E in FIG. 4.
The tissue is highly necrotic and cell debris from dead cells are
present (marked A in the Figure).
[0121] FIG. 8. Tumour tissue at day 7 following administration;
.times.20 magnification.
[0122] FIG. 9. Gut tissue at day 1 following administration;
.times.5 magnification. No HSV staining is present.
[0123] FIG. 10. Gut tissue at day 1 following administration;
.times.20 magnification. Goblet cells producing gut mucosa are
visible (A) and appear to be filled with mucosa that does not
stain. Area B is a fold in the tissue.
[0124] FIG. 11. Spleen tissue at day 1 following administration;
.times.5 magnification.
[0125] FIG. 12. Spleen tissue at day 1 following administration;
.times.5 magnification. A number of light brown stained circular
patches are present (A). They may represent infiltrating blood.
[0126] FIG. 13. Lung tissue at day 1 following administration;
.times.5 magnification. The tissue appears normal and healthy with
no positive staining.
[0127] FIG. 14. Lung tissue at day 1 following administration;
.times.20 magnification. The tissue appears normal and healthy with
no positive staining.
[0128] FIG. 15. Liver tissue at day 1 following administration;
.times.5 magnification.
[0129] FIG. 16. Kidney tissue at day 1 following administration;
.times.5 magnification. No HSV staining is present.
[0130] FIG. 17. Heart tissue at day 1 following administration.
[0131] FIG. 18. Skin tissue at day 1 following administration;
.times.5 magnification.
[0132] FIG. 19. Brain tissue at day 1 following administration;
.times.20 magnification. No HSV staining is present.
[0133] FIG. 20. Gut tissue at day 7 following administration;
.times.5 magnification. No HSV staining is present.
[0134] FIG. 21. Gut tissue at day 7 following administration;
.times.20 magnification. The micrograph shows the villi and
microvilli (A).
[0135] FIG. 22. Spleen tissue at day 7 following administration;
.times.5 magnification. Most of the cells are negative for HSV
staining. There is some background staining. Some cells surrounding
the holes stained bright blue suggesting they are active.
[0136] FIG. 23. Skin tissue at day 7 following administration. Some
positive staining may be present in cells below the basement layer
(indicated with an arrow).
[0137] FIG. 24. Brain tissue at day 7 following administration;
.times.5 magnification. The section is of the cerebellar cortex and
shows several folia. Each folium has a central core of white matter
(A), consisting of nerve processes entering and leaving the
superficial cortex. The cortex has an external pale layer (B) and a
darker staining granular layer beneath (C). No HSV staining is
present.
[0138] FIG. 25. Brain tissue at day 7 following administration;
.times.20 magnification. (A) white matter; (B) granular layer; (C)
cortex. No HSV staining is present.
[0139] FIG. 26. Brain tissue at day 7 following administration;
.times.20 magnification. View of the granular layer between the
white matter and the cortex. No HSV staining is present.
[0140] FIG. 27. Lung tissue at day 7 following administration;
.times.5 magnification. No HSV staining is present.
[0141] FIG. 28. Lung tissue at day 7 following administration;
.times.5 magnification. No HSV staining is present.
[0142] FIG. 29. Heart tissue at day 7 following administration;
.times.5 magnification. No HSV staining is present.
[0143] FIG. 30. Kidney tissue at day 7 following administration.
Background staining in this section is present, but individual
cells are not stained for HSV.
[0144] FIG. 31. Skin tissue at day 7 following administration;
.times.5 magnification. No HSV staining is present.
[0145] FIG. 32. Schematic representation of (A) the HSV17+ genome
and (B) the HSV1716/CMV-NTR/GFP genome.
[0146] BamHI cuts the HSV-1 genome in several places to generate
fragments of different sizes. The RL1.del probe contains the BamHI
`k` fragment and will therefore hybridize to fragments that contain
sequences in the BamHI `k` region. If the foreign DNA had been
inserted into the HSV-1 genome in the location shown in B, the
normal BamHI `k` fragment would disappear and be replaced with a
smaller fragment of 5.2 Kbp. The BamHI `q` fragment would not be
altered and should be 3.4 Kbp. Two new fragments (3.4 Kbp and 2.2
Kbp) would be generated in place of the one `s` fragment that would
hybridize to the RL1.del probe. The fragments are visualized by
Southern Blotting.
[0147] FIG. 33. Western blot analysis of NTR expression in
HSV1716/CMV-NTR/GFP infected cell lines.
[0148] BHK, C8161, VM and 3T6 cells were infected with 10 pfu/cell
HSV1716/CMV-NTR/GFP, HSV17+ or mock infected. 16hrs post infection,
the cells were harvested and protein extracts analyzed in a Western
blot using a polyclonal NTR-specific antibody. Significant NTR
expression was detected in all the HSV1716/CMV-NTR/GFP infected
cells. No NTR expression was detected in the mock or HSV17+
infected cells.
[0149] FIG. 34. Confluent 3T6 cells 24, 48 and 72 hrs post
treatment with HSV 1790, CB1954, or both.
[0150] Confluent 3T6 cells in 60 mm dishes were mock infected (A
and B) or infected with 10 pfu/cell HSV 1790 (C and D). After 45
minutes, the cells were overlaid with media. 50 .mu.M CB1954 was
included in the overlay in B and D. After 24 hrs, no significant
effects were observed in the mock infected cells overlaid with
media containing 50 .mu.M CB1954 (B). However, enhanced cell
killing was evident in HSV1790 infected cells overlaid with media
containing CB1954 (D) compared with HSV1790 infected cells overlaid
with media not containing CB1954 (C). Images were captured under a
10.times. objective.
[0151] FIG. 35. Confluent 3T6 cells were infected with HSV1790,
HSV1716 or no virus. After 45 minutes, the infected cells were
overlaid with media containing CB1954 or with media alone. At 24,
48, 72, 96 and 120 hours, % cell survival (y axis) was determined
relative to that of mock infected cells without prodrug using
CellTiter 96 Aqueous Solution Cell Proliferation Assay (Promega).
Figures shown represent the mean of 3 values.+-.SD.
