U.S. patent application number 14/700036 was filed with the patent office on 2015-08-20 for herpes virus strains.
The applicant listed for this patent is BioVex Limited. Invention is credited to Robert S. COFFIN.
Application Number | 20150232812 14/700036 |
Document ID | / |
Family ID | 27447756 |
Filed Date | 2015-08-20 |
United States Patent
Application |
20150232812 |
Kind Code |
A1 |
COFFIN; Robert S. |
August 20, 2015 |
HERPES VIRUS STRAINS
Abstract
The present invention provides a herpes virus with improved
oncolytic properties which comprises a gene encoding an
immunomodulatory cytokine and which lacks a functional ICP34.5 gene
and a functional ICP47 encoding gene.
Inventors: |
COFFIN; Robert S.; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioVex Limited |
Abingdon |
|
GB |
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|
Family ID: |
27447756 |
Appl. No.: |
14/700036 |
Filed: |
April 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14174521 |
Feb 6, 2014 |
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14700036 |
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13600711 |
Aug 31, 2012 |
8680068 |
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14174521 |
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11738807 |
Apr 23, 2007 |
8277818 |
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13600711 |
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10181697 |
Oct 2, 2002 |
7223593 |
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PCT/GB01/00225 |
Jan 22, 2001 |
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11738807 |
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Current U.S.
Class: |
435/235.1 |
Current CPC
Class: |
C12N 2710/16632
20130101; A61P 31/00 20180101; C12N 2710/16643 20130101; A61K
2039/55522 20130101; A61P 43/00 20180101; C12N 15/869 20130101;
A61K 48/00 20130101; A61P 25/02 20180101; C12N 7/00 20130101; C12N
2710/16033 20130101; A61P 35/00 20180101; C12N 15/86 20130101; A61P
25/00 20180101; C12N 2710/16021 20130101; A61P 37/04 20180101; A61P
25/28 20180101; A61K 35/763 20130101; A61K 2039/55516 20130101;
A61P 35/04 20180101 |
International
Class: |
C12N 7/00 20060101
C12N007/00; A61K 48/00 20060101 A61K048/00; A61K 35/763 20060101
A61K035/763 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 21, 2000 |
GB |
0001475.3 |
Feb 8, 2000 |
GB |
0002854.8 |
Jan 5, 2001 |
GB |
0100288.0 |
Jan 6, 2001 |
GB |
0100430.8 |
Claims
1-43. (canceled)
44. A herpes simplex virus 1 (HSV1) which: (i) lacks a functional
ICP34.5 encoding gene; (ii) lacks a functional ICP47 encoding gene;
and (iii) comprises a gene encoding an immunostimulatory protein,
wherein said gene is under the control of a promoter, and wherein
said promoter is a ubiquitous promoter or a viral promoter.
45. The HSV1 of claim 44, wherein said promoter is a viral
promoter.
46. The viral promoter of claim 45, wherein said viral promoter is
a cytomegalovirus (CMV) promoter.
47. The CMV promoter of claim 46, wherein said CMV promoter is a
CMV immediate early (IE) promoter.
48. The HSV1 of claim 44, wherein said immunostimulatory protein is
a cytokine.
49. The HSV1 of claim 45, wherein said immunostimulatory protein is
a cytokine.
50. The HSV1 of claim 46, wherein said immunostimulatory protein is
a cytokine.
51. The HSV1 of claim 47, wherein said immunostimulatory protein is
a cytokine.
52. The HSV1 of claim 48, wherein said immunostimulatory protein is
human GM-CSF.
53. The HSV1 of claim 49, wherein said immunostimulatory protein is
human GM-CSF.
54. The HSV1 of claim 50, wherein said immunostimulatory protein is
human GM-CSF.
55. The HSV1 of claim 51, wherein said immunostimulatory protein is
human GM-CSF.
56. The HSV1 of claim 44, wherein said herpes simplex virus 1: (iv)
lacks a functional gene encoding ICP6, glycoprotein H or thymidine
kinase.
57. A herpes simplex virus 1 (HSV1) which: (i) lacks a functional
ICP34.5 encoding gene; (ii) lacks a functional ICP47 encoding gene;
and (iii) comprises a gene encoding an immunostimulatory protein,
wherein said gene encoding an immunostimulatory protein is inserted
into the former site of the ICP34.5 encoding gene.
58. The HSV1 of claim 57, wherein said immunostimulatory protein is
a cytokine.
