U.S. patent application number 12/399896 was filed with the patent office on 2009-06-25 for vaccines using nucleic acid-lipid complexes.
This patent application is currently assigned to NATIONAL JEWISH MEDICAL AND RESEARCH. Invention is credited to Steven W. Dow, Robyn E. Elmslie, ERwin W. Gelfand, Jurgen Karl Johannes Schwarze.
Application Number | 20090162427 12/399896 |
Document ID | / |
Family ID | 37071358 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162427 |
Kind Code |
A1 |
Dow; Steven W. ; et
al. |
June 25, 2009 |
VACCINES USING NUCLEIC ACID-LIPID COMPLEXES
Abstract
This invention relates to a vaccine and a method for immune
activation which is effective for eliciting both a systemic,
non-antigen specific immune response and a strong antigen-specific
immune response in a mammal. The method is particularly effective
for protecting a mammal from a disease including cancer, a disease
associated with allergic inflammation, an infectious disease, or a
condition associated with a deleterious activity of a self-antigen.
Also disclosed are therapeutic compositions useful in such a
method.
Inventors: |
Dow; Steven W.; (Littleton,
CO) ; Elmslie; Robyn E.; (Littleton, CO) ;
Schwarze; Jurgen Karl Johannes; (Witten, DE) ;
Gelfand; ERwin W.; (Englewood, CO) |
Correspondence
Address: |
DLA PIPER LLP (US)
4365 EXECUTIVE DRIVE, SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
NATIONAL JEWISH MEDICAL AND
RESEARCH
Denver
CO
|
Family ID: |
37071358 |
Appl. No.: |
12/399896 |
Filed: |
March 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11320019 |
Dec 27, 2005 |
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12399896 |
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10012597 |
Nov 30, 2001 |
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11320019 |
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09104759 |
Jun 25, 1998 |
6693086 |
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10012597 |
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Current U.S.
Class: |
424/450 ;
424/193.1 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 38/208 20130101; A61K 2039/57 20130101; A61P 37/00 20180101;
A61K 39/0011 20130101; A61K 39/12 20130101; A61P 39/00 20180101;
A61K 39/39 20130101; A61K 39/155 20130101; A61P 37/04 20180101;
A61K 2039/5152 20130101; A61K 39/35 20130101; A61K 38/2013
20130101; A61K 38/20 20130101; A61K 38/2086 20130101; A61K 2039/53
20130101; C12N 2760/18834 20130101; A61K 2039/5158 20130101; A61K
2039/55555 20130101; A61P 37/02 20180101; A61K 48/00 20130101; A61K
47/6911 20170801; A61K 38/2086 20130101; A61K 2300/00 20130101;
A61K 38/20 20130101; A61K 2300/00 20130101; A61K 38/208 20130101;
A61K 2300/00 20130101; A61K 38/2013 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/450 ;
424/193.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/00 20060101 A61K039/00; A61P 37/02 20060101
A61P037/02 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was supported in part by NIH Grant No. RO1
CA86224-01, awarded by the National Institutes of Health. The
government has certain rights to this invention.
Claims
1. A vaccine comprising: a. at least one immunogen for vaccinating
a mammal; b. a liposome; and c. an isolated nucleic acid molecule
that does not express said immunogen of (a); wherein said immunogen
and said isolated nucleic acid molecule are complexed to or within
said liposome.
2. The vaccine of claim 1, wherein said immunogen comprises at
least one epitope that elicits a cellular or humoral immune
response in a mammal.
3. The vaccine of claim 1, wherein said immunogen is a peptide.
4. The vaccine of claim 1, wherein said immunogen is selected from
the group consisting of a tumor antigen, an infectious disease
pathogen antigen, allergen, and a self-antigen.
5. The vaccine of claim 1, wherein said vaccine comprises multiple
immunogens.
6. The vaccine of claim 1, wherein said isolated nucleic acid
molecule is an oligonucleotide.
7. The vaccine of claim 1, wherein said isolated nucleic acid
molecule contains a CpG motif that is immunogenic in a mammal.
8. The vaccine of claim 1, wherein said isolated nucleic acid
molecule is demethylated.
9. The vaccine of claim 1, wherein said isolated nucleic acid
molecule is non-coding DNA.
10. The vaccine of claim 1, wherein said isolated nucleic acid
molecule is a plasmid vector that does not contain a gene
insert.
11. The vaccine of claim 1, wherein said liposome is a
multilamellar vesicle.
12. The vaccine of claim 1, wherein said liposome comprises
cationic liposomes.
13. The vaccine of claim 12, wherein said cationic liposomes have
been formulated into multilamellar vesicles (MLVs).
14. The vaccine of claim 13, wherein said liposome further
comprises cholesterol complexed with said cationic lipids.
15. The vaccine of claim 1, wherein said liposome comprises pairs
of lipids selected from the group consisting of DOTMA and
cholesterol; DOTAP and cholesterol; DOTIM and cholesterol; and DDAB
and cholesterol.
16. The vaccine of claim 1, further comprising a pharmaceutically
acceptable excipient.
17. The vaccine of claim 16, wherein said pharmaceutically
acceptable excipient is 5-10% sucrose.
18. The vaccine of claim 1, wherein said composition has a nucleic
acid to lipid ratio of from about 1:1 to about 1:64.
19. A vaccine comprising: a. an antigen; b. a liposome; and c. a
non-coding DNA molecule; wherein said antigen and said non-coding
DNA molecule are complexed to or within said liposome.
20. A method to elicit a systemic, immunogen-specific immune
response in a mammal, comprising administering to said mammal a
vaccine comprising: a. at least one immunogen for vaccinating a
mammal; b. a liposome; and c. an isolated nucleic acid molecule
that does not express said antigen of (a); wherein said antigen and
said isolated nucleic acid molecule are complexed to or within said
liposome.
21. The method of claim 20, wherein said step of administering is
by a route selected from the group consisting of intravenous,
intraperitoneal, subcutaneous, intradermal, intranodal,
intramuscular, transdermal, inhaled, intranasal, rectal, vaginal,
urethral, topical, oral, intraocular, intraarticular, intracranial,
and intraspinal.
22. The method of claim 20, wherein said step of administering is
by a combination of intravenous and intranodal administration.
23. The method of claim 20, wherein said step of administering is
by a combination of intraperitoneal and intranodal
administration.
24. The method of claim 20, wherein said step of administering is
by a combination of intradermal and intranodal administration.
25. The method of claim 20, wherein said antigen is administered at
a dose of from about 1 .mu.g per individual mammal to about 1 mg
per individual mammal.
26. The method of claim 20, wherein said antigen is administered at
a dose of from about 1 .mu.g per individual mammal to about 100
.mu.g per individual mammal.
27. The method of claim 20, wherein said antigen is administered at
a dose of from about 1 .mu.g per individual mammal to about 10
.mu.g per individual mammal.
28. The method of claim 20, wherein administration of said vaccine
to said mammal produces a result selected from the group consisting
of immunization against said disease or condition and stimulation
of effector cell immunity against said disease or condition.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 11/320,019 filed Dec. 27, 2005, now pending;
which is a continuation application of U.S. application Ser. No.
10/012,597 filed Nov. 30, 2001, now abandoned; which is a
continuation-in-part application of U.S. application Ser. No.
09/104,759 filed Jun. 25, 1998, now issued as U.S. Pat. No.
6,693,086. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to a composition and method to
elicit an immune response in a mammal. More particularly, the
present invention includes vaccine compositions and methods for
eliciting systemic, antigen-specific immune responses, as well as
systemic, non-antigen-specific immune responses, in a mammal.
[0004] Three major types of disease in mammals which are amenable
to elicitation and/or is modulation of an immune response include
infectious diseases, allergic inflammatory diseases and cancer.
Infectious diseases are caused by infectious agents (i.e.,
infectious disease pathogens), examples of which include viruses,
bacteria, parasites, prions; yeast and other fungi. In allergic
inflammatory diseases, allergens cause the release of inflammatory
mediators that recruit cells involved in-inflammation in allergic
or sensitized animals, the presence of which can lead to tissue
damage and sometimes death. Cancer can result from an inherited
inability to repair DNA, to prevent DNA damage or to prevent
propagation of cells with damaged DNA, and/or from a biochemical
dysfunction or genetic mutation which leads to uncontrolled cell
proliferation and DNA synthesis.
[0005] Traditional reagents that are used in an attempt to protect
a mammal from such diseases include reagents that destroy
infectious agents or the cells involved in deregulated biological
functions, or that modify the activity of such cells. Such
reagents, however, can result in unwanted side effects. For
example, anti-viral drugs that disrupt the replication of viral DNA
also often disrupt DNA replication in normal cells in the treated
patient. The use of anti-inflammatory and symptomatic relief
reagents in allergic inflammation is a serious problem because of
their side effects or their failure to attack the underlying cause
of an inflammatory response. Other treatments with chemotherapeutic
reagents to destroy cancer cells typically leads to side effects,
such as bleeding, vomiting, diarrhea, ulcers, hair loss and
increased susceptibility to secondary cancers and infections.
[0006] An alternative method of disease treatment includes
modulating the immune system of a patient to assist the patient's
natural defense mechanisms. Traditional reagents and methods used
to attempt to regulate an immune response in a patient also result
in unwanted side effects and have limited effectiveness. For
example, immunopharmacological reagents used to treat cancer (e.g.,
interleukins) are short-lived in the circulation of a patient and
are ineffective except in large doses. Due to the medical
importance of immune regulation and the inadequacies of existing
immunopharmacological reagents, reagents and methods to regulate
specific parts of the immune system have been the subject of study
for many years. Vaccines can be used not only to prevent disease
(prophylactic vaccines), but can also be used to treat established
diseases (i.e., therapeutic vaccines). For example, a number of
tumor antigens which are recognized by T lymphocytes of the immune
system have been recently identified and are being considered as
potential vaccine candidates.
[0007] Conventional vaccines generally include either (1) purified
antigens, or (2) an attenuated form of a pathogen that can be
administered to a patient to generate an immune response, but not
cause serious disease or illness. Genetic vaccines contain a DNA
sequence that encodes an antigen(s) against which the immune
response is to be generated. For genetic vaccines to generate an
antigen-specific immune response, the gene of interest must be
expressed in the mammalian host. Gene expression has been
accomplished by use of viral vectors (e.g., adenovirus, poxvirus)
that express the foreign gene of interest in the vaccinated patient
and induce an immune response against the encoded protein.
Alternatively, plasmid DNA encoding a foreign gene has been used to
induce an immune response. The primary routes of administration of
these so-called "naked" DNA vaccines are intramuscular or
percutaneous. It is generally accepted that viral vector systems
induce better immune responses than naked DNA systems, probably
because the viral delivery systems induce more inflammation and
immune activation than naked DNA vaccines. The propensity of viral
vaccines to induce non-specific immune responses, primarily as a
result of viral component recognition by the complement cascade and
by the elicitation of antigen-specific immune responses against
specific components of the viral vector, also represents a
potential drawback, however, since such immune responses often
prevent readministration of the vaccine.
[0008] Therefore, there is need to provide better vaccines which
can produce an immune response which is safe, antigen-specific, can
be repeatedly administered, and which is effective to prevent
and/or treat diseases amenable to treatment by elicitation of an
immune response, such as infectious disease, allergy and
cancer.
BRIEF SUMMARY OF THE INVENTION
[0009] One embodiment of the present invention relates to a
vaccine. The vaccine includes the following components: (a) at
least one immunogen for vaccinating a mammal; (b) a liposome; and
(c) an isolated nucleic acid molecule that does not express the
immunogen of (a). The immunogen and the isolated nucleic acid
molecule are complexed to or within the liposome.
[0010] Preferably, the immunogen includes at least one epitope that
elicits a cellular or humoral immune response in a mammal. In
another embodiment, the immunogen is selected from the group
consisting of a tumor antigen, an infectious disease pathogen
antigen, an allergen and a self-antigen. The immunogen can include,
but is not limited to, a peptide, a protein or portion thereof, a
cell, a disrupted cell, a pathogenic microorganism, a carbohydrate,
a lipid or any fractions or combinations thereof. In one
embodiment, the vaccine comprises multiple immunogens.
[0011] In one embodiment, the isolated nucleic acid molecule is an
oligonucleotide. In another embodiment, such an oligonucleotide
contains a CpG motif that is immunogenic in a mammal. In yet
another embodiment, such an oligonucleotide is demethylated. In
another embodiment, the isolated nucleic acid molecule is a plasmid
vector that does not contain a gene insert. Other nucleic acid
molecules are also included in the invention, as set forth in the
detailed description.
[0012] In one aspect, the isolated nucleic acid molecule encodes a
cytokine, the nucleic acid sequence being operatively linked to a
transcription control sequence. The cytokine can include, but is
not limited to, hematopoietic growth factors, interleukins,
interferon, immunoglobulin superfamily molecules, tumor necrosis
factor family molecules and chemokines. In one embodiment, the
cytokine is an interleukin. In another embodiment, the cytokine is
selected from the group consisting of interleukin-2 (IL-2),
interleukin-12 (IL-12); interleukin-18 (IL-18), and interleukin-15
(IL-15).
[0013] The liposome can be any suitable liposome, including, but
not limited to, multilamellar vesicles, cationic liposomes,
cholesterol complexed with the cationic lipids, and particularly,
but not limited to, DOTMA and cholesterol; DOTAP and cholesterol;
DOTIM and cholesterol; and DDAB and cholesterol. In one aspect, the
composition has a nucleic acid to lipid ratio of from about 1:1 to
about 1:64.
[0014] In one aspect, the vaccine includes a pharmaceutically
acceptable excipient. Preferably the excipient includes, but is not
limited to, 5-10% sucrose.
[0015] In one embodiment, the vaccine further comprises at least
one cytokine (e.g., provided as a protein). Such a cytokine can
include, but is not limited to, hematopoietic growth factors,
interleukins, interferons, immunoglobulin superfamily molecules,
tumor necrosis factor family molecules and chemokines. Preferred
cytokines include, interleukin-2 (IL-2), interleukin-12 (IL-12),
interleukin-18 (IL-18), and interleukin-15 (IL-15).
[0016] Yet another embodiment of the present invention relates to a
method to elicit a systemic, immunogen-specific immune response in
a mammal. The method includes the step of administering to the
mammal a vaccine comprising: (a) at least one immunogen for
vaccinating a mammal; (b) a liposome; and (c) an isolated nucleic
acid molecule that does not express the immunogen of (a). The
immunogen and the isolated nucleic acid molecule are complexed to
or within the liposome. The step of administering can be by any
route, including, but not limited to, intravenous, intraperitoneal,
subcutaneous, intradermal, intranodal, intramuscular, transdermal,
inhaled, intranasal, rectal, vaginal, urethral, topical, oral,
intraocular, intraarticular, intracranial, and intraspinal. In one
embodiment, the step of administering is by a combination of
intravenous and intranodal administration. In another aspect, the
step of administering is by a combination of intraperitoneal and
intranodal administration. In yet another aspect, the step of
administering is by a combination of intradermal and intranodal
administration.