[0152] FIG. 36. Comparison of tumour volume between HSV1790 treated
tumours and tumour receiving no treatment in A2780 (A) CP70 (B) and
A431 (C).
[0153] (A) A2780 human tumour xenograft model. Mice treated with a
combination of HSV1790 and CB 1954 had smaller tumour burdens and
(D) significantly longer survival that those either not virally
treated or treated with HSV1790 only (p<0.05-log rank test). As
n=3 in this tumour type the reduction in tumour volume seen was not
significant.
[0154] (B) In CP70 xenografts the addition of the prodrug CB1954
had no effect on tumour volume, or (E) survival.
[0155] (C) Mice bearing A431 xenografts. Tumours treated with a
combination of HSV1790 and CB1954 were significantly smaller than
those treated with HSV1790 alone (P<0.05) the difference in
survival (F) between animals treated with HSV1790 alone or
HSV1790+CB 1954 is not statistically significant by log rank
method. However, the difference in the median survival between
animals treated with HSV1790 alone and those treated with
HSV1790+CB1954 is significant (P=0.034, Students t test).
[0156] FIG. 37. Immunohistochemical staining of UVW xenograft
tumours using an HSV antibody.
[0157] (A) Low power field (.times.5) of tumour at day 1 post i.v
viral injection showing small areas of positively stained cells
(brown stained cells).
[0158] (B) .times.20 magnification of the areas with positive
staining cells.
[0159] (C) (.times.40) shows area with a typical HSV
infection--giant multi-nucleated cells have formed holes, where
cells have been killed.
[0160] (D) .times.5 magnification of Day 7 post i.v HSV1790
injection showing more and larger areas of positive staining.
[0161] (E, F) .times.20 magnification of areas from (D). (F) shows
an area of HSV mediated necrosis, with cell debris visible.
[0162] (G) Cross section of the gut showing villi--an area of
active cell division.
[0163] (H) Section of cerebellar cortex. The cortex has an external
pale layer and a darker staining granular layer between it and the
white matter.
[0164] (I) Lung section. A bronchiole is visible on the left of the
section.
[0165] (J) Spleen section.
[0166] (K) Skin section .times.20.
[0167] (L) Liver section .times.20.
[0168] FIG. 38. PCR Results from samples of major organs and
xenograft tumours following i.v injection of HSV1790.
[0169] DNA PCR detects the presence of virus. RNA PCR (reverse
transcriptase PCR) detects mRNA, therefore detecting actively
replicating virus.
[0170] Key:
[0171] +++ very strong band
[0172] ++ strong band
[0173] + weak band
[0174] - no band
[0175] (RNA only) NI--non informative, sample contaminated with
DNA.
[0176] FIG. 39. Kaplan-Meier plots indicate survival of the athymic
nude mice bearing subcutaneous A431 tumours.
[0177] A431 cells were injected subcutaneously and twelve days
later, animals received treatment (arrows) with HSV1790
(1.times.10.sup.6 pfu/mice) on two occasions (day 12 and 14 post
cell injection) with or without 5 further injections of 20 mg/kg
CB1954 at days 15, 16, 17, 18 and 19. Control animals received
media only. Each line represents a group, with at least 6 mice in
each group. The two arrows indicate the days on which the virus was
injected, days 12 and 14. Mice were monitored daily and killed when
their tumour diameter reached 15 mm Tumours were removed at time of
death and immunohistochemistry performed to look for the presence
and/or replication of the virus within the tumour. The median
survival (time at which half the subjects have died) for the group
of mice given no treatment was 19 days; given HSV1790 by i.v, 21
days; and given HSV1790 by i.v and given CB1954 26 days. Median
survival of each group is plotted on FIG. 13b, both virally treated
groups have significantly improved survival (p=0.02 and 0.007)
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0178] Specific details of the best mode contemplated by the
inventors for carrying out the invention are set forth below, by
way of example. It will be apparent to one skilled in the art that
the present invention may be practiced without limitation to these
specific details.
EXAMPLE 1
[0179] Materials and Methods
[0180] Nude mice were subcutaneously injected with
.about.20.times.10.sup.6 UVW tumour cells (a glioma cell line) at
60% confluency per mouse to generate UVW xenografts in the right
flank. The mice were inspected regularly for xenograft
formation.
[0181] Once the xenografts had reached approximately 5mm.times.5mm
the mice were injected intravenously with 10.sup.7 PFU HSV 1790 via
tail vein injection.
[0182] In these experiments HSV 1790 was used as an exemplary HSV-1
strain 17 ICP34.5 null mutant. No prodrug activatable by NTR was
administered.
[0183] One mouse was sacrificed at day 1 post injection and one at
day 7 and the organs harvested. The following tissues were
collected: tumour, blood, liver, lung, spleen, heart, kidney, gut,
brain and skin.
[0184] Half of each sample was flash frozen in LN2 and processed
for polymerase chain reaction (PCR) analysis, the other half was
fixed in neutral buffered formalin for use in
immunohistochemistry.
[0185] Immunohistochemistry
[0186] Sections were stained with an HSV polyclonal antibody (Dako
HSV type 1 polyclonal {Cat. No. B0144} and secondary antibody
Vectastain Elite rabbit IgG kit {Cat. No. PK6101}) and
counterstained with haematoxylin. The sections were observed by
light microscopy.
[0187] Positive staining was indicated by a dark brown colour and
negative staining by a blue colour. Background staining was present
in some samples and showed variation between tissue types, for
example muscle and cardiac cells exhibited a light brown background
stain. The background staining observed was separate and entirely
distinguishable from the dark brown colour indicative of positive
HSV staining.
[0188] The results of the immunohistochemical analysis are shown in
FIGS. 1-31.
[0189] PCR
[0190] DNA and RNA was isolated from the flash frozen samples. The
RNA was used to make cDNA using the ImProm Reverse Transcriptase
kit (Promega). The cDNA obtained was then used for PCR. The
following PCRs were performed:
[0191] HSV
[0192] PCR utilised primers:
TABLE-US-00001 HS13 (ACG ACG ACG TCC GAC GGC GA [SEQ ID No. 1]);
and HS14 (GTG CTG GTG CTG GAC GAC AC [SEQ ID No. 2])
as previously described.sup.10.