59. The HSV1 of claim 58, wherein said immunostimulatory protein is
human GM-CSF.
60. The HSV1 of claim 57, wherein said herpes simplex virus 1: (iv)
lacks a functional gene encoding ICP6, glycoprotein H or thymidine
kinase.
61. A herpes simplex virus 1 (HSV1) which: (i) lacks a functional
ICP34.5 encoding gene; (ii) lacks a functional ICP47 encoding gene;
and (iii) comprises a gene encoding human GM-CSF, wherein said gene
is under the control of a cytomegalovirus (CMV) immediate early
(IE) promoter.
62. The HSV1 of claim 44, wherein said herpes simplex virus 1 is a
clinical isolate.
63. The HSV1 of claim 57, wherein said herpes simplex virus 1 is a
clinical isolate.
64. The HSV1 of claim 61, wherein said herpes simplex virus 1 is a
clinical isolate.
65. A method of treating cancer in a patient, wherein said method
comprises administering to said patient a therapeutically effective
amount of a herpes simplex virus 1 (HSV1), wherein said HSV1: (i)
lacks a functional ICP34.5 encoding gene; (ii) lacks a functional
ICP47 encoding gene; and (iii) comprises a gene encoding an
immunostimulatory protein, wherein said gene is under the control
of a promoter, and wherein said promoter is a ubiquitous promoter
or a viral promoter.
66. The method according to claim 65, wherein said HSV1 is
administered to a tumor via intratumoral injection.
67. The method according to claim 65, wherein said cancer is
selected from the group consisting of prostate cancer, breast
cancer, lung cancer, liver cancer, endometrial cancer, bladder
cancer, colon or cervical carcinoma, adenocarcinoma, melanoma,
lymphoma, glioma and sarcoma.
68. The method according to claim 66, wherein said cancer is
selected from the group consisting of prostate cancer, breast
cancer, lung cancer, liver cancer, endometrial cancer, bladder
cancer, colon or cervical carcinoma, adenocarcinoma, melanoma,
lymphoma, glioma and sarcoma.
69. The method according to claim 65, wherein said cancer is
melanoma.
70. The method according to claim 66, wherein said cancer is
melanoma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation application of U.S. patent
application Ser. No. 13/600,711, filed Aug. 31, 2012, which is a
continuation application of U.S. patent application Ser. No.
11/738,807, filed Apr. 23, 2007, now U.S. Pat. No. 8,277,818, which
is a continuation application of U.S. patent application Ser. No.
10/181,697, filed Oct. 2, 2002, now U.S. Pat. No. 7,223,593, which
is the U.S. National Phase of International Application
PCT/GB01/00225, filed Jan. 22, 2001, which claims priority to
United Kingdom Patent Application Numbers: 0001475.3, filed Jan.
21, 2000; 0002854.8, filed Feb. 8, 2000; 0100288.0, filed Jan. 5,
2001; and 0100430.8, filed Jan. 6, 2001, which are incorporated by
reference herein in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to herpes virus strains with
improved anti-tumour activity as compared to previously known
strains.
BACKGROUND TO THE INVENTION
[0003] Viruses have been shown to have utility in a variety of
applications in biotechnology and medicine on many occasions. Each
is due to the unique ability of viruses to enter cells at high
efficiency. This is followed in such applications by either virus
gene expression and replication and/or expression of an inserted
heterologous gene. Thus viruses can either deliver and express
genes in cells (either viral or other genes) which may be useful in
for example gene therapy or the development of vaccines, or they
may be useful in selectively killing cells by lytic replication or
the action of a delivered gene in for example cancer.
[0004] Herpes simplex virus (HSV) has been suggested to be of use
for the oncolytic treatment of cancer. Here the virus must however
be disabled such that it is no longer pathogenic, i.e. does not
replicate in and kill non-tumor cells, but such that it can still
enter and kill tumor cells. For the oncolytic treatment of cancer,
which may also include the delivery of gene(s) enhancing the
therapeutic effect, a number of mutations to HSV have been
identified which still allow the virus to replicate in culture or
in actively dividing cells in vivo (e.g. in tumors), but which
prevent significant replication in normal tissue. Such mutations
include disruption of the genes encoding ICP34.5, ICP6, and
thymidine kinase. Of these, viruses with mutations to ICP34.5, or
ICP34.5 together with mutation of e.g. ICP6 have so far shown the
most favourable safety profile. Viruses deleted for only ICP34.5
have been shown to replicate in many tumor cell types in vitro and
to selectively replicate in artificially induced brain tumors in
mice while sparing surrounding tissue. Early stage clinical trials
have also shown their safety in man.
