U.S. patent application number 10/772913 was filed with the patent office on 2004-08-12 for systemic immune activation method using nucleic acid-lipid complexes.
Invention is credited to Dow, Steven W., Elmslie, Robyn E., Gelfand, Erwin W., Schwarze, Jurgen Karl Johannes.
Application Number | 20040157791 10/772913 |
Document ID | / |
Family ID | 22302219 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157791 |
Kind Code |
A1 |
Dow, Steven W. ; et
al. |
August 12, 2004 |
Systemic immune activation method using nucleic acid-lipid
complexes
Abstract
This invention relates to a method for systemic 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, or an infectious disease.
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: |
HOGAN & HARTSON LLP
ONE TABOR CENTER, SUITE 1500
1200 SEVENTEENTH ST
DENVER
CO
80202
US
|
Family ID: |
22302219 |
Appl. No.: |
10/772913 |
Filed: |
February 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10772913 |
Feb 5, 2004 |
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09104759 |
Jun 25, 1998 |
|
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6693086 |
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Current U.S.
Class: |
514/44R ;
424/450 |
Current CPC
Class: |
A61K 2039/55555
20130101; A61K 39/0011 20130101; A61K 48/00 20130101; A61K 2039/53
20130101; A61P 37/00 20180101; A61K 39/35 20130101; A61K 38/208
20130101; A61P 39/00 20180101; A61K 38/217 20130101; A61K 9/1272
20130101; A61K 38/2013 20130101 |
Class at
Publication: |
514/044 ;
424/450 |
International
Class: |
A61K 048/00; A61K
009/127 |
Claims
What is claimed is:
1. A method to elicit an immunogen-specific immune response and a
systemic, non-specific immune response in a mammal, comprising
administering to said mammal a therapeutic composition by a route
of administration selected from the group consisting of intravenous
and intraperitoneal, said therapeutic composition comprising: (a) a
liposome delivery vehicle; and, (b) a recombinant nucleic acid
molecule comprising an isolated nucleic acid sequence encoding an
immunogen, said nucleic acid sequence being operatively linked to a
transcription control sequence.
2. The method of claim 1, wherein said route of administration is
intravenous.
3. The method of claim 1, wherein said immunogen is selected from
the group consisting of a tumor antigen, an infectious disease
pathogen antigen and an allergen.
4. The method of claim 1, wherein said therapeutic composition
further comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, said nucleic acid
sequence being operatively linked to a transcription control
sequence.
5. The method of claim 4, wherein said nucleic acid sequence
encoding said immunogen and said nucleic acid sequence encoding
said cytokine are in the same recombinant nucleic acid molecule,
said nucleic acid sequences being operatively linked to at least
one transcription control sequence.
6. The method of claim 4, wherein said nucleic acid sequence
encoding said immunogen and said nucleic acid sequence encoding
said cytokine are operatively linked to different transcription
control sequences.
7. The method of claim 4, wherein said cytokine is selected from
the group consisting of hematopoietic growth factors, interleukins,
interferons, immunoglobulin superfamily molecules, tumor necrosis
factor family molecules and chemokines.
8. The method of claim 4, wherein said cytokine is an
interleukin.
9. The method of claim 4, wherein said cytokine is selected from
the group consisting of interleukin-2, interleukin-7,
interleukin-12, interleukin-15, interleukin-18, and
interferon-*.
10. The method of claim 4, wherein said cytokine is selected from
the group consisting of interleukin-2, interleukin-12,
interleukin-18, and interferon-*.
11. The method of claim 1, wherein said transcription control
sequences are selected from the group consisting of Rous sarcoma
virus (RSV) control sequences, cytomegalovirus (CMV) control
sequences, adenovirus control sequences and Simian virus (SV-40)
control sequences.
12. The method of claim 1, wherein said liposome delivery vehicle
comprises lipids selected from the group consisting of
multilamellar vesicle lipids and extruded lipids.
13. The method of claim 1, wherein said liposome delivery vehicle
comprises multilamellar vesicle lipids.
14. The method of claim 1, wherein said liposome delivery vehicle
comprises cationic liposomes.
15. The method of claim 1, wherein said liposome delivery vehicle
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 method of claim 1, wherein said liposome delivery vehicle
comprises DOTAP and cholesterol.
17. The method of claim 1, wherein expression of said immunogen in
a tissue of said mammal elicits said immunogen-specific immune
response in said mammal.
18. The method of claim 1, wherein administering said nucleic acid
molecule and said liposome elicit said systemic, non-specific
immune response in said mammal.
19. The method of claim 1, wherein said mammal is selected from the
from the group consisting of humans, dogs, cats, mice, rats, sheep,
cattle, horses and pigs.
20. The method of claim 1, wherein said mammal is a human.
21. The method of claim 1, wherein said composition has a nucleic
acid:lipid ratio of from about 1:1 to about 1:64.
22. The method of claim 1, wherein said mammal has cancer and
wherein said immunogen is a tumor antigen.
23. The method of claim 22, wherein said therapeutic composition
further comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, said nucleic acid
sequence being operatively linked to a transcription control
sequence.
24. The method of claim 22, wherein said therapeutic composition
comprises a plurality of recombinant nucleic acid molecules, each
of said recombinant nucleic acid molecules comprising a cDNA
sequence amplified from total RNA isolated from an autologous tumor
sample, each of said cDNA sequences encoding a tumor antigen or a
fragment thereof and being operatively linked to a transcription
control sequence.
25. The method of claim 22, wherein said therapeutic composition
comprises a plurality of recombinant nucleic acid molecules, each
of said recombinant nucleic acid molecules comprising a cDNA
sequence amplified from total RNA isolated from a plurality of
allogeneic tumor samples of the same histological tumor type, each
of said cDNA sequences encoding a tumor antigen or a fragment
thereof and being operatively linked to a transcription control
sequence.
26. The method of claim 22, wherein said cancer is selected from
the group consisting 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.
27. The method of claim 22, wherein said cancer is selected from
the group consisting of a primary lung cancer and a pulmonary
metastatic cancer.
28. The method of claim 22, wherein said tumor antigen is from a
cancer selected from the group consisting 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.
29. The method of claim 22, wherein said tumor antigen is selected
from the group consisting of 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.
30. The method of claim 22, wherein said expression of said tumor
antigen produces a result selected from the group consisting of
alleviation of said cancer, reduction of size of a tumor associated
with said cancer, elimination of a tumor associated with said
cancer, prevention of metastatic cancer, prevention of said cancer
and stimulation of effector cell immunity against said cancer.
31. The method of claim 22, wherein said expression of said tumor
antigen in a pulmonary tissue by administration of said composition
by an intravenous route prevents pulmonary metastatic cancer in
said mammal.
32. The method of claim 1, wherein said mammal has an infectious
disease responsive to an immune response, and wherein said
immunogen is an infectious disease pathogen antigen.
33. The method of claim 32, wherein said therapeutic composition
further comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, said nucleic acid
sequence being operatively linked to a transcription control
sequence.
34. The method of claim 32, wherein said immunogen is from an
infectious disease pathogen selected from the group consisting of
bacteria, viruses, parasites, and fungi.
35. The method of claim 34, wherein said infectious disease
pathogen causes a chronic infectious disease in said mammal.
36. The method of claim 34, wherein said infectious disease
pathogen is selected from the group consisting of human
immunodeficiency virus (HIV), Mycobacterium tuberculosis,
herpesvirus, papillomavirus and Candida.
37. The method of claim 32, wherein said expression of said
pathogen antigen in a tissue of said mammal produces a result
selected from the group consisting of alleviation of said disease,
regression of established lesions associated with said disease,
alleviation of symptoms of said disease, immunization against said
disease and stimulation of effector cell immunity against said
disease.
38. The method of claim 32, wherein said therapeutic composition
comprises a plurality of recombinant nucleic acid molecules, each
of said recombinant nucleic acid molecules comprising a cDNA
sequence amplified from total RNA isolated from an infectious
disease pathogen, each of said cDNA sequences encoding an immunogen
from said infectious disease pathogen or a fragment thereof and
being operatively linked to a transcription control sequence.
39. The method of claim 32, wherein said infectious disease
pathogen is a virus.
40. The method of claim 39, wherein said virus is selected from the
group consisting of human immunodeficiency virus and feline
immunodeficiency virus.
41. The method of claim 32, wherein said infectious disease is
tuberculosis.
42. The method of claim 41, wherein said immunogen is a
Mycobacterium tuberculosis antigen.
43. The method of claim 41, wherein said immunogen is antigen
85.
44. The method of claim 1, wherein said mammal has a disease
associated with allergic inflammation and wherein immunogen is an
allergen.
45. The method of claim 44, wherein said therapeutic composition
further comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, said nucleic acid
sequence being operatively linked to a transcription control
sequence.
46. The method of claim 44, wherein said allergen is selected from
the group consisting of plant pollens, drugs, foods, venoms, insect
excretions, molds, animal fluids, animal hair and animal
dander.
47. The method of claim 44, wherein said disease is selected from
the group consisting of allergic airway diseases, allergic
rhinitis, allergic conjunctivitis, and food allergy.
48. The method of claim 44, wherein said expression of said
allergen in a tissue of said mammal produces a result selected from
the group consisting of alleviation of said disease, alleviation of
symptoms of said disease, desensitization against said disease, and
stimulation of a protective immune response against said
disease.
49. The method of claim 44, wherein said therapeutic composition
comprises a plurality of recombinant nucleic acid molecules, each
of said recombinant nucleic acid molecules comprising a cDNA
sequence amplified from total RNA isolated from an allergen, each
of said cDNA sequences encoding said allergen or a fragment thereof
and being operatively linked to a transcription control
sequence.
50. A method to elicit a tumor antigen-specific immune response and
a systemic, non-specific immune response in a mammal that has
cancer, comprising administering to a mammal a therapeutic
composition by a route of administration selected from the group
consisting of intravenous and intraperitoneal administration, said
therapeutic composition comprising: (a) a liposome delivery
vehicle; and, (b) total RNA isolated from a tumor sample, said RNA
encoding tumor antigens.
51. The method of claim 50, wherein said route of administration is
intravenous.
52. The method of claim 50, wherein said therapeutic composition
further comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, said nucleic acid
sequence being operatively linked to a transcription control
sequence.
53. The method of claim 50, wherein said RNA is enriched for poly-A
RNA prior to said administration to said mammal.
54. A method to elicit a pathogen-antigen-specific immune response
and a systemic, non-specific immune response in a mammal that has
an infectious disease, comprising administering to a mammal a
therapeutic composition by a route of administration selected from
the group consisting of intravenous and intraperitoneal
administration, said therapeutic composition comprising: (a) a
liposome delivery vehicle; and, (b) total RNA isolated from an
infectious disease pathogen, said RNA encoding pathogen
antigens.
55. The method of claim 54, wherein said route of administration is
intravenous.
56. A composition for systemic administration to a mammal to elicit
an immunogen-specific immune response and a systemic, non-specific
immune response, comprising: (a) a liposome delivery vehicle; and
(b) a recombinant nucleic acid molecule comprising an isolated
nucleic acid sequence encoding an immunogen, said nucleic acid
sequence being operatively linked to a transcription control
sequence; wherein said composition has a nucleic acid:lipid ratio
of from about 1:1 to about 1:64.
57. The method of claim 56, wherein said liposome delivery vehicle
comprises lipids selected from the group consisting of
multilamellar vesicle lipids and extruded lipids.
58. The method of claim 56, wherein said composition has a nucleic
acid:lipid ratio of from about 1:10 to about 1:40.
59. The composition of claim 56, wherein said liposome comprises
multilamellar vesicle lipids.
60. The composition of claim 56, wherein said liposome delivery
vehicle comprises cationic liposomes.
61. The composition of claim 56, wherein said liposome delivery
vehicle comprises pairs of lipids selected from the group
consisting of DOTMA and cholesterol; DOTAP and cholesterol; DOTIM
and cholesterol; and DDAB and cholesterol.
62. The composition of claim 56, wherein said liposome delivery
vehicle comprises DOTAP and cholesterol.
63. The composition of claim 56, further comprising a
pharmaceutically acceptable excipient.
64. The composition of claim 63, wherein said excipient comprises a
non-ionic diluent.
65. The composition of claim 64, wherein said excipient is 5
percent dextrose in water (D5W).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 09/104,759, filed Jun. 25, 1998, which is
specifically incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a composition and method to
elicit an immune response in a mammal using a genetic immunization
strategy. More particularly, the present invention includes
compositions and methods for eliciting systemic, non-specific
(i.e., non-antigen-specific) immune responses in a mammal as well
as antigen-specific immune responses, both of which are useful in
immunization protocols.
BACKGROUND OF THE INVENTION
[0003] Vaccines are widely used to prevent disease and to treat
established diseases (therapeutic vaccines). There remains,
however, an urgent need to develop safe and effective vaccines and
adjuvants for a variety of diseases, including those due to
infection by pathogenic agents, cancers and other disorders
amenable to treatment by elicitation of an immune response.
[0004] Three major types of disease in mammals which are amenable
to elicitation and/or modulation of an immune response include
infectious diseases, allergic inflammatory diseases and cancer,
although the present invention is not limited to treatment of these
disease types. Infectious diseases are caused by infectious agents
(i.e., infectious disease pathogens), examples of which include
viruses, bacteria, parasites, 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.
[0007] Vaccines can be used not only to prevent disease, but can
also be used to treat established diseases (i.e., therapeutic
vaccines). A number of tumor antigens that are recognized by T
lymphocytes of the immune system have been recently identified and
are being considered as potential vaccine candidates. Conventional
vaccines generally consist of either (1) purified antigens
administered with an adjuvant, 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.
[0008] Genetic vaccines, by contrast, 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, also represents a potential
drawback, however, since such immune responses often prevent
readministration of the vaccine.
[0009] Therefore, there is need to provide better vaccines which
can produce an immune response which is safe, antigen-specific and
effective to prevent and/or treat diseases amenable to treatment by
elicitation of an immune response, such as infectious disease,
allergy and cancer.
SUMMARY
[0010] One embodiment of the present invention generally relates to
a method to elicit a systemic, non-antigen-specific immune response
in a mammal. The method includes the step of administering to the
mammal a therapeutic composition by a route of administration
selected from intravenous and intraperitoneal administration. The
therapeutic composition includes: (a) a liposome delivery vehicle;
and, (b) an isolated nucleic acid molecule that is not operatively
linked to a transcription control sequence. In another embodiment,
the route of administration is intravenous. In further embodiments
of the method, the isolated nucleic acid molecule comprises a
non-coding sequence. In one embodiment, the isolated nucleic acid
molecule does not comprise a bacterial nucleic acid sequence.
[0011] Accordingly, another embodiment of the present invention is
a composition for eliciting a systemic, non-antigen-specific immune
response in a mammal. Such a composition includes (a) a liposome
delivery vehicle; and (b) an isolated nucleic acid molecule that is
not operatively linked to a transcription control sequence. In one
embodiment, the nucleic acid molecule does not include a bacterial
nucleic acid sequence.