[0193] These primers anneal to HSV-1 sequence co-ordinates
93536-93555 and 93813-93794 (complementary) which lie within the
UL42 region of the genome. This region codes for a sub-unit of the
viral DNA polymerase--the DNA polymerase accessory protein.
[0194] The resulting PCR product is 278 base-pairs in length and
can be visualised by agarose gel electrophoresis.
[0195] The reaction conditions used were a 94.degree. C.
`Hot-Start` for 2 minutes followed by 34 cycles of {94.degree. C.
for 15 seconds (denaturation); 72.degree. C. for 1 min (annealing);
72.degree. C. for 1 minute (extension)} and a final extension step
at 72.degree. C. for 2 minutes.
[0196] NTR
[0197] PCR utilised primers from the nitroreductase (NTR) enzyme
from E. coli B genomic DNA. Sequence information for E. coli NTR
can be found at the NCBI database (http://www.ncbi.nlm.nih.gov/)
under accession numbers BA000007 (GI:47118301)--E. coli complete
genome sequence--and BAB34039 (GI:13360074)--nitroreductase
sequence information.
[0198] Upstream primer 5'-CTTTCACATTGAGTCATTATGG-3'(SEQ ID No.3);
and downstream primer 5'-TTACACTTCGGTTAAGGTGATG-3' (SEQ ID No.4)
were used, based on those of Clark et al.sup.15.
[0199] Following initial denaturation at 94.degree. C. for two
minutes, PCR conditions were 95.degree. C. for 30 s (denaturating),
55.degree. C. for 30 s (annealing), and 72.degree. C. for 60 s
(extension), for 32 cycles.
[0200] Actin (Control)
[0201] The mouse B-actin primers used were:
TABLE-US-00002 5'-cgt gaa aag atg acc cag a-3'; (SEQ ID No. 5) and
5'-agc ata gcc ctc gta gat g-3'. (SEQ ID No. 6)
[0202] Following initial denaturation at 94.degree. C. for two
minutes, PCR conditions were 95.degree. C. for 30 s (denaturating)
57.degree. C. annealing temperature and 72.degree. C. (extension)
for 60 s for 30 cycles.
[0203] The results of the PCR analysis, as visualised by agarose
gel electrophoresis, are contained in Tables 1 and 2.
[0204] Results
[0205] The PCR results are consistent for both DNA and RNA. Whilst
the DNA PCR results may be used to confirm the presence of the HSV
in a particular tissue, RNA PCR provides an indication of virus
activity in that tissue. In particular, the RNA results indicate
whether the HSV is actively replicating and thus provides
information regarding the existence of a pathogenic infection of
the cells of a given tissue. This information may be used in
conjunction with the immunohistochemical analysis for corroboration
of the results.
[0206] A weak positive signal is present in the tumour samples on
day 1 increasing to a strong positive signal by day 7. This is
consistent for both HSV and NTR PCR for both RNA and DNA and
indicates the presence and accumulation of replicating HSV in
tumour tissue.
[0207] The PCR observations are reflected by the
immunohistochemical analysis. FIG. 1 shows positive staining for
HSV confirming the presence of HSV in tumour tissue at day 1. FIGS.
2 and 3 show regions of HSV positive staining. In particular, FIG.
3 shows an island of positive HSV staining in which the cells show
the classic appearance of HSV infection. The cells are large,
multi-nucleated and necrotic.
[0208] By day 7 (FIG. 4) the areas of HSV staining within tumour
tissue are larger and more widespread compared to day 1. FIG. 7
shows an increased magnification view of area E of FIG. 4. The
tissue in this region is highly necrotic and cell debris from dead
cells is present.
[0209] In spleen tissue, the DNA PCR results at day 1 are positive
for HSV, but not NTR. The day 1 RNA PCR result was negative for
both HSV and NTR indicating that any HSV present is not actively
replicating. At day 7 no positive result was obtained either by DNA
or RNA PCR of spleen tissue.
[0210] The immunohistochemical results for spleen tissue are shown
in FIG. 11 (day 1) and FIG. 22 (day 7). In FIG. 11 some positive
HSV staining is present to the right of the line indicated. By day
7 (FIG. 22) this has decreased and most of the cells are negative
for HSV.
[0211] These results are consistent with the PCR data that show a
loss of HSV in spleen between day 1 and day 7. In view of the fact
that the RNA PCR result is negative for spleen tissue at both day 1
and day 7 (indicating that HSV in the spleen are not replicating),
this result is most likely attributable to spleenic filtering of
the blood.
[0212] The DNA PCR data indicates weak positive results in blood,
skin, gut, heart and liver. These results are weak and are not
consistently repeated. Moreover, they are not borne out by the
immunohistochemical analysis except for some possible weak positive
staining for HSV in skin as shown in FIG. 23. Of course, the RNA
PCR results are negative for blood, skin, gut, liver and spleen.
Given that the HSV is administered intravenously in the tail vein,
the presence of some HSV particles in various tissues owing to the
circulation of the HSV particles in the blood is not surprising.
Importantly, the RNA PCR results show that HSV replication
activity, which may lead to lysis and a therapeutic effect, is
exclusively limited to tumour cells by day 7.
[0213] The RNA PCR data indicates a medium positive result in heart
tissue at day 1. However, in contrast to the results in tumour
tissue this result is not consistent for both HSV and NTR,
disappears by day 7 and is not borne out by the immunohistochemical
analysis which does not show any positive staining for HSV.
[0214] Changes in tumour size were not followed in these
experiments but preliminary survival data indicates a median
survival time of 7 days in the absence of virus and 14 days where
virus was administered intravenously. This data supports the
ability of the HSV to not only target the tumour, but to treat the
tumour and improve survival time.
[0215] The results demonstrate that non-neurovirulent HSV-1 mutants
of strain 17 may be administered at a site on the body that is
distal to a tumour requiring treatment such that the HSV
accumulates in the tumour. The results support an increasing
accumulation of HSV in the tumour over time and indicate that the
HSV may self-target the tumour. HSV RNA production is exclusively
limited to tumour cells by day 7 and supports the accumulation of
HSV in tumour tissue by exploiting the ability of the oncolytic HSV
to selectively replicate in dividing tumour cells. The
immunohistochemical analysis provides further support for both HSV
infection of tumour cells and necrosis of tumour cells and is
consistent with a mechanism of lytic HSV replication in tumour
tissue.