[0017] In one aspect, the immunogen is administered at a dose of
from about 1 .mu.g per individual mammal to about 1 mg per
individual mammal. In another aspect, the immunogen is administered
at a dose of from about 1 .mu.g per individual mammal to about 10
.mu.g per individual mammal. In yet another aspect, the immunogen
is administered at a dose of from about 1 g per individual mammal
to about 10 .mu.g per individual mammal. Preferably, administration
of the vaccine to the mammal produces a result selected from the
group consisting of immunization against the disease or condition
and stimulation of effector cell immunity against the disease or
condition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a bar graph showing that immunization with
cationic lipid-DNA complexes is combined with tumor lysates
generates antitumor immunity.
[0019] FIG. 2 is a line graph showing that immunization with a CLDC
plus tumor lysates elicits high levels of CTL activity.
[0020] FIG. 3 is a bar graph showing that immunization with tumor
lysates combined with CLDC elicits lymphocyte proliferative
activity.
[0021] FIG. 4 is a bar graph showing that tumor vaccination
inhibits the growth of established lung tumor metastases.
[0022] FIGS. 5A-5B are line graphs showing that vaccination with
autologous tumor lysates using lipid-antigen-DNA complex (LADC)
induces CTL activity in it dogs with osteosarcoma (FIGS. 5A and 5B
each represent a different dog).
[0023] FIGS. 6A-6D are plots showing that immunization with LADC by
subcutaneous administration (FIG. 6B), intravenous administration
(FIG. 6C), and intraperitoneal administration (FIG. 6D), elicits
large numbers of antigen-specific CD8+T cells (FIG. 6A is a
control).
[0024] FIGS. 7A-7B are plots showing that immunization with
peptide-pulsed dendritic cells by subcutaneous (FIG. 7A) or
intraperitoneal (FIG. 7B) routes fails to elicit strong T cell
responses.
[0025] FIGS. 8A-8B are plots showing that LADC immunization also
elicits CD8+T cell responses against weak antigens.
[0026] FIGS. 9A-9B are plots showing that LADC immunization also
induces strong CD8+T cell responses against protein antigens.
[0027] FIG. 10 is a bar graph showing that LADC efficiently
introduce protein antigens into a class I MHC pathway for
presentation to CD8+T cells.
[0028] FIGS. 11A-11C are plots showing that liposome-DNA complexes,
and not liposomes or DNA alone, are required for effective
immunization.
[0029] FIGS. 12A-12B are bar graphs showing that immunization using
LADC elicits functional activation of T cells and production of
IFN-.gamma..
[0030] FIGS. 13A-13D are line graphs showing that immunization with
LADC elicits functional activation of CD8+T cells and high levels
of cytotoxicity.
[0031] FIG. 14 is a line graph showing that therapeutic vaccination
with LADC controls the growth of established tumors.
[0032] FIG. 15 is a plot showing that immunization with LADC
elicits strong CD4+T cell responses.
[0033] FIG. 16 is a bar graph showing that immunization with LADC
elicits CD4+T cell responses in dogs.
[0034] FIG. 17 is a line graph showing that immunization with LADC
elicits strong antibody responses.
[0035] FIGS. 18A-18B are plots showing that immunization with LADC
elicits virus-specific CD8+T cells.
[0036] FIG. 19 is a bar graph showing that vaccination against
viral antigens using LADC elicits high levels of IFN-.gamma.
production by virus antigen-specific T cells.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention generally relates to a novel
immunization strategy and therapeutic compositions (e.g., vaccines)
for eliciting an immune response in a mammal, and in particular, in
a mammal that has a disease amenable to treatment by elicitation of
an immune response. Diseases which are particularly amenable to
treatment using the method of the present invention include any
disease for which administration of a vaccine can provide
protection against the disease, or reduce the symptoms of ongoing
disease. For example, a vaccine of the present invention can be
used to vaccinate against infectious disease, allergic inflammation
and cancer.
[0038] More particularly, the immunization method of the present
invention comprises the elicitation of an immune response in a
mammal by administration of a therapeutic composition which
includes: (a) at least one immunogen for vaccinating a mammal; (b)
a liposome; and (c) an isolated nucleic acid molecule, as defined
more particularly below, that does not encode the immunogen of (a).
The immunogen and the isolated nucleic acid molecule are complexed
to or within the liposome to form the vaccine.
[0039] The combination of nucleic acids and liposomes is highly
immunostimulatory in vivo when administered by intravenous or
intraperitoneal injection (See U.S. patent application Ser. No.
09/104,759, the entirety of which is incorporated herein by
reference). The potency of this immune response was far greater
than the response induced by administration of either nucleic acids
or liposomes alone, and was particularly effective when
administered by intravenous or intraperitoneal routes, although
other routes could also generate an immune response. Moreover, this
effect was independent of whether or not a protein was encoded by
or expressed by the nucleic acids, and it was also independent of
the source of the nucleic acids (e.g., mammalian, bacterial,
insect, viral), the type of nucleic acids (e.g., DNA or RNA), and
the type of lipids used. In addition, U.S. patent application Ser.
No. 09/104,759 showed that the immune response generated by such a
nucleic acid-lipid complex had potent anti-tumor, anti-allergy and
anti-viral properties. As such, the nucleic acid-lipid complexes
induced a strong, systemic, non-antigen-specific immune response;
which resulted in the activation of multiple different immune
effector cells in vivo. Immune activation induced by such nucleic
acid/lipid complexes was quantitatively more potent than that
induced by either (endotoxin) or poly I/C (a classical inducer of
antiviral immune responses). Furthermore, the type of immune
stimulation induced (e.g., as characterized by the pattern of
cytokines induced) also differed qualitatively from that induced by
LPS or poly I/C. Finally, this effect did not appear to be
associated with the complement cascade problems that have been
experienced using viral delivery systems. In one embodiment of U.S.
patent application Ser. No. 09/104,759, the combination of a
liposome and a nucleic acid molecule that does not express a
protein or peptide (non-coding or not linked to a suitable
regulatory sequence) or have any other sequence-specific
specificity. The non-coding nucleic acid molecule does not have
sequence specific functionality, and includes empty vectors,
non-coding oligonucleotides, and other nucleic acid sequences that
do not encode a protein and do not have other sequence
specificity.
[0040] The present invention relates to the combination of nucleic
acid and liposomes as an effective adjuvant component of a vaccine
for the delivery of an immunogen (e.g., protein, peptide,
carbohydrate, lipid, whole cell immunogens or fractions thereof),
and the use of such compositions for eliciting an immune response,
as detailed below. More particularly, the present inventors
demonstrate herein that the use of the combination of nucleic acids
and lipids to deliver an immunogen (i.e., a protein, peptide,
derivatives of proteins or peptides, carbohydrates, lipids, whole
cells, whole cell lysates or disrupted cells, or organisms) to an
animal provides a surprisingly effective means of generating an
immune response against the immunogen. In particular, the present
inventors have found that vaccination with lipid-DNA complexes
combined with defined antigens can elicit strong antigen-specific
CD8+ and CD4+T cell responses in vivo; using even very low antigen
doses, such that lipid-DNA vaccines of the present invention are
sufficiently potent to elicit T cell responses against even weak
antigens such as endogenous tumor antigens. These vaccines are more
effective in eliciting T cell responses than currently available
vaccine technologies, including dendritic cell immunization. In
addition, the present inventors demonstrate herein that vaccination
using lipid-DNA complexes and pools of tumor antigens prepared from
tumor lysates is an effective means of generating CD8+T cell
responses against established tumors. Indeed, in general, the
present inventors have found that the use of lipid-DNA complexes to
vaccinate against protein antigens elicits surprisingly potent
CD8+T cell responses. This result is quite unexpected given the
inherent difficulties associated with elicitation of CD8+T cell
responses against protein antigens in general. Immunization with
lipid-DNA-antigen complexes according to the present invention also
generates effective humoral immune responses. Importantly, the
effectiveness of lipid-DNA-immunogen vaccines can be readily
translated to large animal spontaneous disease models, including
dogs and cats. Thus, the effectiveness of lipid-DNA-immunogen
vaccines is not restricted to any one species of animal.
[0041] Due to the unexpected immunostimulatory properties of the
nucleic acid:lipid complexes (i.e., the adjuvant portion of the
vaccine), the immunization method of the present invention is
particularly useful in human treatments because traditional
adjuvants can be avoided. This is a particular advantage of the
present method, since some traditional adjuvants can be toxic
(e.g., Freund's adjuvant and other bacterial cell wall components)
and others are relatively ineffective (e.g., aluminum-based salts
and calcium-based salts). Moreover, the only adjuvants currently
approved for use in humans in the United States are the aluminum
salts, aluminum hydroxide and aluminum phosphate, neither of which
stimulates cell-mediated immunity. In addition, traditional naked
DNA delivery, which has been touted as having an adjuvant effect,
is far less effective than the combination of nucleic acid and
liposomes at stimulating a non-antigen-specific immune response.
Finally, the present method can be used to repeatedly deliver the
therapeutic composition described herein without consequences
associated with some specific and non-specific arms of the immune
response, and without the risks associated with some genetic
vaccines, including those using viral vectors.
[0042] In further embodiments of the present invention, the present
inventors have taken advantage of the non-antigen-specific
immunostimulatory effect of the above-described method and have
developed an even more powerful immunization strategy in which a
nucleic acid sequence in the above nucleic acid-lipid complex
encodes a cytokine that is expressed in the tissues of the mammal.
Alternatively, the cytokine is provided in protein form with the
immunogen. The combination of an antigen-specific immune response
elicited by an immunogen, in conjunction with the powerful,
non-antigen specific immune response elicited by the nucleic
acid:lipid complex results in a vaccine that has great in vivo
efficacy (See Examples section). This effect can be additionally
enhanced by co-administration of a nucleic acid molecule encoding a
cytokine such that the cytokine is expressed in the tissues or by
co-administration of a protein cytokine together with the
immunogen.
[0043] Moreover, the method of the present invention is
particularly successful in mammals having cancer, because it
induces a strong enough immune response to reduce or eliminate a
primary tumor and to control any metastatic tumors that are already
present, including large metastatic tumors. The method of the
present invention is also particularly successful for vaccinating
mammals against weak antigens or using very low antigen doses.
[0044] One embodiment of the present invention relates to a vaccine
(used interchangeably with the phrase "therapeutic composition").
The vaccine comprises: (a) at least one immunogen for vaccinating a
mammal; (b) a liposome; and (c) an isolated nucleic acid molecule
that does not encode the immunogen of (a). The immunogen and the
isolated nucleic acid molecule are complexed to or within the
liposome.
[0045] Another embodiment of the present invention is a method to
elicit a systemic, immunogen-specific immune response in a mammal.
In this method, a vaccine of the present invention as described
above is administered to a mammal. Administration of such a vaccine
composition by the method of the present invention results in the
elicitation of a systemic, immunogen-specific immune response in
the mammal to which the vaccine is administered. As discussed
above, this immune response has strong, systemic, anti-tumor,
anti-allergic inflammation (i.e., protective), and anti-viral
properties. Such properties include, but are not limited to, the
elicitation of surprisingly potent CD8+T cell responses, CD4+T cell
responses, and humoral immune responses.
[0046] Elicitation of an immune response in a mammal can be an
effective treatment for a wide variety of medical disorders, and in
particular, for cancer, allergic inflammation and/or infectious
disease. As used herein, the term "elicit" can be used
interchangeably with the terms "activate", "stimulate", "generate"
or "upregulate". According to the present invention, "eliciting an
immune response" in a mammal refers to specifically controlling or
influencing the activity of the immune response, and can include
activating an immune response, upregulating an immune response,
enhancing an immune response and/or altering an immune response
(such as by eliciting a type of immune response which in turn
changes the prevalent type of immune response in a mammal from one
which is harmful or ineffective to one which is beneficial or
protective). For example, elicitation of a Th1-type response in a
mammal that is undergoing a Th2-type response, or vice versa, may
change the overall effect of the immune response from harmful to
beneficial. Eliciting an immune response which alters the overall
immune response in a mammal can be particularly effective in the
treatment of allergic inflammation, mycobacterial infections, or
parasitic infections. According to the present invention, a disease
characterized by a Th2-type immune response (alternatively referred
to as a Th2 immune response), can be characterized as a disease
which is associated with the predominant activation of a subset of
helper T lymphocytes known in the art as Th2-type T lymphocytes (or
Th2 lymphocytes), as compared to the activation of Th1-type T
lymphocytes (or Th1 lymphocytes). According to the present
invention, Th2-type T lymphocytes can be characterized by their
production of one or more cytokines, collectively known as Th2-type
cytokines. As used herein, Th2-type cytokines include interleukin-4
(IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-9
(IL-9), interleukin-10 (IL-10), interleukin-13 (IL-13) and
interleukin-15 (IL-15). In contrast, Th1-type lymphocytes produce
cytokines which include IL-2, TNF.alpha. and IFN.gamma..
Alternatively, a Th2-type immune response can sometimes be
characterized by the predominant production of antibody isotypes
which include IgG1 (the approximate human equivalent of which is
IgG4) and IgE, whereas a Th1-type immune-response can sometimes be
characterized by the production of an IgG2a or an IgG3 antibody
isotype (the approximate human equivalent of which is IgG1, IgG2 or
IgG3).
[0047] Preferably, the method of the present invention elicits an
immune response against a tumor, an allergen or an infectious
disease pathogen. In particular, eliciting an immune response in a
mammal refers to regulating cell-mediated immunity (i.e., helper T
cell (Th) activity, cytotoxic T lymphocyte (CTL) activity, NK cell
activity) and/or humoral immunity (i.e., B cell/immunoglobulin
activity), including Th1-type and/or Th2-type cellular and/or
humoral activity. In a preferred embodiment, the method of the
present invention increases or elicits effector cell immunity
against a tumor, an allergen or an infectious disease pathogen: As
discussed above, the vaccine and method of the present invention
are particularly effective at eliciting a CD8+T cell response. As
used herein, effector cell immunity refers to increasing the number
and/or the activity of effector cells in the mammal to which a
composition is administered. In particular, T cell activity refers
to increasing proliferation, cytokine production and/or cytoxicity
of T cells in the area of the tumor cell or pathogen. Similarly, NK
cell activity refers to increasing the proliferation, cytokine
production and cytotoxic activity of NK cells. In the method of the
present invention, effector cell immunity is elicited both
systemically and in the area of the mammal in which the vaccine is
primarily targeted, if any. According to the present invention, an
effector cell includes a helper T cell, a cytotoxic T cell, a B
lymphocyte, a macrophage, a monocyte and/or a natural killer cell.