[0012] Another embodiment of the present invention relates to a
composition for eliciting a systemic, non-antigen-specific immune
response in a mammal which comprises (a) a liposome delivery
vehicle and (b) an isolated non-coding nucleic acid sequence.
[0013] A composition of the present invention can further comprise
a pharmaceutically acceptable excipient. A pharmaceutically
acceptable excipient can include, for example a non-ionic diluent,
and more preferably, 5 percent dextrose in water (D5W).
[0014] The above-mentioned method and compositions of the present
invention have the advantages of eliciting a systemic, non-antigen
specific immune response in a mammal, and more particularly, of
eliciting a systemic, anti-viral immune response in a mammal.
Additionally, the method and composition of the present invention
can elicit a systemic, anti-tumor immune response in a mammal. Such
an anti-tumor immune response can result in the reduction of a
tumor in the mammal. The method and composition of the present
invention can also elicit a systemic, protective immune response
against allergic inflammation in a mammal. The systemic,
non-antigen-specific immune response elicited by the method and
composition of the present invention result in an increase in
effector cell activity, and particularly, natural killer (NK) cell
activity in the mammal, and additionally can result in increased
production of IFN* in the mammal.
[0015] Yet another embodiment of the present invention relates to a
method to elicit an immunogen-specific immune response and a
systemic, non-specific immune response in a mammal. The method
includes administering to the mammal a therapeutic composition by a
route of administration selected from intravenous and
intraperitoneal. The therapeutic composition comprises: (a) a
liposome delivery vehicle; and, (b) a recombinant nucleic acid
molecule comprising an isolated nucleic acid sequence encoding an
immunogen, wherein the nucleic acid sequence is operatively linked
to a transcription control sequence. Particularly suitable
transcription control sequences include Rous sarcoma virus (RSV)
control sequences, cytomegalovirus (CMV) control sequences,
adenovirus control sequences and Simian virus (SV-40) control
sequences. This method of the present invention has the particular
advantage of eliciting both a systemic, non-immunogen-specific
immune response in a mammal, as well as an immunogen-specific
immune response that have a potent therapeutic effect in the
mammal. In one embodiment, the route of administration is
intravenous. In other preferred embodiments, the immunogen is a
tumor antigen, an infectious disease pathogen antigen or an
allergen.
[0016] When the mammal has cancer, this immunogen is preferably a
tumor antigen. In one embodiment of this method, the therapeutic
composition can include a plurality of recombinant nucleic acid
molecules, each of the recombinant nucleic acid molecules
comprising a cDNA sequence amplified from total RNA isolated from
an autologous tumor sample, each of the cDNA sequences encoding a
tumor antigen or a fragment thereof and being operatively linked to
a transcription control sequence. In another embodiment, the
therapeutic composition comprises a plurality of recombinant
nucleic acid molecules, each of the recombinant nucleic acid
molecules comprising a cDNA sequence amplified from total RNA
isolated from a plurality of allogeneic tumor samples of the same
histological tumor type, each of the cDNA sequences encoding a
tumor antigen or a fragment thereof and being operatively linked to
a transcription control sequence.
[0017] The methods and compositions of the present invention are
particularly useful for treating a cancer which includes 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. The compositions and
methods of the present invention are especially useful for treating
primary lung cancer or pulmonary metastatic cancer.
[0018] Accordingly, a tumor antigen useful in the present
composition is preferably 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. The tumor antigen preferably is selected from the group of
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/or tumor antigens that are expressed by tumor cells and by
non-tumor cells.
[0019] When the immunogen is a tumor antigen which is expressed in
the mammal, the method of the present invention produces a result
selected from alleviation of the cancer, reduction of size 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. When the tumor antigen is administered intravenously, the
antigen is expressed in a pulmonary tissue of the mammal and
prevents pulmonary metastatic cancer in the mammal.
[0020] When the immunogen is an infectious disease pathogen
antigen, the methods and composition of the present invention are
useful for mammals having an infectious disease, and particularly
for mammals having a chronic infectious disease. Such immunogens
can be from infectious disease pathogens which include bacteria,
viruses, parasites and fungi. Such infectious disease pathogens
include, for example, human immunodeficiency virus (HIV),
Mycobacterium tuberculosis, herpesvirus, papillomavirus and
Candida. The present method is particularly useful when the
infectious disease pathogen is a virus, and more particularly,
human immunodeficiency virus and feline immunodeficiency virus. In
another embodiment, the present method is particularly useful when
the infectious disease is tuberculosis. In this embodiment, the
immunogen can be, for example, a Mycobacterium tuberculosis
antigen, or more specifically, antigen 85.
[0021] Expression of the pathogen antigen in a tissue of the mammal
produces a result selected from the group of 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.
[0022] In one embodiment of this method, the therapeutic
composition comprises a plurality of recombinant nucleic acid
molecules, each of the recombinant nucleic acid molecules
comprising a cDNA sequence amplified from total RNA isolated from
an infectious disease pathogen, each of the cDNA sequences encoding
an immunogen from the infectious disease pathogen or a fragment
thereof and being operatively linked to a transcription control
sequence.
[0023] When the mammal has a disease associated with allergic
inflammation, the immunogen is an allergen. Suitable allergens
include, plant pollens, drugs, foods, venoms, insect excretions,
molds, animal fluids, animal hair and animal dander. This method is
particularly useful when the mammal has a disease selected from
allergic airway diseases, allergic rhinitis, allergic
conjunctivitis, and food allergy. Expression of the allergen in a
tissue of the mammal produces a result selected from the group
consisting of alleviation of the disease, alleviation of symptoms
of the disease, desensitization against the disease, and
stimulation of a protective immune response against the
disease.
[0024] In another embodiment of this method, the therapeutic
composition comprises a plurality of recombinant nucleic acid
molecules, each of the recombinant nucleic acid molecules
comprising a cDNA sequence amplified from total RNA isolated from
an allergen, each of the cDNA sequences encoding the allergen or a
fragment thereof and being operatively linked to a transcription
control sequence.
[0025] Yet another embodiment of the present invention relates to a
method to elicit a systemic, non-specific immune response in a
mammal, which includes administering to the mammal a therapeutic
composition by a route of administration selected from intravenous
and intraperitoneal, wherein the therapeutic composition comprises:
(a) a liposome delivery vehicle; and, (b) a recombinant nucleic
acid molecule comprising an isolated nucleic acid sequence encoding
a cytokine, the nucleic acid sequence being operatively linked to a
transcription control sequence. The method of the present invention
is particularly useful for eliciting a systemic, anti-viral immune
response or a systemic; an anti-tumor immune response; a systemic,
protective immune response against allergic inflammation in the
mammal; and/or for reduction of a tumor in the mammal.
Additionally, the method increases production of IFN* in the mammal
and/or increases natural killer (NK) cell activity in the mammal.
In one embodiment, the route of administration is intravenous. The
cytokine can include hematopoietic growth factors, interleukins,
interferons, immunoglobulin superfamily molecules, tumor necrosis
factor family molecules and/or chemokines. In one embodiment, the
cytokine is an interleukin, and in a more preferred embodiment, the
interleukin is selected from the group of interleukin-2 (IL-2),
interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15
(IL-15), interleukin-18 (IL-18) or interferon-* (IFN*), and in an
even more preferred embodiment, the interleukin is selected from
the group of interleukin-2 (IL-2), interleukin-12 (IL-12),
interleukin-18 (IL-18) or interferon-* (IFN*).
[0026] Another embodiment of the present invention relates to a
method to elicit a tumor antigen-specific immune response and a
systemic, non-specific immune response in a mammal that has cancer.
The method includes administering to a mammal a therapeutic
composition by a route of administration selected from intravenous
and intraperitoneal administration. The therapeutic composition
comprises: (a) a liposome delivery vehicle; and, (b) total RNA
isolated from a tumor sample, the RNA encoding tumor antigens. In
one embodiment, the route of administration is intravenous. In
another embodiment, the RNA is enriched for poly-A RNA prior to
administration to the mammal.
[0027] Yet another embodiment of the present invention relates to a
method to elicit a pathogen-antigen-specific immune response and a
systemic, non-specific immune response in a mammal that has an
infectious disease. Such method includes administering to a mammal
a therapeutic composition by a route of administration selected
from intravenous and intraperitoneal administration, the
therapeutic composition comprising: (a) a liposome delivery
vehicle; and, (b) total RNA isolated from an infectious disease
pathogen, the RNA encoding pathogen antigens. In another
embodiment, the route of administration is intravenous.
[0028] Another embodiment of the present invention relates to a
composition for systemic administration to a mammal to elicit an
immunogen-specific immune response and a systemic, non-specific
immune response. The composition includes (a) a liposome delivery
vehicle; and (b) a recombinant nucleic acid molecule comprising an
isolated nucleic acid sequence encoding an immunogen, the nucleic
acid sequence being operatively linked to a transcription control
sequence. The composition has a nucleic acid:lipid ratio of from
about 1:1 to about 1:64.
[0029] In one embodiment, any of the above compositions of the
present invention administered to a mammal by the present methods
can include a recombinant nucleic acid molecule having a nucleic
acid sequence encoding a cytokine. In this embodiment, the nucleic
acid sequence encoding a cytokine is operatively linked to a
transcription control sequence. In the compositions which include a
nucleic acid sequence encoding an immunogen, the nucleic acid
sequence encoding a cytokine can be in the same or separate
recombinant nucleic acid molecule which contains the nucleic acid
sequence encoding the immunogen. The nucleic acid sequence encoding
a cytokine and the nucleic acid sequence encoding an immunogen can
be operatively linked to the same or different transcription
control sequences. In preferred embodiments, the cytokine is
selected from the group of hematopoietic growth factors,
interleukins, interferons, immunoglobulin superfamily molecules,
tumor necrosis factor family molecules and/or chemokines. In one
embodiment, the cytokine is an interleukin, and in a more preferred
embodiment, the interleukin is selected from the group of
interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12),
interleukin-15 (IL-15), interleukin-18 (IL-18) or interferon-*
(IFN*), and in an even more preferred embodiment, the interleukin
is selected from the group of interleukin-2 (IL-2), interleukin-12
(IL-12), interleukin-18 (IL-18) or interferon-* (IFN*).
[0030] Liposome delivery vehicles suitable for use in any of the
compositions and methods of the present invention can include any
liposomes. Particularly preferred liposomes are cationic liposomes.
Other preferred liposomes include multilamellar vesicle lipids and
extruded lipids, with multilamellar vesicle lipids being more
preferred. Liposome compositions can include, but are not limited
to, pairs of lipids selected from DOTMA and cholesterol, DOTAP and
cholesterol, DOTIM and cholesterol, and DDAB and cholesterol, with
DOTAP and cholesterol being particularly preferred.
[0031] The compositions of the present invention administered by
the present methods have a nucleic acid:lipid ratio of from about
1:1 to about 1:64. In some embodiments, the compositions have a
nucleic acid:lipid ratio of from about 1:10 to about 1:40. Other
suitable ratios are additionally set forth below.
[0032] The methods and compositions of the present invention are
preferably used to elicit an immune response in a mammal, which
includes humans, dogs, cats, mice, rats, sheep, cattle, horses or
pigs, and more preferably, humans.
[0033] Additional advantages and novel features of this invention
shall be set forth in part in the description that follows, and in
part will become apparent to those skilled in the art upon
examination of the following specification or may be learned by the
practice of the invention. The advantages of the invention may be
realized and attained by means of the instrumentalities,
combinations, compositions, and methods particularly pointed out in
the appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 is a bar graph illustrating that intravenous
injection of CLDC induces marked activation of 5 different immune
effector populations in vivo.
[0035] FIG. 2A is a bar graph showing that intravenous injection of
CLDC, but not lipid or DNA alone, induces immune activation of CD8+
cells in vivo.
[0036] FIG. 2B is a bar graph showing that intravenous injection of
CLDC, but not lipid or DNA alone, induces immune activation of
NK1.1+ cells in vivo.
[0037] FIG. 3 is a bar graph comparing the immune activating
potencies of LPS, poly I/C and CLDC in vivo.
[0038] FIG. 4 is a bar graph is a bar graph showing in vivo dose
responses for immune activation by CLDC.
[0039] FIG. 5 is a bar graph illustrating the influence of route of
administration of CLDC on immune activation.
[0040] FIG. 6 is a bar graph showing that immune activation can be
induced by CLDC formed with several different lipids.
[0041] FIG. 7 is a bar graph demonstrating that immune activation
by CLDC is independent of the DNA source.
[0042] FIG. 8 is a bar graph illustrating that IFN* release by
immune cells is induced by administration of CLDC, but not lipid or
DNA alone.
[0043] FIG. 9 is a bar graph showing that administration of CLDC,
but not poly I/C or LPS, induces IFN* production by splenocytes in
vivo.
[0044] FIG. 10A is a bar graph showing that NK cells are the source
of IFN* production in splenocytes elicited by intravenous
administration of CLDC injection.
[0045] FIG. 10B is a bar graph showing that NK cells are the source
of IFN* production in lung mononuclear cells elicited by
intravenous administration of CLDC injection.
[0046] FIG. 11 is a line graph illustrating that administration of
CLDC induces high levels of NK activity in splenocytes.
[0047] FIG. 12A is a bar graph showing that intraperitoneal
administration of CLDC induces immune activation in CD8+
splenocytes in vivo.
[0048] FIG. 12B is a bar graph showing that intraperitoneal
administration of CLDC induces immune activation in
NK1.1+splenocytes in vivo.
[0049] FIG. 13A is a bar graph demonstrating that CLDC exert potent
antitumor effects against fibrosarcoma tumor cells in vivo.
[0050] FIG. 13B is a bar graph demonstrating that CLDC exert potent
antitumor effects against melanoma tumor cells in vivo.
[0051] FIG. 13C is a bar graph demonstrating that CLDC exert potent
antitumor effects against colon carcinoma tumor cells in vivo.
[0052] FIG. 13D is a bar graph demonstrating that CLDC exert potent
antitumor effects against breast cancer tumor cells in vivo.
[0053] FIG. 14 is a bar graph showing that systemic administration
of CLDC, but not DNA or lipid alone, induces antitumor activity in
vivo.
[0054] FIG. 15 is a bar graph demonstrating that the antitumor
activity of CLDC is independent of the DNA source.
[0055] FIG. 16 is a bar graph showing that the type of CLDC
administered significantly influences antitumor activity.
[0056] FIG. 17A is a bar graph illustrating that intravenous
administration of CLDC induces selective gene expression in
pulmonary tissues.
[0057] FIG. 17B is a bar graph illustrating that intravenous
administration of CLDC encoding IL-2 induces intrapulmonary IL-2
expression.
[0058] FIG. 17C is a bar graph illustrating that intravenous
administration of CLDC encoding IFN* induces intrapulmonary IFN*
expression.
[0059] FIG. 18A is a bar graph showing that day 3 administration of
CLDC encoding 3 different cytokine genes improves the antitumor
activity against fibrosarcoma tumor cells in vivo over empty vector
alone.
[0060] FIG. 18B is a bar graph showing that day 3 administration of
CLDC encoding 3 different cytokine genes improves the antitumor
activity against colon carcinoma tumor cells in vivo over empty
vector alone.