TABLE-US-00003 TABLE 1 DNA PCR Results HSV HSV NTR Sample 21 May 7
Jun. 21 May ID No. info. Day 2005 2005 2005 Actin 350 Tumour 1 + +
+ - 351 Blood 1 - + + - 352 Brain 1 - - - - 353 Skin 1 - - - + 354
Lung 1 - - - + 355 Kidney 1 - - - ++ 356 Gut 1 - - - +++ 357 Spleen
1 +++ +++ - ++ 358 Heart 1 - - - + 359 Liver 1 - - - ++ 360 Tumour
7 +++ +++ +++ + 361 Blood 7 - ++ - - 367 Brain 7 - - - + 369 Skin 7
+ - ++ + 364 Lung 7 - - - + 366 Kidney 7 - - - + 368 Gut 7 - ++ -
+++ 362 Spleen 7 - - - ++ 365 Heart 7 + - - + 363 Liver 7 - ++ -
++
TABLE-US-00004 TABLE 2 RNA PCR Results Sample HSV NTR Actin ID No.
info. Day 30 Apr. 2005 06 Jun. 2005 26 May 2005 350 Tumour 1 ++ -
+++ 351 Blood 1 NI - +++ 352 Brain 1 - - +++ 353 Skin 1 - - +++ 354
Lung 1 - - +++ 355 Kidney 1 - - +++ 356 Gut 1 - - +++ 357 Spleen 1
- - +++ 358 Heart 1 ++ - +++ 359 Liver 1 - - +++ 360 Tumour 7 +++
++ +++ 361 Blood 7 NI NI +++ 367 Brain 7 - - +++ 369 Skin 7 - - +++
364 Lung 7 - - +++ 366 Kidney 7 - - +++ 368 Gut 7 - - +++ 362
Spleen 7 NI - +++ 365 Heart 7 - - +++ 363 Liver 7 NI - +++ 362
Spleen 7 - - +++ Key to Tables 1 and 2: +++ Strong band on gel ++
Medium band on gel + Weak band on gel - No band NI Non-informative
- contaminated with DNA (RNA only)
EXAMPLE 2
Evaluation of the Anti-Tumor Activity of a Selectively Replication
Competent Herpes Simplex Virus in Combination with Enzyme Prodrug
Therapy
[0216] HSV1790 is a second generation oncolytic virus generated by
inserting the bacterial enzyme nitroreductase (NTR) into the
oncolytic virus HSV 1716, under the control of the CMV IE promoter.
NTR converts the inactive prodrug CB 1954 into an active alkylating
agent which has an anti-tumor effect. The purpose of this study was
to determine the anti-tumor efficacy of the combination of HSV1790
and CB 1954 in vitro and in vivo, and to explore the efficacy of
this combination after systemic (intravenous) administration of
HSV1790.
[0217] Experimental Design:
[0218] In vitro, cells which are known to be non permissive for HSV
1790 replication were used in order to distinguish between an
oncolytic effect due to viral replication, and cell death due to
activated prodrug.
[0219] In vivo, intratumoural administration of HSV1790
(10.sup.5-10.sup.9 PFU) with, or without administration of CB 1954
(max 80 mg/kg) by the intraperitoneal route was performed on mouse
xenograft models of A2780, CP70 and A431 cell lines, and tumor
volume measured regularly. Tumor and organ distribution of HSV1790
following intravenous administration was determined by
immunohistochemistry and by analysis of DNA and RNA from harvested
tumor tissues and organs.
[0220] Administration of HSV 1790, followed by CB1954, enhanced
tumor cell killing in 3T6 cells compared to HSV1790 alone. In vivo,
the combination of intra-tumoral administration of HSV1790,
followed by intraperitoneal CB1954, enhanced tumor reduction and
improved survival compared to administration of virus alone.
Following systemic administration of HSV1790, viral replication is
detected in tumor tissue, but not in normal organs.
[0221] HSV1790, when used in combination with CB1954, can enhance
tumor cell killing in vitro and enhance tumor reduction and
survival in vivo without toxicity in normal tissues and organs.
[0222] Introduction
[0223] Cancer is a genetic disease, and the hallmarks of individual
cancer cells are mutations in genes related to growth control,
apoptosis, immortality and also functional aberrations that support
the ability of cancer cells to invade and metastasize (1). Genetic
therapies in cancer are designed to produce one of several types of
outcome. Firstly, the genetic material introduced into the host or
tumor may result directly in cancer cell death, for example by the
intratumoral administration of a replication competent virus.
Secondly, the genes introduced into the host or tumor cells are
expressed, and can induce an immune response directed against the
tumor (2). Thirdly, the gene product may be toxic to the tumor cell
or may activate a subsequently administered drug into a cytotoxic
agent that results in cancer cell death. This approach is
frequently described as `gene directed enzyme pro-drug therapy`
(GDEPT) or `suicide gene therapy` (3).
[0224] Herpes simplex virus type 1 (HSV-1) has a number of
pertinent characteristics that support its use in cancer therapy.
It infects a broad range of cell types, it is cytolytic by nature
(the life cycle of the virus results in host destruction), and it
has a well characterized and, in the case of Glasgow strain 17+, a
fully sequenced genome (4).
[0225] Furthermore, its large genome (152 kb) contains non
essential genes that can be replaced by therapeutic transgenes of
up to 30 kb (5). HSV1716 is a selectively replication competent
mutant of the HSV-1 in which both copies of the RL1 gene has been
deleted (6). The RL1 gene encodes the protein ICP34.5, which is a
specific determinant of virulence (7). ICP34.5 functions by
complexing with proliferating cell nuclear antigen (PCNA), which is
involved in DNA replication and repair (8). In most tumor cells,
PCNA levels are high, and ICP34.5 is not required for productive
HSV replication. In contrast, in normal, terminally differentiated
cells, PCNA levels are low and ICP34.5 is required to recruit any
available PCNA to initiate virus replication. Thus HSV1716
replicates in actively dividing but not terminally differentiated
cells (9), and has an antitumor effect in vitro in a range of tumor
cell types including gliomas (10).