[0005] However, while promise has been shown for various viruses
including HSV for the oncolytic treatment of cancer, the majority
of this work has used virus strains which do not carry a
heterologous gene which may enhance the anti-tumor effect. We
propose that the combined use of HSV with an inactivating mutation
in the gene encoding ICP34.5 together with the delivery of the gene
encoding an immunomodulatory protein such as granulocyte macrophage
colony stimulating factor (GM-CSF) encoded in the disabled virus
genome may have optimal immune stimulating properties against the
tumor to be treated, particularly if functions in the virus which
usually reduce immune responses to HSV infected cells have also
been inactivated. For example the HSV ICP47 protein specifically
inhibits antigen presentation in HSV infected cells (Hill et al
1995), and the product of the UL43 gene and the vhs protein reduce
the immune-stimulating abilities of dendritic cells infected with
HSV. ICP47 and/or dendritic cell-inactivating genes might therefore
usefully be deleted from an oncolytic HSV mutant virus used for the
treatment of cancer, particularly if immune effects are to be
enhanced through the use of GM-CSF or other immunostimulatory
cytokine or chemokine. GM-CSF has recently been shown to give an
enhanced anti-tumor immune effect if expressed from within a tumor
cell rather than administered systemically (Shi et al 1999). Thus
in such use an oncolytic HSV mutant would be inoculated into a
primary or secondary tumor where replication and oncolytic
destruction of the tumor would occur Immune responses would also be
stimulated against the HSV infected cells, and also to tumor cells
elsewhere which had spread from the primary tumor site.
SUMMARY OF THE INVENTION
[0006] The present invention provides viruses with improved
capabilities for the lytic destruction of tumor cells in vivo. Here
herpes simplex virus strains are constructed using a strain of HSV1
or HSV2 in which the genes encoding ICP34.5 and ICP47 have been
inactivated such that a functional ICP34.5 or ICP47 protein cannot
be expressed and which also carries a gene encoding an
immunomodulatory protein. The virus may also be mutated for any
additional gene(s) which may be involved in inhibiting the function
of dendritic cells including the UL43 gene and/or the gene encoding
vhs. The present invention therefore provides viruses capable of
the oncolytic destruction of tumor cells and in which anti-tumor
immune effects will have been maximised.
[0007] Accordingly the invention provides: [0008] a herpes virus
which comprises a gene encoding an immunomodulatory protein, which
lacks a functional ICP34.5 encoding gene and a functional ICP47
encoding gene and which is a replication competent in tumour cells;
[0009] a herpes virus of the invention for use in a method of
treatment of the human or animal body by therapy; [0010] use of a
virus of the invention in the manufacture of a medicament for the
treatment of cancer; [0011] a pharmaceutical composition comprising
as active ingredient a virus according to the invention and a
pharmaceutically acceptable carrier or diluent; and [0012] HSVI
strain JSI as deposited at the European Collection of Cell Cultures
(ECACC) under provisional accession number 01010209, or an HSV1
strain derived therefrom.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Viruses
[0014] From top to bottom, diagrams show: laboratory HSV1 strain
17+, clinical HSV1 strain JS1, strain 17+/ICP34.5-, strain
JS1/ICP34.5-, strain JS1/ICP34.5-/ICP47-/hGMCSF, strain
JS1/ICP34.5-/ICP47-/mGMCSF.
DETAILED DESCRIPTION OF THE INVENTION
A. Viruses
[0015] A herpes virus of the invention is capable of efficiently
infecting target tumor cells and the genes encoding ICP34.5 and
ICP47 are inactivated in the virus. Mutation of ICP34.5 allows
selective oncolytic activity. Such mutations are described in Chou
et al 1990 and Maclean et al 1991, although any mutation in which
ICP34.5 is non-functional may be used. The genes encoding ICP6
and/or thymidine kinase may additionally be inactivated, as may
other genes if such inactivation does significantly reduce the
oncolytic effect, or if such deletion enhances oncolytic or other
desirable properties of the virus. ICP47 usually functions to block
antigen presentation in HSV-infected cells so its disruption leads
to a virus that does not confer on infected tumour cells properties
that might protect them from the host's immune system when infected
with HSV. Viruses of the invention additionally encode an
immunomodulatory protein, preferably GM-CSF, but may also encode
other cytokines, chemokines such as RANTES, or other
immune-modulatory proteins such as B7.1, B7.2 or CD40L. Genes
encoding immunomodulatory proteins may be included individually or
in combination.