For example, the method of the present invention can be performed
to increase the number of effector cells in a mammal that are
capable of killing a target cell or releasing cytokines when
presented with antigens derived from a tumor cell, an allergen or a
pathogen. According to the present invention, elicitation of an
immune response (i.e., a non-specific immune response) includes
stimulation of non-specific immune cells, such as macrophages and
neutrophils, as well as activation of effector cells such as NK
cells and antigen-specific cells, including B lymphocytes and/or T
lymphocytes, and induction of cytokine production.
[0048] Accordingly, the method of the present invention preferably
elicits an immune response in a mammal such that the mammal is
protected from a disease that is amenable to elicitation of an
immune response, including cancer, allergic inflammation and/or an
infectious disease. As used herein, the phrase "protected from a
disease" refers to reducing the symptoms of the disease; reducing
the occurrence of the disease, and/or reducing the severity of the
disease. Protecting a mammal can refer to the ability of a
therapeutic composition of the present invention, when administered
to a mammal, to prevent a disease from occurring and/or to cure or
to alleviate disease symptoms, signs or causes. As such, to protect
a mammal from a disease includes both preventing disease occurrence
(prophylactic treatment) and treating a mammal that has a disease
or that is experiencing initial symptoms of a disease (therapeutic
treatment). In particular, protecting a mammal from a disease is
accomplished by eliciting an immune response in the mammal by
inducing a beneficial or protective immune response which may, in
some instances, additionally suppress (e.g., reduce, inhibit or
block) an overactive or harmful immune response. The term,
"disease" refers to any deviation from the normal health of a
mammal and includes a state when disease symptoms are present, as
well as conditions in which a deviation (e.g., infection, gene
mutation, genetic defect, etc.) has occurred, but symptoms are not
yet manifested.
[0049] More specifically, a vaccine as described herein, when
administered to a mammal by the method of the present invention,
preferably produces a result which can include alleviation of the
disease (e.g., reduction of at least one symptom or clinical
manifestation of the disease), elimination of the disease,
reduction of a tumor or lesion associated with the disease,
elimination of a tumor or lesion associated with the disease,
prevention or alleviation of a secondary disease resulting from the
occurrence of a primary disease (e.g. metastatic cancer resulting
from a primary cancer), prevention of the disease, and stimulation
of effector cell immunity against the disease.
[0050] One component of a vaccine or therapeutic composition of the
present invention includes an immunogen for vaccinating an animal.
According to the present invention, the terms "immunogen" and
"antigen" can be used interchangeably, although the term "antigen"
is primarily used herein to describe a protein, cellular
composition (whole cell, cell lysate or disrupted cells) or
organism (whole organism, lysate or disrupted cells) which elicits
a humoral and/or cellular immune response (i.e., is antigenic), and
the term "immunogen" is primarily used herein to describe a protein
(including peptides and glycoproteins), cellular composition or
organism, which elicits a humoral and/or cellular immune response
in vivo, such that administration of the immunogen to a mammal
mounts an immunogen-specific (antigen-specific) immune response
against the same or similar proteins, cellular compositions or
organisms that are encountered within the tissues of the mammal.
Therefore, to vaccinate an animal means that an immune response is
elicited against the immunogen as a result of administration of the
immunogen. Vaccination preferably results in a protective or
therapeutic effect, wherein subsequent exposure to the immunogen
(or a source of the immunogen) elicits an immune response against
the immunogen (or source) that reduces or prevents a disease or
condition in the animal. The concept of vaccination is well known
in the art. The immune response that is elicited by administration
of a vaccine of the present invention can be any detectable
increase in any facet of the immune response (e.g., cellular
response, humoral response, cytokine production), as compared to in
the absence of the administration of the vaccine. Preferably, an
immunogen-specific immune response is detectable.
[0051] According to the present invention, the general use herein
of the term "immunogen" refers to any portion of a protein
(peptide, partial protein, full-length protein), wherein the
protein is naturally occurring or synthetically derived, to
carbohydrate antigens, to lipid antigens, to a whole cell, cellular
lysate, or portion thereof, or to a microorganism, extract thereof,
or other portion thereof, or to any combination of any of the
above-mentioned immunogens, wherein the immunogen elicits a humoral
and/or cellular immune response. An epitope is defined herein as a
single antigenic site within a given immunogen that is sufficient
to elicit an immune response. Those of skill in the art will
recognize that T cell epitopes are different in size and
composition from B cell epitopes, and that epitopes presented
through the Class I MHC pathway differ from epitopes presented
through the Class II MHC pathway. An immunogen can be as small as a
single epitope, or larger, and can include multiple epitopes. As
such, the size of an antigen or immunogen can be as small as about
5-12 amino acids (e.g., a peptide) and as large as: a full length
protein, including a multimer and fusion proteins, chimeric
proteins, whole cells, whole microorganisms, or portions thereof
(e.g., lysates of whole cells or extracts of microorganisms). In
preferred embodiments, the immunogen is selected from the group of
a tumor antigen, an allergen or an antigen of an infectious disease
pathogen (i.e., a pathogen antigen). According to the present
invention, an immunogen suitable for use in the present composition
or vaccine can include two or more epitopes from the same antigen,
two or more epitopes or antigens from the same cell, tissue or
organism, two or more different epitopes or antigens from different
cells, tissues or organisms.
[0052] A tumor antigen that is useful as an immunogen or source of
immunogen according to the present invention includes tumor
antigens having epitopes that are recognized by T cells, tumor
antigens having epitopes that are recognized by B cells, tumor
antigens that are exclusively expressed by tumor cells, and tumor
antigens that are expressed by tumor cells and by non-tumor cells.
Tumor antigens can a tumor antigen including a protein or
glycoprotein from a tumor cell, an epitope from a tumor antigen, an
entire tumor cell, mixtures of tumor cells, and portions thereof
(e.g., lysates). Immunogens useful in a to composition according to
the present invention can include any combination of epitopes,
including from the same tumor antigen, from different tumor
antigens, from different tumor cells, and from different
individuals. Preferably, tumor immunogens useful in the present
method have at least one T cell and/or B cell epitope. Therefore,
delivery of the tumor immunogen to a tissue of a mammal elicits a
tumor antigen-specific immune response against the tumor in the
tissue of the mammal.
[0053] In one embodiment, tumor antigens useful in the present
invention can be isolated or derived from an autologous tumor
sample. An autologous tumor sample is derived from the mammal to
whom the therapeutic composition is to be administered. Therefore,
such antigens will be present in the cancer against which an immune
response is to be elicited. In this embodiment, if all antigens
from a given autologous source are administered together, it is not
necessary to know which of the antigens in a given tumor sample is
the most immunogenic (i.e., the best immunogens), since
substantially all of the antigens expressed by the tumor sample can
be administered to the mammal. In addition, eliciting an immune
response against multiple tumor antigens/immunogens is likely to
have the benefit of enhancing the therapeutic efficacy of the
immune response against the cancer.
[0054] In another embodiment, the tumor antigen is isolated or
derived from at least one, two or from a plurality of allogeneic
tumor samples of the same histological tumor type. According to the
present invention, a plurality of allogeneic tumor samples are
tumor samples of the same histological tumor type, isolated from
two or more mammals of the same species who differ genetically at
least within the major histocompatibility complex (MHC), and
typically at other genetic loci. Therefore, if administered
together, the plurality of tumor antigens can be representative of
the substantially all of the tumor antigens present in any of the
individuals from which antigen is derived. This embodiment of the
method of the present invention provides a vaccine which
compensates for natural variations between individual patients in
the expression of tumor antigens from tumors of the same
histological tumor type. Therefore, administration of this
therapeutic composition is effective to elicit an immune response
against a variety of tumor antigens such that the same therapeutic
composition can be administered to a variety of different
individuals. In some embodiments, antigens from tumors of different
histological tumor types can be administered to a mammal, in order
to provide a very broad vaccine.
[0055] In a preferred embodiment, a tumor immunogen includes a
tumor immunogen from a cancer selected from the group of melanomas,
squamous cell carcinoma, breast cancers, head and neck carcinomas,
thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular
cancers, prostatic cancers, ovarian cancers, bladder cancers, skin
cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell
tumors, primary hepatic cancers, lung cancers, pancreatic cancers,
gastrointestinal cancers, renal cell carcinomas, hematopoietic
neoplasias and metastatic cancers thereof.
[0056] According to the present invention, a pathogen immunogen
that is useful in the present invention includes any immunogen from
an infectious disease pathogen that can include pathogen immunogens
having epitopes that are recognized by T cells, pathogen antigens
having epitopes that are recognized by B cells, pathogen immunogens
that are exclusively expressed by pathogens, and pathogen
immunogens that are expressed by pathogens and by other cells.
Pathogen immunogens can include whole cells and the entire pathogen
organism, as well as lysates, extracts or other fractions thereof,
including secreted toxins and/or spores produced by the pathogen.
Preferably, pathogen immunogens useful in the present method have
at least one T cell and/or B cell epitope and are exclusively
expressed by pathogens (i.e., and not by the endogenous tissues of
the infected mammal). Therefore, delivery of the pathogen immunogen
to a tissue of a mammal elicits an antigen specific immune response
against the pathogen in the tissues of the mammal as well as
systemically. In some instances, an immunogen can include organisms
which may not be ordinarily considered to be pathogenic in a
mammal, but against which immunization is nonetheless desired.
[0057] In one embodiment, the immunogen(s) can include at least two
or preferably, a plurality of pathogen antigens that is
representative of the substantially all of the antigens present in
the infectious disease pathogen against which the vaccine is to be
administered. In this embodiment, it is not necessary to know which
of the antigens in a given pathogen is the most immunogenic (i.e.,
the best immunogens), since substantially all of the antigens
expressed by the pathogen are administered to the mammal. In
addition, eliciting an immune response against multiple pathogen
antigens/immunogens is likely to have the benefit of enhancing the
therapeutic efficacy of the immune response against the infectious
disease. In other embodiments, immunogens from two or more
different strains of the same pathogen or from different pathogens
can be administered to increase the therapeutic efficacy and/or
efficiency of the vaccine.
[0058] According to the present invention, a pathogen immunogen
includes, but is not limited to, an immunogen that is expressed by
a bacterium, a virus, a parasite, a prion or a fungus. Preferred
pathogen immunogens for use in the method of the present invention
include immunogens which cause a chronic or an acute infectious
disease in a mammal. For example, some preferred pathogen
immunogens for use in the present method are immunogens from
pathogens that cause chronic infections, including, but not limited
to, immunodeficiency virus (HIV), Mycobacterium tuberculosis,
herpesvirus, papillomavirus, Leishmania, Toxoplasma, Cryptococcus,
Blastomyces, Histoplasma, and Candida. Also included would be
antibiotic resistant strains of bacteria that can cause chronic
infections, such as Staphylococcus, Pseudomonas, Streptococcus,
Enterococcus, and Salmonella. Additionally, for immunization
against acute, disease, preferred pathogens from which immunogens
can be derived include, but are not limited to, Bacillus anthracis,
Francisella, Yersenia, Pasteurella, small pox, and other gram
negative and gram positive bacterial pathogens.
[0059] In another embodiment of the present invention, a pathogen
immunogen for use in the method or composition of the present
invention includes an immunogen from a virus. As discussed above,
the present inventors have found that the composition and method of
the present invention are particularly useful in the treatment of
and protection against viral infections. Specifically, the nucleic
acid:lipid complex administered by the method of the present
invention elicits a strong, systemic, non-antigen-specific,
anti-viral response in vivo, and the viral antigen further elicits
a strong, viral immunogen-specific immune response in addition to
the above-described systemic immune response. In a preferred
embodiment, the immunogen is from a virus selected from the group
of human immunodeficiency virus, Epstein Barr virus, other Herpes
viruses, papilloma virus, and Hepatitis viruses.
[0060] According to the present invention, an allergen immunogen
includes a full-length allergen, a portion of the allergen or a
homologue of the allergen protein, and includes allergens having
epitopes that are recognized by T cells, allergens having epitopes
that are recognized by B cells, and any allergens that are a
sensitizing agent in diseases associated with allergic
inflammation. Preferred allergens to use in the therapeutic
composition of the present invention include plant pollens, drugs,
foods, venoms, insect excretions, molds, animal fluids, animal hair
and animal dander. Sensitization to an allergen refers to being
previously exposed one or more times to an allergen such that an
immune response is developed against the allergen. Responses
associated with an allergic reaction (e.g., histamine release,
rhinitis, edema, vasodilation, bronchial constriction, airway
inflammation), typically do not occur when a naive individual is
exposed to the allergen for the first. time, but once a cellular
and humoral immune response is produced against the allergen, the
individual is "sensitized" to the allergen. Allergic reactions then
occur when the sensitized individual is re-exposed to the same
allergen (e.g., an allergen challenge). Once an individual is
sensitized to an allergen, the allergic reactions can become worse
with each subsequent exposure to the allergen, because each
re-exposure not only produces allergic symptoms, but further
increases the level of antibody produced against the allergen and
the level of T cell response against the allergen.
[0061] In one embodiment, the immunogen(s) useful in the present
composition can be isolated or derived from multiple allergens. In
this embodiment of the present invention, the plurality allergens
and/or allergen epitopes is representative of the substantially all
of the epitopes present in one allergen or from a class of
allergens, for example.
[0062] In another embodiment of the present invention, an immunogen
for use in the method or composition of the present invention
includes a normal, self-antigen, wherein immunization against such
an antigen induces a therapeutic outcome. The present inventors
have found that the vaccination composition and method of the
present invention is effective to break self tolerance, and
therefore, when elicitation of an immune response against a
self-antigen (one to which the mammal's immune system is normally
tolerant) would be therapeutic, the immunogen can be a
self-antigen. Such self-antigens include, but are not limited to,
growth factors, signaling molecules, and normal cells. For example,
in conditions where abnormal blood vessel growth is experienced,
the present invention can be used to immunize the patient against
an angiogenic growth factor or receptor thereof, such as the VEGF
receptor. As another example, immunization against a signaling
molecule is used to treat a neurologic disease. Such immunogens
include proteins, portions thereof, carbohydrates, lipid molecules,
normal cells, and fractions thereof. A normal cell useful as an
immunogen includes, but is not limited to, an endothelial cell (or
a fraction thereof). Such a cell may be particularly useful in an
immunization strategy against cancer.
