U.S. patent application number 15/784520 was filed with the patent office on 2018-03-08 for method of vaccination comprising a histone deacetylase inhibitor.
This patent application is currently assigned to Turnstone Limited Partnership. The applicant listed for this patent is Turnstone Limited Partnership. Invention is credited to John Bell, Byram Bridle, Jean-Simon Diallo, Chantal Lemay, Brian Lichty, Yonghong Wan.
Application Number | 20180064805 15/784520 |
Document ID | / |
Family ID | 46829980 |
Filed Date | 2018-03-08 |
United States Patent
Application |
20180064805 |
Kind Code |
A1 |
Bridle; Byram ; et
al. |
March 8, 2018 |
Method of Vaccination Comprising a Histone Deacetylase
Inhibitor
Abstract
A vaccination method is provided. The method comprises
administering to a mammal a histone deacytelase inhibitor in
conjunction with a vaccine that expresses an antigen to which the
mammal has a pre-existing immunity.
Inventors: |
Bridle; Byram; (Guelph,
CA) ; Lichty; Brian; (Brantford, CA) ; Wan;
Yonghong; (Hamilton, CA) ; Diallo; Jean-Simon;
(Ottawa, CA) ; Lemay; Chantal; (Ottawa, CA)
; Bell; John; (Ottawa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Turnstone Limited Partnership |
Toronto |
|
CA |
|
|
Assignee: |
Turnstone Limited
Partnership
|
Family ID: |
46829980 |
Appl. No.: |
15/784520 |
Filed: |
October 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14004546 |
Mar 25, 2014 |
9821054 |
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PCT/CA2012/000212 |
Mar 9, 2012 |
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15784520 |
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61451794 |
Mar 11, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001181 20180801;
A61P 31/18 20180101; A61K 39/0011 20130101; A61P 35/00 20180101;
A61K 39/001188 20180801; A61P 31/06 20180101; A61P 31/12 20180101;
A61K 39/29 20130101; C12N 2760/20243 20130101; A61K 31/18 20130101;
A61K 31/4406 20130101; A61K 31/4745 20130101; A61K 38/15 20130101;
C12N 2710/10343 20130101; A61K 39/285 20130101; A61K 39/001191
20180801; A61K 39/04 20130101; A61P 31/22 20180101; A61K 31/4045
20130101; A61K 2039/545 20130101; A61P 31/04 20180101; A61P 43/00
20180101; A61K 31/167 20130101; A61K 38/12 20130101; A61K 39/165
20130101; A61K 39/17 20130101; A61K 31/506 20130101; A61K 39/12
20130101; A61K 39/001186 20180801; A61P 37/04 20180101; A61K 31/713
20130101; A61K 38/164 20130101; A61K 45/06 20130101; A61K 31/19
20130101; A61K 31/192 20130101; A61K 39/001192 20180801; C12N
2760/20232 20130101; A61K 39/001106 20180801; A61K 39/001182
20180801; A61K 31/165 20130101; A61K 35/766 20130101; A61K 39/02
20130101; A61K 39/205 20130101; A61K 38/164 20130101; A61K 2300/00
20130101; A61K 38/12 20130101; A61K 2300/00 20130101; A61K 38/15
20130101; A61K 2300/00 20130101; A61K 31/4406 20130101; A61K
2300/00 20130101; A61K 31/167 20130101; A61K 2300/00 20130101; A61K
31/18 20130101; A61K 2300/00 20130101; A61K 31/165 20130101; A61K
2300/00 20130101; A61K 31/4045 20130101; A61K 2300/00 20130101;
A61K 31/192 20130101; A61K 2300/00 20130101; A61K 31/19 20130101;
A61K 2300/00 20130101; A61K 31/4745 20130101; A61K 2300/00
20130101; A61K 31/506 20130101; A61K 2300/00 20130101; A61K 31/713
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/29 20060101
A61K039/29; A61K 38/16 20060101 A61K038/16; A61K 31/167 20060101
A61K031/167; A61K 31/18 20060101 A61K031/18; A61K 31/19 20060101
A61K031/19; A61K 45/06 20060101 A61K045/06; A61K 39/285 20060101
A61K039/285; A61K 39/205 20060101 A61K039/205; A61K 39/17 20060101
A61K039/17; A61K 39/165 20060101 A61K039/165; A61K 39/12 20060101
A61K039/12; A61K 39/04 20060101 A61K039/04; A61K 39/02 20060101
A61K039/02; A61K 39/00 20060101 A61K039/00; A61K 31/165 20060101
A61K031/165; A61K 38/15 20060101 A61K038/15; A61K 38/12 20060101
A61K038/12; A61K 31/713 20060101 A61K031/713; A61K 31/506 20060101
A61K031/506; A61K 31/4745 20060101 A61K031/4745; A61K 31/4406
20060101 A61K031/4406; A61K 31/4045 20060101 A61K031/4045; A61K
31/192 20060101 A61K031/192 |
Claims
1. A vaccination method comprising administering to a mammal a
histone deacetylase inhibitor and a vaccine that delivers an
antigen to which the mammal has a pre-existing immunity.
2. The method of claim 1, wherein the antigen is selected from the
group consisting of include tumour antigens, antigens from viral
pathogens and antigens from bacterial pathogens.
3. The method of claim 2, wherein the tumour antigen is selected
from the group consisting of alphafetoprotein (AFP),
carcinoembryonic antigen (CEA), CA 125, Her2, dopachrome
tautomerase (DCT), GP100, MART1, MAGE protein, NY-ESO1, HPV E6 and
HPV E7.
4. The method of claim 1, wherein the antigen is from a pathogenic
organism selected from the group consisting of HIV, HepC, FIV,
LCMV, Ebola virus and mycobacterium tuberculosis.
5. The method of claim 1, wherein the histone deacetylase inhibitor
is selected from the group consisting of hydroxamic, benzamides,
cyclic tetrapeptides, depsipeptides, electrophilic ketones, and
aliphatic acid compounds.