[0061] FIG. 18C is a bar graph showing that day 3 administration of
CLDC encoding 3 different cytokine genes improves the antitumor
activity against melanoma tumor cells in vivo over empty vector
alone.
[0062] FIG. 18D is a bar graph showing that day 6 administration of
CLDC encoding 3 different cytokine genes improves the antitumor
activity against fibrosarcoma tumor cells in vivo over empty vector
alone.
[0063] FIG. 18E is a bar graph showing that day 6 administration of
CLDC encoding 3 different cytokine genes improves the antitumor
activity against colon carcinoma tumor cells in vivo over empty
vector alone.
[0064] FIG. 18F is a bar graph showing that day 6 administration of
CLDC encoding 3 different cytokine genes improves the antitumor
activity against melanoma tumor cells in vivo over empty vector
alone.
[0065] FIG. 19A is a line graph illustrating that intravenous
administration of CLDC encoding ovalbumin induces strong, systemic,
antigen-specific immune responses in vivo.
[0066] FIG. 19B is a line graph demonstrating that intravenous
immunization with CLDC encoding an antigen is at least 10 times
more potent immune inducer of immune activation than intramuscular
injection of DNA encoding an antigen.
[0067] FIG. 20 is a bar graph showing that systemic immunization
with CLDC encoding a tumor antigen induces strong antitumor
activity.
[0068] FIG. 21 is a bar graph illustrating that intravenous
administration of CLDC encoding a tumor antigen induces effective
antitumor immunity, whereas administration of DNA encoding a tumor
antigen intramuscularly or intradermally does not.
[0069] FIG. 22 is a line graph showing that intravenous
administration of CLDC encoding a tumor antigen induces a potent
humoral immune response against the tumor antigen in vivo.
[0070] FIG. 23 is a bar graph showing that CLDC-mediated
immunization with a tumor antigen induces antigen-specific
production of IFN* by splenocytes.
[0071] FIG. 24 is a bar graph demonstrating that CLRC-mediated
immunization with tumor RNA with and without DNA encoding a
cytokine induces strong antitumor activity in vivo.
[0072] FIG. 25 is a bar graph illustrating that immunization with
CLRC containing tumor-specific RNA induces tumor-specific CTL
responses in vivo.
[0073] FIG. 26 is a line graph showing that intraperitoneal
immunization with CLDC containing DNA encoding IL-2 induces a
reduction in FeLV viral titer.
[0074] FIG. 27 is a line graph illustrating that intravenous
pulmonary transfection with CLDC containing DNA encoding IFN*
inhibits the development of airway hyperresponsiveness in allergen
sensitized and challenged mice.
[0075] FIG. 28 is a bar graph demonstrating that intravenous
pulmonary transfection with CLDC containing DNA encoding IFN*
inhibits eosinophil influx to the airways in mice sensitized and
challenged with allergen.
[0076] FIG. 29A is a bar graph illustrating that intravenous
administration of CLDC induces IFN* release from spleen as compared
to intratracheal administration.
[0077] FIG. 29B is a bar graph illustrating that intravenous
administration of CLDC induces IFN* release from lung as compared
to intratracheal administration.
DETAILED DESCRIPTION OF THE INVENTION
[0078] The present invention generally relates to a novel genetic
immunization strategy and therapeutic compositions 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 cancer, allergic
inflammation and infectious disease. In one embodiment, the method
and composition of the present invention are particularly useful
for the prevention and treatment of primary lung cancers, pulmonary
metastatic diseases, allergic asthma and viral diseases. In another
embodiment, the method and composition of the present invention are
useful for treating chronic obstructive pulmonary diseases. In
addition, elicitation of an immune response according to the method
of the present invention can be useful for the development and
implementation of immunological diagnostic and research tools and
assays.
[0079] More particularly, the genetic immunization method of the
present invention comprises the elicitation of an immune response
in a mammal by intravenous or intraperitoneal administration (i.e.,
systemic administration) of a therapeutic composition that includes
an isolated nucleic acid molecule complexed with a liposome
delivery vehicle. The present inventors have made the surprising
discovery that the combination of nucleic acids and liposomes is
highly immunostimulatory in vivo when administered by intravenous
or intraperitoneal injection. The potency of this immune response
is far greater than the response induced by administration of
either nucleic acids or liposomes alone (See Examples 1b, 1h and
2b), and is dependent upon the intravenous or intraperitoneal
administration of the complex (See Examples 5 and 6b). Moreover,
this effect is independent of whether or not a protein is encoded
by or expressed by the nucleic acids (See Examples 1 and 2), and it
is also independent of the source of the nucleic acids (e.g.,
mammalian, bacterial, insect, viral; see Examples 1g and 2c), the
type of nucleic acids (e.g., DNA or RNA; see Examples 7a-b), and to
some extent, the type of lipids used (See Example 1f). As such, the
nucleic acid-lipid complexes of the present invention induce a
strong, systemic, non-antigen-specific immune response when
administered intravenously or intraperitoneally, which results in
the activation of multiple different immune effector cells in vivo.
The present inventors have additionally discovered that the immune
response generated by such a nucleic acid-lipid complex
administered by the present method has potent anti-tumor,
anti-allergy and anti-viral properties (See Examples 1a-c,
1h-1,2a-d, 8 and 9). Immune activation induced by such a
therapeutic composition of the present invention is quantitatively
more potent than that induced by either LPS (endotoxin) or poly I/C
(a classical inducer of antiviral immune responses; see Examples 1c
and 1i). Furthermore, the type of immune stimulation induced (e.g.,
as characterized by the pattern of cytokines induced) also differs
qualitatively from that induced by LPS or poly I/C. Finally, this
effect does not appear to be associated with the complement cascade
problems that have been experienced using viral delivery
systems.
[0080] These findings are surprising because, prior to the present
invention, liposome delivery vehicles, which are often used in gene
therapy protocols, were touted by many in the art as being
relatively non-immunogenic, particularly as compared to viral
vector delivery vehicles (e.g., adenovirus vectors), and have thus
been considered safe and useful for delivering a gene to a site in
a mammal while substantially avoiding an immune inflammatory
response (See, for example, Liu et al., 1997, Nature Biotechnology
15:167-173, Stewart et al., 1992, Hum. Gene Ther. 3:267-275; Zhu et
al., 1993, Science 261:209-211; Canonico et al., 1994, J. Appl.
Phys. 77:415-419). This recognized relative non-immunogenicity of
liposomes has motivated those of skill in the art to use liposomes
to deliver genes with the confidence that the delivery vehicle is
relatively innocuous in vivo. The present invention provides
evidence that contradicts this principle.
[0081] The discovery of the present inventors is further surprising
because, although it was previously recognized that administration
of naked DNA (i.e., by intramuscular or percutaneous delivery),
which comprises a bacterially derived vector ligated to a target
gene, provides an adjuvant effect (i.e., due to the bacterially
derived vector DNA), the nucleic acid:lipid complexes of the
present invention are significantly more immunostimulatory than DNA
administered alone (i.e., naked DNA) (See Examples section). This
discovery by the present inventors is quite unexpected and thus
represents a new frontier in genetic vaccine design. Previously
described naked DNA vaccines are typically designed to use
bacterial plasmid DNA, since a vast body of literature has reported
that bacterial and some insect nucleic acids may be immunogenic
(See, for example, Pisetsky et al., 1996, Immunity, 5:303-310;
Pisetsky, 1996, Journal of Immunology 156:421-423; Yamamoto, et
al., 1994, Microbiol. Immunol. 38(10):831-836; Roman, et al., 1997,
Nature Medicine, 3(8):849-854; Krieg, 1996, Trends in Microbiology,
4(2):73-77; Sun, et al., 1996, Immunity, 4:555-564; Stacey et al.,
1996, The Journal of Immunology, 157:2116-2122; Sato, et al., 1996,
Science, 273:352-354; or Ballas, 1996, The Journal of Immunology,
157:1840-1845). Significantly, this literature has specifically
excluded mammalian nucleic acids for use in naked DNA vaccines,
asserting that mammalian nucleic acids are not immunogenic.
Therefore, it is completely unpredicted by the art at the time of
the present invention that nucleic acids from mammalian sources
would have immunostimulatory properties, and it is even more
unexpected that the effect of nucleic acids from any source
complexed with lipids at very low doses would synergize to provide
such a strong immunostimulatory effect demonstrated by the present
inventors, particularly in comparison to lipids or nucleic acids
alone.
[0082] In view of the present inventors' discoveries, previous
investigators in the art may be misdirecting the use of liposome
delivery vehicles for gene therapy when elicitation of an immune
response is not desirable. Moreover, with regard to genetic
immunization, which is the primary focus of the present invention,
previous investigators have not taken advantage of the superior
immunostimulatory effect of nucleic acid:lipid complexes in
designing genetic vaccines. In fact, most of the disclosed specific
genetic immunization strategies do not make use of liposome
delivery and/or are administered by intramuscular, intradermal,
oral or aerosol delivery routes, for the reasons discussed
above.
[0083] The present inventors disclose herein that alternate,
non-systemic routes of administration (i.e., other than intravenous
or intraperitoneal) significantly decrease both the
immunostimulatory effect and the therapeutic efficacy of the
present composition in comparison with administration by the
present method. Specifically, the present inventors have found that
the efficacy of the genetic immunization method of the present
invention is unattainable using previously described genetic
immunization protocols wherein naked DNA is delivered
intramuscularly or percutaneously, even when such protocols use 10
to 100 times more DNA than the present method (See Example 5 and
6b-c). The present inventors' discovery is surprising, because
there was no suggestion in any genetic immunization disclosure that
the particular genetic immunization protocol of the present
invention would be considerably more efficacious than other
possible protocols.
[0084] When the route of administration is intravenous, the primary
site of immunization (i.e., elicitation of an immune response) is
the lung, which is a very active organ immunologically, containing
large numbers of both effector cells (e.g., T cells, B cells, NK
cells) and antigen presenting cells (e.g., macrophages, dendritic
cells). Similarly, when the route of administration is
intraperitoneal, the primary sites of immunization are the spleen
and liver, both of which are also immunologically active organs.
Without being bound by theory, the present inventors believe that
these organs are capable of mounting a robust, non-antigen-specific
immune response both in the tissues and systemically, due to the
mode of administration. Additionally, when the nucleic acid
molecules of the nucleic acid:lipid complex encode and express an
immunogen, these organs are further capable of expressing the
immunogen and mounting a strong antigen-specific immune response
against antigens that are encountered within the tissues. These
activated immune cells are then capable of eliciting an immune
response in other areas of the body in which the appropriate
antigen is encountered. Administration of the nucleic acid:lipid
complexes can be at any site in the mammal wherein systemic
administration (i.e., intravenous or intraperitoneal
administration) is possible, including to sites in which the target
site for immune activation is not the first organ having a
capillary bed proximal to the site of administration.
[0085] As discussed above, the use of genetic vaccines and gene
therapy vehicles has generally been described in the art (See for
example, U.S. Pat. No. 5,593,972, issued Jan. 14, 1997, to Weiner
et al.; U.S. Pat. No. 5,580,859, issued Dec. 3, 1996, to Felgner et
al.; U.S. Pat. No. 5,589,466, issued Dec. 31, 1996, to Felgner et
al.; U.S. Pat. No. 5,641,662, issued Jun. 24, 1997, to Debs et al.
and U.S. Pat. No. 5,676,954, issued Oct. 14, 1997, to Brigham).
Such publications have broadly disclosed genetic vaccine and/or
gene therapy protocols which include administration of nucleic acid
molecules (e.g., DNA) encoding any of a variety of antigens and
other proteins, which are administered to an animal by a variety of
administration routes, and using a variety of delivery mechanisms.
These disclosures have failed, however, to appreciate the
surprising advantages and unexpected efficacy of the particular
genetic immunization compositions and methods discovered by the
present inventors. Indeed, in view of the above discussion, many of
the methods and compositions for genetic immunization and/or gene
therapy disclosed by the above publications are predicted to be
inoperable, unsafe, and/or significantly less effective in vivo
than the specific compositions and methods of the present
invention. The present inventors' discoveries provide strong
evidence that the development of both genetic vaccines designed to
immunize an animal and gene therapy protocols designed to deliver a
gene to a site in an animal should be reevaluated to avoid
previously unknown safety and efficacy concerns.
[0086] Due to the unexpected immunostimulatory properties of the
nucleic acid:lipid complexes administered by the present method,
the genetic 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, as will be shown in
the Examples below, traditional naked DNA delivery, which has been
touted as having an adjuvant effect, is far less effective than the
present compositions at stimulating a non-antigen-specific immune
response. Finally, unlike many protocols for administration of
viral vector-based genetic vaccines, the present method can be used
to repeatedly deliver the therapeutic composition described herein
without consequences associated with some non-specific arms of the
immune response, such as the complement cascade.
[0087] 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 genetic immunization strategy in
which a nucleic acid sequence in the above nucleic acid-lipid
complex encodes an immunogen and/or a cytokine that is expressed in
the tissues of the mammal (i.e., is operatively linked to a
transcription control sequence; see Examples 4-9). The present
inventors have also found that the combination of an
antigen-specific immune response elicited by expression of 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 significantly greater in vivo efficacy than
previously described genetic vaccines (See Examples 5, 6b-c, 9).
This effect can be additionally enhanced by coadministration of a
nucleic acid molecule encoding a cytokine such that the cytokine is
expressed in the tissues (See Examples 4 and 7a).
[0088] Moreover, with regard to intravenous administration of the
present composition, in cancer patients, the lung is the principal
site to which metastatic tumors spread. 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. Therefore,
the genetic immunization method and compositions of the present
invention, unlike previously described genetic immunization
methods, elicit both a systemic, non-antigen-specific immune
response (similar to a conventional adjuvant) and, when the nucleic
acid encodes a tumor antigen, a strong, antigen-specific,
intrapulmonary (intravenous administration; see Examples 1e, 3 and
5) or splenic and/or hepatic (intraperitoneal administration; see
Examples 1e and 1l) immune response in a mammal which is effective
to significantly reduce or eliminate established tumors in
vivo.
[0089] One embodiment of the present invention is a method to
elicit a systemic, non-antigen-specific immune response in a mammal
immune response in a mammal. In this method, a therapeutic
composition which includes: (a) a liposome delivery vehicle; and
(b) an isolated nucleic acid molecule that is not operatively
linked to a transcription control sequence, is administered by
intravenous or intraperitoneal administration to a mammal.
Administration of such a composition by the method of the present
invention results in the elicitation of a systemic,
non-antigen-specific immune response in the mammal to which the
composition is administered. As discussed above, this immune
response additionally has strong, systemic, anti-tumor,
anti-allergic inflammation (i.e., protective), and anti-viral
properties. Such properties include the activation of NK cells (as
measured by upregulation of NK cell markers, such as NK1.1, for
example, or by production of IFN*), production of Th1-type
cytokines (e.g., IFN*) and the non-antigen-specific recruitment and
upregulation of activity in mononuclear cells and T
lymphocytes.