[0226] HSV 1716 has demonstrated selective tumor cell killing, with
minimal toxicity, and its administration has resulted in improved
survival in a number of xenograft tumor models in mice, including
glioma (11), melanoma (12-14), mesothelioma (15), ovarian (161,
lung (17, 18) and breast (19) carcinomas. Clinical trials of
intra-lesional administration of HSV1716 in patients with glioma,
melanoma and squamous cell carcinoma of the head and neck have been
performed (20-23) and have demonstrated the safety of this
approach, and with evidence that the virus is capable of directly
destroying human tumor cells while leaving normal cells intact.
[0227] One potential limitation of the intra-lesional
administration of HSV1716 for the treatment of human tumors is that
there is heterogeneity of cell type and growth state within a
tumor, and consequently not all cells within a tumor will be
permissive for lytic replication by HSV1716. One strategy to
overcome this limitation is to combine the oncolytic effects of
HSV1716 with a gene-directed enzyme prodrug therapy approach. A
number of enzyme pro-drug systems have been proposed for cancer
gene therapy (24), including the E. Coli nitroreductase (NTR) with
the pro-drug CB1954 (25-27). CB1954
[5(aziridin-1-yl)-2,4-dinitrobenzamide] is a mono-functional
alkylating agent that is poorly metabolized in human cells and thus
has low toxicity. The enzyme, NTR, converts the inactive CB 1954
pro-drug into its active form, which is a functional cytotoxic
alkylating agent that introduces poorly repaired inter-strand
cross-links into DNA and these lesions kill cells regardless of
their cell cycle state (28). In addition, the active metabolite is
diffusible and membrane permeable--this results in an efficient
bystander effect (29,30). We have generated and characterized a
second generation virus (HSV1790) which contains the E. coli
nitroreductase gene inserted into the RL1 locus of the HSV1716
genome. As the virus should only replicate and produce NTR in tumor
cells, toxicity to normal cells should be avoided. In this
manuscript, we report the generation of this second generation
virus, and demonstrate that the combination of HSV 1790 and the
prodrug CB 1954 has enhanced tumor cell killing in vitro, and
results in improved tumor reduction and survival in vivo compared
to the oncolytic effect of HSV 1790 alone.
[0228] Another potential drawback in the application of genetic
therapies in cancer medicine is that administration of the genetic
therapeutic usually requires direct injection into the patient's
tumor. Consequently many of the clinical trials of these therapies
have been restricted to patients with localized tumors that are
accessible by direct injection or by injection under radiological
guidance or post operatively. However, patients with advanced
cancer invariably have metastatic disease or tumors that are
inaccessible for direct injection. In this manuscript we report
safety and efficacy data after systemic (intravenous)
administration of HSV1790 in athymic mice bearing human tumor
xenografts. We demonstrate that the virus selectively locates to
tumor tissues, replicates and produces NTR within tumors without
affecting other organs. Furthermore, tumour reduction, and enhanced
survival, is observed in vivo in tumor bearing mice following
pro-drug administration. These studies indicate that systemic
administration of HSV1790 and CB1954 should be explored further in
human clinical trials.
[0229] Materials and Methods
[0230] Construction of HSV-1 Recombinant Virus Expressing ntr:
[0231] The plasmid pPS949, containing the ntr gene downstream of
the CMV IE promoter (pCMV-NTR) in a pLNCX (Clonetech) backbone, was
a kind donation from Professor Lawrence Young (University of
Birmingham). The pCMV-NTR fragment was excised from pPS949 and
cloned into BglII digested, CIP treated RLI-.dIRES-GFP. Clones were
screened for the pCMV-NTR insert using BglIIXhol restriction enzyme
analysis and one clone was found to contain the insert in the
correct orientation (data not shown). The plasmid RLI.dCMV-NTR-GFP
was digested with ScaI which cuts in a region outwith the flanking
sequences and the PCMV-NTR-IRES-GFP-PolyA fragment. BW cells were
co-transfected with the linearised plasmid and HSV17.sup.+ DNA.
Recombinant virus was identified using GFP fluorescence and several
green plaques were plaque purified as described in (31). To ensure
that recombination had taken place in the correct location and that
the endogenous copy of the RL1 gene had been replaced by the
pCMV-NTR-IRES-GFP-Poly A cassette, DNA from HSV1790 was purified,
digested with BamHI and analysed by Southern Blot (data not shown).
FIG. 32 shows a schematic representation of HSV1716/CMV/NTR
(designated HSV 1790 and referred to as such hereafter) genome and
relevant fragment sizes expected from BamHI digestion of HSV 1790
DNA.
[0232] Cell Cultures:
[0233] BHK (baby hamster kidney 21 clone 13) cells and mouse embryo
3T6 cells were obtained from the European Collection of Cell
Culture (ECACC). BHK cells were grown in Eagle's medium
supplemented with 10% newborn calf serum and 10% (v/v) tryptose
phosphate broth. This will be referred to subsequently as ETC 10.
For virus titrations and plaque purification EMC 10 (Eagle's medium
containing 1.5% methylcellulose and 10% newborn calf serum) was
used to overlay the cells. The ovarian cell lines A2780 and CP70
(obtained from ECACC) were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum. Cell cultures were
incubated in a humidified atmosphere of 5% CO.sub.2/95% O.sub.2 at
37.degree. C. The squamous cell carcinoma cell line A431, cervical
carcinoma C33a and the glioma line U373MG were all obtained from
the American Tissue Culture Collection (--ATCC) and were cultured
in DMEM containing 10% fetal calf serum and incubated in a
humidified atmosphere of 10% CO.sub.2/90% O.sub.2 at 37.degree. C.
All cultures were tested for Mycoplasma using VenorGeM.RTM.
Mycoplasma PCR Detection Kit (Cambio, Cambridge, UK). Titration of
virus stocks was carried out as previously described in (32).