6. The method of claim 5, wherein the hydroxamic acids are selected
from vorinostat (SAHA), belinostat (PXD101), LAQ824, trichostatin A
and panobinostat (LBH589); the benzamides are selected from
entinostat (MS-275), CI994 and mocetinostat (MGCD0103); the cyclic
tetrapeptide is trapoxin B); and the aliphatic acid compounds may
be phenylbutyrate or valproic acid.
7. The method of claim 1, wherein the vaccine is a viral vaccine
that expresses the antigen.
8. The method of claim 1, wherein the viral vaccine is an oncolytic
virus.
9. The method of claim 8, wherein the oncolytic virus is selected
from the group consisting of rhabdoviruses, measles, vaccinia,
herpes, myxoma, parvoviral, Newcastle disease, adenovirus and
semliki forest virus.
10. The method of claim 8, wherein the oncolytic virus is a
vesiculovirus selected from the group consisting of vesicular
stomatitis virus (VSV), Maraba virus, Ephemerovirus,
Cytorhabdovirus, Nucleorhabdovirus and Lyssavirus virus.
11. The method of claim 1, wherein the vaccine induces expression
of type I interferon.
12. The method of claim 1, wherein the vaccine is an
antigen-presenting B-cell.
13. The method of claim 1, wherein the vaccine is administered hi
combination with an agent that induces expression of type I
interferon.
14. A composition comprising a vaccine and a histone deacetylase
inhibitor.
15. The composition of claim 14, wherein the vaccine is a viral
vaccine.
16. The composition of claim 14, wherein the vaccine induces type I
interferon.
17. The composition of claim 14, wherein the histone deacetylase
inhibitor is selected from the group consisting of hydroxamic,
benzamides, cyclic tetrapeptides, depsipeptides, electrophilic
ketones, and aliphatic acid compounds.
18. The composition of claim 17, wherein the hydroxamic acids are
selected from vorinostat (SAHA), belinostat (PXD101), LAQ824,
trichostatin A and panobinostat (LBH589); the benzamides are
selected from entinostat (MS-275), CI994 and mocetinostat
(MGCD0103); the cyclic tetrapeptide is trapoxin B); and the
aliphatic acid compounds may be phenylbutyrate or valproic
acid.
19. The composition of claim 14, additionally comprising an agent
that induces expression of type I interferon.
20. The composition of claim 19, wherein the agent is selected from
the group consisting of a toll-like receptor ligand, imiquimod,
polyinosine-polycytidylic acid (polyI:C), CpG ODN,
imidazoquinoline, monophosphoryl lipid A, flagellin, FimH and
N-glycolyted muramyldipeptide
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a method of
vaccination, and in particular, relates to a vaccination method in
which a viral vaccine is co-administered with a histone deacetylase
inhibitor.
BACKGROUND OF THE INVENTION
[0002] Histone deacetylase inhibitors (HDACi) are epigenetic
modifier drugs having broad effects on gene expression by virtue of
impairing histone modifications required for controlling gene
transcription. HDACi can modify interferon signalling in tumor
cells and thus can be utilized as viral sensitizers to enhance
oncolysis. By accentuating the inherent defects in interferon
responsiveness of cancer cells, these drugs are able to increase
the effectiveness of tumor-tropic viruses without rendering normal
cells susceptible. Thus HDACi can alter innate immunity to
facilitate viral oncolysis but their impact on acquired immune
responses has not been investigated in this therapeutic
setting.
[0003] Viral oncolysis and cancer immunotherapy exhibit clinical
efficacy as stand-alone treatments. There is an ever-growing body
of literature suggesting successful oncolytic virotherapy depends
on its inherent ability to induce anti-tumor immunity, leading some
to go so far as to define it as a form of immunotherapy. Several
promising clinical candidates are viruses that have been engineered
to express immunostimulatory transgenes. However, debate continues
as to whether stimulating the immune system is of net benefit to
oncolytic virotherapy. Indeed, if immune responses against the
oncolytic vector were inadvertently promoted, this could compromise
viral replication and harm the induction of tumor-specific
responses, especially when self-antigens are targeted, via
mechanisms such as antigen competition, where foreign viral
antigens would have a marked advantage, and reduced antigen release
due to less oncolysis. Therefore, optimal strategies to combine
direct oncolysis with immunotherapy should aim to promote both
anti-tumor immunity and oncolytic virus replication.
[0004] Oncolytic viruses have recently been shown to be
particularly potent boosters of anti-tumor immune responses. This
therapeutic approach combines conventional and oncolytic viral
vaccines, both expressing the same tumor antigen. Boosting with an
oncolytic vaccine can lead to both tumor debulking by the virus and
a large increase in the number of tumor-specific CTL (cytotoxic
T-lymphocytes) in primed animals. Paradoxically, this methodology
actually generates larger anti-tumor immune responses in
tumor-bearing, as compared to tumor-free, animals since the
replicating oncolytic vector is amplified in the tumor leading to a
very large increase in the number of antigen-specific TILs and
eradication of established intracranial melanomas in some
cases.
[0005] Several HDACi, including valproic acid (VPA),
suberoylanilide hydroxamic acid (SAHA) and MS-275, are currently
undergoing clinical investigations as anti-cancer drugs for various
solid and hematological malignancies. Initial promising results
have been obtained in acute myelogenous leukemia, T cell lymphomas
and renal cell carcinoma. Interestingly, in addition to their
direct anti-tumor activity, these HDACi have immunomodulatory
properties. For instance, it has been shown that VPA, SAHA and
MS-275 all can promote immunogenicity and immune recognition of
cancer cells.
SUMMARY OF THE INVENTION
[0006] It has now been found that co-administration of HDACi with a
boosting vaccine exhibits an enhanced effect.