[0090] Therapeutic compositions useful in the method of the present
invention include compositions containing nucleic acids having any
nucleic acid sequence, including coding (i.e. encoding at least a
portion of a protein or peptide) and/or non-coding (i.e., not
encoding any portion of a protein or peptide) sequences, and
including DNA and/or RNA. In the above-described embodiment of the
present invention, since expression of a protein encoded by the
nucleic acid molecule is not required for elicitation of a
systemic, non-antigen-specific immune response, the molecule is not
necessarily operatively linked to a transcription control sequence.
It is to be noted, however, that further advantages can be obtained
(i.e., antigen-specific and enhanced immunity) by including in the
composition a nucleic acid sequence (DNA or RNA) which encodes an
immunogen and/or a cytokine.
[0091] In another embodiment of the present invention, the present
method of eliciting an immune response can be modified to include
the intravenous or intraperitoneal administration to a mammal of a
therapeutic composition comprising: (a) a liposome delivery
vehicle; and (b) a recombinant nucleic acid molecule comprising a
nucleic acid sequence which encodes an immunogen. 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 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 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 that are encountered within the tissues of
the mammal. According to the present invention, an immunogen or an
antigen can be any portion of a protein, naturally occurring or
synthetically derived, which elicits a humoral and/or cellular
immune response. As such, the size of an antigen or immunogen can
be as small as about 5-12 amino acids and as large as a full length
protein, including a multimer and fusion proteins. The terms,
"immunogen" and "antigen", as used to describe the present
invention, do not include a superantigen. 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).
[0092] 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). In this
embodiment, the nucleic acid sequence is operatively linked to a
transcription control sequence, such that the immunogen is
expressed in a tissue of a mammal, thereby eliciting an
immunogen-specific immune response in the mammal, in addition to
the non-specific immune response discussed above.
[0093] In a further embodiment of the method of the present
invention, the therapeutic composition to be administered to a
mammal includes an isolated nucleic acid molecule encoding a
cytokine (also referred to herein as a "cytokine-encoding nucleic
acid molecule"), in which the nucleic acid molecule is operatively
linked to one or more transcription control sequences. The result
of administration of such a therapeutic composition to the mammal
is that the nucleic acid molecule encoding the cytokine is
expressed in the pulmonary tissues of the mammal, when
administration is intravenous, and in the spleen and liver tissues
of the mammal when administration is peritoneal. 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. The nucleic acid sequence encoding
a cytokine can be on the same recombinant nucleic acid molecule as
a nucleic acid sequence encoding an immunogen, or on a different
recombinant nucleic acid molecule.
[0094] A composition useful in the method of the present invention,
as discussed in detail below, comprises: (a) a liposome delivery
vehicle; and (b) a nucleic acid molecule, such molecule including:
(1) an isolated nucleic acid sequence that is not operatively
linked to a transcription control sequence; (2) an isolated
non-coding nucleic acid sequence; (3) an isolated recombinant
nucleic acid molecule encoding an immunogen operatively linked to a
transcription control sequence, wherein the nucleic acid:lipid
complex has a ratio of from about 1:1 to about 1:64; and/or (4) an
isolated recombinant nucleic acid molecule encoding a cytokine. In
preferred embodiments, the nucleic acid:lipid complex has a ratio
of from about 1:10 to 1:40. Various components of such a
composition are described in detail below.
[0095] 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 and IFN*. 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).
[0096] 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
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 the number and/or the activity of T cells in the area
of the tumor cell or pathogen. Similarly, NK cell activity refers
to increasing the number and/or 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 therapeutic
composition is primarily targeted (i.e., intrapulmonary for
intravenous administration and in the spleen or liver for
intraperitoneal administration, although the present composition is
effective at other sites in the body as well). 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.
[0097] According to the present invention, elicitation of a
non-antigen-specific immune response (i.e., a non-specific immune
response) includes stimulation of non-specific immune cells, such
as macrophages and neutrophils, as well as induction of cytokine
production, particularly IFN* production, and non-antigen-specific
activation of effector cells such as NK cells, B lymphocytes and/or
T lymphocytes. More specifically, the systemic,
non-antigen-specific immune response elicited by the method and
composition of the present invention result in an increase in
natural killer (NK) cell function and number in the mammal, wherein
an increase in NK function is defined as any detectable increase in
the level of NK cell function compared to NK cell function in
mammals not immunized with a composition of the present invention,
or in mammals immunized with a composition of the present invention
by a non-systemic (i.e., non-intravenous, non-intraperitoneal)
route of administration, with the amount of nucleic acid delivered
and the ratio of nucleic acid:lipid being equal. NK function (i.e.,
activity) can be measured by cytotoxicity assays against a suitable
target cell. An example of a suitable target cell by which to
measure NK cell cytotoxic activity is YAC-1. An example of an NK
cell cytotoxicity assay is presented in Example 1 (FIG. 11). NK
cell activation can be measured by determining an upregulation of
NK1.1/CD69 on cells in various organs, including spleen, lymph
node, lung and liver, by flow cytometric analysis (See Example 1,
FIGS. 1 and 2). Additionally, the systemic, non-antigen-specific
immune response elicited by the method and composition of the
present invention can result in an increase in production of IFN*
by the NK cells in the mammal in various organs including spleen
and lung, wherein an increase in IFN* production is defined as any
detectable increase in the level of IFN* production compared to
IFN* production by NK cells in mammals not administered with a
composition of the present invention, or in mammals administered
with a composition of the present invention by a non-systemic route
of administration, with the amount of nucleic acid delivered and
the ratio of nucleic acid:lipid being equal. IFN* production can be
measured by a IFN* ELISA (as is known in the art; Example 1, FIG.
10). Preferably, a composition of the present invention
administered by the method of the present invention elicits at
least about 100 .mu.g/ml of IFN* per 5.times.10.sup.6 mononuclear
cells from blood, spleen or lung, and more preferably, at least
about 500 .mu.g/ml of IFN*, and more preferably at least about 1000
.mu.g/ml of IFN*, and even more preferably, at least about 5000
.mu.g/ml of IFN*, and even more preferably, at least about 10,000
.mu.g/ml of IFN*.
[0098] 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
(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.
[0099] More specifically, a therapeutic composition 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, 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 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.
[0100] One component of the therapeutic composition used in the
present method is a nucleic acid sequence, which can include coding
and/or non-coding nucleic acid sequences, and both oligonucleotides
(described below) and larger nucleic acid sequences. 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 is operatively linked to a transcription control
sequence, so that the peptide or protein can be expressed. 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. The term, "empty vector" can be used
interchangeably with the term "non-coding," and particularly refers
to a nucleic acid sequence in the absence of a protein coding
portion, such as a plasmid vector without a gene insert. The phrase
"operatively linked" refers to linking a nucleic acid molecule to a
transcription control sequence in a manner such that the molecule
can be expressed when transfected (i.e., transformed, transduced or
transfected) into a host cell. Therefore, a nucleic acid sequence
that is "not operatively linked to a transcription control
sequence" refers to any nucleic acid sequence, including both
coding and non-coding nucleic acid sequences, which are not linked
to a transcription control sequence in a manner such that the
molecule is able to be expressed when transfected into a host cell.
It is noted that this phrase does not preclude the presence of a
transcription control sequence in the nucleic acid molecule.
[0101] In some embodiments of the present invention, a nucleic acid
sequence included in a therapeutic composition of the present
invention is incorporated into a recombinant nucleic acid molecule,
and encodes an immunogen and/or a cytokine. As discussed in detail
below, preferred immunogens include a tumor antigen, an allergen or
an antigen from an infectious disease pathogen (i.e., a pathogen
antigen). The phrase "recombinant 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.
[0102] According to the present invention, an isolated, or
biologically pure, 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" and "biologically pure" do
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
derivatives of either DNA or RNA. 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, and nucleic acid molecules that
comprise regulatory regions, introns, or other non-coding DNA or
RNA. 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. Immune activation by nucleic
acid:lipid complexes of the present invention can be induced by
eukaryotic as well as prokaryotic nucleic acids, indicating that
there is some property of the nucleic acid:lipid complexes that is
inherently immune activating, regardless of the source of the
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 by the nucleic acid-lipid
complex. 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.
[0103] An isolated immunogen-encoding (e.g., a tumor antigen-,
allergen-, or pathogen antigen-) or cytokine-encoding nucleic acid
molecule can be obtained from its natural source, either as an
entire (i.e., complete) gene or a portion thereof capable of
encoding: a tumor antigen protein having a B cell and/or T cell
epitope, an allergen having a B cell and/or T cell epitope, a
pathogen antigen having a B cell and/or a T cell epitope, or a
cytokine protein capable of binding to a complementary cytokine
receptor. A nucleic acid molecule can also be produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Nucleic acid
molecules include natural nucleic acid molecules and homologues
thereof, including, but not limited to, natural allelic variants
and modified nucleic acid molecules in which nucleotides have been
inserted, deleted, substituted, and/or inverted in such a manner
that such modifications do not substantially interfere with the
nucleic acid molecule's ability to encode an immunogen or a
cytokine useful in the method of the present invention.
[0104] A nucleic acid molecule homologue can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Labs Press, 1989), which is incorporated herein
by reference in its entirety. For example, nucleic acid molecules
can be 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. Nucleic acid molecule homologues can be
selected from a mixture of modified nucleic acids by screening for
the function of the protein encoded by the nucleic acid (e.g.,
tumor antigen, allergen or pathogen antigen immunogenicity, or
cytokine activity, as appropriate). Techniques to screen for
immunogenicity, such as tumor antigen, allergen or pathogen antigen
immunogenicity, or cytokine activity, are known to those of skill
in the art and include a variety of in vitro and in vivo
assays.
[0105] As heretofore disclosed, immunogen or cytokine proteins of
the present invention include, but are not limited to, proteins
encoded by nucleic acid molecules having full-length immunogen or
cytokine coding regions; proteins encoded by nucleic acid molecules
having partial immunogen regions which contain at least one T cell
epitope and/or at least one B cell epitope; proteins encoded by
nucleic acid molecules having cytokine coding regions capable of
binding to a complementary cytokine receptor; fusion proteins; and
chimeric proteins comprising combinations of different immunogens
and/or cytokines.
[0106] One embodiment of the present invention is an isolated
nucleic acid molecule that encodes at least a portion of a
full-length immunogen, including a tumor antigen, allergen or
pathogen antigen, or a homologue of such immunogens. As used
herein, "at least a portion of an immunogen" refers to a portion of
an immunogen protein containing a T cell and/or a B cell epitope.
In one embodiment, an immunogen-encoding nucleic acid molecule
includes an entire coding region of such an immunogen. As used
herein, a homologue of an immunogen is a protein having an amino
acid sequence that is sufficiently similar to a natural immunogen
amino acid sequence (i.e., a naturally occurring, endogenous, or
wild-type immunogen) that a nucleic acid sequence encoding the
homologue encodes a protein capable of eliciting an immune response
against the natural immunogen.
[0107] A tumor antigen-encoding nucleic acid molecule of the
present invention encodes an antigen that can include 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.
Preferably, tumor antigens useful in the present method have at
least one T cell and/or B cell epitope. Therefore, expression of
the tumor antigen in a tissue of a mammal elicits a tumor
antigen-specific immune response against the tumor in the tissue of
the mammal. As discussed above, the present inventors have found
that administration of the nucleic acid:lipid complex of the
present invention elicits a strong, systemic, non-antigen-specific,
anti-tumor response in vivo, and this effect enhances the
antigen-specific immune response to a tumor antigen expressed by
the nucleic acid molecule.
[0108] In a preferred embodiment, a nucleic acid molecule of the
present invention encodes a tumor antigen 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.
[0109] According to the present invention, a pathogen
antigen-encoding nucleic acid molecule of the present invention
encodes an antigen from an infectious disease pathogen that can
include pathogen antigens having epitopes that are recognized by T
cells, pathogen antigens having epitopes that are recognized by B
cells, pathogen antigens that are exclusively expressed by
pathogens, and pathogen antigens that are expressed by pathogens
and by other cells. Preferably, pathogen antigens 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, expression
of the pathogen antigen in a tissue of a mammal elicits an
antigen-specific immune response against the pathogen in the
tissues of the mammal as well as systemically.
[0110] According to the present invention, a pathogen antigen
includes an antigen that is expressed by a bacterium, a virus, a
parasite or a fungus. Preferred pathogen antigens for use in the
method of the present invention include antigens which cause a
chronic infectious disease in a mammal. Particularly preferred
pathogen antigens for use in the present method are immunogens from
immunodeficiency virus (HIV), Mycobacterium tuberculosis,
herpesvirus, papillomavirus and Candida.
[0111] In one embodiment, a pathogen antigen for use in the method
or composition of the present invention includes an antigen from a
pathogen associated with an infectious pulmonary disease, such as
tuberculosis. In a more preferred embodiment, such a pathogen
antigen includes an antigen from Mycobacterium tuberculosis, and
even more preferably, is Mycobacterium tuberculosis antigen 85.
[0112] In another embodiment of the present invention, a pathogen
antigen 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, regardless of whether or not the
nucleic acid encodes or expresses an immunogen. When the nucleic
acid sequence does encode a viral antigen that is operatively
linked to a transcription control sequence such that the viral
antigen is expressed in a tissue of a mammal, the present
composition further elicits a strong, viral antigen-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 and feline
immunodeficiency virus.
[0113] Another embodiment of the present invention includes an
allergen-encoding nucleic acid molecule that encodes at least a
portion of a full-length 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 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.
[0114] Another embodiment of the present invention includes a
cytokine-encoding nucleic acid molecule that encodes at least a
portion of a full-length cytokine or a homologue of the cytokine
protein. 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). 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.
[0115] A cytokine-encoding nucleic acid molecule of the present
invention encodes a cytokine that is capable of affecting the
biological function of a cell, 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.
[0116] A preferred cytokine nucleic acid molecule of the present
invention encodes a 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 nucleic acid molecule
of the present invention encodes an interleukin. An even more
preferred cytokine nucleic acid molecule useful in the method of
the present invention encodes interleukin-2 (IL-2), interleukin-7
(IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15),
interleukin-18 (IL-18), and/or interferon-* (IFN*). A most
preferred cytokine nucleic acid molecule useful in the method of
the present invention encodes interleukin-2 (IL-2), interleukin-12
(IL-12), interleukin-18 (IL-18) and/or interferon-* (IFN*).
[0117] 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.
[0118] According to the present invention, a cytokine-encoding
nucleic acid molecule of the present invention 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. The present
invention includes a nucleic acid molecule of the present invention
operatively linked to one or more transcription control sequences
to form a recombinant molecule. As discussed above, the phrase
"operatively linked" refers to linking a nucleic acid molecule to a
transcription control sequence in a manner such that the molecule
can be expressed when transfected (i.e., transformed, transduced or
transfected) into a host cell. Preferably, a nucleic acid molecule
used in a composition of the present invention is operatively
linked to a transcription control sequence that 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 an immunogen or 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.
[0119] 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), *- actin, retroviral
long terminal repeat (LTR), Rous sarcoma virus (RSV),
cytomegalovirus (CMV), tac, lac, trp, trc, oxy-pro, omp/lpp, miB,
bacteriophage lambda (*) (such as *PL and *PR and fusions that
include such promoters), bacteriophage T7, T7lac, bacteriophage T3,
bacteriophage 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 an immunogen, including tumor
antigen, an allergen, a pathogen antigen or a cytokine.