[0234] Western Blotting:
[0235] Western blotting was carried out using standard techniques.
For NTR detection a rabbit polyclonal anti-NTR antibody was kindly
provided by Professor Lawrence Young (University of Birmingham, UK)
and used at a concentration of 1/1000. The secondary antibody, goat
anti-rabbit IgG peroxidase conjugate (Sigma) was also used at
1/1000.
[0236] In Vivo Tumor Reduction and Biodistribution Studies.
[0237] Female 6-8 week old athymic nude mice (Charles River Labs)
were maintained under specific pathogen free conditions and in
isolation after virus injection.
[0238] Actively growing A2780, CP70 and A431 cells were harvested
and resuspended in phosphate buffered saline (PBS). Cells
(1.times.10.sup.7 for A2780 and A431, 5.times.10.sup.6 CP70 per
mouse) were injected into the flanks of athymic mice. The mice were
examined regularly for tumor growth. When the mean tumor diameters
were approximately 5 mm, the animals were randomized into groups.
HSV 1790 or HSV1716 or PBS (maximum volume 100 .mu.l) was
administered by direct intratumoral injection. Virus was diluted in
PBS+10% fetal calf serum and kept at -70.degree. C. until use.
CB1954, resuspended in archais oil with 10% acetone was then
administered by intraperitoneal injection (maximum volume 100
.mu.l, maximum dose 3.times.80 mg/kg) at least 48 hrs after viral
injection. For general examination of toxicity, animals were
weighed regularly and tumor volumes were calculated from caliper
measurements (volume=d.sup.3.times..pi./6). For statistical
analysis, unpaired Student's t-tests were used. P values of
<0.05 were considered significant. All animal experimentation
was performed according to United Kingdom Home Office regulations
and UKCCR guidelines adhered to at all times.
[0239] Immunohistochemistry:
[0240] Organs and xenograft tumor samples were removed immediately
from individual mice after sacrifice and fixed in neutral buffered
formalin (NBF) for at least 24 hrs before embedding in paraffin
using standard procedures, Sections were prepared for
immunohistochemistry by standard protocols. Briefly, paraffin
embedded sections were dewaxed, dehydrated and endogenous
peroxidase was quenched. Non-specific binding was reduced with 10%
normal goat serum before sections were incubated with primary
antibody overnight at 4.degree. C. (HSV-1 polyclonal, Dako
1:1000).
[0241] After incubation with biotinylated secondary antibody
(Vector anti-rabbit Elite kit 1:500) and avidin biotin complex
(ABC) solution (Elite Kit, Vector Laboratories) color was developed
using diaminobenzidine (DAB) (Vector Labs) as the chromogen. The
slides were counterstained, dehydrated, mounted and visualized
using a light microscope. HSV-1 infected liver sections were used
as a positive control, omission of primary antibody and tumor
sections from mice uninfected with virus constituted the negative
controls.
[0242] Extraction of RNA and DNA from Tissues--(RNA):
[0243] Tumor tissue and mouse internal organs were collected from
individual mice at time of sacrifice and frozen at -70.degree. C. A
small (<0.5 g) tissue sample was resuspended in buffer and the
cells were disrupted with a Retsch MM200 homogenizer. RNA was
extracted using the Promega (SV) RNA extraction kit following the
manufacturers recommended procedure (Promega, Southampton, UK).
[0244] DNA:
[0245] Tissue (<0.5g) samples were homogenized using the Retsch
MM200 homogenizer and DNA extracted using the Nucleon ST DNA
extraction Kit (Thistle Scientific Ltd, Glasgow, UK) using the
manufacturers instructions.
[0246] Reverse Transcriptase Reactions:
[0247] Reverse transcription was preformed using ImProm Reverse
transcriptase kit (Promega, Southampton, UK) using the random
hexanucleotide primers under manufacturers recommended
conditions.
[0248] PCR:
[0249] PCRs were carried out with Ready-Mix (Abgene, Surrey, UK)
and 1 .mu.l of RT reaction mix in a 20 .mu.l reaction volume. For
HSV PCR the primers HS 13 (ACG ACG ACG TCC GAC GGC GA) [SEQ ID
No.7] and HS 14 (GTG CTG GTG CTG GAG GAC AC) (34) [SEQ ID No.8]
were used. These anneal to HSV-1 sequence co-ordinates 93536-93555
and 93813-93794 (complementary) which lie within the UL42 region of
the genome. This region codes for a sub-unit of the viral DNA
polymerase--the DNA polymerase accessory protein. The resulting PCR
product is 278 base-pairs in length and was visualized by agarose
gel electrophoresis. The reaction conditions used are a 94.degree.
C. `Hot-Start` for 2 minutes followed by 34 cycles of {94.degree.
C. for 15 seconds (denaturation); 72.degree. C. for 1 min
(annealing); 72.degree. C. for 1 minute (extension)} and a final
extension step at 72.degree. C. for 2 minutes. For NTR: The PCR
used the primers sequence of the nitroreductase (NTR) enzyme from
E. coli B genomic DNA. Upstream primer 5-TTTCACATTGAGTCATTATGG-3
[SEQ ID No.9] and downstream primer 5-TTACACTTCGGTTAAGGTGATG-3 [SEQ
ID No.10] were used (35). Following initial denaturation at
94.degree. C. for two minutes, PCR conditions were 95.degree. C.
for 30 s, 55.degree. C. for 30 s and 72.degree. C. for 60 s, for 32
cycles.
[0250] Results
[0251] Expression of NTR in Cell Lines Infected with HSV1790.
[0252] To demonstrate that HSV 1790 expresses the NTR protein a
Western blot was performed using a polyclonal NTR antibody.
Although the recombinant virus strongly expressed GFP (observed
during the plaque purification process) indicating that it should
express NTR, this was not conclusive proof. Four cell lines--BHK,
C8161, VM and 3T6 were infected with HSV1790 and protein expression
analysed by Western blotting, using an NTR-specific antibody. FIG.
33 shows that the 24 KDa NTR protein was expressed in all the cell
lines infected with HSV1790. Even though the virus does not
replicate efficiently in confluent 3T6 cells, reasonably strong
expression of NTR was detected demonstrating that productive
replication of the virus is not necessary for NTR expression in
infected cells.