[0007] Accordingly, in one aspect of the invention, a vaccination
method is provided comprising the step of administering to a mammal
a histone deacetylase inhibitor and a vaccine that delivers an
antigen to which the mammal has a pre-existing immunity.
[0008] In another aspect of the invention, a composition is
provided comprising a vaccine and a histone deacetylase
inhibitor.
[0009] These and other aspects of the invention will become
apparent in the detailed description that follows, by reference to
the following figures.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 illustrates the survival curves of a cancer model
treated with PBS, Ad-BHG (negative control, 1.times.10.sup.8 PFU
1M), Ad-hDCT (TAA transgene, 1.times.10.sup.8 PFU IM),
Ad-hDCT+VSV-hDCT or Ad-hDCT+VSV-hDCT+MS-275 beginning at 1- or
5-days post-engraftment (A and 13, respectively);
[0011] FIG. 2 graphically illustrates that co-administration of an
oncolytic vaccine booster with an HDACi maintains tumour-antigen
specific responses (A), tumour antigen-specific antibody responses
(B), number of activated NK cells (C), enhances the co-expression
of IFN.gamma. and TNF.alpha. (D), enhances the amount of IFN.gamma.
(E) and TNF.alpha. (F) expressed by tumour antigen-specific T cells
following oncolytic vaccine boosting, and enhances the T cell
avidity (G);
[0012] FIG. 3 graphically illustrates that co-administration of an
oncolytic vaccine booster with an HDACi produces a profound
lymphopenia as evidenced by a reduction of total lymphocyte counts
in the peripheral blood attributed to a reduction in naive CD8+
cells (B), naive NK cells (C), CD4+ T cells (D) and B cells
(E);
[0013] FIG. 4 graphically illustrates that co-administration of an
oncolytic vaccine booster with an HDACi reduces anti-viral
responses while maintaining anti-tumour responses;
[0014] FIG. 5 graphically illustrates that co-administration of an
oncolytic vaccine booster with an HDACi reduces Treg frequencies
(A), increases tumour antigen-specific T cell:Treg ratio (B) and
reduces Foxp3 expression levels (C);
[0015] FIG. 6 graphically illustrates the lymphopenia induced by
co-administration of an oncolytic vaccine booster with an HDACi is
not strain-specific as similar reductions in total lymphocytes (A),
naive CD8+ T cells (B), total NK cells (C), CD4+ T cells (D) and B
cells (E) occurred in Balb/c mice as in C57/B6 mice;
[0016] FIG. 7 graphically illustrates that VSV induces a transient
lymphopenia that is significantly extended by MS-275
co-administration as evidenced by total lymphocyte counts (A),
naive CD4+ cells (B), CD8+ T cells (C) and B cells (D). Horizontal
dotted line represents average count for untreated mice;
[0017] FIG. 8 graphically illustrates lymphopenia induced by
co-administration of Poly I:C with MS-275 as compared with
administration of Poly I:C alone and VSV/MS-275 as evidenced by
total lymphocyte counts; and
[0018] FIG. 9 illustrates the structure of MS-275 (A) and an
inactive MS-275 analogue (B).
DETAILED DESCRIPTION OF THE INVENTION
[0019] A vaccination method is provided comprising administering to
a mammal a histone deacytelase inhibitor in combination with a
vaccine adapted to express an antigen to which the mammal has a
pre-existing immunity.
[0020] The term "vaccine" is used herein to refer to a biological
preparation that induces an immunogenic response to a target
antigen. Examples of vaccines include viral, bacterial, protein and
nucleic acid vaccines. The term "viral vaccine" refers to a virus
that induces an immunogenic response to a target antigen.
[0021] The term "mammal" refers to human as well as non-human
mammals.
[0022] The present method includes administration to the mammal of
a vaccine that delivers or expresses an antigen to which the mammal
has a pre-existing immunity. As used herein, the term "pre-existing
immunity" is meant to encompass an immunity induced by vaccination
with an antigen, as well as a naturally existing immunity within
the mammal resulting from a prior exposure to a given antigen.
[0023] To establish a pre-existing immunity, the present method may
include a step of vaccinating a mammal with an antigen appropriate
to induce an immune reaction against target cells, e.g. a priming
step. Suitable antigens include tumour antigens, viral antigens,
and in particular, antigens derived from viral pathogenic organisms
such as HIV, HepC, FIV, LCMV, Ebola virus, as well as bacterial
pathogens such as mycobacterium tuberculosis.
[0024] In one embodiment, the antigen is a tumour antigen, such as
a tumor-associated antigen (TAA), e.g. a substance produced in a
tumor cell which triggers an immune response in the mammal.
Examples of such antigens include oncofetal antigens such as
alphafetoprotein (AFP) and carcinoembryonic antigen (CEA), surface
glycoproteins such as CA-125 and mesothelin, oncogenes such as
Her2, melanoma-associated antigens such as dopachrome tautomerase
(DCT), GP100 and MART1, cancer-testes antigens such as the MAGE
proteins and NY-ESO1, viral oncogenes such as HPV E6 and E7,
proteins ectopically expressed in tumours that are usually
restricted to embryonic or extraembryonic tissues such as PLAC1. As
one of skill in the art will appreciate, an antigen may be selected
based on the type of cancer to be treated using the present method
as one or more antigens may be particularly suited for use in the
treatment of certain cancers. For example, for the treatment of
melanoma, a melanoma-associated antigen such as DCT may be used.
The term "cancer" is used herein to encompass any cancer, including
but not limited to, melanoma, sarcoma, lymphoma, carcinoma such as
brain, breast, liver, stomach and colon cancer, and leukaemia.
[0025] The antigen may be administered per se, or, preferably,
administered via a vector, e.g. rhabdoviral, adenoviral (Ad),
poxviral or retroviral vector, a plasmid or loaded
antigen-presenting cells such as dendritic cells. Methods of
introducing the antigen into the vector are known to those of skill
in the art. Generally, the vector will be modified to express the
antigen. In this regard, nucleic acid encoding the selected antigen
is incorporated into the selected vector using well-established
recombinant technology.