[0120] 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 protein 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.
This is yet another point of difference between the method of the
present invention and previously described gene therapy/gene
replacement protocols. Suitable promoters for use with nucleic acid
molecules encoding immunogens and/or 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.
[0121] 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 immunogen or cytokine protein to be secreted from the
cell that produces the protein. Suitable signal segments include:
(1) an immunogen signal segment (e.g., a tumor antigen, allergen or
pathogen antigen signal segment); (2) a cytokine signal segment;
(3) or any heterologous signal segment capable of directing the
secretion of an immunogen and/or cytokine protein according to the
present invention.
[0122] Preferred recombinant molecules of the present invention
include a recombinant molecule containing a nucleic acid sequence
encoding an immunogen, a recombinant molecule containing a nucleic
acid sequence encoding a cytokine, or a recombinant molecule
containing both a nucleic acid sequence encoding an immunogen and a
nucleic acid sequence encoding a cytokine to form a chimeric
recombinant molecule (i.e., the nucleic acid sequence encoding the
immunogen and the nucleic acid sequence encoding the cytokine are
in the same recombinant molecule). The nucleic acid molecules
contained in such recombinant chimeric molecules are operatively
linked to one or more transcription control sequences, in which
each nucleic acid molecule contained in a chimeric recombinant
molecule can be expressed using the same or different transcription
control sequences.
[0123] One or more recombinant molecules of the present invention
can be used to produce an encoded product (i.e., an immunogen
protein or a cytokine protein) useful in the method of the present
invention. In one embodiment, an encoded product is produced by
expressing a nucleic acid molecule as described herein under
conditions effective to produce the protein. A preferred method to
produce an encoded protein is by transfecting a host cell with one
or more recombinant molecules to form a recombinant cell. Suitable
host cells to transfect include any mammalian cell that can be
transfected. Host cells can be either untransfected cells or cells
that are already transformed with at least one nucleic acid
molecule. Host cells according to the present invention can be any
cell capable of producing an immunogen (e.g., tumor, allergen or
pathogen) and/or a cytokine according to the present invention. A
preferred host cell includes a mammalian lung cells, lymphocytes,
muscle cells, hematopoietic precursor cells, mast cells, natural
killer cells, macrophages, monocytes, epithelial cells, endothelial
cells, dendritic cells, mesenchymal cells, Langerhans cells, cells
found in granulomas and tumor cells of any cellular origin. An even
more preferred host cell of the present invention includes
mammalian mesenchymal cells, epithelial cells, endothelial cells,
macrophages, monocytes, lung cells, muscle cells, T cells and
dendritic cells.
[0124] According to the method of the present invention, a host
cell is preferably transfected in vivo (i.e., in a mammal) as a
result of intravenous or intraperitoneal administration to a mammal
of a nucleic acid molecule complexed to a liposome delivery
vehicle. Transfection of a nucleic acid molecule into a host cell
according to the present invention can be accomplished by any
method by which a nucleic acid molecule administered with a
liposome delivery vehicle can be inserted into the cell in vivo,
and includes lipofection.
[0125] It may be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve expression of transfected
nucleic acid molecules by manipulating, for example, the duration
of expression of the transgene (i.e., recombinant nucleic acid
molecule), the number of copies of the nucleic acid molecules
within a host cell, the efficiency with which those nucleic acid
molecules are transcribed, the efficiency with which the resultant
transcripts are translated, and the efficiency of
post-translational modifications. Recombinant techniques useful for
increasing the expression of nucleic acid molecules of the present
invention include, but are not limited to, operatively linking
nucleic acid molecules to high-copy number plasmids, integration of
the nucleic acid molecules into one or more host cell chromosomes,
addition of vector stability sequences to plasmids, increasing the
duration of expression of the recombinant molecule, substitutions
or modifications of transcription control signals (e.g., promoters,
operators, enhancers), substitutions or modifications of
translational control signals (e.g., ribosome binding sites,
Shine-Dalgarno sequences), modification of nucleic acid molecules
of the present invention to correspond to the codon usage of the
host cell, and deletion of sequences that destabilize transcripts.
The activity of an expressed recombinant protein of the present
invention may be improved by fragmenting, modifying, or
derivatizing nucleic acid molecules encoding such a protein.
Additionally, a nucleic acid molecule, and particularly a plasmid
portion, including transcription control sequences, can be modified
to make the nucleic acids more immunostimulatory, such as by the
addition of CpG moieties to the nucleic acids.
[0126] One embodiment of the method of the present invention, when
the mammal has cancer, a therapeutic composition to be
intravenously administered to the mammal comprises a plurality of
recombinant nucleic acid molecules, wherein each of the recombinant
nucleic acid molecules comprises a cDNA sequence, each of the cDNA
sequences encoding a tumor antigen or a fragment thereof (i.e., at
least a portion of a tumor antigen as defined above, preferably a
portion containing a T or B cell epitope). The cDNA sequences are
amplified from total RNA that has been isolated from an autologous
tumor sample. Each of the plurality of cDNA sequences is
operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal that
has cancer results in the expression of the cDNA sequences encoding
the tumor antigens in the tissue of the mammal (pulmonary tissue by
intravenous administration and spleen and liver by intraperitoneal
administration). In a further embodiment, such a therapeutic
composition comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, wherein the nucleic acid
sequence is operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal
results in the expression of the nucleic acid sequence encoding the
cytokine in the above-mentioned tissues of the mammal. According to
this embodiment of the present invention, an autologous tumor
sample is derived from the mammal to whom the therapeutic
composition is to be administered. Therefore, the cDNA sequences in
the therapeutic composition will encode tumor antigens present in
the cancer against which an immune response is to be elicited. In
this embodiment, 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 are 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.
[0127] In this embodiment of the method of the present invention, a
plurality of recombinant nucleic acid molecules as described can
also be referred to as a library of nucleic acid molecules, and
more particularly, a cDNA library. Methods to produce cDNA
libraries are well known in the art. Such methods are disclosed,
for example, in Sambrook et al., supra. More particularly, in this
embodiment, a therapeutic composition includes a plurality of
recombinant cDNA molecules encoding tumor antigens, or fractions
thereof, which represents the genes that are expressed by an
autologous tumor sample. Such a plurality of recombinant nucleic
acid molecules can be produced, for example by isolating total RNA
from an autologous tumor sample, converting (i.e., amplifying) the
RNA into a plurality of cDNA molecules, and then preparing a cDNA
library by cloning the cDNA molecules into recombinant vectors to
form a plurality of recombinant molecules. As used herein, total
RNA refers to all of the RNA isolatable from a cellular sample
using standard methods known in the art, and typically includes
mRNA, hnRNA, tRNA and rRNA. Methods for isolating total RNA from a
cellular sample, such as a tumor sample, are known in the art (See
for example, Sambrook et al., supra). In a further embodiment,
prior to amplification of cDNA from the total RNA, the RNA can be
selected to isolate poly-A RNA (i.e., RNA comprising a poly-A tail
at the 3' terminus, reflective of mRNA, the primary RNA transcript
which encodes a protein expressed by a cell). In yet another
embodiment, such a cDNA library can be "subtracted" against a cDNA
library from a normal cellular sample in the mammal in order to
remove nucleic acid molecules encoding antigens present in
non-tumor cells (i.e., normal cells) of the mammal, thereby
enriching the tumor-specific immune response and preventing
deleterious immune responses. Methods for subtraction of a nucleic
acid library are also known in the art (See Sambrook et al.,
supra).
[0128] In yet another embodiment of the present invention of the
method to elicit an immune response in a mammal that has cancer, a
therapeutic composition to be intravenously or intraperitoneally
administered to a mammal comprises a plurality of recombinant
nucleic acid molecules, wherein each of the recombinant nucleic
acid molecules comprises a cDNA sequence, each of the cDNA
sequences encoding a tumor antigen or a fragment thereof (i.e., at
least a portion of a tumor antigen as defined above). In this
embodiment, the cDNA sequences are amplified from total RNA that
has been isolated from a plurality of allogeneic tumor samples of
the same histological tumor type. Each of the plurality of cDNA
sequences is operatively linked to a transcription control
sequence. Administration of such a therapeutic composition to a
mammal that has cancer results in the expression of the cDNA
sequences encoding the tumor antigens in the tissue of the mammal
(according to the route of administration, as previously
discussed). In a further embodiment, such a therapeutic composition
comprises a recombinant nucleic acid molecule having a nucleic acid
sequence encoding a cytokine, wherein the nucleic acid sequence is
operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal
results in the expression of the nucleic acid sequence encoding the
cytokine in the tissues of the mammal.
[0129] In this embodiment of the present invention, a plurality of
recombinant nucleic acid molecules comprising cDNA sequences
encoding tumor antigens (i.e., a cDNA library) is prepared from the
total RNA isolated 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, the plurality of recombinant
molecules encoding tumor antigens is representative of the
substantially all of the tumor antigens present in any of the
individuals from which the RNA was isolated. This embodiment of the
method of the present invention provides a genetic 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. Such a therapeutic composition delivered by the
present method is particularly useful as a treatment, but may also
be useful as a preventative (i.e., prophylactic) therapy. Methods
to prepare such a cDNA library from a plurality of allogeneic tumor
samples are the same as those described above for autologous tumor
samples.
[0130] In yet another embodiment of the present invention of the
method to elicit an immune response in a mammal, a therapeutic
composition to be intravenously or intraperitoneally administered
to a mammal comprises a plurality of recombinant nucleic acid
molecules, wherein each of the recombinant nucleic acid molecules
comprises a cDNA sequence, each of the cDNA sequences encoding an
immunogen from an infectious disease pathogen or a fragment thereof
(i.e., at least a portion of a pathogen antigen as defined above).
In this embodiment, the cDNA sequences are amplified from total RNA
that has been isolated from an infectious disease pathogen. Each of
the plurality of cDNA sequences is operatively linked to a
transcription control sequence. Administration of such a
therapeutic composition to a mammal that has or might contract an
infectious disease results in the expression of the cDNA sequences
encoding the pathogen antigens in the tissue of the mammal
(according to the route of administration, as previously
discussed). In a further embodiment, such a therapeutic composition
comprises a recombinant nucleic acid molecule having a nucleic acid
sequence encoding a cytokine, wherein the nucleic acid sequence is
operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal
results in the expression of the nucleic acid sequence encoding the
cytokine in the tissues of the mammal.
[0131] In this embodiment of the present invention, the plurality
of recombinant molecules encoding pathogen antigens is
representative of the substantially all of the antigens present in
the infectious disease pathogen from which the RNA was isolated. 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. Methods to
prepare such a cDNA library from an infectious disease pathogen are
the same as those described above for tumor samples.
[0132] In yet another embodiment of the present invention of the
method to elicit an immune response in a mammal, a therapeutic
composition to be intravenously or intraperitoneally administered
to a mammal comprises a plurality of recombinant nucleic acid
molecules, each of the recombinant nucleic acid molecules
comprising a cDNA sequence amplified from total RNA isolated from
at least one allergen. In this embodiment, the cDNA sequences are
amplified from total RNA, or a fragment thereof, that has been
isolated from at least one, and preferably, multiple, allergens.
Each of the plurality of cDNA sequences is operatively linked to a
transcription control sequence. Administration of such a
therapeutic composition to a mammal that has or might contract a
disease associated with allergic inflammation results in the
expression of the cDNA sequences encoding the allergens in the
tissue of the mammal (according to the route of administration, as
previously discussed). In a further embodiment, such a therapeutic
composition comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, wherein the nucleic acid
sequence is operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal
results in the expression of the nucleic acid sequence encoding the
cytokine in the tissues of the mammal. In this embodiment of the
present invention, the plurality of recombinant molecules encoding
allergens is representative of the substantially all of the
epitopes present in the allergen from which the RNA was isolated.
Additionally, more than one allergen can be administered
simultaneously.
[0133] Another embodiment of the present invention relates to a
method to elicit a tumor antigen-specific immune response and a
systemic, non-specific immune response in a mammal that has cancer,
which includes the step of intravenously or intraperitoneally
administering to the mammal a therapeutic composition which
includes: (a) a liposome delivery vehicle; and (b) total RNA
isolated from a tumor sample, wherein the RNA encodes tumor
antigens or fragments thereof. Administration of such a therapeutic
composition to the mammal results in the expression of the RNA
encoding tumor antigens or fragments thereof in the tissue of the
mammal. In a preferred embodiment, the RNA is enriched for poly-A
RNA prior to administration of the therapeutic composition to the
mammal, as described above. In a further embodiment, the
therapeutic composition comprises a recombinant nucleic acid
molecule having a nucleic acid sequence encoding a cytokine,
wherein the nucleic acid sequence is operatively linked to a
transcription control sequence. Administration of such a
therapeutic composition to a mammal results in expression of the
nucleic acid sequence encoding the cytokine in the tissue of the
mammal.
[0134] In this embodiment of the present invention, total RNA or
more preferably, poly-A enriched RNA, is isolated from a tumor
sample as previously described (See Sambrook et al., supra),
complexed with a liposome delivery vehicle and administered
intravenously or intraperitoneally to a mammal that has cancer. The
RNA encoding substantially all of the tumor antigens of the tumor
sample is then expressed in the tissues of the mammal. Although RNA
is normally degraded rapidly in serum by RNAses, the present
inventors believe that RNA complexed to cationic lipids are
protected from such RNAses until it reaches the tissues, where gene
expression occurs. The advantage of administering RNA directly to a
mammal according to this particular embodiment of the method of the
present invention is that an immune response can be elicited
against multiple tumor antigens directly in vivo, without requiring
any substantial in vitro manipulations of the tumor tissues or host
immune cells. Specific examples of this embodiment of the present
invention are described in Examples 7a and 7b.
[0135] Another embodiment of the present invention relates to a
method to elicit a pathogen antigen-specific immune response and a
systemic, non-specific immune response in a mammal that has an
infectious disease, which includes the step of intravenously or
intraperitoneally administering to the mammal a therapeutic
composition which includes: (a) a liposome delivery vehicle; and
(b) total RNA isolated from an infectious disease pathogen, wherein
the RNA encodes pathogen antigens or fragments thereof.
Administration of such a therapeutic composition to the mammal
results in the expression of the RNA encoding pathogen antigens or
fragments thereof in the tissue of the mammal. In a preferred
embodiment, the RNA is enriched for poly-A RNA prior to
administration of the therapeutic composition to the mammal, as
described above. In a further embodiment, the therapeutic
composition comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, wherein the nucleic acid
sequence is operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal
results in expression of the nucleic acid sequence encoding the
cytokine in the tissue of the mammal.