[0016] Viral regions altered for the purposes described above may
be either eliminated (completely or partly), or made
non-functional, or substituted by other sequences, in particular by
a gene for an immunomodulatory protein such as GM-CSF.
[0017] The virus of the invention may be derived from a HSV1 or
HSV2 strain, or from a derivative thereof, preferably HSV1.
Derivatives include inter-type recombinants containing DNA from
HSV1 and HSV2 strains. Such inter-type recombinants are described
in the art, for example in Thompson et al, 1988 and Meignier et al,
1988. Derivatives preferably have at least 70% sequence homology to
either the HSV1 or HSV2 genomes, more preferably at least 80%, even
more preferably at least 90 or 95%. More preferably, a derivative
has at least 70% sequence identity to either the HSV1 or HSV2
genome, more preferably at least 80% identity, even more preferably
at least 90%, 95% or 98% identity.
[0018] For example the UWGCG Package provides the BESTFIT program
which can be used to calculate homology (for example used on its
default settings) (Devereux et al. (1984) Nucleic Acids Research
12, p387-395). The PILEUP and BLAST algorithms can be used to
calculate homology or line up sequences (typically on their default
settings), for example as described in Altschul (1993) J. Mol.
Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.
215:403-10.
[0019] Software for performing BLAST analyses is publicly available
through the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighbourhood word score threshold (Altschul et al., 1990).
These initial neighbourhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program uses as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10, M=5, N=4, and a comparison of both strands.
[0020] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0021] A derivative may have the sequence of a HSV1 or HSV2 genome
modified by nucleotide substitutions, for example from 1, 2 or 3 to
10, 25, 50 or 100 substitutions. The HSV1 or HSV2 genome may
alternatively or additionally be modified by one or more insertions
and/or deletions and/or by an extension at either or both ends.
[0022] Virus strains of the invention may be "non-laboratory"
strains. These can also be referred to as "clinical" strains. A
person of skill in the art will readily be able to distinguish
between a laboratory strain and a non-laboratory, or clinical,
strain. Further guidance on the properties likely to be exhibited
by virus strains is given below.
[0023] The key distinction between a laboratory and non-laboratory
strain is that laboratory strains currently in common use have been
maintained for long periods, many years in some cases, in culture.
The culture of viruses such as HSV involves a technique known as
serial passage. To grow and maintain viruses, suitable cells are
infected with the virus, the virus replicates within the cell and
the virus is then harvested; fresh cells are then re-infected, this
process constitutes one cycle of serial passage. Each such cycle
may take, for example, a few days in the case of HSV. As discussed
above, such serial passaging may lead to changes in the properties
of the virus strain, in that selection takes places for properties
that favour growth in culture (e.g. rapid replication), as opposed
to properties useful for practical applications, e.g. maintenance
of the capacity to travel along axons in the case of HSV or to
infect human cells.
[0024] Virus strains of the invention are may be non-laboratory
strains in that they are derived from strains recently isolated
from infected individuals. Strains of the invention are modified
compared to the original clinical isolates, and may have spent a
time in culture, but any time spent in culture will be
comparatively short. Strains of the invention are prepared in such
a manner as to retain substantially the desirable properties of the
original clinical isolates from which they are derived.
[0025] A virus strain of the invention is derived from a parental
virus strain if the parental virus strain is mutated to produce the
virus. For example, a virus of the invention may be derived from
the clinical isolate JSI. The parental strain of such a JSI-derived
virus may be JSI or another HSV1 strain derived from JSI. Thus a
virus of the invention may be a JSI virus comprising a gene
encoding an immunomodulatory protein and which lacks a functional
ICP34.5 encoding gene and a functional ICP47 encoding gene. In
addition, such a virus may contain any other mutation as mentioned
herein.
[0026] A virus of the invention is capable of efficiently infecting
target human cancer cells. When such a virus is a non-laboratory or
clinical strain it will have been recently isolated from an HSV
infected individual and then screened for the desired ability of
enhanced replication, infection or killing of tumour and/or other
cells in vitro and/or in vivo in comparison to standard laboratory
strains. Such viruses of the invention with improved properties as
compared to laboratory virus strains are then engineered such that
they lack functional ICP34.5 and ICP47 genes and encode a gene(s)
for an immunomodulatory protein(s) such as GM-CSF under the control
of a suitable promoter(s). Other genes encoding proteins which
interfere with the function of dendritic cells such as UL43 and/or
vhs may also be inactivated.