[0026] The antigen is administered to the mammal in any one of
several ways including, but not limited to, intravenously,
intramuscularly, or intranasally. As will be appreciated by one of
skill in the art, the antigen, or vector incorporating the antigen,
will be administered in a suitable carrier, such as saline or other
suitable buffer. Following vaccination with a selected antigen, an
immune response is generated by the mammal within an immune
response interval, e.g. at least about 24 hours, preferably at
least about 2-4 days or longer, e.g. at least about 1 week and
possibly extending for months, years, or potentially life.
[0027] To establish an immune response to the antigen, vaccination
using the antigen is conducted using well-established techniques.
Accordingly, a selected antigen, or a vector expressing the
antigen, may be administered to the mammal, in an amount sufficient
to generate an immune response. As one of skill in the art will
appreciate, the amount required to generate an immune response will
vary with a number of factors, including, for example, the selected
antigen, the vector used to deliver the antigen, and the mammal to
be treated, e.g. species, age, size, etc. In this regard, for
example, intramuscular administration of a minimum of at least
about 10.sup.7 PFU of adenoviral vector to a mouse, or at least
about 10.sup.9 PFU in a human, is sufficient to generate an immune
response.
[0028] In another embodiment, the immune response to the antigen
may be naturally-occurring within the mammal and a priming
vaccination step is not necessary to induce the immune response.
Naturally-occurring immune response to an antigen may result from
any prior exposure to the antigen.
[0029] Once an immune response has been generated in the mammal to
a given antigen, within a suitable immune response interval, a
boosting vaccine adapted to deliver or express the antigen, such as
a viral vaccine or an antigen-presenting cell, is then administered
to the mammal in conjunction with an HDACi.
[0030] A viral vaccine expressing a selected antigen may be
prepared by incorporating a transgene encoding the antigen into a
suitable virus using standard recombinant technology. For example,
the transgene may be incorporated into the genome of the virus, or
alternatively, may be incorporated into the virus using a plasmid
incorporating the transgene. Suitable viruses for use in this
regard include oncolytic viruses, as well as both replicating (e.g.
poxviral) and non-replicating (e.g. retroviral or adenoviral)
vaccine vectors. The present method is not particularly restricted
with respect to the oncolytic virus that may be utilized and may
include any oncolytic virus capable of destroying tumour, while
being appropriate for administration to a mammal. Examples of
oncolytic viruses that may be utilized in the present method
include rhabdoviruses such as vesieuloviruses, e.g. vesicular
stomatitis virus (VSV) and Maraba viruses, Ephemerovirus,
Cytorhabdovirus, Nucleorhabdovirus and Lyssavirus viruses, as well
as measles, vaccinia, herpes, myxoma, parvoviral, Newcastle
disease, adenoviral and semliki forest viruses.
[0031] The antigen-expressing virus is administered in an amount
suitable to boost the immune response resulting from the
pre-existing immunity in conjunction with an HDACi. In one
embodiment, a tumour antigen-expressing oncolytic virus is
administered in an amount suitable for oncolytic viral therapy in
conjunction with an effective amount of an HDACi. The amount of
each will vary with at least the selected virus, the selected HDACi
and the mammal to be treated, as will be appreciated by one of
skill in the art. For example, a minimum of 10.sup.8 PFU of
oncolytic VSV administered IV to a mouse is sufficient for
oncolytic therapy. A corresponding amount would be sufficient for
use in a human.
[0032] The viral vaccine is administered in conjunction with an
HDACi. Suitable histone deacetylase inhibitors (HDACi) in
accordance with the invention include, but are not limited to,
hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101),
LAQ824, trichostatin A and panobinostat (LBH589); benzamides such
as entinostat (MS-275), 01994, and mocetinostat (MGCD0103), cyclic
tetrapeptides (such as trapoxin B), and the depsipeptides,
electrophilic ketones, and the aliphatic acid compounds such as
phenylbutyrate and valproic acid. A therapeutic amount of HDACi is
administered to a mammal in the present method, e.g. an amount
sufficient to enhance the immunological response to the viral
vaccine. The HDACi may be administered using any suitable
administrable form, including for example, oral, subcutaneous,
intravenous, intraperitoneal, intranasal, enteral, topical,
sublingual, intramuscular, intra-arterial, intramedullary,
intrathecal, inhalation, ocular, transdermal, vaginal or rectal
means.