[0063] Other immunogens useful in the present invention and
combinations of immunogens will be apparent to those of skill in
the art. The present invention is not restricted to the use of the
immunogens as described above.
[0064] One component of the vaccine of the present invention is a
nucleic acid molecule that does not encode the immunogen used in
the vaccine. This does not preclude the possibility that the
nucleic acid molecule encodes a protein or peptide that is not the
immunizing antigen (immunogen), although it may be, in some
aspects, preferable that the nucleic acid molecule does not encode
any protein or peptide that would be substantially immunogenic
(e.g., sufficient to elicit a substantial immune response), or
alternatively that the nucleic acid molecule does not encode a
protein, or that the nucleic acid molecule has no sequence specific
functionality (discussed below). In one embodiment, the nucleic
acid molecule encodes a protein, which could even include the
immunizing antigen, but the molecule is not operatively linked to a
transcription control sequence so that as a result, no protein or
peptide is expressed by the molecule. In another embodiment, the
vaccine can include a nucleic acid molecule that encodes an
immunogen in addition to the immunogen described above, or in place
of the immunogen described above.
[0065] According to the present invention, an isolated nucleic acid
molecule or nucleic acid sequence, is a nucleic acid molecule or
sequence that has been removed from its natural milieu. As such,
"isolated" does not necessarily reflect the extent to which the
nucleic acid molecule has been purified. An isolated nucleic acid
molecule useful in the present composition can include DNA, RNA, or
any derivatives of either DNA or RNA. An isolated nucleic acid
molecule can be double stranded (i.e., containing both a coding
strand and a complementary strand) or single stranded. An isolated
nucleic acid molecule useful in the present composition can include
oligonucleotides and larger sequences, including both nucleic acid
molecules that encode a protein or a fragment thereof (in one
aspect, such protein is not the immunogen to be used with the given
nucleic acid molecule in a vaccine according to the present
invention), and nucleic acid molecules that comprise regulatory
regions, introns, or other non-coding DNA or RNA. An isolated
nucleic acid molecule can include a nucleic acid vector (e.g., a
plasmid vector), which is preferably an "empty vector", described
in more detail below, or any fragment. of such a vector. An
isolated nucleic acid molecule can include naturally occurring
nucleic acid sequences, and sequences that are not from a naturally
occurring sequence, such as a sequence of randomly generated
nucleotides. In one embodiment, the nucleic acid molecule is not an
antisense nucleic acid molecule (i.e., is not complementary to a
sequence in a coding strand of DNA or to mRNA in the cells of a
patient and/or does not specifically hybridize under stringent
conditions to a coding strand of DNA or to mRNA in the cells of a
patient), or is not a functional antisense molecule (e.g., does not
have an ability to inhibit gene function even if it is a
complementary sequence). In another embodiment, the nucleic acid
molecule is not a viral nucleic acid, such as a viral vector or a
nucleic acid encoding a viral protein(s). In another embodiment,
the nucleic acid molecule is not a ribozyme.
[0066] Nucleic acid molecules useful in a therapeutic composition
of the present invention can be eukaryotic or prokaryotic nucleic
acids. Therefore, the nucleic acid molecule can be derived from any
source, including mammalian, bacterial, insect, or viral sources,
since the present inventors have discovered that the source of the
nucleic acid does not have a significant effect on the ability to
elicit an immune response when the nucleic acid is complexed with
liposomes. In one embodiment of the present invention, the nucleic
acid molecule used in a therapeutic composition of the present
invention is not a bacterial nucleic acid molecule.
[0067] Although the phrase "nucleic acid molecule" primarily refers
to the physical nucleic acid molecule and the phrase "nucleic acid
sequence" primarily refers to the sequence of nucleotides on the
nucleic acid molecule, the two phrases can be used interchangeably.
As used herein, a "coding" nucleic acid sequence refers to a
nucleic acid sequence which encodes at least a portion of a peptide
or protein (e.g. a portion of an open reading frame), and can more
particularly refer to a nucleic acid sequence encoding a peptide or
protein which, when operatively linked to a transcription control
sequence, can express the peptide or protein. A "non-coding"
nucleic acid sequence refers to a nucleic acid sequence which does
not encode any portion of a peptide or protein. According to the
present invention, "non-coding" nucleic acids can include
regulatory regions of a transcription unit, such as a promoter
region. According to the present invention, a nucleic acid molecule
that "does not express" a peptide or protein can include both
coding and non-coding nucleic acids, but in the case of the coding
nucleic acids, the nucleic acid sequence is not provided in a form
that can readily express the peptide or protein. For example, the
sequence may not be operatively linked to an appropriate
transcription control sequence so that the peptide or protein is
not expressed when the nucleic acid molecule enters a cell. The
term, "empty vector" is generally considered to be a type of
"non-coding" nucleic acid molecule, and particularly refers to a
nucleic acid sequence in the absence of a protein coding portion
other than a selectable marker, such as a plasmid vector without a
gene insert. Some empty vectors, and particularly those that are
commercially available, may encode one or more selection markers,
but such markers are not important to the inclusion of the nucleic
acid molecule in the vaccine of the invention. Moreover, it is not
believed that the selectable markers present in such empty vectors,
even if expressed, would elicit a significant immune response, if
any. Therefore, selectable markers encoded in vectors are not
considered to be immunogens according to the present invention.
Preferably, non-coding nucleic acids used in the present invention
do not include antisense molecules, ribozymes, or other such
nucleic acid molecules which may not encode a protein, but which
have another function that is related to the sequence specificity
of the molecule. Essentially, the nucleic acid molecule of the
present invention preferably does not have any sequence specific
functionality, in that the nucleic acids are used as a chemical
composition in conjunction with a liposome, rather than for the
purpose of encoding a particular protein, or executing a
sequence-specific function associated with antisense or
ribozymes.
[0068] A nucleic acid molecule that is a component of the vaccine
or therapeutic composition of the present invention does not encode
a superantigen. It is noted, however, that a protein superantigen
may be added to the vaccine of the present invention, if desired to
further enhance an immune response. A superantigen is defined
herein as the art-recognized term. More particularly, a
superantigen is a molecule within a family of proteins that binds
to the extracellular portion of an MHC molecule (i.e., not in the
peptide binding groove) to form and MHC:superantigen complex. The
activity of a T cell can be modified when a TCR binds to an
MHC:superantigen complex. Under certain circumstances, an
MHC:superantigen complex can have a mitogenic role (i.e., the
ability to stimulate the proliferation of T cells) or a suppressive
role (i.e., deletion of T cell subsets).
[0069] A nucleic acid molecule can be isolated from a natural
source, or it can be produced using recombinant DNA technology
(e.g., polymerase chain reaction (PCR) amplification, cloning) or
chemical synthesis. Nucleic acid molecules can be generated or
modified using a variety of techniques including, but not limited
to, classic mutagenesis techniques and recombinant DNA techniques,
such as site-directed mutagenesis, chemical treatment of a nucleic
acid molecule to induce mutations, restriction enzyme cleavage of a
nucleic acid fragment, ligation of nucleic acid fragments,
polymerase chain reaction (PCR) amplification and/or mutagenesis of
selected regions of a nucleic acid sequence, synthesis of
oligonucleotide mixtures and ligation of mixture groups to "build"
a mixture of nucleic acid molecules and combinations thereof.
[0070] Typically, an oligonucleotide has a nucleic acid sequence
from about 1 to about 500 nucleotides, and more typically, is at
least about 5 nucleotides in length, or any length increasing by
whole integers (e.g., 6, 7, 8, 9, 10 and so on), up to about 500
nucleotides. In a preferred embodiment, an oligonucleotide for use
in the present invention includes an oligonucleotide containing a
cytosine-guanine (CpG) motif that is immunogenic in a mammal. In
another embodiment, the oligonucleotide is demethylated.
Methylation of CpG motifs in DNA is involved in the control of gene
expression and in several other epigenic effects. It suppresses the
immuno-stimulation properties of bacterial or viral DNAs that
contain CpGs. It is further known in the art that bacterial DNA and
synthetic oligodeoxynucleotides containing unmethylated CpG-motifs
in a particular sequence context can activate vertebrate immune
cells.
[0071] A vaccine or therapeutic composition of the present
invention also includes a liposome component. According to the
present invention, a liposome comprises a lipid composition that is
capable of fusing with the plasma membrane of a cell, thereby
allowing the liposome to deliver a nucleic acid molecule and/or a
protein composition into a cell. A liposome is also capable of
either incorporating an immunogen on its surface or incorporating
the immunogen internally. Suitable liposomes for use with the
present invention include any liposome. In fact, the present
inventors have demonstrated that the immune stimulatory effect of
the combination of liposomes and nucleic acids is not limited to a
particular type of liposome. Some preferred liposomes of the
present invention include is those liposomes commonly used in, for
example, gene delivery methods known to those of skill in the art.
Some preferred liposome delivery vehicles comprise multilamellar
vesicle (MLV) lipids and extruded lipids, although the invention is
not limited to such liposomes. Methods for preparation of MLV's are
well known in the art and are described, for example, in U.S.
patent application Ser. No. 09/104,759, ibid. According to the
present invention, "extruded lipids" are lipids which are prepared
similarly to MLV lipids, but which are subsequently extruded
through filters of decreasing size, as described in Templeton et
al., 1997, Nature Biotech., 15:647-652, which is incorporated
herein by reference in its entirety. Small unilamellar vesicle
(SUV) lipids can also be used in the composition and method of the
present invention and have been shown to be effective in
combination with nucleic acids for eliciting an immune response
(see U.S. patent application Ser. No. 09/104,759, ibid.). Other
preferred liposome delivery vehicles comprise liposomes having a
polycationic lipid composition (i.e., cationic liposomes). For
example, cationic liposome compositions include, but are not
limited to, any cationic liposome complexed with cholesterol, and
without limitation, include DOTAA and cholesterol, DOTAP and
cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.
Liposomes of the present invention can be any size, including from
about 10 and 1000 nanometers (nm), or any size in between.
[0072] Complexing a liposome with a nucleic acid molecule can be
achieved using methods standard in the art (see, for example,
methods in U.S. patent application Ser. No. 09/104,759, ibid.).
According to the present invention a cationic lipid:DNA complex is
also referred to herein as a CLDC, and a cationic lipid:RNA complex
is also referred to herein as CLRC. A cationic lipid:DNA complex,
wherein the DNA is an empty vector can be referred to as EV/CLDC. A
CLDC that is further complexed with an immunogen according to the
present invention can be referred to as a lipid-antigen-DNA complex
(LADC) or as a vaccine (Vacc) or therapeutic composition.
[0073] A suitable concentration of a nucleic acid molecule to add
to a liposome includes a concentration effective for delivering a
sufficient amount of nucleic acid molecule into a mammal such that
a systemic immune response is elicited. Preferably, from about 0.1
.mu.g to about 10 .mu.g of nucleic acid molecule of the present
invention is combined with about 8 nmol liposomes, more preferably
from about 0.5 .mu.g to about 5 .mu.g of nucleic acid molecule is
combined with about 8 nmol liposomes, and even more preferably
about 1.0 .mu.g of nucleic acid molecule is combined with about 8
nmol liposomes. In one embodiment, the ratio of nucleic acids to
lipids (.mu.g nucleic acid:nmol lipids) in a composition of the
present invention is preferably at least about 1:1 nucleic
acid:lipid by weight (i.e., 1 .mu.g nucleic acid:1 nmol lipid), and
more preferably, at least about 1:5, and more preferably at least
about 1:10, and even more preferably at least about 1:20. Ratios
expressed herein are based on the amount of cationic lipid in the
composition, and not on the total amount of lipid in the
composition. In another embodiment, the ratio of nucleic acids to
lipids in a composition of the present invention is preferably from
about 1:1 to about 1:64 nucleic acid:lipid by weight; and more
preferably, from about 1:5 to about 1:50 nucleic acid:lipid by
weight; and even more preferably, from about 1:10 to about 1:40
nucleic acid:lipid by weight; and even more preferably, from about
1:15 to about 1:30 nucleic acid:lipid by weight. Another
particularly preferred ratio of nucleic acid:lipid is from about
1:8 to 1:16, with 1:8 to 1:32 being more preferred. Typically,
while non-systemic routes of nucleic acid administration (e.g.,
intramuscular, intratracheal, intradermal) would use a ratio of
about 1:1 to about 1:3, systemic routes of administration according
to the present invention can use much less nucleic acid as compared
to lipid and achieve equivalent or better results than non-systemic
routes. Moreover, compositions designed for gene therapy/gene
replacement, even when is administered by intravenous
administration, typically use more nucleic acid (e.g., from 6:1 to
1:10, with 1:10 being the least amount of DNA used) as compared to
the systemic immune activation composition and method of the
present invention.
[0074] Complexing an immunogen with a liposome and nucleic acid
molecule is accomplished in a straightforward manner. The immunogen
can be complexed with a preformed complex of nucleic acid and
liposome, or it can be complexed with the liposome at the same time
as the nucleic acid molecule. The immunogen can be effectively
complexed with the liposome simply by gently mixing the immunogen
and the liposome (and the nucleic acid) together, preferably in a
suitable excipient (e.g., 5-10% sucrose or 5-10% lactose). The
immunogen can also be incorporated into the liposome as the
liposome is formulated (e.g., rehydrated). The immunogen can be
mixed with the preformed lipid and nucleic acid complexes; mixed
with the preformed lipid, followed by adding the nucleic acid; or
can be mixed with the nucleic acid and then, together, added to
preformed liposomes.
[0075] A suitable concentration of an immunogen to add to a
liposome includes a concentration effective for delivering a
sufficient amount of immunogen into a mammal such that an
immunogen-specific immune response is elicited, at least at or near
the site of administration, and preferably, systemically.
Preferably, from about 1 .mu.g immunogen per individual mammal to
about 1 mg immunogen per individual mammal is combined with about 8
nmol liposomes (or other suitable amount of liposomes which can be
determined by the skilled artisan), more preferably from about 1
.mu.g immunogen per individual mammal to about 100 .mu.g immunogen
per individual mammal is combined with about 8 nmol liposomes, and
even more preferably from about 1 .mu.g immunogen per individual
mammal to about 10 .mu.g immunogen per individual mammal is
combined with about 8 nmol liposomes. In one embodiment, at least
about 0.1 .mu.g immunogen per individual mammal, and more
preferably at least about 1 .mu.g immunogen per individual mammal,
and more preferably, at least about 5 .mu.g immunogen per
individual mammal, and more preferably at least about 10 .mu.g
immunogen per individual mammal is added to a liposome composition
of the present invention.