[0027] Preferably, a non-laboratory virus strain of the invention
has undergone three years or less in culture since isolation of its
unmodified clinical precursor strain from its host. More
preferably, the strain has undergone one year or less in culture,
for example nine months or less, six months or less, three months
or less, or two months or less, one month or less, two weeks or
less, or one week or less. By these definitions of time in culture,
is meant time actually spent in culture. Thus, for example, it is a
common practice to freeze virus strains in order to preserve them.
Evidently, preserving by freezing or in an equivalent manner does
not qualify as maintaining the strain in culture. Thus, time spent
frozen or otherwise preserved is not included in the above
definitions of time spent in culture. Time spent in culture is
typically time actually spent undergoing serial passage, i.e. time
during which selection for undesirable characteristics can
occur.
[0028] Preferably, a non-laboratory virus strain has undergone
1,000 or less cycles or serial passage since isolation of its
unmodified clinical precursor strain from its host. More
preferably, it has undergone 500 or less, 100 or less, 90 or less,
80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or
less, 20 or less, or 10 or less such cycles.
[0029] Preferably, a non-laboratory virus has a greater ability, as
measured by standard statistical tests, than a reference laboratory
strain with the equivalent modifications to perform certain
functions useful in the application at hand. In the case of an
oncolytic virus for tumour treatment, a non-laboratory virus strain
of the invention will preferably have a greater ability than a
reference laboratory strain with equivalent modifications to infect
or replicate in tumour cells, to kill tumour cells or to spread
between cells in tissue. More preferably, such greater ability is a
statistically significantly greater ability. For example, according
to the invention, a non-laboratory strain of the invention may have
up to 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20
fold, 50 fold, or 100 fold the capacity of the reference strain in
respect of the property being tested.
[0030] Statistical analysis of the properties described herein may
be carried out by standard tests, for example, t-tests, ANOVA, or
Chi squared tests. Typically, statistical significance will be
measured to a level of p=0.05 (5%), more preferably p=0.01p,
p=0.001, p=0.0001, p=0.000001.
[0031] Viruses of the invention infect and replicate in tumour
cells, subsequently killing the tumour cells. Thus, such viruses
are replication competent. Preferably, they are selectively
replication competent in tumour cells. This means that either they
replicate in tumour cells and not in non-tumour cells, or that they
replicate more effectively in tumour cells than in non-tumour
cells. Cells in which the virus is able to replicate are permissive
cells. Measurement of selective replication competence can be
carried out by the tests described herein for measurement of
replication and tumour cell-killing capacity, and also analysed by
the statistical techniques mentioned herein if desired.
[0032] A virus of the invention preferably has a greater ability
than an unmodified parent strain to infect or replicate in a tumour
cell, to kill tumour cells or to spread between cells in tissues.
Preferably this ability is a statistically significant greater
ability. For example, a virus according to the invention may have
up to 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20
fold, 50 fold or 100 fold the capacity of the unmodified parent
strain in respect of the property being tested.
[0033] The properties of the virus strain in respect of tumour
cells can be measured in any manner known in the art. For example,
the capacity of a virus to infect a tumour cell can be quantified
by measuring the dose of virus required to measure a given
percentage of cells, for example 50% or 80% of cells. The capacity
to replicate in a tumour cell can be measured by growth
measurements such as those carried out in the Examples, e.g. by
measuring virus growth in cells over a period of 6, 12, 24, 36, 48
or 72 hours or longer.
[0034] The ability of a virus to kill tumour cells can be roughly
quantitated by eye or more exactly quantitated by counting the
number of live cells that remain over time for a given time point
and MOI for given cell type. For example, comparisons may be made
over 24, 48 or 72 hours and using any known tumour cell type. In
particular, HT29 colorectal adenocarcinoma, LNCaP.FGC prostate
adenocarcinoma, MDA-MB-231 breast adenocarcinoma, SK-MEL-28
malignant melanoma or U-87 MG glioblastoma astrocytoma cells can be
used. Any one of these cell types or any combination of these cell
types can be used, as may other tumour cell types. It may be
desirable to construct a standard panel of tumour cell types for
this purpose. To count the number of live cells remaining at a
given time point, the number of trypan blue-excluding cells (i.e.
live cells) can be counted. Quantitation may also be carried out by
fluorescence activated cell sorting (FACS) or MTT assay. Tumour
cell-killing ability may also be measured in vivo, e.g. by
measuring the reduction in tumour volume engendered by a particular
virus.