[0136] Another embodiment of the present invention relates to a
method to elicit an allergen-specific immune response and a
systemic, non-specific immune response in a mammal that has a
disease associated with allergic inflammation, which includes the
step of intravenously or intraperitoneally administering to the
mammal a therapeutic composition which includes: (a) a liposome
delivery vehicle; and (b) total RNA isolated from an allergen,
wherein the RNA encodes at least one allergen protein or a fragment
thereof. Administration of such a therapeutic composition to the
mammal results in the expression of the RNA encoding at least one
allergen or a fragment thereof in the tissue of the mammal. In a
preferred embodiment, the RNA is enriched for poly-A RNA prior to
administration of the therapeutic composition to the mammal, as
described above. In a further embodiment, the therapeutic
composition comprises a recombinant nucleic acid molecule having a
nucleic acid sequence encoding a cytokine, wherein the nucleic acid
sequence is operatively linked to a transcription control sequence.
Administration of such a therapeutic composition to a mammal
results in expression of the nucleic acid sequence encoding the
cytokine in the tissue of the mammal.
[0137] A therapeutic composition of the present invention includes
a liposome delivery vehicle. According to the present invention, a
liposome delivery vehicle comprises a lipid composition that is
capable of preferentially delivering a therapeutic composition of
the present invention to the pulmonary tissues in a mammal when
administration is intravenous, and to the spleen and liver tissues
of a mammal when administration is intraperitoneal. The phrase
"preferentially delivering" means that although the liposome can
deliver a nucleic acid molecule to sites other than the pulmonary
or spleen and liver tissue of the mammal, these tissues are the
primary site of delivery.
[0138] 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. Other
targeting mechanisms, such as targeting by addition of exogenous
targeting molecules to a liposome (i.e., antibodies) are not a
necessary component of the liposome delivery vehicle of the present
invention, since effective immune activation at immunologically
active organs is already provided by the composition and route of
delivery of the present compositions without the aid of additional
targeting mechanisms. Additionally, for efficacy, the present
invention does not require that a protein encoded by a given
nucleic acid molecule be expressed within the target cell (e.g.,
tumor cell, pathogen, etc.). The compositions and method of the
present invention are efficacious when the proteins are expressed
in the vicinity of (i.e., adjacent to) the target site, including
when the proteins are expressed by non-target cells.
[0139] 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 of the present invention to a
preferred site in the mammal. A liposome delivery vehicle 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 hours.
[0140] A liposome delivery vehicle of the present invention
comprises a lipid composition that is capable of fusing with the
plasma membrane of the targeted cell to deliver a nucleic acid
molecule into a cell. Preferably, when a nucleic acid:liposome
complex of the present invention is administered intravenously, the
transfection efficiency of a nucleic acid:liposome complex of the
present invention is at least about 1 picogram (pg) of protein
expressed per milligram (mg) of total tissue protein per microgram
(.mu.g) of nucleic acid delivered. More preferably, the
transfection efficiency of a nucleic acid:liposome complex of the
present invention is at least about 10 pg of protein expressed per
mg of total tissue protein per .mu.g of nucleic acid delivered; and
even more preferably, at least about 50 pg of protein expressed per
mg of total tissue protein per .mu.g of nucleic acid delivered; and
most preferably, at least about 100 pg of protein expressed per mg
of total tissue protein per .mu.g of nucleic acid delivered. When
the route of delivery of a nucleic acid:lipid complex of the
present invention is intraperitoneal, the transfection efficiency
of the complex can be as low as 1 fg of protein expressed per mg of
total tissue protein per .mu.g of nucleic acid delivered, with the
above amounts being more preferred.
[0141] A preferred liposome delivery vehicle of the present
invention is between about 100 and 500 nanometers (nm), more
preferably between about 150 and 450 nm and even more preferably
between about 200 and 400 nm in diameter.
[0142] Suitable liposomes for use with the present invention
include any liposome. Preferred liposomes of the present invention
include those liposomes commonly used in, for example, gene
delivery methods known to those of skill in the art. Preferred
liposome delivery vehicles comprise multilamellar vesicle (MLV)
lipids and extruded lipids. Methods for preparation of MLV's are
well known in the art and are described, for example, in the
Examples section. 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. Although small unilamellar vesicle (SUV) lipids can be
used in the composition and method of the present invention, the
present inventors have found that multilamellar vesicle lipids are
significantly more immunostimulatory than SUVs when complexed with
nucleic acids in vivo (See Example 2d). More preferred liposome
delivery vehicles comprise liposomes having a polycationic lipid
composition (i.e., cationic liposomes) and/or liposomes having a
cholesterol backbone conjugated to polyethylene glycol. Preferred
cationic liposome compositions include, but are not limited to
DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and
cholesterol, and DDAB and cholesterol. A most preferred liposome
composition for use as a delivery vehicle in the method of the
present invention includes DOTAP and cholesterol.
[0143] Complexing a liposome with a nucleic acid molecule of the
present invention can be achieved using methods standard in the art
(see, for example, methods Section A described in the Examples).
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 suitable concentration of a
nucleic acid molecule of the present invention 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. When the nucleic acid molecule encodes
an immunogen or a cytokine, a suitable concentration of nucleic
acid molecule to add to a liposome includes a concentration
effective for delivering a sufficient amount of nucleic acid
molecule into a cell such that the cell can produce sufficient
immunogen and/or cytokine protein to regulate effector cell
immunity in a desired manner. 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 mmol 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 (i.e.,
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 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.
[0144] In another embodiment of the present invention, a
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 site. Preferred pharmaceutically
acceptable excipients are capable of maintaining a nucleic acid
molecule of the present invention in a form that, upon arrival of
the nucleic acid molecule to a cell, the nucleic acid molecule is
capable of entering the cell and being expressed by the cell if the
nucleic acid molecule encodes a protein to be expressed. Suitable
excipients of the present invention include excipients or
formularies that transport, but do not specifically target a
nucleic acid molecule 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
a preferred non-ionic buffer being 5% dextrose in water (DW5).
[0145] 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.
[0146] According to the present invention, an effective
administration protocol (i.e., administering a 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.
[0147] 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. Doses of a therapeutic
composition of the present invention suitable for use with
intravenous or intraperitoneal administration techniques can be
used by one of skill in the art to determine appropriate single
dose sizes for systemic administration based on the size of a
mammal.
[0148] In a preferred embodiment, an appropriate single dose of a
nucleic acid:liposome complex 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 11g 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.
[0149] Preferably, when nucleic acid:liposome complex of the
present invention contains a nucleic acid molecule which is to be
expressed in the mammal, an appropriate single dose of a nucleic
acid:liposome complex of the present invention results in at least
about 1 pg of protein expressed per mg of total tissue protein per
.mu.g of nucleic acid delivered. More preferably, an appropriate
single dose of a nucleic acid:liposome complex of the present
invention is a dose which results in at least about 10 pg of
protein expressed per mg of total tissue protein per .mu.g of
nucleic acid delivered; and even more preferably, at least about 50
pg of protein expressed per mg of total tissue protein per .mu.g of
nucleic acid delivered; and most preferably, at least about 100 pg
of protein expressed per mg of total tissue protein per .mu.g of
nucleic acid delivered. When the route of delivery of a nucleic
acid:lipid complex of the present invention is intraperitoneal, an
appropriate single dose of a nucleic acid:liposome complex of the
present invention is a dose which results in as low as 1 fg of
protein expressed per mg of total tissue protein per .mu.g of
nucleic acid delivered, with the above amounts being more
preferred.
[0150] A suitable single dose of a therapeutic composition of the
present invention to elicit a systemic, non-antigen-specific immune
response in a mammal is a sufficient amount of a nucleic acid
molecule complexed to a liposome delivery vehicle, when
administered intravenously or intraperitoneally, to elicit 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). Preferred dosages of nucleic acid molecules to be included
in a nucleic acid:lipid complex of the present invention have been
discussed above.
[0151] A suitable single dose of a therapeutic composition to
elicit an immune response against a tumor is a sufficient amount of
a tumor antigen-encoding recombinant molecule, alone or in
combination with a cytokine-encoding recombinant molecule, to
reduce, and preferably eliminate, the tumor following lipofection
of the recombinant molecules into cells of the tissue of the mammal
that has cancer.
[0152] According to the present invention, a single dose of a
therapeutic composition useful to elicit an immune response against
an infectious disease and/or against a lesion associated with such
a disease, comprising a pathogen-encoding recombinant molecule
combined with liposomes, alone or in combination with a
cytokine-encoding recombinant molecule with liposomes, is
substantially similar to those doses used to treat a tumor (as
described in detail above). Similarly, a single dose of a
therapeutic composition useful to elicit an immune response against
an allergen, comprising an allergen-encoding recombinant molecule
combined with liposomes, alone or in combination with a
cytokine-encoding recombinant molecule with liposomes, is
substantially similar to those doses used to treat a tumor.
[0153] 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 required to treat a
given disease.
[0154] 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 3-4 weeks after the initial
administration, optionally followed by subsequent booster
immunizations every 3-4 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).
[0155] A preferred number of doses of a therapeutic composition
comprising a tumor antigen-encoding recombinant molecule, alone or
in combination with a cytokine-encoding recombinant molecule,
complexed with a liposome delivery vehicle in order to elicit an
immune response against a metastatic cancer, 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 3-4 weeks, as described above,
until signs of remission appear, and then once a month until the
disease is gone.
[0156] According to the present invention, the number of doses of a
therapeutic composition to elicit an immune response against an
infectious disease and/or a lesion associated with such disease,
comprising a pathogen antigen-encoding recombinant molecule, alone
or in combination with a cytokine-encoding recombinant molecule,
complexed with a liposome delivery vehicle, is substantially
similar to those number of doses used to treat a tumor (as
described in detail above).
[0157] A therapeutic composition is administered to a mammal in a
fashion to elicit a systemic, non-antigen-specific immune response
in a mammal, and when the nucleic acid molecule in the composition
encodes an immunogen, to enable expression of the administered
recombinant molecule of the present invention into an immunogenic
protein (in the case of the tumor, pathogen antigen or allergen) or
immunoregulatory protein (in the case of the cytokine) in the
mammal to be treated for disease. According to the method of the
present invention, a therapeutic composition is administered by
intravenous or intraperitoneal injection, and preferably,
intravenously. Intravenous injections can be performed using
methods standard in the art. According to the method of the present
invention, administration of the nucleic acid:lipid complexes can
be at any site in the mammal wherein systemic administration (i.e.,
intravenous or intraperitoneal administration) is possible,
particularly when the liposome delivery vehicle comprises cationic
liposomes. Administration at any site in a mammal will elicit a
potent immune response when either intravenous or intraperitoneal
administration is used, and particularly, when intravenous
administration is used. 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. When a specific
anti-tumor effect is desired (i.e., reduction or elimination of a
tumor) and the route of administration is intravenous, the site of
administration again can be at any site by which a composition can
be administered intravenously, regardless of the location of the
tumor relative to the site of administration. For intraperitoneal
administration with regard to anti-tumor efficacy (but not immune
activation/immunization), it is preferable to use this mode of
administration when the tumor is in the peritoneal cavity, or when
the tumor is a small tumor. For immunization and immune activation,
as discussed above, intraperitoneal administration is a suitable
mode of administration, particularly in comparison to non-systemic
routes, as demonstrated in the Examples section.
[0158] In the method of the present invention, 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.
[0159] As discussed above, a 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. A therapeutic composition of the present
invention, when delivered intravenously or intraperitoneally, 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 a nucleic acid:lipid complex of the
present invention, wherein the nucleic acid either is not
operatively linked to a transcription control sequence, or more
preferably, encodes a tumor antigen-encoding recombinant molecule
operatively linked to a transcription control sequence, alone or in
combination with a cytokine-encoding recombinant molecule
(separately or together). A therapeutic composition of the present
invention, elicits a systemic, non-specific immune response in the
mammal and, upon entering targeted pulmonary or spleen and liver
cells, leads to the production of tumor antigen (and, in particular
embodiments, cytokine protein) that activate cytotoxic T cells,
natural killer cells, T helper cells and macrophages. Such cellular
activation overcomes the otherwise relative lack of immune response
to cancer cells, leading to the destruction of such cells.
[0160] A therapeutic composition of the present invention which
includes a nucleic acid molecule encoding a tumor antigen is useful
for eliciting an immune response in a mammal that has cancer,
including both 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 target the vesicles of tumors, so that
expression of a tumor antigen or cytokine within the tumor cell
itself is not necessary to provide efficacy against the tumor.
Indeed, a general advantage of the present invention is that
delivery of the composition itself elicits a powerful immune
response and expression of the nucleic acid molecule at least in
the vicinity of the target site (at or adjacent to the site)
provides effective immune activation and efficacy against the
target.
[0161] A therapeutic composition of the present invention which
includes a nucleic acid molecule encoding a tumor antigen is
preferably used to elicit an immune response in a mammal that has a
cancer which includes, but is 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 pulmonary
tissue of a mammal that has cancer (i.e., by intravenous delivery)
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.
[0162] A therapeutic composition of the present invention which
includes a nucleic acid molecule encoding an immunogen from an
infectious disease pathogen is advantageous for eliciting an immune
response in a mammal that has infectious diseases responsive to an
immune response. 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. A therapeutic
composition for use in the elicitation of an immune response in a
mammal that has an infectious disease comprises a pathogen
antigen-encoding recombinant molecule, alone or in combination with
a cytokine-encoding recombinant molecule of the present invention,
combined with a liposome delivery vehicle. 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 with or without cytokines can
result in increased T cell, natural killer cell, and macrophage
cell activity that overcome the relative lack of immune response to
a lesion formed by a pathogen. Preferably, expression of the
immunogen in 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 stimulation of effector cell immunity against the
disease.
[0163] A therapeutic composition of the present invention is
particularly useful for eliciting an immune response in a mammal
that has an infectious diseases caused by pathogens, including, but
not limited to, bacteria (including intracellular bacteria which
reside in host cells), viruses, parasites (including internal
parasites), fungi (including pathogenic fungi) and endoparasites.
Preferred infectious diseases to treat with a therapeutic
composition of the present invention include chronic infectious
diseases, and more preferably, pulmonary infectious diseases, such
as tuberculosis. Particularly preferred infectious diseases to
treat with a therapeutic composition of the present invention
include human immunodeficiency virus (HIV), Mycobacterium
tuberculosis, herpesvirus, papillomavirus and Candida.
[0164] In one embodiment, an infectious disease a therapeutic
composition of the present invention is a viral disease, and
preferably, is a viral disease caused by a virus which includes,
human immunodeficiency virus, and feline immunodeficiency
virus.
[0165] A therapeutic composition of the present invention which
includes a nucleic acid molecule encoding an immunogen that is an
allergen is advantageous for eliciting an immune response in a
mammal that has 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, as described in detail in the Examples section,
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 allergen-encoding recombinant molecule,
alone or in combination with a cytokine-encoding recombinant
molecule, combined with a liposome delivery vehicle. 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 with allergens with or without cytokines can
result in increased Th1-type T cell, natural killer cell, and
macrophage cell activity that overcome the harmful effects of a
Th2-type immune response against the same allergen. Preferably,
expression of the allergen in a tissue of a mammal that has a
disease associated with allergic inflammation 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.
[0166] 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.
[0167] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
[0168] For the following Examples 1-7, the following experimental
methods and materials were used.