[0076] The vaccine of the present invention is capable of
delivering the immunogen to a multitude of tissues, including
spleen, lymph node, pulmonary tissues, and liver. The method of the
present invention is not limited to delivery by intravenous or
intraperitoneal routes, since the present inventors have found that
other routes and combinations of routes are also quite effective at
eliciting an immunogen-specific immune response. For example, the
inventors have found that the combination of any of intravenous,
intraperitoneal or intradermal administration with intranodal
administration (into the lymph node) is particularly effective for
eliciting an immune response.
[0077] A liposome delivery vehicle of the present invention can be
modified to target a particular site in a mammal, thereby targeting
and making use of a nucleic acid molecule of the present invention
at that site. Suitable modifications include manipulating the
chemical formula of the lipid portion of the delivery vehicle.
Manipulating the chemical formula of the lipid portion of the
delivery vehicle can elicit the extracellular or intracellular
targeting of the delivery vehicle. For example, a chemical can be
added to the lipid formula of a liposome that alters the charge of
the lipid bilayer of the liposome so that the liposome fuses with
particular cells having particular charge characteristics. In one
embodiment, other targeting mechanisms, such as targeting by
addition of exogenous targeting molecules to a liposome (i.e.,
antibodies) may not be a necessary component of the liposome of the
present invention, since effective immune activation at
immunologically active organs can already be provided by the
composition when the route of delivery is intravenous or
intraperitoneal, without the aid of additional targeting
mechanisms. However, in some embodiments, a liposome can be
directed to a particular target cell or tissue by using a targeting
agent, such as an antibody, soluble receptor or ligand,
incorporated with the liposome, to target a particular cell or
tissue to which the targeting molecule can bind. Targeting
liposomes are described, for example, in Ho et al., 1986,
Biochemistry 25: 5500-6; Ho et al., 1987a, J Biol Chem 262:
13979-84; Ho et al., 1987b, J Biol Chem 262: 13973-8; and U.S. Pat.
No. 4,957,735 to Huang et al., each of which is incorporated herein
by reference in its entirety). In one embodiment, if avoidance of
the efficient uptake of injected liposomes by reticuloendothelial
system cells due to opsonization of liposomes by plasma proteins or
other factors is desired, hydrophilic lipids, such as gangliosides
(Allen et al., 1987, FEBS Lett 223: 42-6) or polyethylene glycol
(PEG)-derived lipids (Klibanov et al., 1990, FEBS Lett 268: 235-7),
can be incorporated into the bilayer of a conventional liposome to
form the so-called sterically-stabilized or "stealth" liposomes
(Woodle et al., 1992, Biochim Biophys Acta 1113: 171-99).
Variations of such liposomes are described, for example, in U.S.
Pat. No. 5,705,187 to Unger et al., U.S. Pat. No. 5,820,873 to Choi
et al., U.S. Pat. No. 5,817,856 to Tirosh et al.; U.S. Pat. No.
5,686,101 to Tagawa et al.; U.S. Pat. No. 5,043,164 to Huang et
al., and U.S. Pat. No. 5,013,556 to Woodle et al., all of which are
incorporated herein by reference in their entireties).
[0078] A liposome delivery vehicle is preferably capable of
remaining stable in a mammal for a sufficient amount of time to
deliver a nucleic acid molecule and immunogen to a preferred site
in the mammal. A liposome component of the vaccine or therapeutic
composition of the present invention is preferably stable in the
mammal into which it has been administered for at least about 30
minutes, more preferably for at least about 1 hour and even more
preferably for at least about 24-72 hours.
[0079] In another embodiment of the present invention, a vaccine or
therapeutic composition further comprises a pharmaceutically
acceptable excipient. As used herein, a pharmaceutically acceptable
excipient refers to any substance suitable for delivering a
therapeutic composition useful in the method of the present
invention to a suitable in vivo or ex vivo site. Preferred
pharmaceutically acceptable excipients are capable of maintaining a
nucleic acid molecule and/or immunogen in a form that, upon arrival
of the nucleic acid molecule and/or immunogen to a cell, the
nucleic acid molecule and/or protein are capable of contacting
and/or entering the cell and eliciting an immune response at or
near the site of the cell. Suitable excipients of the present
invention include excipients or formularies that transport, but do
not specifically target the vaccine to a cell (also referred to
herein as non-targeting carriers). Examples of pharmaceutically
acceptable excipients include, but are not limited to water,
phosphate buffered saline, Ringer's solution, dextrose solution,
serum-containing solutions, Hank's solution, other aqueous
physiologically balanced solutions, oils, esters and glycols.
Aqueous carriers can contain suitable auxiliary substances required
to approximate the physiological conditions of the recipient, for
example, by enhancing chemical stability and isotonicity.
Particularly preferred excipients include non-ionic diluents, with
preferred non-ionic buffer being 5%-10% sucrose or 5-10% lactose in
water.
[0080] Suitable auxiliary substances include, for example, sodium
acetate, sodium chloride, sodium lactate, potassium chloride,
calcium chloride, and other substances used to produce phosphate
buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances
can also include preservatives, such as thimerosal, m- or o-cresol,
formalin and benzol alcohol. Therapeutic compositions of the
present invention can be sterilized by conventional methods and/or
lyophilized.
[0081] In one embodiment of the invention, the vaccine or
therapeutic composition can additionally include either a cytokine
or a nucleic acid molecule that encodes a cytokine (also referred
to herein as a "cytokine-encoding nucleic acid molecule"). The
nucleic acid molecule encoding a cytokine is typically provided as
a recombinant molecule. The phrase "recombinant molecule" or
"recombinant nucleic acid molecule" primarily refers to a nucleic
acid molecule or nucleic acid sequence operatively linked to a
transcription control sequence, but can be used interchangeably
with the phrase "nucleic acid molecule" which is administered to a
mammal. The nucleic acid molecule can encode a full-length
cytokine, at least a portion of a full-length cytokine that is
biologically active (i.e., has substanstantially similar biological
activity as the naturally occurring cytokine), or a homologue of
the cytokine that is biologically active. As used herein, "at least
a portion of a cytokine" refers to a portion of a cytokine protein
having cytokine activity and being capable of binding to a cytokine
receptor. Preferably, a cytokine-encoding nucleic acid molecule
includes an entire coding region of a cytokine. As used herein, a
homologue of a cytokine is a protein having an amino acid sequence
that is sufficiently similar to a natural cytokine amino acid
sequence so as to have cytokine activity (i.e. activity associated
with naturally occurring, or wild-type cytokines). A homologue can
include natural allelic variants and modified cytokines in which at
least one or a few amino acids have been inserted, deleted,
substituted, and/or derivatized in such a manner that such
modifications do not substantially effect the ability of the
cytokine homologue to function as a cytokine.
[0082] In accordance with the present invention, a cytokine
includes a protein that is capable of affecting the biological
function of another cell. A biological function affected by a
cytokine can include, but is not limited to, cell growth, cell
differentiation or cell death. Preferably, a cytokine of the
present invention is capable of binding to a specific receptor on
the surface of a cell, thereby affecting the biological function of
a cell. Cytokines can be capable of affecting the biological
function of cells, including, but not limited to, a lymphocyte, a
muscle cell, a hematopoietic precursor cell, a mast cell, a natural
killer cell, a macrophage, a monocyte, an epithelial cell, an
endothelial cell, a dendritic cell, a mesenchymal cell, a
Langerhans cell, cells found in granulomas and tumor cells of any
cellular origin, and more preferably a mesenchymal cell, an
epithelial cell, an endothelial cell, a muscle cell, a macrophage,
a monocyte, a T cell and a dendritic cell.
[0083] Preferred cytokines for use in the present invention
include: hematopoietic growth factor, an interleukin, an
interferon, an immunoglobulin superfamily molecule, a tumor
necrosis factor family molecule and/or a chemokine (i.e., a protein
that regulates the migration and activation of cells, particularly
phagocytic cells). A more preferred cytokine is an interleukin. An
even more preferred cytokine is interleukin-2 (IL-2), interleukin-7
(IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15),
interleukin-18 (IL-18), granulocyte-macrophage colony stimulating
factor (GM-CSF) and/or interferon-.gamma. (IFN.gamma.). A
most-preferred cytokine is interleukin-2 (IL-2), interleukin-12
(IL-12), interleukin-18 (IL-18), and/or interleukin-15 (IL-15).
[0084] As will be apparent to one of skill in the art, the present
invention is intended to apply to cytokines derived from all types
of mammals. A preferred mammal from which to derive cytokines
includes a mouse, a human and a domestic pet (e.g., dog, cat). A
more preferred mammal from which to derive cytokines includes a dog
and a human. An even more preferred mammal from which to derive
cytokines is a human. According to the present invention, a
cytokine or cytokine-encoding nucleic acid molecule is preferably
derived from the same species of mammal as the mammal to be
treated. For example, a cytokine-encoding nucleic acid molecule
derived from a canine (i.e., dog) nucleic acid molecule is
preferably used to treat a disease in a canine. It is to be noted
that the term "a" or "an" entity refers to one or more of that
entity; for example, a cytokine refers to one or more cytokines. As
such, the terms "a" (or "an"), "one or more" and "at least one" can
be used interchangeably herein.
[0085] When the cytokine is provided by a nucleic acid molecule
encoding the protein, the nucleic acid sequence encoding the
cytokine is operatively linked to one or more transcription control
sequences to form a recombinant molecule. The phrase "operatively
linked" refers to linking a nucleic acid molecule to a
transcription control sequence in a manner such that the molecule
is able to be expressed when transfected (i.e., transformed,
transduced or transfected) into a host cell. Preferably, a nucleic
acid molecule encoding a cytokine for use in a composition of the
present invention is operatively linked to a transcription control
sequence which allows for transient expression of the molecule in
the recipient mammal. To avoid adverse affects of prolonged immune
activation (e.g., shock, excessive inflammation, immune tolerance),
it is a preferred embodiment of the present invention that a
cytokine encoded by a nucleic acid molecule be expressed in the
immunized mammal for about 72 hours to about 1 month, and
preferably, from about 1 week to about 1 month, and more
preferably, from about 2 weeks to about 1 month. Expression of a
longer period of time than 1 month is not desired in instances
where undesirable effects associated with prolonged immune
activation occur. However, if such effects do not occur for a
particular composition or can be avoided or controlled, then
extended expression is acceptable. In one embodiment, transient
expression can be achieved by selection of suitable transcription
control sequences, for example. Transcription control sequences
which are suitable for transient gene expression are discussed
below.
[0086] Transcription control sequences are sequences which control
the initiation, elongation, and termination of transcription.
Particularly important transcription control sequences are those
which control transcription initiation, such as promoter, enhancer,
operator and repressor sequences. Suitable transcription control
sequences include any transcription control sequence that can
function in at least one of the recombinant cells useful in the
method of the present invention. A variety of such transcription
control sequences are known to those skilled in the art. Preferred
transcription control sequences include those which function in
mammalian, bacteria, insect cells, and preferably in mammalian
cells. More preferred transcription control sequences include, but
are not limited to, simian virus 40 (SV-40); .beta.-actin,
retroviral long terminal repeat (LTR); Rous sarcoma virus (RSV),
cytomegalovirus (CMV), tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB,
bacteriophage lambda (.lamda.) (such as .lamda.p.sub.L and
.lamda.p.sub.R and fusions that include such promoters),
bacteriophage T7, T7lac, bacteriophage T3, bacteri ophage SP6,
bacteriophage SP01, metallothionein, alpha mating factor, Pichia
alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis
virus subgenomic promoters); baculovirus, Heliothis zea insect
virus, vaccinia virus and other poxviruses, herpesvirus, and
adenovirus transcription control sequences, as well as other
sequences capable of controlling gene expression in eukaryotic
cells. Additional suitable transcription control sequences include
tissue-specific promoters and enhancers (e.g., T cell-specific
enhancers and promoters). Transcription control sequences of the
present invention can also include naturally occurring
transcription control sequences naturally associated with a gene
encoding a cytokine.
[0087] Particularly preferred transcription control sequences for
use in the present invention include promoters which allow for
transient expression of a nucleic acid molecule that is to be
expressed, thereby allowing for expression of the cytokine encoded
by the nucleic acid molecule to be terminated after a time
sufficient to elicit an immune response. Adverse effects related to
prolonged activation of the immune system can be avoided by
selection of promoters and other transcription control factors
which allow for transient expression of a nucleic acid molecule.
Suitable promoters for use with nucleic acid molecules encoding
cytokines for use in the present invention include cytomegalovirus
(CMV) promoter and other non-retroviral virus-based promoters such
as RSV promoters, adenovirus promoters and Simian virus promoters.
LTR, tissue-specific promoters, promoters from self-replication
viruses and papillomavirus promoters, which may be quite desirable
in gene therapy/gene replacement protocols because they provide
prolonged expression of a transgene, are not preferred
transcription control sequences for use in the present
invention.
[0088] Recombinant molecules of the present invention, which can be
either DNA or RNA, can also contain additional regulatory
sequences, such as translation regulatory sequences, origins of
replication, and other regulatory sequences that are compatible
with the recombinant cell. In one embodiment, a recombinant
molecule of the present invention also contains secretory signals
(i.e., signal segment nucleic acid sequences) to enable an
expressed cytokine protein to be secreted from the cell that
produces the protein. Suitable signal segments include: (1) a
cytokine signal segment; or (2) any heterologous signal segment
capable of directing the secretion of a cytokine protein according
to the present invention.
[0089] It is noted that the description of recombinant production
of a cytokine presented above can be readily applied to the
encoding and production of any protein to be expressed by a nucleic
acid molecule of the present invention.
[0090] According to the present invention, an effective
administration protocol (i.e., administering a vaccine or
therapeutic composition in an effective manner) comprises suitable
dose parameters and modes of administration that result in
elicitation of an immune response in a mammal that has a disease,
preferably so that the mammal is protected from the disease.
Effective dose parameters can be determined using methods standard
in the art for a particular disease. Such methods include, for
example, determination of survival rates, side effects (i.e.,
toxicity) and progression or regression of disease. In particular,
the effectiveness of dose parameters of a therapeutic composition
of the present invention when treating cancer can be determined by
assessing response rates. Such response rates refer to the
percentage of treated patients in a population of patients that
respond with either partial or complete remission. Remission can be
determined by, for example, measuring tumor-size or microscopic
examination for the presence of cancer cells in a tissue
sample.