[0033] The viral vaccine and histone deacetylase inhibitor may be
administered in accordance with methods of the invention alone or
combined together in a composition, and may also be combined with
one or more pharmaceutically acceptable adjuvants or carriers. The
expression "pharmaceutically acceptable" means acceptable for use
in the pharmaceutical arts, i.e. not being unacceptably toxic, or
otherwise unsuitable for administration to a mammal. Examples of
pharmaceutically acceptable adjuvants include, but are not limited
to, diluents, excipients and the like. Reference may be made to
"Remington's: The Science and Practice of Pharmacy", 21st Ed.,
Lippincott Williams & Wilkins, 2005, for guidance on drug
formulations generally. The selection of adjuvant depends on the
intended mode of administration of the composition. In one
embodiment of the invention, the compounds are formulated for
administration by infusion, or by injection either subcutaneously
or intravenously, and are accordingly utilized as aqueous solutions
in sterile and pyrogen-free form and optionally buffered or made
isotonic. Thus, the compounds may be administered in distilled
water or, more desirably, in saline, phosphate buffered saline or
5% dextrose solution. Compositions for oral administration via
tablet, capsule, lozenge, solution or suspension in an aqueous or
non-aqueous liquid, an oil-in-water or water-in-oil liquid
emulsion, an elixir or syrup are prepared using adjuvants including
sugars, such as lactose, glucose and sucrose; starches such as corn
starch and potato starch; cellulose and derivatives thereof,
including sodium carboxymethylcellulose, ethylcellulose and
cellulose acetates; powdered tragancanth; malt; gelatin; tale;
stearic acids; magnesium stearate; calcium sulfate; vegetable oils,
such as peanut oils, cotton seed oil, sesame oil, olive oil and
corn oil; polyols such as propylene glycol, glycerine, sorbital,
mannitol and polyethylene glycol; agar; alginic acids; water;
isotonic saline and phosphate buffer solutions. Wetting agents,
lubricants such as sodium lauryl sulfate, stabilizers, tableting
agents, disintegrating agents, anti-oxidants, preservatives,
colouring agents and flavouring agents may also be present. In
another embodiment, the composition may be formulated for
application topically as a cream, lotion or ointment. For such
topical application, the composition may include an appropriate
base such as a triglyceride base. Such creams, lotions and
ointments may also contain a surface-active agent and other
cosmetic additives such as skin softeners and the like as well as
fragrance. Aerosol formulations, for example, for nasal delivery,
may also be prepared in which suitable propellant adjuvants are
used. Compositions of the present invention may also be
administered as a bolus, electuary, or paste. Compositions for
mucosal administration are also encompassed, including oral, nasal,
rectal or vaginal administration for the treatment of infections,
which affect these areas. Such compositions generally include one
or more suitable non-irritating excipients or carriers comprising,
for example, cocoa butter, polyethylene glycol, a suppository wax,
a salicylate or other suitable carriers. Other adjuvants may also
be added to the composition regardless of how it is to be
administered, which, for example, may aid to extend the shelf-life
thereof.
[0034] The present method provides an effective synergistic
vaccination in a mammal in which primary immune responses are
impaired, while the secondary immune response to a given antigen is
enhanced, e.g. enhanced by at least about 2-fold or greater, e.g.
about 4-fold or greater, e.g. about 6 to 8-fold or greater, in
comparison to the response induced by the viral vaccine alone. A
contributing factor to this effect is the selective lymphopenia
induced by the method whereby naive lymphocytes are selectively
depleted by the combination. The combination of histone deacetylase
inhibitor with the vaccine booster also reduces the autoimmune
sequelae resulting from vaccination against an autoantigen without
reducing the effects, e.g. anti-tumor effects, of such
vaccination.
[0035] In one embodiment, a method of boosting an immune response
in a mammal having a pre-existing immunity to an antigen is
provided in which the antigen is administered to the mammal, for
example intravenously, via a vector that is capable of infecting
B-cells, via antigen-presenting cells such as B cells, by a vector
that induces expression of type I interferon or via a vector in
combination with an agent that induces expression of type I
interferon to achieve a vaccination in which the antigen immune
response is enhanced and the primary immune response is impaired.
The term "pre-existing immunity" is as defined above and may be
achieved as described above. This method may be utilized to boost
immunity with respect to any antigen, including for example, tumour
antigens, viral antigens and particularly antigens derived from
viral pathogenic organisms such as HIV, HepC, FIV, LCMV, Ebola
virus, as well as bacterial pathogens such as mycobacterium
tuberculosis.
[0036] As one of skill in the art will appreciate, the vector may
be prepared to express a selected antigen using well-established
recombinant technology. Appropriate vectors for use in delivering
an antigen to the mammal preferably include vectors that induce
expression of type I interferon, such as, for example,
rhabdoviruses as set out above, including vesiculoviruses and
Maraba-based viruses. Mutant viral vectors are also appropriate for
use in the present method. Mutant attenuated virus, including
replication incompetent forms, are particularly advantageous for
use in the present method.
[0037] The antigen-expressing vector may be combined with an agent
that induces expression of type I interferon. Examples of such
agents include toll-like receptor (TLR) ligands or adjuvants
including, but not limited to, imiquimod, polyinosine-polycytidylic
acid (polyI:C), CpG ODN, imidazoquinoline, monophosphoryl lipid A,
flagellin, FimH and N-glycolyted muramyldipeptide. To achieve a
vaccination in which the antigen immune response is enhanced and
the primary immune response is impaired, the vector is combined
with an amount of type I interferon-inducing agent sufficient to
induce interferon and cause the lympopenia.
[0038] Once the vector is prepared to express the selected antigen,
it is administered, e.g. intravenously, to the mammal for optimal
immunity boosting in conjunction with an HDACi as described above.
The amount of vector administered will again vary with the selected
vector, as well as the mammal. In relation to the pre-existing
immunity, the antigen-expressing vector may be administered to the
mammal prior to or coinciding with the peak immune response of the
pre-existing immunity. The antigen-expressing vector is optimally
administered to the mammal to boost the pre-existing immunity
following the effector phase of the priming of the pre-existing
immunity.
[0039] Embodiments of the invention are described by reference to
the following specific examples which are not to be construed as
limiting.
Example
Methods
Mice
[0040] Female, age-matched (8-10 weeks old at initiation of
experiments) C57BL/6 mice were purchased from Charles River
Laboratories (Wilmington, Mass.) and housed in a controlled
environment in the Central Animal Facility at McMaster University
with food and water provided ad libitum. All animal experimentation
was approved by McMaster University's Animal Research Ethics Board
and complied with the Canadian Council on Animal Care
guidelines.
Viral Vectors
[0041] The replication-deficient rHuAd5-hDCT vector had E1/E3
deleted, expressed the full-length hDCT gene and was propagated in
293 cells and purified on a CsCl gradient. Replication-competent
rVSV-hDCT and rVSV-GFP have been described (Stojdl et al. (2003).
Cancer Cell, 4(4), 263-275). The rHuAd5-BHG and rVSV-MT were
control vectors lacking a transgene.