[0169] A. Preparation of Cationic Lipid DNA Complexes (CLDC):
[0170] The cationic liposomes used in the following experiments
(unless otherwise indicated) consisted of DOTAP (1,2
dioleoyl-3-trimethylammonium- -propane) and cholesterol mixed in a
1:1 molar ratio, dried down in round bottom tubes, then rehydrated
in 5% dextrose solution (D5W) by heating at 50.degree. C. for 6
hours, as described previously (Solodin et al., 1995, Biochemistry
34:13537-13544, incorporated herein by reference in its entirety).
Other lipids (e.g., DOTMA) were prepared similarly for some
experiments as indicated. This procedure results in the formation
of liposomes that consists of multilamellar vesicles (MLV), which
the present inventors have found give optimal transfection
efficiency as compared to small unilamellar vesicles (SUV). The
production of MLVs and related "extruded lipids" is also described
in Liu et al., 1997, Nature Biotech. 15:167-173; and Templeton et
al., 1997, Nature Biotech. 15:647-652; both of which are
incorporated herein by reference in their entirety. Plasmid DNA
(pCR3.1, Invitrogen) was purified from E. coli as described
previously, using modified alkaline lysis and polyethylene glycol
precipitation (Liu et al., 1997, supra). DNA for injection was
resuspended in distilled water. Eukaryotic DNA (salmon testis and
calf thymus) was purchased from Sigma Chemical Company. For many of
the experiments reported here, the plasmid DNA did not contain a
gene insert (unless otherwise noted), and is thus referred to as
"non-coding" or "empty vector" DNA.
[0171] The cationic lipid DNA complexes (CLDC) used in the
experiments below were prepared by gently adding DNA to a solution
of lipid in 5% dextrose solution (D5W) at room temperature, then
gently pipetting up and down several times to assure proper mixing.
The DNA:lipid ratio was 1:8 (1.0 ug DNA to 8 nmol lipid). The CLDC
were used within 30-60 minutes of preparation. To prepare small
unilamellar vesicles (SUV) used in some experiments (as indicated),
the CLDC that were formed using MLV liposomes as described above
were subjected to sonication for 5 minutes, as described previously
(Liu et al., 1997, supra).
[0172] B. Gene Constructs
[0173] For antigen-specific immunization experiments,
plasmid-based, eukaryotic expression vectors were utilized to
express genes in vivo. Expression vectors (using pCR3.1,
Invitrogen) for the cytokine cDNAs (IL-2, IFN*, 1L-12) were all
constructed using PCR amplification of RNA prepared from normal
mouse spleens as described, for example in Sambrook et al., supra.
The *-gal expression construct was provided by Dr. Cori Gorman. For
immunization with these gene constructs, CLDC containing the
desired gene constructs were injected by tail vein (i.e.,
intravenous delivery) or intraperitoneally (as indicated) to
deliver a total DNA amount of 5.0 to 10.0 ug DNA.
[0174] For RNA immunization experiments, tumor cells (either B 16
cells or CT-26 cells; see below) were grown in vitro, followed by
extraction of the poly-A enriched RNA using standard procedures
(Sambrook, supra). The RNA was resuspended in water and frozen
prior to formation of complexes with liposomes. The same lipid:RNA
ratios as described above for lipid:DNA complexes were used to
prepare cationic lipid RNA complexes (CLRC).
[0175] When more than one gene was injected simultaneously into the
same animal, the plasmid DNAs were first mixed and then added to
liposomes to form CLDC.
[0176] C. In Vivo Evaluation of Immune Activation
[0177] Mice (3 per group, unless otherwise indicated) were injected
intravenously or intraperitoneally, as indicated in the individual
experiments, once with 100 ul of CLDC (prepared as described above)
in D5W. Control mice were injected with 100*1 of D5W only. Three
different strains of mice were evaluated in these experiments
(C57B1/6, BALB/c, ICR), but most of the data was generated using
C57B1/6 mice. The total amount of DNA injected was 10 .mu.g per
mouse, unless specified otherwise. At various time points
post-injection (as indicated), the spleen and lung tissues were
collected, mononuclear cell preparations were made, and the cells
were assayed for expression of activation markers or cytokine
release (see below).
[0178] D. In Vitro Evaluation of Immune Activation
[0179] Spleen cells obtained from normal (untreated mice) were
incubated in modified Eagles cell culture medium with 10% FBS with
either lipid alone, DNA alone, or cationic lipid DNA complexes
(CLDC) to assess the effects on immune activation. The final DNA
concentration in these experiments was 1.0 .mu.g/ml medium. Cell
activation was assessed by flow cytometry and cytokine release was
quantitated by ELISA (see below).
[0180] E. Flow Cytometry
[0181] Upregulation of the early activation marker, CD69, which is
upregulated on activated T cells, B cells, macrophages and NK
cells, was used to assess early immune cell activation. Single cell
suspensions were prepared from spleens of mice by NH.sub.4Cl lysis
procedure (Sambrook, supra), and lung mononuclear cells were
prepared from lung tissues by collagenase digestion. Briefly, lung
tissues were digested in 0.02% collagenase at 37.degree. C. for one
hour. Lung mononuclear cells were purified from the digested tissue
by Ficoll gradient centrifugation. For each experiment, spleen and
lung cells were prepared from 3 animals per treatment group, unless
noted otherwise. Cells were analyzed using a Becton-Dickinson
FACSCalibur flow cytometer, with analysis gates set by first gating
on spleen lymphocytes. Between 10,000 and 30,000 gated events were
analyzed for each cell type. For analysis of cell activation,
3-color flow cytometric analysis was done, using anti-CD69
phycoerythrin (Pharmingen, San Diego, Calif.) to quantitate the
number of CD69 positive cells. Cells were also dual-labeled to
evaluate T cells (anti-**TCR antibody (biotin H57.597; Pharmingen)
plus antibodies to either CD4 (FITC RM4-5; Pharmingen) or CD8 (FITC
53-6.7; Pharmingen). B cells were dual-labeled with anti-B220
(Pharmingen) and anti-IA.sup.b (FITC 3F12.35; provided by Dr. John
Freed, National Jewish) or anti-IA.sup.d (FITC 14.44); NK cells
were dual-labeled using anti NK 1.1 (biotin PK136; Pharmingen) and
anti CD3 (FITC 2C11); macrophages were evaluated using anti-CR3
(biotin Mac-1; Pharmingen) and FITC anti-IA.sup.b or anti-IA.sup.d.
The percentage of double positive cells expressing CD69 was
determined for each cell type, and the mean (.+-.SD) CD69+ cells
plotted.
[0182] F. Cytotoxicity Assay
[0183] A standard 4-hour .sup.51Cr-release assay was used to
quantitate cytotoxic activity present in freshly isolated lung and
spleen mononuclear cells, using YAC-1 cells as targets. Briefly,
effector cells from lung or spleen were added in decreasing
concentrations to duplicate wells of a Linbro plate, to which was
then added 5.times.10.sup.3 target cells that had been previously
labeled for 1 hour with .sup.51Cr. The plates were incubated at
37.degree. C. for 4 hours, then supernatants from each well were
harvested and the amount of radioactive Cr present was determined
by automated gamma counter. The percentage specific lysis was
calculated as follows: 1 ( observed 51 Cr release ) - ( spontaneous
51 Cr release ) ( maximum 51 Cr release ) - ( spontaneous 51 Cr
release ) .times. 100
[0184] G. NK Cell Depletion In Vivo
[0185] Mice were depleted of NK cells in vivo by a single
intraperitoneal (i.p.) injection of 50 .mu.l rabbit anti-asialoGM1
antiserum (Wako BioProducts, Richmond, Va.). Control animals were
injected with 50 .mu.l non-immune rabbit serum. In other
experiments, mice were depleted of NK cells by i.p. injection of a
monoclonal antibody to NK cells (PK-136), and control mice were
injected with an irrelevant, isotype-matched antibody. It was
confirmed that these treatments eliminated detectable NK cells in
spleen and lung (as determined by flow cytometry) and also
eliminated cytotoxic activity in spleen cells (data not shown).
[0186] H. Cytokine Assays
[0187] Cytokine release was measured in spleen cell supernatants
after either in vivo or in vitro stimulation. For assay of cytokine
release after in vivo stimulation, spleen or lung mononuclear cells
were prepared from mice either 6 or 24 hours after i.v. injection,
then cultured at a concentration of 5.times.10.sup.6 cells/ml for
an additional 18 hours before supernatants were harvested. For in
vitro stimulation of cytokine release, spleen cells were incubated
in vitro with DNA, lipid, or DNA plus lipid at a final DNA
concentration of 1.0 .mu.g DNA per ml for 18 hours, at which time
the supernatants were harvested for cytokine assay.
Interferon-gamma (IFN*) was assayed using a sandwich ELISA as is
known in the art.
[0188] I. Tumor Challenge Experiments
[0189] The B16 (clone F1O) cells were obtained from Dr. Isiah
Fidler (M D Anderson, Houston, Tex.); MCA-205 cells were provided
by Dr Jack Routes (National Jewish); CT-26 cells were provided by
Dr. Nicholas Restifo (National Cancer Institute); 4T1 cells were
provided by Dr. Susan Rosenberg). All cell lines were maintained at
37.degree. C. in Modified Eagles medium supplemented with essential
and non-essential amino acids, penicillin and glutamine, and 5%
fetal bovine serum, and were treated periodically with
ciprofloxacin (101g/ml) to maintain mycoplasma-free conditions. The
*-gal transfected CT-26 tumor cell line (known as CL-25) was also
provided by Dr. Nicholas Restifo.
[0190] To establish experimental pulmonary metastases, mice (4 per
treatment group) were injected once via the lateral tail vein with
2.5.times.10.sup.5 tumor cells. Treatment with DNA-lipid complexes
was initiated 3 days after tumor injection, and was repeated once
on day 10 after tumor injection; control mice were injected i.v.
with D5W alone. Mice were sacrificed on day 17 to 20 after tumor
injection, and the number of tumor nodules per lung was determined
by insufflating lungs with India ink solution and manually counting
total nodules per lung under a tumor dissecting microscope (Wexter
et al., 1966, J. Natl. Cancer Inst. 36:641-645, incorporated herein
by reference in its entirety).
Example 1
[0191] The following experiments a-l and FIGS. 1-12 show that
systemically administered cationic liposome DNA complexes (CLDC)
formed with non-coding DNA (empty vector) elicit potent immune
responses in vivo.
[0192] (a) The following experiment shows that intravenous (i.v.)
injection of CLDC containing empty vector DNA induces marked
activation of 5 different immune effector cell populations in vivo.
In this experiment, CLDC were prepared which consisted of DOTAP and
cholesterol mixed in a 1:1 molar ratio complexed with empty vector
plasmid DNA (see Section A above). C57B1/6 mice were injected
intravenously with 100*1 of CLDC (10 .mu.g empty vector DNA per
mouse) in DW5 as described (Section C). 24 hours post-injection,
spleen cells were harvested from control mice injected with diluent
(D5W), and from mice injected with CLDC. Cells were labeled with
specific antibodies to evaluate CD4+ and CD8+ T cells, NK cells, B
cells, and macrophages and with an antibody to CD69 (early
activation marker) and analyzed by flow cytometry (Section E). FIG.
1 shows the results from CD69/immune effector cell staining with
control mice (open bars) and 3 CLDC-injected mice (black bars).
Injection of CLDC (empty vector) induced pronounced upregulation of
CD69 expression on all relevant immune effector cell populations,
and similar results were observed as early as 6 hours
post-administration (data not shown). These results indicate that
systemic administration of CLDC (empty vector) induces massive and
rapid immune activation.
[0193] (b) The following experiment shows that CLDC, but not lipid
or DNA alone, induce immune activation in vivo. C57B1/6 mice were
injected intravenously with DNA alone (empty vector; 10 .mu.g),
lipid alone (DOTAP:cholesterol), or DNA+ lipid (CLDC-empty vector)
as described above (Sections A & C) and upregulation of CD69
expression (immune activation) on T cells, NK cells was evaluated
24 hours later by flow cytometry (Section E). The data presented in
FIG. 2A (CD69+/CD8+ cells) and 2B (CD69+/NK1.1+ cells) clearly
illustrate the synergistic immune stimulatory interaction that
occurs when DNA and cationic lipids are complexed together. Similar
results were also obtained for CD4+ T cells, B cells and
macrophages (data not shown).
[0194] (c) The following experiment compares the immune activating
potencies of LPS, poly I/C, and CLDC (empty vector). C57B1/6 mice
were injected i.v. with 10 .mu.g each of LPS, poly I/C, or CLDC (10
.mu.g DNA) and spleen cells were analyzed for upregulation of CD69
by flow cytometry 24 hours later (as described in Sections A, C,
and E). FIG. 3 shows that injection of CLDC induced substantially
greater immune activation than either of the classical immune
activating stimuli, LPS or poly I/C, indicative of the extreme
immune activating potency of CLDC.
[0195] (d) The following experiment shows that even low dose CLDC
administered by the present method induces significant immune
activation. C57B1/6 mice were injected i.v. with decreasing doses
of CLDC (empty vector), and immune activation (CD69 upregulation on
NK cells) was assessed 24 hours later (see Sections A, C, and E).
FIG. 4 shows that even an extremely low dose of CLDC (100 ng) was
capable of inducing significant immune activation.
[0196] (e) The following experiment demonstrates that both
intraperitoneal and intravenous administration of CLDC induce
potent immune activation. CLDC (empty vector) were administered to
C57B1/6 mice either intravenously (i.v.) or intraperitoneally
(i.p.), and immune activation (CD69 upregulation) on splenic NK
cells was assessed by flow cytometry (See Sections A, C, and E).
FIG. 5 shows that administration of CLDC by either route induced
substantial immune activation, although the i.v. route was more
potent than the i.p. route.
[0197] (f) The following experiment shows that the immune
activation elicited by administration of CLDC according to the
present method can be induced by different lipid formulations.
C57B1/6 mice were injected i.v. with CLDC (empty vector) prepared
using liposomes of several different lipid compositions, but all
formulated as MLVs (as described in Sections A and C). At 24 hours
post injection, the degree of immune activation (CD69 upregulation)
on spleen cells was assessed (Section E). FIG. 6 shows that
equivalent immune activation was induced by lipids having 3
different chemical compositions, indicating that the immune
activating properties of CLDC is a general property and is not
dependent on any one particular lipid composition.
[0198] (g) The following experiment demonstrates that immune
activation by CLDC is independent of the DNA source. It has been
previously established that bacterial DNA is immunostimulatory in
mammals, whereas DNA from eukaryotic sources is not (See, for
example, Pisetsky et al., 1996, supra; Pisetsky, 1996, supra;
Yamamoto, et al., 1994, supra; Roman, et al., 1997, supra; Krieg,
1996, supra; Sun, et al., 1996, supra; Stacey et al., 1996, supra;
Sato, et al., 1996, supra; or Ballas, 1996, supra). Therefore, the
ability of CLDC formulated with either bacterial DNA (empty vector
plasmid DNA) or eukaryotic DNA from 2 different sources (salmon
sperm or calf thymus) was evaluated in vivo. C57B1/6 mice were
injected i.v. with CLDC containing DNA from one of these sources
(each formulated to deliver 10 ug DNA per mouse) (See Section A
& C). Twenty-four hours after i.v. injection of CLDC, the
degree of CD69 upregulation on splenic NK cells was assessed by
flow cytometry (Section E). FIG. 7 illustrates that immune
activation was observed when mice were injected with CLDC comprised
of either eukaryotic or bacterial DNA. Injection of salmon sperm or
calf thymus DNA alone did not induce CD69 upregulation (data not
shown). Thus, the immune activating properties of CLDC are
surprisingly independent of the DNA source, and immune activation
can also be induced by complexes of cationic lipids and RNA (see
Example 7 below).