[0091] In accordance with the present invention, a suitable single
dose size is a dose that is capable of eliciting an immune response
in a mammal with a disease when administered one or more times over
a suitable time period. Doses can vary depending upon the disease
being treated. In the treatment of cancer, a suitable single dose
can be dependent upon whether the cancer being treated is a primary
tumor or a metastatic form of cancer. One of skill in the art can
readily determine appropriate single dose sizes for systemic
administration based on the size of a mammal and the route of
administration. One of skill in the art can monitor the
effectiveness of the immunization by measuring, for example,
proliferative response, cytokine responses, cytotoxicity, antibody
production, by enumerating antigen-specific T cells, or monitoring
delayed type hypersensitivity (DTH) responses.
[0092] In a preferred embodiment, an appropriate single dose of the
nucleic acid:liposome, portion of the composition of the present
invention is from about 0.1 .mu.g to about 100 .mu.g per kg body
weight of the mammal to which the complex is being administered. In
another embodiment, an appropriate single dose is from about 1
.mu.g to about 10 .mu.g per kg body weight. In another embodiment,
an appropriate single dose of nucleic acid:lipid complex is at
least about 0.1 .mu.g of nucleic acid to the mammal, more
preferably at least about 1 .mu.g of nucleic acid, even more
preferably at least about 10 .mu.g of nucleic acid, even more
preferably at least about 50 .mu.g of nucleic acid, and even more
preferably at least about 100 .mu.g of nucleic acid to the
mammal.
[0093] As discussed above, an appropriate single dose size for the
composition based on the amount of immunogen is an amount of the
vaccine that delivers from about 1 .mu.g immunogen per individual
mammal to about 1 mg immunogen per individual mammal, and more
preferably from about 1 .mu.g immunogen per individual mammal to
about 100 .mu.g immunogen per individual mammal, and even more
preferably from about 10 .mu.g immunogen per individual mammal to
about 100 .mu.g immunogen per individual mammal. In one embodiment,
an appropriate single dose size for the composition based on the
amount of immunogen is an amount of the vaccine that delivers at
least about 0.1 .mu.g immunogen per individual mammal, and more
preferably at least about 1 .mu.g immunogen per individual mammal,
and more preferably, at least about 5 .mu.g immunogen per
individual mammal, and more preferably at least about 10 .mu.g
immunogen per individual mammal. One of skill in the art will
appreciate that the dose amount will depend to some extent on the
size of the mammal to which the composition is being
administered.
[0094] Preferably, when the composition of the present invention
contains a nucleic acid molecule encoding a cytokine which is to be
expressed in the mammal, an appropriate single dose of the nucleic
acid molecule encoding the cytokine results in at least about 1
.mu.g of protein expressed per mg of total tissue protein per .mu.g
of nucleic acid delivered; and preferably, at least about 10 .mu.g
of protein expressed per mg of total tissue protein per .mu.g of
nucleic acid delivered; and even more preferably, at least about 50
.mu.g of protein expressed per mg of total tissue protein per .mu.g
of nucleic acid delivered; and most preferably, at least about 100
.mu.g of protein expressed per mg of total tissue protein per .mu.g
of nucleic acid delivered.
[0095] A suitable single dose of a vaccine or therapeutic
composition of the present invention to elicit a systemic,
immunogen-specific immune response in a mammal is an amount that,
when administered by any route of administration, elicits a
cellular and/or humoral immune response in vivo in a mammal, as
compared to a mammal which has not been administered with the
therapeutic composition of the present invention (i.e., a control
mammal).
[0096] A suitable single dose of a vaccine or therapeutic
composition to elicit an immune response against a tumor is an
amount that is sufficient to reduce, stop the growth of, and
preferably eliminate, the tumor following administration of the
composition into the tissue of the mammal that has cancer. A single
dose of a vaccine or therapeutic composition useful to elicit an
immune response against an infectious disease and/or against a
lesion associated with such a disease is substantially similar to
those doses used to treat a tumor, wherein the amount is sufficient
to reduce, eliminate, or prevent at least one symptom of an
infectious disease or lesion associated with such disease.
Similarly, a single dose of a therapeutic composition useful to
elicit an immune response against an allergen is substantially
similar to those doses used to treat a tumor, wherein the amount is
sufficient to reduce, eliminate or prevent at least one symptom of
allergic inflammation. Also, a single dose of a therapeutic
composition useful to elicit an immune response against a
self-antigen is substantially similar to those doses used to treat
a tumor, wherein the amount is sufficient produce a desired
therapeutic effect in the mammal as a result of eliciting an immune
response against the self-antigen.
[0097] It will be obvious to one of skill in the art that the
number of doses administered to a mammal is dependent upon the
extent of the disease and the response of an individual patient to
the treatment. For example, a large tumor may require more doses
than a smaller tumor. In some cases, however, a patient having a
large tumor may require fewer doses than a patient with a smaller
tumor, if the patient with the large tumor responds more favorably
to the therapeutic composition than the patient with the smaller
tumor. Thus, it is within the scope of the present invention that a
suitable number of doses includes any number require to treat a
given disease.
[0098] It is to be noted that the method of the present invention
further differs from previously described gene therapy/gene
replacement protocols, because the time between administration and
boosting of the nucleic acid:lipid complex is significantly longer
than the typical administration protocol for gene therapy/gene
replacement. For example, elicitation of an immune response using
the compositions and methods of the present invention typically
includes an initial administration of the therapeutic composition,
followed by booster immunizations at 2-4 weeks after the initial
administration, optionally followed by subsequent booster
immunizations every 2-4 weeks after the first booster, as needed to
treat a disease according to the present invention. In contrast,
gene therapy/gene replacement protocols typically require more
frequent administration of a nucleic acid in order to obtain
sufficient gene expression to generate or replace the desired gene
function (e.g., weekly administrations).
[0099] A preferred number of doses of a vaccine or therapeutic
composition of the present invention is from about 2 to about 10
administrations patient, more preferably from about 3 to about 8
administrations per patient, and even more preferably from about 3
to about 7 administrations per patient. Preferably, such
administrations are given once every 2-4 weeks, as described above,
until signs of a therapeutic improvement appear, and then once a
month until the disease is gone, or until sufficient memory immune
response is established to be considered to be effective for
prevention of the disease or condition. Variation of the dose and
frequency of administration can be determined by those of skill in
the art, and will depend on the immunogen being administered, the
type of condition being targeted, and whether prevention or
treatment of the condition is desired.
[0100] As discussed above, a vaccine or therapeutic composition of
the present invention is administered to a mammal in a manner
effective to deliver the composition to a cell, a tissue, and/or
systemically to the mammal, whereby elicitation of an
immunogen-specific immune response is achieved as a result of the
administration of the composition. It is noted that it is not
necessary to specifically target the composition to a particular
cell or tissue, since the inventors have found that several
different modes of administration in the absence of specific
targeting is effective to elicit the desired immune response.
Suitable administration protocols include any in vivo or ex vivo
administration protocol. According to the present invention,
suitable methods of administering a vaccine or therapeutic
composition of the present invention to a patient include any route
of in vivo administration that is suitable for delivering the
composition into a patient. The preferred-routes of administration
will be apparent to those of skill in the art, depending on the
type of condition to be prevented or treated, the immunogen used,
and/or the target cell population. Preferred methods of in vivo
administration include, but are not limited to, intravenous
administration, intraperitoneal administration, intramuscular
administration, intranodal administration, intracoronary
administration, intraarterial administration (e.g., into a carotid
artery), subcutaneous administration, transdermal delivery,
intratracheal administration, subcutaneous administration,
intraarticular administration, intraventricular administration,
inhalation (e.g., aerosol), intracranial, intraspinal, intraocular,
intranasal, oral, bronchial, rectal, topical, vaginal, urethral,
pulmonary, administration, impregnation of a catheter, and direct
injection into a tissue. In particular, any routes of delivery
which elicit an immune response in the mucosal tissues is
preferred. Such routes include bronchial, intradermal,
intramuscular, intranodal, other inhalatory, rectal, subcutaneous,
topical, transdermal, vaginal and urethral routes. Some
particularly preferred routes of administration include,
intravenous, intraperitoneal, subcutaneous, intradermal,
intranodal, intramuscular, transdermal, inhaled, intranasal,
rectal, vaginal, urethral, topical, oral, intraocular,
intraarticular, intracranial, and intraspinal. As discussed
previously, combinations of routes of delivery can be used and in
some instances, may enhance the therapeutic effects of the vaccine
or composition. Therefore, any combination of two or more routes of
administration, performed simultaneously, within a short time
period one after another, or at different time intervals relative
to the immunization schedule (e.g., initial administration versus
boosters), are contemplated by the present inventors. In one
embodiment, a preferred route of administration is a combination of
any one or more of intravenous, intraperitoneal or intradermal
administration with intranodal administration. In another
embodiment where the target cells are in or near a tumor, a
preferred route of administration is by direct injection into the
tumor or tissue surrounding the tumor.
[0101] Ex vivo administration refers to performing part of the
regulatory step outside of the patient, such as administering a
composition of the present invention to a population of cells
removed from a patient under conditions such that the composition
contacts and/or enters the cell, and returning the lipofected cells
to the patient. Ex vivo methods are particularly suitable when the
target cell can easily, be removed from and returned to the
patient.
[0102] Many of the above-described routes of administration,
including intravenous, intraperitoneal, intradermal, and
intramuscular administrations can be performed using methods
standard in the art. Aerosol (inhalation) delivery can also be
performed using methods standard in the art (see, for example,
Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992,
which is incorporated herein by reference in its entirety). Oral
delivery can be performed by complexing a therapeutic composition
of the present invention to a carrier capable of withstanding
degradation by digestive enzymes in the gut of an animal. Examples
of such carriers, include plastic capsules or tablets, such as
those known in the art. One method of local administration is by
direct injection. Direct injection techniques are particularly
useful for administering a composition to a cell or tissue that is
accessible by surgery, and particularly, on or near the surface of
the body. Administration of a composition locally within the area
of a target cell refers to injecting the composition centimeters
and preferably, millimeters from the target cell or tissue.
Suitable sites for administration include sites in which the target
site for immune activation is not restricted to the first organ
having a capillary bed proximal to the site of administration
(i.e., compositions can be administered at an administration site
that is distal to the target immunization site). In other words,
for example, intravenous administration of a composition of the
present invention which is used to treat a kidney tumor in a mammal
can be administered intravenously at any site in the mammal and
will still elicit a strong anti-tumor immune response and be
efficacious at reducing or eliminating the tumor, even though the
kidney is not the first organ having a capillary bed proximal to
the site of administration.
[0103] In the method of the present invention, vaccines and
therapeutic compositions can be administered to any member of the
Vertebrate class, Mammalia, including, without limitation,
primates, rodents, livestock and domestic pets. Livestock include
mammals to be consumed or that produce useful products (e.g., sheep
for wool production). Preferred mammals to protect include humans,
dogs, cats, mice, rats, sheep, cattle, horses and pigs, with humans
and dogs being particularly preferred, and humans being most
preferred. While a therapeutic composition of the present invention
is effective to elicit an immune response against a disease in
inbred species of mammals, the composition is particularly useful
for eliciting an immune response in outbred species of mammals. As
shown in the Examples, the method of the present invention can be
readily translated to large animal spontaneous disease models,
including dogs and cats. Thus, the effectiveness of
lipid-DNA-antigen vaccines is not restricted to any one species of
animal and is predicted to be effective in humans.
[0104] As discussed above, a vaccine or therapeutic composition of
the present invention administered by the present method is useful
for eliciting an immune response in a mammal having a variety of
diseases, and particularly cancer, allergic inflammation and
infectious diseases, as well as diseases that can be ameliorated by
elicitation of an immune response against a self-antigen.
[0105] A therapeutic composition of the present invention is
advantageous for eliciting an immune response in a mammal that has
cancer in that the composition overcomes the mechanisms by which
cancer cells avoid immune elimination (i.e., by which cancer cells
avoid the immune response effected by the mammal in response to the
disease). Cancer cells can avoid immune elimination by, for
example, being only slightly immunogenic, modulating cell surface
antigens and inducing immune suppression. A suitable therapeutic
composition for use in eliciting an immune response in a mammal
that has cancer comprises an immunogen useful for eliciting an
immune response against the cancer, including individual tumor
antigens or epitopes, tumor lysates, or whole tumor cells, and any
combinations thereof. The immunogen(s) can be derived from
autologous tumors or tumor antigens, from allogeneic tumors or
tumor antigens, from xenogenic tumors or tumor antigens, or
combinations thereof. Multiple different types of tumors may also
serve as the source for the immunogens. The composition further
comprises a liposome and a nucleic acid molecule that does not
encode the immunogen. A therapeutic composition of the present
invention, elicits a systemic, immunogen-specific immune response
in the mammal and in particular, activates cytotoxic T cells,
natural killer cells, T helper cells and macrophages, as well as
humoral immunity. Such cellular activation overcomes the otherwise
relative lack of immune response to cancer cells, leading to the
destruction of such cells.
[0106] A therapeutic composition of the present invention which
includes an immunogen that elicits an immune response against a
tumor is useful for eliciting an immune response in a mammal that
has cancer, including both primary tumors and metastatic forms of
cancer. Treatment with the therapeutic composition overcomes the
disadvantages of traditional treatments for metastatic cancers. For
example, compositions of the present invention can target dispersed
metastatic cancer cells that cannot be treated using surgical
methods. In addition, administration of such compositions do not
result in the harmful side effects caused by chemotherapy and
radiation therapy, and can be administered repeatedly. Moreover,
the compositions administered by the method of the present
invention typically elicit a powerful immune response such that
delivery of the composition at least in the vicinity of the target
site (at or adjacent to the site) provides effective immune
activation and efficacy against the target.
[0107] Cancers to be treated or prevented using the method and
composition of the present invention include, but are not limited
to, melanomas, squamous cell carcinoma, breast cancers, head and
neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone
sarcomas, testicular cancers, prostatic cancers, ovarian cancers,
bladder cancers, skin cancers, brain cancers, angiosarcomas,
hemangiosarcomas, mast cell tumors, primary hepatic cancers, lung
cancers, pancreatic cancers, gastrointestinal cancers, renal cell
carcinomas, hematopoietic neoplasias, and metastatic cancers
thereof. Particularly preferred cancers to treat with a therapeutic
composition of the present invention include primary lung cancers
and pulmonary metastatic cancers. A therapeutic composition of the
present invention is useful for eliciting an immune response in a
mammal to treat tumors that can form in such cancers, including
malignant and benign tumors. Preferably, expression of the tumor
antigen in a tissue of a mammal that has cancer produces a result
selected from the group of alleviation of the cancer, reduction of
a tumor associated with the cancer, elimination of a tumor
associated with the cancer, prevention of metastatic cancer,
prevention of the cancer and stimulation of effector cell immunity
against the cancer.