[0035] In order to determine the properties of viruses of the
invention, it will generally be desirable to use a standard
laboratory reference strain for comparison. Any suitable standard
laboratory reference strain may be used. In the case of HSV, it is
preferred to use one or more of HSV1 strain 17+, HSV1 strain F or
HSV1 strain KOS. The reference strain will typically have
equivalent modifications to the strain of the invention being
tested. Thus, the reference strain will typically have equivalent
modifications gene deletions and, such as heterologous gene
insertions. In the case of a virus of the invention, where the
ICP34.5 and ICP47-encoding genes have been rendered non-functional,
then they will also have been rendered non-functional in the
reference strain. The modifications made to the reference strain
may be identical to those made to the strain of the invention. By
this, it is meant that the gene disruptions in the reference strain
will be in exactly equivalent positions to those in the strain of
the invention, e.g. deletions will be of the same size and in the
same place. Similarly, in these embodiments, heterologous genes
will be inserted in the same place, driven by the same promoter,
etc. However, it is not essential that identical modifications be
made. What is important is that the reference gene has functionally
equivalent modifications, e.g. that the same genes are rendered
non-functional and/or the same heterologous gene or genes is
inserted.
B. Methods of Mutation
[0036] The various genes referred to may be rendered functionally
inactive by several techniques well known in the art. For example,
they may be rendered functionally inactive by deletion(s),
substitution(s) or insertion(s), preferably by deletion. Deletions
may remove one or more portions of the gene or the entire gene. For
example, deletion of only one nucleotide may be made, resulting in
a frame shift. However, preferably a larger deletion( ) is made,
for example at least 25%, more preferably at least 50% of the total
coding and non-coding sequence (or alternatively, in absolute
terms, at least 10 nucleotides, more preferably at least 100
nucleotides, most preferably, at least 1000 nucleotides). It is
particularly preferred to remove the entire gene and some of the
flanking sequences. Where two or more copies of the gene are
present in the viral genome it is preferred that both copies of the
gene are rendered functionally inactive.
[0037] Mutations are made in the herpes viruses by homologous
recombination methods well known to those skilled in the art. For
example, HSV genomic DNA is transfected together with a vector,
preferably a plasmid vector, comprising the mutated sequence
flanked by homologous HSV sequences. The mutated sequence may
comprise a deletion(s), insertion(s) or substitution(s), all of
which may be constructed by routine techniques. Insertions may
include selectable marker genes, for example lacZ or green
fluorescent protein (GFP), for screening recombinant viruses, for
example, .E-backward.-galactosidase activity or fluorescence.
C. Heterologous Genes and Promoters
[0038] The viruses of the invention may be modified to carry a
heterologous gene encoding an immunomodulatory protein. Preferably
the immunomodulatory protein will enhance the anti-tumour activity
of the virus. More preferably the protein is GM-CSF or another
cytokine, a chemokine such as RANTES, or another immunomodulatory
molecule such as B7.1, B7.2 or CD40L. Most preferably the
immunomodulatory molecule is GM-CSF. The immunomodulatory gene may
be any allelic variant of a wild-type gene, or it may be a mutant
gene. The immunomodulatory gene will be derived from a mammal,
preferably a rodent or primate, more preferably a human. The
immunomodulatory gene is preferably operably linked to a control
sequence permitting expression of said gene in a cell in vivo.
Viruses of the invention may thus be used to deliver the
immunomodulatory gene (or genes) to a cell in vivo where it will be
expressed.
[0039] The immunomodulatory gene may be inserted into the viral
genome by any suitable technique such as homologous recombination
of HSV strains with, for example, plasmid vectors carrying the gene
flanked by HSV sequences. The GM-CSF gene, or other
immunomodulatory gene, may be introduced into a suitable plasmid
vector comprising herpes viral sequences using cloning techniques
well-known in the art. The gene may be inserted into the viral
genome at any location provided that oncolytic properties are still
retained Immunomodulatory genes may be inserted at multiple sites
within the virus genome. For example, from 2 to 5 genes may be
inserted into the genome.