[0253] Enhanced Cell Kill In Vitro in HSV1790 Infected Cells
Treated with CB1954.
[0254] Previous experiments had shown the replication kinetics of
the virus to be identical to that of the parental strain HSV 1716
indicating no alteration in replication potential due to the
insertion of the ntr gene (Paul Dunn, PhD Thesis, University of
Glasgow, 2003). To determine whether NTR expression would result in
enhanced cell killing after HSV 1790 infection and addition of the
prodrug CB1954, cytotoxicity assays were performed in 3T6 cells, a
cell line in which the HSV did not replicate efficiently. As almost
no HSV1790 replication occurs in 3T6 cells infected at a low MOI
(>0.1), any significant cell death observed in the cells
infected with HSV1790 after CB1954 administration would be due to
NTR expression in the cells, and the subsequent activation of
CB1954. The addition of 50 pM CB1954 alone had previously been
shown to cause less than 5% cell death--(data not shown). The
effects of HSV1790 infection, with or without 50 pM CB1954, were
examined and the results shown in FIGS. 34 and 35. Five days after
treatment, 75% of the cells treated with 10 plaque forming units
(pfu)/cell HSV1790+CB1954 were dead compared to only 2% in those
treated with HSV1790 only (FIGS. 34 and 35). It can therefore be
concluded that almost 70% of the cell death observed in the HSV1790
infected cells was due to NTR converting CB1954 to its toxic form
and not from cell lysis from viral replication.
[0255] HSV1790 Toxicity and Efficacy In Vivo.
[0256] No formal toxicity studies were performed. However in
preliminary experiments, the dose of HSV1790, administered by a
single intratumoral injection, was escalated in groups of mice
bearing A2780 human tumour xenografts (n=3) to determine the
acceptable dose for subsequent experiments, based on toxicity. A
dose of 1.times.10.sup.9 pfu HSV1790 by intratumoural injection was
not tolerated well; the mice lost more than 10% of their body
weight and had to be sacrificed. At doses of 1.times.10.sup.8 and
below, the treatment was well tolerated and the mice did not show
any signs of ill health or adverse effects.
[0257] To determine the efficacy of HSV1790+CB1954 in treating
established subcutaneous human tumour xenografts, serial tumour
volume measurement were taken regularly after administration of
HSV1790 alone, HSV1790+CB1954 or no treatment. Administration of
CB1954 alone was not performed as previous experiments had shown
that CB1954 administration as a single agent has no anti-tumoral
effect (data not shown). Two doses of virus were administered as
giving multiple intratumoral injections appears to be more
effective than the administration of the same total dose on one
occasion (36). CB1954 treatment was commenced 48 hrs after the last
virus injection (max 80 mg/kg on 3 occasions). Previous experiments
in vitro (data not shown) and (36) suggested that administering the
prodrug too soon after viral administration kills the cells in
which the virus is replicating, effectively thereby reducing the
number of infectious virus particles within the tumor.
Administration of HSV1790 and CB1954 had no effect on body weight
(data not shown) and there were no signs of toxicity in the mice.
In mice bearing A2780 (FIG. 36A) and A431 (FIG. 36C) xenografts,
there was a marked reduction in tumor volume when the xenografts
are treated with HSV1790+CB1954 compared to HSV 1790 alone. For
mice bearing A431 tumours, administration of HSV1790+CB1954
resulted in significantly smaller tumour volumes (P=0.03) and
significantly longer median survival (P<0.05) (FIGS. 36C and F)
than in the mice treated with HSV1790 alone. In mice bearing CP70
tumor xenografts, administration of HSV1790 reduced tumor volume
compared to control mice (FIG. 36B) but the addition of prodrug had
no further anti-tumor effect.
[0258] Tumor and Organ Distribution of HSV1790 After Systemic
(Intravenous) Administration.
[0259] To explore the tumor and organ distribution of HSV1790 after
systemic (intravenous) administration, 1.times.10.sup.7 pfu of
HSV1790 was administered by tail vein injection to athymic nude
mice bearing established UVW and A431 tumour xenografts overlying
their right hind flank. Mice were sacrificed either on day 1 or day
7 post injection and the tumors, blood and major organs (brain,
heart, lung, liver, spleen, intestine, kidney and skin) were
collected. Tissue from these organs was analysed by PCR and
immunohistochemistry for the presence of HSV1790.
Immunohistochemical staining with an anti-HSV antibody revealed
active replication of the virus within the xenograft tumours (FIG.
37A-F) Necrosis was also widespread in the areas of positive
staining (FIG. 37F). There was no indication of positive staining
in any other organ (FIG. 37G-J) with the exception of skin; in
which some positive staining was visible in cells below the dermal
and epidermal layer (FIG. 37K).
[0260] DNA and RNA, extracted from the organs and tumours were also
analysed by PCR, to determine presence of virus and the replication
of the virus (FIG. 38). Both viral DNA and RNA are detected in the
tumour tissue at day 1 post i.v injection at a low level. By day 7
post i.v injection the PCR band intensity has increased,
demonstrating that HSV is replicating within the tumor tissue.
Furthermore ntr DNA and RNA is detectable in the tumor
demonstrating that HSV1790 is replicating within the tumor and is
also producing the ntr protein. A high level of viral DNA is seen
in the spleen at day 1. However there is no replicating virus
detectable by RT-PCR. This suggests that the virus is not actively
replicating and the positive PCR result is likely to be due to
spleenic clearance of the virus from the circulating blood.
[0261] Tumor Growth Inhibition in Athymic Nude Mice After
Administration of HSV1790 by Intravenous Injection.
[0262] To determine whether HSV1790 replication within tumors seen
after intravenous injection has any anti-tumor effects, mice with
A431 xenografts were randomly allocated into groups: [0263] (a)
2.times.(1.times.10.sup.6) PFU of HSV1790 injected i.v
(intravenous) at Days 1 and 3 followed by 20 mg/kg injection CB1954
i.p (intraperitoneal) daily for 5 days; [0264] (b)
2.times.(1.times.10.sup.6) PFU of HSV1790 injected i.v at Days 1
and 3; [0265] (c) No virus injections (injection of PBS only).