Prime-Boost Protocol
[0042] Mice were primed by intramuscular injection of
1.times.10.sup.8 pfu of rHuAd5. For boosting, 1.times.10.sup.9 pfu
of rVSV was injected i.v. at a 14-day interval. For the HDACi
treatment, MS-275 (dissolved in DMSO and diluted in saline) was
co-administered with VSV administration and for the following 4
days, 0.1 mg given IP.
Cancer Model
[0043] To establish brain tumors, mice received intracranial
injections of 1.times.10.sup.3 B16-F10 cells in 1 .mu.l of PBS.
Mice were placed in a stereotaxis (Xymotech Biosystems Inc, Quebec,
Canada) and an incision made in the scalp with a scalpel blade to
expose the skull under anaesthesia. A small burr hole was drilled
through the skull at the injection site. Cells were injected with a
26-gauge needle mounted on a 10 .mu.l Hamilton syringe (Hamilton
Company, Reno, Nev.) at the following site in the right hemisphere
of the brain (relative to bregma): 0.62 mm anterior, 2.25 mm
lateral and 4.0 mm deep. Cells were injected over a period of 1
minute and the needle was left in place for 2 minutes prior to
withdrawal to minimize reflux along the injection tract. The scalp
incision was closed with stainless steel clips that were removed
7-10 days later.
Peptides
[0044] The immunodominant peptide from DCT that binds to H-2K.sup.b
(DCT.sub.180-188, SVYDFFVWL) was synthesized by PepSean Systems
(Lelystad, The Netherlands). The H-2K.sup.b-restricted epitope from
the N protein of VSV (RGYVYQGL) was purchased from Biomer
Technologies (Hayward, Calif.).
Antibodies/Tetramers
[0045] Monoclonal antibodies recognizing the following targets were
used for flow cytometry assays: CD16/CD32 (Fc Block), CD3
(145-2C11), CD4 (RM4-5), CD8 (53-6.7), IFN-.gamma. (XMG1.2),
TNF-.alpha. (MP6-XT22), CD19 (1D3), 13220 (RA3-6B2), NK1.1 (PK136),
KLRG-1, CD44 (IM7), CD62L (MEL-14), CD107a (ID4B), H-2K.sup.b
(AF688.5) and I-A.sup.b (25-9-17) (from BD Biosciences,
Mississauga, ON, Canada) and Foxp3 (FJK-16s) (eBioscience, San
Diego, Calif., USA).
Detection of Antigen-Specific T Cell Responses
[0046] Single cell suspensions prepared from different tissues were
re-stimulated with peptides (1 .mu.g/ml) at 37.degree. C. for 5 hrs
and brefeldin A (Golgi Plug, 1 .mu.g/ml; BD Biosciences) was added
during the last 4-hrs of incubation. Cells were treated with Fc
block and stained for surface expression of CD3 and CD8. Cells were
subsequently fixed, permeabilized (Cytofix/Cytoperm, BD
Biosciences) and stained for intracellular IFN-.gamma. and
TNF-.alpha.. Data were acquired using a FACSCanto with FACSDiva
software (BD Biosciences) and analyzed with FlowJo software (Tree
Star, Ashland, Oreg.).
T Cell Functional Avidity Assay
[0047] Splenocytes were exposed to a dilution series of the SVY
peptide such that lower and lower concentrations were provided. The
cells were treated with Golgi plug during this stimulation and
assessed by flow cytometry to measure IFN.gamma. by intracellular
staining as above.
Quantification of DCT-Specific Antibodies
[0048] U2OS cells engineered to express DCT were plated into
multiwell plates and grown to confluence. The cells were then fixed
and permeabilized. Sera from treated mice were serially diluted and
used to probe these fixed monolayers. Following incubation the
unbound antibody was washed away and the bound DCT-specific
antibodies were detected with an anti-mouse secondary bearing a
fluorescent tag. The fluorescent signal was detected to measure the
titre of anti-DCT antibody present in the sera.
Statistical Analyses
[0049] GraphPad Prism for Windows (GraphPad Software, San Diego,
Calif., USA) was used for graphing. For statistical analyses,
GraphPad Prism and Minitab Statistical Software (Minitab Inc.,
State College, Pa., USA) were used. If required, data were
normalized by log transformation. Student's two-tailed t-test, one-
or two-way ANOVA or general linear modeling was used to query
immune response data. Differences between means were considered
significant at p<0.05. Means plus standard error bars are shown.
Survival data were analyzed using the Kaplan-Meier method and the
logrank test.
Results
[0050] MS-275 Dramatically Improves the Therapeutic Outcome in
Combination with an Oncolytic Booster Vaccine
[0051] To determine the potential synergistic effect of MS-275 with
a prime-boost regimen as described in WO2010/105347 A1, an
aggressive brain melanoma model was used with a defined
melanoma-associated antigen, dopachrome tautomerase (DCT), which is
expressed by the melanoma cell line B16-F10 as well as normal
melanocytes (Bridle JI/MT). One- or 5-days after intracranial
inoculation of B16-F10 cells, mice were treated sequentially with a
recombinant human type 5 adenoviral vector expressing human DCT
(rHuAd5-hDCT) and an oncolytic recombinant VSV expressing the same
antigen (rVSV-hDCT) at a 14-day interval. With the goal of
enhancing oncolysis, MS-275 was administered in the context of
boosting with rVSV. This also coincided with the persistence of
rVSV and the peak of the boosted CTL response. The data in FIGS. 1a
and b show that the average survival in untreated animals was 15
days confirming the aggressiveness of this model with a small
treatment window. Vaccination with rHuAd5-hDCT alone prolonged
animal survival to a median of 28 and 25 days in the 1- and 5-day
therapeutic models, respectively. Subsequent delivery of the
oncolytic vaccine, rVSV-hDCT, significantly enhanced animal
survival (FIG. 1a). Despite the improvement of the survival rate,
however, most animals treated with the prime-boost regimen
ultimately succumbed to tumor progression, especially in mice
bearing 5-day-old tumours (FIG. 1b), consistent with previous
observations. Concomitant treatment with MS-275 at the time of
rVSV-hDCT delivery dramatically enhanced the efficacy of the
combination treatment and cured 85% (n=13) and 64% (n=11) of mice
bearing one- or 5-day-old tumors, respectively, at the initiation
of treatment. MS-275 alone had no effect on efficacy in this cancer
model despite in vitro inhibition of B16-F10 cell growth.