[0108] A vaccine or therapeutic composition of the present
invention which includes an immunogen that is associated with an
infectious disease (e.g., from a pathogenic microorganism that
causes the disease) is advantageous for eliciting an immune
response in a mammal that has an infectious disease that is
beneficially effected by an immune response or to elicit an immune
response m a mammal that will protect the mammal against subsequent
infection by the pathogen. An infectious disease responsive to an
immune response is a disease caused by a pathogen in which the
elicitation of an immune response against the pathogen can result
in a prophylactic or therapeutic effect as previously described
herein. Such a method provides a long term, targeted therapy for
primary lesions (e.g., granulomas) resulting from the propagation
of a pathogen. As used herein, the term "lesion" refers to a lesion
formed by infection of a mammal with a pathogen. The infectious
disease can be either, chronic or acute. A therapeutic composition
for use in the elicitation of an immune response in a mammal that
has an infectious disease comprises an immunogen that is selected
from an antigen from a pathogen, an immunogenic epitope from a
pathogen, a whole pathogen organism, or a lysate or other fraction
of a pathogen organism, a secreted toxin or a spore from the
pathogen, or any combination thereof. Immunogens derived from
different strains of the same or similar pathogens, or from
different pathogens may be combined. The composition further
comprises a liposome and a nucleic acid molecule that does not
encode the immunogen against the pathogen. Similar to the mechanism
described above for the treatment of cancer, eliciting an immune
response in a mammal that has an infectious disease with immunogens
from. the infectious disease pathogens can result in increased T
cell, natural killer cell, macrophage cell activity and humoral
immune responses that overcome the relative lack of immune response
to a lesion formed by a pathogen. Preferably, delivery of the
immunogen to a tissue of a mammal that has an infectious disease
produces a result which includes alleviation of the disease,
regression of established lesions associated with the disease,
alleviation of symptoms of the disease, immunization against the
disease and/or stimulation of effector cell immunity against the
disease.
[0109] Pathogens from which immunogens useful in the present
invention can be derived include, but are not limited to, bacteria
(including intracellular bacteria which reside in host cells),
viruses, parasites (including internal parasites), fungi (including
pathogenic fungi); endoparasites, ectoparasites, and prions (e.g.,
bovine spongiform encephalopathy; BSE). Preferred infectious
diseases to treat or prevent with a vaccine of therapeutic
composition of the present invention include chronic infectious
diseases, including pulmonary infectious diseases, such as
tuberculosis. Particularly preferred infectious diseases to treat
with a therapeutic composition of the present invention include
viral infections such as those caused by human immunodeficiency
virus, Epstein Barr virus, other Herpes viruses, papilloma virus,
and Hepatitis viruses; mycobacterial infections such as that caused
by Mycobacterium tuberculosis, and; fungal infections such as those
caused by Candida, Blastomyces, and Histoplasma; parasitic
infections such as those caused by Toxoplasma; and bacterial
infectious diseases such as those caused by Cryptococcus, Bacillus
anthracis and Yersenia pestis, Staphylococcus, Pseudomonas,
Streptococcus, Enterococcus, Salmonella, Pasteurella, Fransicella
and other gram negative and gram positive bacterial pathogens.
[0110] A therapeutic composition of the present invention which
includes an immunogen that elicits an immune response against an
allergen is advantageous for eliciting an immune response in a
mammal that has or is at risk of developing a disease associated
with allergic inflammation. A disease associated with allergic
inflammation is a disease in which the elicitation of one type of
immune response (e.g., a Th2-type immune response) against a
sensitizing agent, such as an allergen, can result in the release
of inflammatory mediators that recruit cells involved in
inflammation in a mammal, the presence of which can lead to tissue
damage and sometimes death. The method of the present invention
typically elicits a Th1-type response, which, without being bound
by theory, the present inventors believe can have prophylactic or
therapeutic effects such that allergic inflammation is alleviated
or reduced. A therapeutic composition for use in the elicitation of
an immune response in a mammal that has a disease associated with
allergic inflammation comprises an immunogen that is an allergen,
an immunogenic portion of an allergen, an epitope of an allergen,
or combinations thereof. The composition can include multiple
allergens, portions thereof, epitopes thereof, or combinations of
the same. The composition further comprises a liposome and a
nucleic acid molecule that does not encode an immunogen that
elicits an immune response against the target allergen(s). Similar
to the mechanism described above for the treatment of cancer,
eliciting an immune response in a mammal that has a disease
associated with allergic inflammation can result in increased
Th1-type T cell, natural killer cell, or macrophage cell activity
or humoral immune responses, that overcome the harmful effects of a
Th2-type immune response against the same allergen. Preferably,
delivery of the allergen to a tissue of a mammal that has a disease
associated with allergic inflammation or is at risk of developing
the disease, produces a result which includes alleviation of the
disease, alleviation of symptoms of the disease, desensitization
against the disease and stimulation a protective immune response
against the disease. Preferred diseases associated with allergic
inflammation which are preferable to treat using the method and
composition of the present invention include, allergic airway
diseases, allergic rhinitis, allergic conjunctivitis and food
allergy. Vaccines comprising allergen immunogens are, in one
embodiment, administered by a route including aerosol, intranasal,
subcutaneous, intramuscular, or oral administration.
[0111] A therapeutic composition of the present invention which
includes an immunogen that elicits an immune response against a
self-antigen is advantageous for eliciting an immune response in a
mammal that has or is at risk of developing a disease wherein an
immune response against such an antigen will have a therapeutic
benefit in the patient. For example, in conditions where abnormal
blood vessel growth is experienced, the present invention can be
used to immunize the patient against an angiogenic growth factor or
receptor thereof, such as the VEGF receptor. As another example,
immunization against a signaling molecule is used to treat a
neurologic disease. As yet another example, immunization against an
endothelial cell or a fraction thereof is used to treat cancer.
Such immunogens include proteins, portions thereof, carbohydrates,
lipid molecules, normal cells (e.g., non-tumor cells) and fractions
thereof.
[0112] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
Example 1
[0113] The following example shows that immunization with cationic
lipid-DNA complexes combined with tumor lysates generates antitumor
immunity.
[0114] The ability of cationic lipid-DNA complexes (CLDC) to
enhance the effectiveness of vaccination with tumor antigens was
assessed in BALB/c mice using the syngeneic CT26 tumor cell line.
The cationic lipid-DNA complexes were prepared using DOTIM and
cholesterol liposomes and empty vector (EV; non-coding) plasmid
DNA. The tumor cell vaccine consisted of lysates of CT26 tumor
cells, which were prepared by first incubating live tumor cells in
vitro with EV/CLDC for 30 minutes, then subjecting the cells to 4
freeze-thaw cycles. The tumor lysate solution was then added to 1
ml of pre-formed EV/CLDC to prepare lipid-antigen-DNA complexes
(LADC), where the antigen was comprised of tumor cell lysates.
Control tumor cell lysates were prepared by freeze-thaw of tumor
cells without addition of any EV/CLDC. BALB/c mice (4 per group)
were immunized with either: (1) control tumor cell lysates only
("cells"; equivalent to 10.sup.6 tumor cells per mouse); (2) 10
.mu.g EV/CLDC only ("CLDC"); or (3) the tumor vaccine ("Vacc"),
which consisted of tumor lysates plus 10 .mu.g EV/CLDC. Untreated
mice served as controls. Mice were boosted once 7 days later, then
spleen cells were collected 7 days after that and re-stimulated in
vitro with autologous tumor cell lysates.
[0115] Twenty-four hours later, release of IFN-.gamma. into, tissue
culture supernatants was quantitated by specific ELISA assay (FIG.
1). Immunization with the tumor lysate vaccine elicited
significantly greater release of IFN-.gamma. than did immunization
with tumor lysates only or with CLDC only. These data indicate that
EV/CLDC have potent adjuvant activity when combined with tumor
antigens and can generate substantial antitumor immunity against
even syngeneic tumors.
Example 2
[0116] The following example demonstrates that immunization with a
CLDC plus tumor lysates elicits high levels of cytotoxic T
lymphocyte (CTL) activity.
[0117] The effect of tumor vaccination on tumor-specific cytotoxic
T lymphocyte activity (CTL) was assessed using BALB/c mice and the
CT26 tumor line, as described in Example 1 above. BALB/c mice (4
per group) were immunized twice with either tumor lysates only
("cells"), with EV/CLDC only ("CLDC"), or with tumor lysates plus
CLDC ("Vacc"). Untreated mice served as controls. One week after
the second immunization, spleen cells were harvested and
re-stimulated in vitro for 5 days using irradiated CT26 cells. On
day 5, cytotoxic activity of the effector T cells was assessed
using CT26 .sup.51Cr-labeled target cells. As shown in FIG. 2,
vaccination with tumor lysates plus CLDC elicited much greater
levels of CTL activity against autologous tumor cells than
immunization with either tumor lysates alone or CLDC alone.
Example 3
[0118] The following example demonstrates that immunization with
tumor lysates combined with CLDC elicits lymphocyte proliferative
activity.
[0119] BALB/c mice were immunized with either CT26 tumor lysates
alone ("cells"), CLDC alone, or tumor lysates plus CLDC ("Vacc"),
as described in Example 1. One week after a booster vaccination,
spleen cells were harvested and incubated with tumor cell lysates
for 3 days. The proliferative response was assessed by pulsing with
.sup.3H thymidine for 18 h. The mean proliferative response
(.+-.SE) was then plotted. Immunization with tumor lysates plus
CLDC elicited significantly greater proliferative responses than
immunization with tumor lysates alone. These results and the
results from Example 2 (FIG. 2) indicate that a tumor vaccine
prepared using tumor lysates combined with CLDC is capable of
eliciting both CD4+T cell responses (proliferation) and CD8+T cell
responses (CTL activity).
Example 4
[0120] The following example shows that tumor vaccination inhibits
the growth of established lung tumor metastases.
[0121] BALB/c mice (5 per group) with day 3 established CT26 lung
metastases were treated by vaccination with tumor lysates only
("cells"), CLDC only, or tumor lysates plus CLDC ("Vacc"),
administered intravenously, as described in Example 1. The
vaccination was repeated twice at 7-day intervals. Five days later,
the mice were sacrificed and the lung tumor burden was determined
by determining the weight of lung tumor nodules (lung weight minus
weight of normal control lungs). As shown in FIG. 4, therapeutic
vaccination with tumor lysates plus CLDC induced a significantly
greater reduction in lung tumor burden than either tumor cell
lysates alone or CLDC alone. These results demonstrate that the
combination of CLDC plus tumor antigens creates a potent vaccine
for generating therapeutic antitumor immunity.
Example 5
[0122] This example demonstrates that vaccination with autologous
tumor lysates using LADC induces CTL activity in dogs with
osteosarcoma.
[0123] The ability of lipid-antigen-DNA complexes (LADC) to elicit
antitumor immunity was investigated in dogs with spontaneous
osteosarcoma. Tumor lysates and primary cultures of the tumor
tissues were prepared from tumor tissues immediately after
amputation of the limb for control of the primary tumor
(osteosarcoma). The tumor lysates were then added to CLDC to form
LADC. In addition to tumor lysates, a second antigen KLH (keyhole
limpet hemocyanin) was also added to the LADC vaccine. Each dog was
immunized intravenously with LADC once every other week for 6
treatments. Peripheral blood mononuclear cells were collected after
3 immunizations, restimulated in vitro with irradiated autologous
tumor cells for 5 days, then assayed for their ability to lyse
autologous tumor cells or MHC and tissue-type mismatched (melanoma)
tumor cells ("allogeneic"), using a chromium release assay. PBMC
from both patients (FIGS. 5A and 5B, each representing a single
patient) demonstrated increased CTL activity (specific lysis)
against autologous tumor cells, whereas there was little killing of
allogeneic target cells. These results indicate that LADC
vaccination using autologous tumor lysates can generate antitumor
immunity in a large animal model of spontaneous neoplasia.
Example 6
[0124] This example demonstrates that immunization with LADC by
different routes elicits large numbers of antigen-specific CD8+T
cells.
[0125] Mice were immunized twice, 7 days apart, with
liposome-antigen-DNA complexes (LADC) comprised of empty
vector/CLDC (EV/CLDC) and a peptide (SIINFEKL) (SEQ ID NO:1)
derived from ovalbumin. Each mouse received 5 g peptide per
immunization. The T cell response to LADC vaccination was assessed
using MHC-peptide tetramers and flow cytometry. Spleen cells were
harvested after the second immunization and stained with soluble
H-2 Kb molecules-bound to the peptide SIINFEKL. The MHC-peptide
tetrameric complexes bind specifically only to T cells expressing a
T cell receptor that recognizes the SIINFEKL peptide. This
technique allows direct and accurate quantitation of the exact
number of antigen-specific T cells in vivo, without the need for
cell culture or other manipulations. The effect of immunization by
different routes (subcutaneous {SC vacc}, FIG. 6B; intravenous {IV
vacc}, FIG. 6C; and intraperitoneal {IP vacc}, FIG. 6D) was also
assessed. The percentage of tetramer+T cells ("tet+"; upper right
quadrant of each figure) was calculated as a percentage of total
CD8+T cells in the spleen. FIGS. 6A-6D show that, compared to
unvaccinated animals, animals that were immunized with LADC had a
significant increase in the percentage of tet+T cells. The results
indicate that vaccination using LADC can elicit large numbers of
antigen-specific CD8+T cells, even using low doses of peptide
antigens. The intraperitoneal route of immunization (FIG. 6D) was
particularly effective in eliciting T cell responses.
Example 7
[0126] The following example shows that immunization with
peptide-pulsed dendritic cells fails to elicit strong T cell
responses.
[0127] The inventors compared the effectiveness of LADC
immunization with other standard immunization techniques, including
immunization with peptide in Freund's adjuvant, immunization with a
vaccinia virus vector, and immunization with peptide-pulsed
dendritic cells. Dendritic cell immunization is considered
currently to be the most effective means of eliciting T cell
immunity. Mice (3 per group) were immunized with 5.times.10.sup.5
dendritic cells pulsed with SIINFEKL (SEQ ID NO:1) peptide twice,
one week apart. One group of mice was immunized by the SC route
(FIG. 7A) and one group by the IP route (FIG. 7B). Spleen cells
were immunostained with Kb tetramers, as described in Example 6.
The results demonstrate that dendritic cell immunization by either
route elicited only small increases in numbers of antigen-specific
CD8+T cells, and was much less effective than LADC immunization.
Moreover, immunization using peptide in Freund's adjuvant or
vaccinia virus was also much less effective than LADC immunization
(data not shown). Thus, LADC immunization was much more effective
in eliciting strong T cell responses than any of the currently
available techniques for immunization against peptide or protein
antigens.