[0040] The transcribed sequence of the immunomodulatory gene is
preferably operably linked to a control sequence permitting
expression of the gene in a tumour cell. The term "operably linked"
refers to a juxtaposition wherein the components described are in a
relationship permitting them to function in their intended manner.
A control sequence "operably linked" to a coding sequence is
ligated in such a way that expression of the coding sequence is
achieved under conditions compatible with the control sequence.
[0041] The control sequence comprises a promoter allowing
expression of the immunomodulatory gene and a signal for
termination of transcription. The promoter is selected from
promoters which are functional in mammalian, preferably human
tumour cells. The promoter may be derived from promoter sequences
of eukaryotic genes. For example, the promoter may be derived from
the genome of a cell in which expression of the heterologous gene
is to occur, preferably a mammalian, preferably a human tumour
cell. With respect to eukaryotic promoters, they may be promoters
that function in a ubiquitous manner (such as promoters of
.E-backward.-actin, tubulin) or, alternatively, in a
tumour-specific manner. They may also be promoters that respond to
specific stimuli, for example promoters that bind steroid hormone
receptors. Viral promoters may also be used, for example the
Moloney murine leukaemia virus long terminal repeat (MMLV LTR)
promoter or other retroviral promoters, the human or mouse
cytomegalovirus (CMV) IE promoter, or promoters of herpes virus
genes including those driving expression of the latency associated
transcripts.
[0042] Expression cassettes and other suitable constructs
comprising the immunomodulatory gene and control sequences can be
made using routine cloning techniques known to persons skilled in
the art (see, for example, Sambrook et al., 1989, Molecular
Cloning--A laboratory manual; Cold Spring Harbor Press).
[0043] It may also be advantageous for the promoters to be
inducible so that the levels of expression of the immunomodulatory
gene(s) can be regulated during the life-time of the tumour cell.
Inducible means that the levels of expression obtained using the
promoter can be regulated. For example, a virus of the invention
may further comprise a hererologous gene encoding the tet
repressor/VP16 transcriptional activator fusion protein under the
control of a strong promoter (e.g. the CMV IE promoter) and the
immunomodulatory gene may be under the control of a promoter
responsive to the tet repressor VP16 transcriptional activator
fusion protein previously reported (Gossen and Bujard, 1992, Gossen
et al, 1995). Thus, in this example, expression of the
immunomodulatory gene would depend on the presence or absence of
tetracycline.
[0044] Multiple heterologous genes can be accommodated in the
herpes virus genome. Therefore, a virus of the invention may
comprise two or more immunomodulatory genes, for example from 2 to
3, 4 or 5 immunomodulatory genes. More than one gene and associated
control sequences could be introduced into a particular HSV strain
either at a single site or at multiple sites in the virus genome.
Alternatively pairs of promoters (the same or different promoters)
facing in opposite orientations away from each other, each driving
the expression of an immunomodulatory gene may be used.
D. Therapeutic Uses
[0045] Viruses of the invention may be used in methods of cancer
therapy of the human or animal body. In particular, viruses of the
invention may be used in the oncolytic treatment of cancer, either
with or without additional pro-drug therapy or stimulation of an
anti-tumour immune response. Viruses of the invention may be used
in the therapeutic treatment of any solid tumour in a mammal,
preferably in a human. For example viruses of the invention may be
administered to a subject with prostate, breast, lung, liver,
endometrial, bladder, colon or cervical carcinoma; adenocarcinoma;
melanoma; lymphoma; glioma; or sarcomas such as soft tissue and
bone sarcomas.
E. Administration
[0046] The invention provides the use of a herpes virus according
to the invention in the manufacture of a medicament for the
treatment of cancer. The medicament may be used in a patient,
preferably a human patient, in need of treatment. A patient in need
of treatment is an individual suffering from cancer, preferably an
individual with a solid tumour. The aim of therapeutic treatment
using the medicament is to improve the condition of a patient.
Typically therapeutic treatment using a virus of the invention
alleviates the symptoms of the cancer. Administration of the
medicament to an individual suffering from a tumour will typically
kill the cells of the tumour thus decreasing the size of the tumour
and/or preventing spread of malignant cells from the tumour.
[0047] The invention provides a pharmaceutical composition
comprising the virus and a pharmaceutically acceptable carrier or
diluent. Suitable carriers and diluents include isotonic saline
solutions, for example phosphate-buffered saline.