The Magnitude of NK Cell and Secondary Tumor-Specific CTL and
Antibody Responses is Preserved in the Presence of MS 275
[0052] The efficacy of prime-boost vaccination in this model
directly correlated with the magnitude of tumor-specific CD8.sup.+
T cell responses. Given that the impact of HDAC inhibition on
immune responses during oncolytic viral therapy has not been
investigated it was determined whether or not this crucial
component of the therapy was enhanced, leading to the dramatic
improvement in efficacy. DCT-specific, IFN-.gamma.-producing
CD8.sup.+ T cells were quantified in the circulation at days 5 and
12 post-rVSV booster vaccination, based on the previous observation
where the secondary T cell response induced by rVSV reached its
peak at day 5 and declined after 12 days. The magnitude of the
DCT-specific CD8.sup.+ T cell response was unaffected by MS-275
(FIG. 2a). In parallel, DCT-specific IgG antibodies in plasma were
measured using an in-cell Western blotting assay (FIG. 2b). These
results revealed a previously unappreciated aspect of the therapy;
namely, that tumor-specific antibodies, like the T cells, were
significantly boosted. Similar to the T cells, the secondary
antibody response was not affected by MS-275. It was also found
that booster vaccination with rVSV increased the number of
circulating NK cells that were capable of producing IFN-.gamma. and
undergoing degranulation (based on CD107a expression) but MS-275
did not affect this (FIG. 2c). Overall, these findings suggest that
the improved therapeutic effect of MS-275 was not due to induction
of higher-magnitude effector responses.
Enhanced Efficacy with MS-275 Correlates with Improved CTL
Quality
[0053] Compared to rHuAd5/rVSV alone, the addition of MS-275
increased the frequency of CD8.sup.+ T cells that co-expressed
TNF-.alpha. (FIG. 2d) and the intensity of their TNF-.alpha. (FIG.
2e) and IFN-.gamma. production (FIG. 2f). This suggests that MS-275
may improve the quality of activated CD8.sup.+ T cells. Better
quality T cells are often associated with higher avidity cognate
interactions with MHC-peptide complexes (ref). Therefore, the
functional avidity of T cells from mice treated with or without
MS-275 was determined. To acquire enough cells for this assay,
splenocytes were used. These cells were exposed to serial dilutions
of the immunodominant peptide from DCT (DCT.sub.180-188;
concentration range: 1 ug/ml to 10 pg/ml). Interestingly,
5.6.times. more CD8.sup.+ T cells could respond to the lowest
concentration of peptide when mice received HDACi treatment (FIG.
2g).
MS-275 Causes Lymphopenia During VSV Booster Vaccination
[0054] Surprisingly, although the number of tumor-specific CTL and
activated NK cells were not influenced by MS-275 treatment, a
transient but severe lymphopenia in the treated mice was observed
(FIG. 3a). Indeed, a closer examination indicated that MS-275
provoked a profound loss of circulating naive CD8.sup.+ T cells
(CD8.sup.highCD44.sup.-CD62L.sup.+KLRG-1.sup.-) (FIG. 3b) and
resting NK cells (CD107a.sup.-IFN-.gamma..sup.-) (FIG. 3c) were
substantially reduced during treatment with MS-275. Furthermore, a
77% reduction of total CD4.sup.+ T cells was seen at the same
timepoint (FIG. 3d). These affected cell populations started to
recover one week after the peak of the boosted CTL response likely
due to cessation of MS-275 treatment and VSV clearance. Most
strikingly, 97% of B cells were eliminated and their recovery was
much slower than other cell populations (FIG. 3e). This lymphopenic
effect was not dependent on the presence of a tumor (data not
shown) nor was it strain-specific (FIG. 6), suggesting that it is a
general phenomenon. These results reveal a novel property of MS-275
that allows secondary expansion of CD8.sup.+ T cells and antibodies
while simultaneously eliminating other lymphocyte populations
including naive T, B and NK cells. This lymphopenic environment may
not only provide more physical space and growth factors to promote
expansion and function of effector cells but may also modulate the
outcome of immune responses against VSV and its oncolytic
effect.
[0055] Similar results were achieved using the HDACi, CI-994.
[0056] FIG. 7 illustrates total lymphocyte coats measured in the
peripheral blood of mice over a 30-day period. Number of cells per
.mu.l of blood were determined over a 30 day period post-treatment.
The virus alone induces a very transient lymphopenia that is
significantly extended by co-administration of an HDACi drug
(MS-275), while the drug alone has a modest effect. Importantly a
drug analogue that lacks HDAC inhibitory properties has no effect
here indicating the requirement for HDAC inhibition. These effects
extend to CD4+ and CD8+ T cells as well as B cells.
[0057] The ability of MS-275 to impair primary immune responses
prompted a determination of whether or not it could attenuate the
immune response against the rVSV boosting vector. To evaluate this,
CD8.sup.+ T cell responses against a K.sup.b-restricted
immunodominant epitope from the N-protein of rVSV were measured at
day 7 post-rVSV inoculations. As shown in FIG. 4a, while the number
of DCT-specific CTL was not affected by MS-275 treatment,
rVSV-reactive CTLs were significantly reduced suggesting that
MS-275 differentially influences expansion of memory and naive
CD8.sup.+ T cells.
[0058] Altogether, these results point to a great benefit of
combining MS-275 with an oncolytic virus-based booster vaccine that
leads to a focused immune response against the tumor while delaying
the response against the oncolytic virus, allowing for extended
viral oncolysis.