[0266] FIG. 39 show that HSV1790 either alone or in combination
with CB1954 results in significantly prolonged survival of tumor
bearing mice.
[0267] Discussion
[0268] We have previously described the safety and potential
efficacy of herpes simplex virus therapy using the selectively
replication competent mutant HSV1716 both in vivo (11-19) and in
clinical studies (20-23). However, it is anticipated that HSV1716
although able to infect all cells will not lytically replicate in
all cells within tumors due, predominantly, to the heterogeneity of
the cell state within a tumor mass. To overcome this, we have
generated HSV1790, a second generation herpes simplex virus derived
from the ICP34.5 null mutant HSV1716 and in which the E. Coli ntr
gene has been inserted under the control of the CMV IE
promoter.
[0269] Western blot analyses demonstrated that the ntr protein was
expressed in all four cell lines tested following infection with
HSV1790. Furthermore, the addition of the prodrug CB1954 to 3T6
cells infected with HSV1790 resulted in a significantly enhanced
cell kill compared to infection with HSV1790 alone. Administration
of the prodrug CB1954 to athymic mice, bearing tumor xenografts of
either A431 or A2780 cells, 48 hours after intratumoral
administration of HSV1790 resulted in a marked reduction of tumor
volumes, and also resulted in significantly improved survival for
mice bearing A431 tumors, compared to administration of HSV1790
without administration of CB1954. In contrast, administration of
CB1954 following administration of HSV1790 to mice bearing CP70
tumor xenografts had no additional anti-tumor effect compared to
administration of HSV1790 alone. CP70 is a derivative of the
ovarian carcinoma cell line A2780, and has a drug-resistant
phenotype due to the loss of MLH1, a key protein involved in
mismatch repair (37, 38). These cells display resistance to a
variety of DNA damaging agents and alkylating agents. CP70 has a
higher tolerance of CB1954 than the parental A2780, with IC.sub.50
values in vitro of 29 .mu.M and 60 .mu.M respectively (39).
[0270] Expression of NTR in these cells results in similar-fold
sensitisation to the prodrug, with the value for A2780 remaining
approximately half of that for CP70 cells (39). As the active form
of CB 1954 is a bi-functional alkylating agent, it is possible that
the intratumoral activation of CB 1954 to its active form by NTR
expression, following HSV1790 administration, is insufficient to
overcome the drug resistant phenotype of these CP70 cells.
[0271] Expression of a prodrug activating enzyme early in the virus
replication cycle risks killing the virus (40, 41). In addition,
the insertion of a transgene could theoretically worsen virus
efficacy in vivo. However, neither of these potential drawbacks is
likely with HSV1790 based on the results reported in this
manuscript. Another potential drawback of combining an oncolytic
virus with a `suicide-gene therapy` approach is that if the virus
alone can induce efficient cytopathic effects, then the transgene
may not be expressed in target cells prior to the cell death, and
there may be no additional therapeutic benefit to the combined
approach. Again, the data presented in this manuscript suggests
that the second generation virus HSV1790, when combined with the
addition of the prodrug, has an enhanced anti-tumor efficacy
compared to HSV1716.
[0272] Most human clinical trials of genetic therapies for cancer,
including those using oncolytic viruses, continue to use direct
intra-tumoral injection as the route of administration of the
therapeutic virus. However, as most patients have metastatic
disease, the preferred route of delivery of oncolytic viruses is
through intravenous administration. Intravenous administration of
viruses can cause significant systemic side effects, due to the
acute release of cytokines. These symptoms can be effectively
minimised by appropriate pre-radiation (42-44) and intravenous
administration of Newcastle Disease Virus (42, 43) and Onyx 015
(44, 45) have been well tolerated in initial clinical studies with
minimal toxicity.
[0273] Our results demonstrate replication of HSV1790 and
expression of the ntr transgene within tumor tissue following
intravenous administration, but with no evidence of viral
replication in normal organ tissues. However, as athymic nude mice
have a compromised immune system, the effect of a normal immune
system on viral toxicity and efficacy is unknown. Viral infection
of tumors can attribute an anti-tumor immune response which is
beneficial (13, 46, and 47). In contrast, the host's immune system
has been shown to neutralise virus and inhibit oncolytic activity
(48). However prior immunity to HSV does not appear to
significantly impair the therapeutic efficacy of herpes simplex
therapy in immunocompetent models (49, 50). Further studies of
systemic administration HSV 1790 in immunocompetent models are
required.
[0274] In conclusion, we have demonstrated that the combination of
the oncolytic herpes simplex virus HSV1716 with a `suicide gene
therapy` approach can enhance anti-tumor efficacy in comparison
with the effects of the oncolytic virus alone. Intravenous
administration of HSV1790 results in expression of the ntr
transgene in tumor tissues, but not in normal organs, with
efficient anti-tumor efficacy following administration of prodrug.
As the prototype oncolytic virus HSV1716 has been shown to be
totally non-toxic in human patients and has recently entered a
Phase III trial, the results presented in this paper lead us to the
conclusion that clinical studies of HSV1790 and CB1954 are
warranted in patients with otherwise refractory tumors.
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Sequence CWU 1
1
10120DNAArtificial SequenceSynthetic 1acgacgacgt ccgacggcga
20220DNAArtificial SequenceSynthetic 2gtgctggtgc tggacgacac
20322DNAArtificial SequenceSynthetic 3ctttcacatt gagtcattat gg
22422DNAArtificial SequenceSynthetic 4ttacacttcg gttaaggtga tg
22519DNAArtificial SequenceSynthetic 5cgtgaaaaga tgacccaga
19619DNAArtificial SequenceSynthetic 6agcatagccc tcgtagatg
19720DNAArtificial SequenceSynthetic 7acgacgacgt ccgacggcga
20820DNAArtificial SequenceSynthetic 8gtgctggtgc tggaggacac
20921DNAArtificial SequenceSynthetic 9tttcacattg agtcattatg g
211022DNAArtificial SequenceSynthetic 10ttacacttcg gttaaggtga tg
22
* * * * *
References