Example 8
[0128] The following example demonstrates that LADC immunization
also elicits CD8+T cell responses against weak antigens.
[0129] Most tumor antigens that have been identified to date are
actually normal or "self" antigens. These antigens are therefore
difficult to immunize against because the immune system is tolerant
to these antigens. They are therefore considered to be "weak"
antigens. The inventors therefore assessed the ability of LADC
immunization to induce immune responses against an endogenous tumor
antigen (tyrosinase-related protein 2, or trp-2). It has been
difficult in the past to immunize against trp-2 using conventional
vaccine approaches. Mice were immunized twice with 1 .mu.g trp-2
peptide using LADC. T cell responses were assessed using Kb-trp-2
tetramers and flow cytometry. Compared to unvaccinated control mice
(FIG. 8A), mice immunized with LADC+trp2 (FIG. 8B) had a
significant increase in the percentage and number of trp2+CD8+T
cells in the spleen. Thus, LADC immunization is sufficiently potent
to elicit T cell responses against both foreign antigens (e.g.,
ovalbumin) and endogenous tumor antigens such as trp-2.
Example 9
[0130] The following example shows that LADC immunization also
induces strong CD8+T cell responses against protein antigens.
[0131] A major goal for vaccines against viruses, tumors, and
intracellular bacterial infections is induction of CD8+T cell
responses, in addition to antibody and CD4+T cell responses.
Because of the nature of antigen processing and presentation, it is
very difficult to immunize against protein antigens and elicit
CD8+T cell responses. The inventors therefore assessed the ability
of LADC to introduce antigens into a class I MHC pathway and elicit
CD8+T cell responses. Mice were immunized against the ovalbumin
(Ova) protein using LADC (5 .mu.g Ova per mouse) and the CD8+T cell
response was quantitated in spleen cells and lymph nodes using
tetramers (see Example 6). It was found, unexpectedly, that LADC
were capable of eliciting very strong antigen-specific CD8+T cell
responses against SIINFEKL (the dominant Ova peptide; SEQ ID NO: 1)
following LADC immunization (FIG. 9B). In fact, the response to
protein vaccination was in many cases superior to that elicited by
peptide immunization (e.g., see Example 6 and FIG. 6). In contrast,
immunization using dendritic cells pulsed with Ova protein did not
elicit any detectable CD8+T cell responses (data not shown). Thus,
the LADC vaccine approach is particularly effective in eliciting
CD8+T cell responses not only against peptides but alto against
whole proteins. This is very important from the standpoint of
vaccine development because it is much easier to prepare vaccines
using whole proteins than to use peptide antigens.
Example 10
[0132] The following example shows that LADC efficiently introduce
protein antigens into a class I MHC pathway for presentation to
CD8+T cells.
[0133] The ability to introduce protein antigens into an MHC class
I pathway represents a very desirable attribute for a vaccine. The
inventors therefore assessed the ability of LADC to introduce
antigens into a class I pathway in vitro. EL-4 cells were incubated
with the Ova protein at a concentration of 1 .mu.g/ml in vitro for
3 h. EL-4 cells were incubated with either ovalbumin protein alone
(OVA), ovalbumin in liposomes alone (OVA+lipos), or ovalbumin in
LADC (OVA+LADC). Cells incubated with the SIINFEKL peptide alone
(SEQ ID NO:1) served as a positive control, while EL-4 cells
incubated with nothing served as a negative control. After
incubation, the cells were washed, then irradiated for 30 minutes.
Lymph node T cells were obtained from a mouse that was transgenic
for the T cell receptor that recognizes SIINFEKL (these T cells
respond by vigorous proliferation when pulsed with the SIINFEKL
peptide). These T cells were then added in triplicate cultures to
the EL-4 cells incubated with Ova. After 48 hours of culture,
proliferation was assessed by pulsing with 3H thymidine. As shown
in FIG. 10, strong proliferation was elicited by pulsing with
SIINFEKL (positive control) and also by pulsing with Ova in LADC,
but not by Ova alone or by Ova with liposomes alone. Thus, LADC
appear to be uniquely effective in efficiently introducing protein
antigens into a MHC class I pathway for antigen presentation to
CD8+T cells. This responses also occurs efficiently in vivo, as
demonstrated by the results presented in Example 9 (FIG. 9).
Example 11
[0134] The following example shows that liposome-DNA complexes, and
not liposomes or DNA alone, are required for effective
immunization.
[0135] The inventors conducted experiments to determine whether the
liposome and DNA complex was necessary for efficient induction of T
cell responses, or whether immunization with antigen plus liposomes
alone or antigen plus bacterial DNA alone could also produce
effective immunization. Mice (3 per group) were immunized twice
intraperitoneally with ova (5 .mu.g per mouse) plus empty vector
(EV) plasmid DNA alone (FIG. 11A), ova plus liposomes alone (FIG.
11B), or ova plus liposomes and DNA (LADC, FIG. 11C. Spleen cells
were then collected and immunostained to quantitate
antigen-specific CD8+T cell responses, using Kb-SIINFEKL tetramers,
as described in Example 6. The inventors found that only the
combination of liposomes, DNA, and antigen was able to elicit
effective T cells responses. These results were also confirmed
using functional T cell assays (cytokine release and cytotoxicity;
data not shown). Thus, the synergistic immune stimulatory activity
of liposomes plus DNA is required for effective immunization using
liposomes and DNA as vaccine adjuvants.
Example 12
[0136] The following example shows that immunization using LADC
elicits functional activation of T cells and production of
IFN-.gamma.. Generation of functionally active T cells following
LADC immunization was assessed using cytokine release assays. Mice
were immunized intraperitoneally with either SIINFEKL-LADC
(LADC-peptide) or ova-LADC (FIG. 12A), or trp-2 LADC or CLDC alone
(LDC) (FIG. 12B). Spleens from immunized mice (4 per group) were
then restimulated in vitro for 24 h with the appropriate peptides
and the concentration of IFN-.gamma. in the supernatants was
assessed by ELISA. Immunization with LADC elicited high levels of
the key immunoregulatory cytokine IFN-.gamma., indicative of
functional activation. In contrast, IL-4 was not produced (data not
shown). Thus, immunization with LADC generates functionally active
T cells of the Th1 phenotype.
Example 13
[0137] The following example demonstrates that immunization with
LADC elicits functional activation of CD8+T cells and high levels
of cytotoxicity.
[0138] The ability of immunization with LADC to elicit functionally
active T cells was also assessed using cytotoxicity assays. Spleen
cells from mice (3-4 per group) immunized with ova-LADC by the IV
route (FIG. 13A) or the SC route (FIG. 13B), or with trp-2-LADC by
the IV route (FIG. 13C) or unvaccinated control mice (FIG. 13D)
were restimulated with relevant peptides for 5 days in the presence
of recombinant human interleukin-2 (rhuIL-2). The effector T cells
were then incubated for 4 h with chromium labeled target cells that
were either pulsed with relevant SIINFEKL (SEQ ID NO: 1) or trp-2
peptides ("peptide+") or were unpulsed controls ("peptide-"). The
percentage specific lysis was calculated and plotted for each
group. FIGS. 13A-D show that immunization with ova or trp-2
elicited high levels of peptide-specific cytotoxic activity. Thus,
LADC immunization elicits high levels of functionally active T
cells with strong cytotoxic activity.
Example 14
[0139] The following example demonstrates that therapeutic
vaccination with LADC controls the growth of established
tumors.
[0140] The ability of LADC immunization to elicit therapeutic
anti-tumor immunity was assessed using the B16 tumor model. Mice (5
per group) with day 7 established B16 SC tumors were immunized with
LADC prepared with trp-2 peptide (5 .mu.g per mouse) by either the
IV or SC routes. Three immunizations were administered at 7 day
intervals and the effect on tumor growth was assessed by serial
measurements of tumor volume. Untreated mice served as controls.
FIG. 14 shows that immunization with trp-2 LADC by either the SC or
IV routes significantly inhibited the growth of established B16
tumors. Thus, LADC immunization, using even a weak tumor antigen,
elicited sufficient T cell immunity to control the growth of even a
very aggressive tumor such as B16.
Example 15
[0141] The following example shows that immunization with LADC
elicits strong CD4+T cell responses.
[0142] The inventors assessed the ability of LADC immunization to
elicit CD4+T cell responses by using a peptide (pigeon cytochrome
c; PCC) that binds to the class II MHC. CD4+T cell responses were
enumerated using MHC class II tetramers (I-Ek), as described for
MHC class I tetramers in Example 6. Mice (3 per group) were
immunized with LADC containing either the PCC protein or the PCCV
MHC class II-binding peptide. Mice were immunized by the SC or IP
routes twice, 7d apart, then spleen cells were collected and
immunostained with tetramer and analyzed by flow cytometry. FIG. 15
shows that immunization with either peptide or whole protein
elicited a significant increase in numbers of antigen-specific
CD4+T cells. Untreated control mice did not have detectable
PCC-specific T cells (data not shown). The IP route of immunization
was consistently most effective, consistent with the results
observed previously for CD8+T cell responses. Moreover, the CD4+T
cell response to LADC immunization was also much stronger than the
CD4+T cell response to either peptide and Freund's adjuvant or
peptide-pulsed dendritic cells (data not shown). These results
indicated that LADC can be used to efficiently elicit both CD8+ and
CD4+T cell responses, thus giving balanced T cell immune
responses.
Example 16
[0143] The following example shows that immunization with LADC
elicits CD4+T cell responses in dogs.
[0144] The inventors assessed the ability of LADC vaccination to
elicit CD4+ cell responses in dogs. A dog with osteosarcoma was
immunized with an autologous tumor lysate vaccine prepared using
LADC. A novel antigen (keyhole limpet hemacyanin; KLH) was also
added to the vaccine in order to elicit CD4+T cell responses. The
dog was immunized 3 times with the LADC vaccine, receiving 50 .mu.g
KLH with each vaccine. PBMC were collected after the third
immunization and incubated for 5 days in 10 .mu.g/ml KLH in
quadruplicate wells in vitro and proliferation was assessed using
3H uptake. Unstimulated PBMC served as a control. Significant
proliferative responses to KLH were observed, indicative of CD4+T
cell immunity. In addition, the dog mounted a strong DTH response
to intradermal skin testing with the KLH antigen (data not shown).
These results indicate that LADC can be used to induce CD4+T cell
responses in a large, outbred animal, using even low antigen
doses.
Example 17
[0145] The following example demonstrates that immunization with
LADC elicits strong antibody responses.
[0146] The present inventors assessed the ability of LADC to elicit
humoral immune responses by immunizing mice with low doses (10
.mu.g per mouse) or ovalbumin protein. In addition, the inventors
compared the antibody response to LADC immunization to that
elicited by immunization with ova protein in conventional Freund's
complete adjuvant (CFA). Mice (4 per group) were immunized once,
then boosted 2 weeks later. Serum was collected prior to
immunization then at one week intervals after immunization.
Antibody titers to ova were quantitated using an ELISA assay.
Immunization with LADC was very effective in eliciting antibody
responses when compared to a standard method of immunization
(antigen in CFA). These data indicate that LADC-based vaccines, in
addition to eliciting T cell immunity, are also very effective in
eliciting humoral immunity.
Example 18
[0147] The following example demonstrates that immunization with
LADC elicits virus-specific CD8+T cells.
[0148] The ability of the LADC vaccine to elicit immunity against
viral antigens was assessed using an antigen from the Sendai virus.
Mice were immunized twice with 5 .mu.g peptide in the LADC vaccine.
Five days after the second vaccination, spleen cells were collected
and immunostained with Kb-Sendai peptide tetramers and analyzed by
flow cytometry. As shown in FIG. 18, there was a substantial
expansion in the number of Sendai specific CD8+T cells in Sendai
vaccinated mice (FIG. 18A), compared to control mice (FIG. 18B).
These data illustrate that the LADC vaccine is versatile and can
also be used to generate effective antiviral immunity, in addition
to antitumor immunity.
Example 19
[0149] The following example demonstrates that vaccination against
viral antigens using LADC elicits high levels of IFN-.gamma.
production by virus antigen-specific T cells.
[0150] Mice (3 per group) were immunized twice with LADC containing
5 .mu.g of Sendai virus peptide. Spleen cells were collected and
re-stimulated for 18 h in vitro, either with ("+pep") or without
("-pep") Sendai virus peptide. Supernatants from the spleen
cultures were then assayed for production of IFN-.gamma. by ELISA.
As shown in FIG. 19, immunization with LADC Sendai peptide elicited
high levels of virus-antigen specific production of IFN-.gamma.. In
contrast, re-stimulation of spleen cells from non-vaccinated
(control) mice in vitro with virus peptide did not elicit
IFN-.gamma. production. These data indicate that LADC immunization
against viral antigens elicits functionally active,
antigen-specific T cells.
[0151] In summary, the examples presented above show:
[0152] 1. Vaccination using lipid-DNA complexes and pools of tumor
antigens prepared from tumor lysates is an effective means of
generating CD8+T cell responses against established tumors.
Induction of antitumor activity has been observed in both
experimental rodent models and in dogs with spontaneous tumors.
[0153] 2. Lipid-DNA complexes combined with defined antigenic
peptides can elicit strong antigen-specific CD8+ and CD4+T cell
responses following immunization in vivo, using even very low
antigen doses.
[0154] 3. Vaccines comprised of antigens plus lipid-DNA complexes
are more effective in eliciting T cell response that currently
available vaccine technologies, including dendritic cell
immunization.
[0155] 4. Lipid-DNA vaccines are sufficiently potent to elicit T
cell responses against even weak antigens such as endogenous tumor
antigens.
[0156] 5. Use of lipid-DNA complexes to vaccinate against protein
antigens elicits surprisingly potent CD8+T cell responses. This
result is quite unexpected given the inherent difficulties
associated with elicitation of CD8+T cell responses against protein
antigens in general.
[0157] 6. Immunization with lipid-DNA-antigen complexes also
generates effective humoral immune responses.
[0158] 7. Lipid-antigen-DNA complexes can also be used to generate
effective T cell immunity against viruses. The immune response
elicited includes high levels of virus-specific CD8+T cells; these
virus-specific T cells also produce high levels of IFN-.gamma..
[0159] 8. The effectiveness of lipid-DNA-protein/peptide vaccines
can be readily translated to large animal spontaneous disease
models, including dogs and cats. Thus, the effectiveness of
lipid-DNA-antigen vaccines is not restricted to any one species of
animal. This result is consistent with the broad cross-species
immune activating properties of cationic lipid-DNA complexes in
general.
[0160] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
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