[0048] Therapeutic treatment may be carried out following direct
injection of the virus composition into target tissue which may be
the tumour or a blood vessel supplying the tumour. The amount of
virus administered is in the case of HSV in the range of from
10.sup.4 to 10.sup.10 pfu, preferably from 10.sup.5 to 10.sup.8
pfu, more preferably about 10.sup.6 to 10.sup.8 pfu. Typically up
to 500:1, typically from 1 to 200 .mu.l preferably from 1 to 10
.mu.l of a pharmaceutical composition consisting essentially of the
virus and a pharmaceutically acceptable suitable carrier or diluent
would be used for injection. However for some oncolytic therapy
applications larger volumes up to 10 ml may also be used, depending
on the tumour and the inoculation site.
[0049] The routes of administration and dosages described are
intended only as a guide since a skilled practitioner will be able
to determine readily the optimum route of administration and
dosage. The dosage may be determined according to various
parameters, especially according to the location of the tumour, the
size of the tumour, the age, weight and condition of the patient to
be treated and the route of administration. Preferably the virus is
administered by direct injection into the tumour. The virus may
also be administered systemically or by injection into a blood
vessel supplying the tumour. The optimum route of administration
will depend on the location and size of the tumour.
[0050] The following Examples illustrates the invention.
[0051] Herpes simplex type-1 virus (HSV1) in which the
neurovirulence factor ICP34.5 is inactivated has previously been
shown to direct tumour specific cell lysis in tumour models both in
vitro and in vivo. Such viruses have also been shown to be safe in
Phase I clinical trials by direct intra-cerebral injection in late
stage glioma patients.
[0052] Previous work has used serially passaged laboratory isolates
of HSV1 (viruses derived from HSV1 strain 17+ or HSV1 strain F)
which might be anticipated to be attenuated in their lytic
capability in human tumour cells as compared to more recent
clinical isolates.
[0053] In work aimed at producing ICP34.5 deleted HSV with enhanced
oncolytic and anti-tumour potential, we have deleted ICP47 and
ICP34.5 from HSV1 strain JS1 and have inserted the immunomodulatory
gene for GM-CSF.
Virus Construction (see FIG. 1)
[0054] The viruses used were either based on HSV1 strain 17+(a
standard laboratory strain) or a clinical isolate derived from cold
sores from a frequent re-activator of HSV1. This clinical, or
"non-laboratory", strain is named JS1. ICP34.5 was completely
deleted from strain 17+ and JS1 together with the insertion of a
CMV-GFP cassette. JS1 was then also further engineered by the
insertion of human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) so as
to replace the ICP34.5 gene and by the deletion of ICP47. The
derivatives of JS1 discussed herein are also non-laboratory
strains, i.e. modified non-laboratory strains of the invention.
Lytic Capabilities of Viruses
[0055] Lytic (cell killing) capabilities were enhanced with the
JS1-derived non-laboratory strains derived virus in all tumour cell
lines tested as compared with the 17+ derived strains. More
particularly, the JS1/34.5-virus, i.e. JS1 with ICP34.5 removed by
deletion, showed enhanced lytic capabilities in HT29 colorectal
adenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-MB-231
breast adenocarcinoma, SK-MEL-28 malignant melanoma and U-87 MG
glioblastoma astrocytoma cells.
[0056] Thus, to provide increased oncolytic activity, the use of
recent clinical virus strains is likely to enhance the anti-tumour
capabilities of such viruses when used in human patients for cancer
treatment.
Further Enhanced Anti-Tumour Activity
[0057] Further enhanced activity may also be anticipated if these
viruses are then used to deliver genes with anti-tumour activity.
Such genes include those encoding pro-drug activators or
immunostimulatory proteins.
[0058] An ICP34.5 deleted clinical isolated of HSV1 which expresses
human or mouse GM-CSF was produced from JS1. GM-CSF is a potent
immune stimulator. These virus are designed to enhance anti-tumour
immune responses following intratumoral injection. These viruses
were demonstrated to express human or mouse GM-CSF using ELISA
assay kits (Biotrak, RTM Amersham) when the viruses are produced in
BHK cells in culture. Individual wells of a six well plate produced
0.56 or 0.54 microgrammes of human or mouse GM-CSF respectively 24
hrs after infection of confluent BHK cells at MOI=0.5.
Deposit Information
[0059] HSV1 strain JS1 has been deposited at the European
Collection of Cell Cultures (ECACC), CAMR, Sailsbury, Wiltshire SP4
0JG, United Kingdom, on 2 Jan. 2001 under provisional accession
number 01010209.
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* * * * *
References