Agents that Induce Interferon Type I Expression Induce a
Lymphopenia that is Extended by MS-275 Co-Administration.
[0059] Female mice (8-10 weeks old C57BL/6) were treated with a
single dose of PolyI:C (200 .mu.g in 100 .mu.l of
phosphate-buffered saline, Sigma) and were treated with 0.1 mg of
MS-275 once a day for 5 days via intraperitoneal injection as the
PolyI:C combination MS-275 treatment group. PolyI:C is a classic
inducer of type I interferon expression. Mice treated with a single
dose of PolyI:C only were regarded as PolyI:C treatment group,
whereas mice treated with five doses of MS-275 for 5 days represent
the drug only treatment group. Blood was taken from the periorbital
sinus and red blood cells were lysed with ACK lysis buffer.
Peripheral blood lymphocyte counts were assessed at 2 h, 6 h, 24 h,
48 h, 72 h and 120 h after PolyI:C injection (N=3) as shown in FIG.
8. Data were collected by a FACSCanto flow cytometer with FACSDiva
5.0.2 software (BD Pharmingen) and analyzed with FlowJo Mac
(Treestar, Ashland, Oreg.). In the absence of virus, PolyI:C
generates an identical lymphopenia that is extended by the drug.
Thus, the drug induces these effects in the presence of inducers of
interferon.
MS-275 Reduces Tregs, Especially Those that Express a High Level of
Foxp3 and Up-Regulates MHC Expression on Tumor Cells
[0060] The lymphopenia, especially the reduction of total CD4.sup.+
T cells, induced by MS-275 in the model led to the assessment of
its direct impact on CD4.sup.+Foxp3.sup.+ Tregs. Data in FIG. 5a
show that the number of Tregs was significantly decreased (75%
reduction) during booster immunization, though it appeared to
bounce back faster than other cell populations (FIG. 3a-e). This
led to more than a 3-fold increase in the DCT-specific CD8.sup.+ to
Treg cell ratio (FIG. 5b). Notably, the intensity of Foxp3
expression by Tregs was significantly lower in mice upon MS-275
treatment (FIG. 5c) suggesting the drug may selectively remove
Foxp3 high Tregs that have stronger suppressive function. Together,
these data demonstrate that MS-275 can directly down-regulate Treg
activities, at least in the context of an oncolytic booster
vaccine, allowing the massive secondary CD8.sup.+ T cell responses
to function in a less stringently regulated environment.
MS-275 Prevents Vaccine-Induced Autoimmune Vitiligo
[0061] The oncolytic vaccine vector utilized here leads to a very
potent immune response against an auto-antigen expressed in normal
melanocytes, leading to severe autoimmune vitiligo in those mice
treated with both rHuAd5-hDCT and rVSV-hDCT (FIG. 6, representative
of multiple mice in each group from 4 experiments). Given that
MS-275 co-administration significantly reduces Treg frequencies in
these mice one might predict that the drug would exacerbate this
autoimmune pathology. Remarkably, the induction of systemic
vitiligo by prime-boost vaccination was almost completely abolished
by concomitant treatment with MS-275, in stark contrast to its
enhancement of anti-tumor efficacy. This suggests that a
pharmacological drug may achieve separation of unwanted autoimmune
sequelae from anti-tumor immunity during vaccination therapy
against self-tumor antigens.
Co-Administration of VSV and MS-275 Depleted Immature Lymphocyte
Precursors in Bone Marrow and Thymus.
[0062] Mice were infected with a single tail-vein injection (i.v.)
dose of VSV (2.times.10.sup.9 PFU VSV in 200 .mu.l of
phosphate-buffered saline) and were treated with 0.1 mg of MS-275
via intraperitoneal injection once a day for 3 days as VSV
combination MS-275 treatment group. Mice infected with a single
dose of VSV only were regarded as VSV treatment group, whereas mice
treated with three doses MS-275 or MS-275 analogue (as shown in
FIG. 9) for 3 days were drug only or analogue only treatment groups
respectively, naive mice were not treated with virus or drug. (N=3)
Lymphocytes from thymus or bone marrow (femur and tibia) were
harvested 3 days after VSV injection. Cells were then treated with
anti-CD16/32 and surface markers fluorescently labelled by
antibodies for CD4/CD8 or B220/IgM (BD Pharmingen). Lymphocyte
progenitors were depleted by the combination therapy in both thymus
(immature T cells) and bone marrow (immature B cells). Thus, the
extended lymphopenia is due in part to reduction in progenitors
that can replace depleted lymphocytes in periphery.
Discussion
[0063] When used in conjunction with an oncolytic vaccine therapy,
MS-275, a benzamide class inhibitor of type 1 HDACs, not only
enhances viral replication and MHC expression within the tumor but
also has profound effects on the acquired arm of the immune system.
This combination therapy leads to a selective lymphopenia that
impairs both cellular and humoral immune responses against the
oncolytic virus while significantly reducing Tregs thus generating
a focused and derepressed immune response versus the tumour. By
deleting the undesirable immune cells and maintaining those that
are beneficial, this combination provides the best of both worlds,
where the immune system is impaired in its ability to respond to
the therapeutic virus but continues to attack the tumor, thus
enhancing the therapy dramatically, leading to a 60-80% durable
cure rate in a very challenging cancer model. This represents the
first time that anti-melanoma efficacy was dramatically enhanced
with a simultaneous and equally dramatic reduction in vitiligo.
[0064] In summary, an oncolytic vaccine therapy was combined with
an HDACi to impair innate immunity and mediate significant
modification of both anti-viral and anti-tumoral acquired immunity.
By delaying anti-viral responses while focusing the immune response
on the tumor, viral oncolysis was extended, anti-tumor efficacy was
enhanced and autoimmune sequelae were reduced.
[0065] All references referred to herein are incorporated by
reference.
* * * * *