[0199] (h) The following experiment shows that cytokine release is
induced by CLDC, but not by DNA or lipid alone. Spleen cells were
incubated for 24 hours in vitro with CLDC (empty vector), DNA alone
(empty vector), or lipid alone (DOTAP:cholesterol) and the
supernatants were assayed for IFN* (as well as other cytokines,
data not shown) (See Sections D and H). FIG. 8 shows the results of
an IFN* ELISA. As was observed for CD69 upregulation, cytokine
release is also triggered only by the CLDC and not by either
component alone. Thus, formation of the DNA-lipid complex clearly
markedly accentuates any immune stimulatory properties that plasmid
DNA and lipid alone might possess.
[0200] (i) The following experiment demonstrates that injection of
CLDC, but not poly I/C or LPS, induces IFN* production in vivo.
C57B1/6 mice (3 per group) were injected i.v. with 10 .mu.g of
either CLDC (empty vector), poly I/C, or LPS (as described in
Sections A & C). Six hours later, spleen cells were harvested
and cultured in vitro for an additional 12 hours. Then, cytokine
levels in the supernatants were measured (Section H). FIG. 9 shows
that the in vivo cytokine response to CLDC injection was clearly
different than the response to 2 other classical immune activating
stimuli (LPS, poly I/C), thereby illustrating a marked difference
between CLDC and other so-called non-specific immune
stimulators.
[0201] (j) The following experiment shows that NK cells are the
source of IFN* production elicited by i.v. CLDC injection. To
determine the cell type producing IFN* after injection of CLDC
(empty vector), C57B1/6 mice were depleted of NK cells using an
anti-NK cell antibody (EV/aNK), or were untreated (control), or
injected with CLDC and untreated (EV/-) or injected with CLDC and
treated with an irrelevant antiserum (EV/NRS) (as described in
Section G). The amount of IFN* elaborated by spleen (FIG. 10A) and
lung cells (FIG. 10B) 24 hours after injection of CLDC was
quantitated (Section H). This experiment demonstrates that NK cells
are the primary source of IFN* induced by i.v. administration of
CLDC.
[0202] (k) The following experiment shows that intravenous
injection of CLDC induces high levels of NK activity in spleen
cells. FIG. 11 illustrates that spleen cells harvested 24 hours
after i.v. injection of CLDC (empty vector) exhibit high levels of
killing of tumor target cells (tumor cell cytotoxicity) (See
Section F). To identify the cell type responsible for this tumor
cell killing activity, C57B1/6 mice were depleted of NK cells 48
hours prior to injection of CLDC (asialo GM1) or were treated with
an irrelevant antiserum (NRS) or were untreated (control) (as
described in Section G). This experiment indicates that NK cells
are the primary cell type responsible for the tumor cell killing
activity elicited by injection of CLDC.
[0203] (l) The following experiment demonstrates that
intraperitoneal injection of CLDC induces immune activation. Spleen
cells were harvested from C57BI/6 mice 24 hours after
intraperitoneal injection of 10 .mu.g CLDC (10 .mu.g DNA) complexes
encoding either nothing (empty vector; EV) or the IL-2 gene (IL-2),
and assayed for CD69 upregulation in both CD8+ and NK1.1+ cells
(Sections A, B, C and E). FIG. 12A (CD8+) and FIG. 12B (NK1.1+)
shows that intraperitoneal injection of CLDC with either empty
vector or the IL-2 gene induced immune activation, although the
effect was not as great as that induced by i.v. delivery (see FIG.
5). CLDC encoding IL-2 also demonstrated an enhanced immune
activation as compared to CLDC (empty vector).
Example 2
[0204] The following experiments a-d and FIGS. 13-16 demonstrate
that CLDC formed with non-coding DNA (empty vector) exert potent
antitumor effects in vivo when administered according to the method
of the present invention.
[0205] (a) The following experiment demonstrates that CLDC exert
potent antitumor effects when administered to a mammal by the
present method. The antitumor efficacy of CLDC (empty vector) was
evaluated in 4 different murine models of metastatic cancer:
MCA-205 (C57B1/6; fibrosarcoma; FIG. 13A); B16 (C57BI/6; melanoma;
FIG. 13B); CT26 (BALB/c; colon carcinoma; FIG. 13C); and 4T1
(BALB/c; breast cancer; FIG. 13D). In each model, tumors were
established in the lungs of mice (4 per group) by i.v. injection of
2.5.times.10.sup.5 tumor cells per mouse (as described in Section
I). Three days after the tumor cells were injected, treatment with
i.v. administration of 100 .mu.l CLDC was administered (10 .mu.g
empty vector DNA complexed to MLV liposomes as described in
Sections A and C), and repeated once in 7 days. Control mice were
injected with diluent (D5W). Seven days after the second injection
(17 days after the tumor cells were first injected), the mice were
sacrificed and the number of tumor nodules in the lungs determined
by manual counting, as described above (Wexter et al., 1966,
supra). FIGS. 13A-D illustrates the potent antitumor activity
exerted by systemically administered CLDC, using 4 different tumor
models and 2 different strains of mice (C57B1/6 and BALB/c).
[0206] (b) The following experiment shows that systemic
administration of CLDC, but not administration of DNA or lipid
alone, induces antitumor activity. C57B1/6 mice (4 per group) with
day 3 established MCA-205 tumors (Section I) were treated twice
with i.v. injections of either MLV liposomes alone, empty vector
DNA alone, or CLDC (empty vector) (See Sections A and I). The
number of lung tumor metastases was determined on day 17 post-tumor
injection and the results are shown in FIG. 14. This experiment
demonstrates that the CLDC, but neither of the 2 constituents (DNA
or lipid) alone, induces antitumor activity.
[0207] (c) The following experiment shows that the antitumor
activity of CLDC (empty vector) is independent of the DNA source.
To determine whether the antitumor activity observed with CLDC in
experiments (a) and (b) above was only a property of CLDC
formulated with bacterial DNA, mice with day 3 established MCA-205
lung metastases were treated with CLDC that were formed using
either plasmid (bacterial) DNA, or eukaryotic DNA (from calf thymus
or salmon testis). FIG. 15 shows clearly that CLDC formulated with
either bacterial or eukaryotic DNA induced antitumor activity,
though the bacterial DNA had slightly more potent activity.
[0208] (d) The following experiment demonstrates that the type of
CLDC administered significantly influences antitumor activity of
the composition. Previous investigators have used CLDC formulated
as SUV (small unilamellar vesicles) to target systemic gene
transfer to the lungs. The present inventors have found that
systemic administration of CLDC formulated as MLV, however, induce
much greater antitumor activity, even when only empty vector DNA is
administered. FIG. 16 clearly illustrates this difference. FIG. 16
shows that, where day 3 MCA-205 lung metastases were treated with
101g empty vector DNA administered using CLDC formulated as either
SUVs or MLVs, the MLV formulations provided significantly greater
antitumor effects.
Example 3
[0209] The following experiment and FIGS. 17A-C show that
intravenous injection of CLDC induces selective gene expression in
pulmonary tissues. C57B1/6 mice were injected i.v. with CLDC
encoding a reporter gene, courteously provided by Dr. Robert Debs
(luciferase; panel a), and the location of gene expression in
various organs was determined 24 hours later (See Sections A, B and
C). As shown in FIG. 17A, luciferase gene expression was almost
exclusively confined to pulmonary tissues. In FIGS. 17B and 17C,
i.v. injection of CLDC encoding IL-2 or IFN* resulted in efficient
intrapulmonary expression of IL-2 and IFN*, as demonstrated by
determination of cytokine expression in lung tissues extracted from
the mice. Injection of non-coding CLDC (EV) was included as an
additional control.
Example 4
[0210] The following experiment and FIGS. 18A-F demonstrates that
administration of cytokine genes using CLDC delivery improves the
antitumor effect over empty vector alone. Using 3 different tumor
models as described in Example 2 (MCA-205, FIGS. 18A and 18D; CT26,
FIGS. 18B and 18E; B16, FIGS. 18C and 18F), we evaluated the
antitumor effects of i.v. delivery of cytokine genes (IL-2, IFN*,
and IL-12) using CLDC containing plasmid DNA expressing these
genes, and compared the antitumor effects to those induced by empty
vector DNA (See Sections A, B, C, and I). In both the day 3
treatment models (FIGS. 18A, 18B and 18C) and the day 6 treatment
models (FIGS. 18D, 18E and 18F), addition of a cytokine gene that
stimulates NK cells induced greater antitumor activity than the
empty vector DNA alone, and this additional antitumor effect was
particularly pronounced in the day 6 treatment models. It is
believed that the added antitumor effect induced by the cytokine
genes enhances and depends to a large degree on the initial immune
activation inherent to administration of CLDC.
Example 5
[0211] The following experiment and FIGS. 19A and 19B show that
administration of CLDC having DNA encoding ovalbumin induces strong
systemic antigen-specific immune responses.
[0212] The following experiment shows that intravenous injection of
CLDC encoding an antigen gene induces strong systemic
antigen-specific immune responses and that intravenous (i.v.) DNA
immunization is more potent than intramuscular (i.m.) DNA
immunization. C57BI/6 mice (3 per group) were immunized either
intramuscularly (IM) with 100 .mu.g DNA encoding the ovalbumin
(OVA) gene, or intravenously (IV) with 10 .mu.g CLDC encoding the
OVA gene (Sections A, B, C). Three weeks later, spleen cells were
harvested and assayed for their ability (i.e., CTL activity) to
lyse OVA-expressing target cells (Section F). The results are shown
in FIGS. 19A and 19B. To detect OVA-specific CTL, lymphocytes from
immunized mice were assayed for cytotoxic activity against a
control cell line (open circles) or an OVA-expressing target cell
(filled circles). FIG. 19A illustrates that there was significantly
greater killing of the OVA-expressing target cells, indicating that
immunization with CLDC encoding an antigen is an efficient means of
inducing antigen-specific immune responses in vivo. FIG. 19B shows
that administration of one-tenth of the amount of DNA using CLDC by
intravenous administration induces equivalent levels of
antigen-specific CTL activity observed with intramuscular
injection.
Example 6
[0213] The following experiments a-d and FIGS. 20-23 demonstrate
that the administration of CLDC having DNA encoding a tumor antigen
induces strong anti-tumor activity and antigen-specific immune
responses in vivo.
[0214] (a) The following experiment shows that systemic
immunization with CLDC encoding a tumor antigen induces strong
antitumor activity in vivo. BALB/c mice (4 per group) were given
2.5.times.10 CL-25 tumor cells i.v. to establish pulmonary
metastases (Section I). The CL-25 tumor line is derived from the
CT26 colon carcinoma cell line and has been modified to express the
*-gal antigen. Three days after administration of the CL-25 tumor
cells, mice were treated with 2 i.v. administrations of CLDC
encoding either nothing (EV) or the *-gal gene (B-gal), one week
apart (Sections A, B, and C). One week after the second treatment,
the mice were sacrificed and the antitumor effect was quantitated
by counting the number of lung tumor nodules. FIG. 20 shows that
the number of tumors was significantly reduced by administration of
empty vector CLDC (EV), but was even further reduced by
administration of CLDC encoding the specific tumor antigen, *-gal
(B-gal). This experiment illustrates the principle that i.v.
administration of CLDC encoding a tumor antigen (or antigen(s)) is
an effective approach to eliciting immune responses against
established tumors.
[0215] (b) The following experiment demonstrates that i.v.
administered CLDC-mediated immunization against a tumor antigen
induces effective antitumor immunity, whereas intramuscular (IM) or
intradermal (ID) immunization does not. Mice (4 per treatment
group) with day 3 established CL25 lung tumors were treated by
intravenous DNA immunization with *-gal DNA (Sections A, B, C, and
I). FIG. 21 shows that mice treated with intramuscular (B-gal/IM)
or intradermal (B-gal/ID) administration of 100 .mu.g B-gal DNA
showed no detectable antitumor effect as compared to control mice.
By contrast, mice treated with *-gal CLDC (B-gal/IV; either 10
.mu.g (10) or 1 .mu.g (1) total DNA per mouse), had significantly
reduced lung tumor burdens compared to control mice or to mice
treated with i.v. administration of empty vector (EV/IV) CLDC,
although i.v. administration of empty vector CLDC had a clear
antitumor effect as compared to i.m. or i.d. administration of DNA.
Thus, administration of {fraction (1/10)}th or {fraction (1/100)}th
the amount of tumor antigen DNA using CLDC by i.v. administration
was much more effective than conventional DNA immunization
approaches.
[0216] (c) The following experiment demonstrates that CLDC-mediated
intravenous immunization with a tumor antigen induces an
antigen-specific humoral response in vivo. The relative efficiency
of immunization via different routes of DNA administration was
evaluated in BALB/c mice (4 per group) using plasmid DNA encoding
the *-galactosidase gene (*-gal). At 2 week intervals, serum was
collected from each mouse and assayed for antibodies against the
*-gal protein, using an antibody ELISA assay. Mice immunized by the
intradermal and intramuscular route were injected once with 50
.mu.g *-gal plasmid DNA. Mice immunized once by the intravenous
route and intraperitoneal routes received 101g DNA that was
complexed to a cationic liposome (CLDC). Control animals were not
treated. The mean *-gal-specific antibody level (at a 1:1000 serum
dilution) was determined for each group of mice and plotted for
each of 4 different time points evaluated. FIG. 22 shows that
intravenous administration of CLDC containing 10 .mu.g DNA elicited
a similar antigen-specific humoral immune response to intradermal
administration of 50 .mu.g DNA, and both intravenous and
intradermal administration elicited a more potent humoral immune
response than either intraperitoneal or intramuscular injection of
*-gal DNA.
[0217] (d) The following experiment demonstrates that CLDC-mediated
immunization with a tumor antigen induces antigen-specific
production of IFN * by spleen cells. As another means of assessing
the effectiveness of CLDC-mediated immunization, the release of
IFN* (a cytokine with antitumor effects) was quantitated in spleen
cells of mice that were immunized twice, one week apart, with
either empty vector CLDC (EV), IL-2 CLDC (i.e., DNA encoding IL-2),
or *-gal CLDC (DNA encoding *-gal) (Sections A, B, C & H). FIG.
23 demonstrates that, mice immunized with the *-gal CLDC mounted a
strong antigen specific immune response when re-challenged in vitro
with the CL25 (*-gal transfected) cell line, as measured by IFN*
production by splenocytes. In contrast, splenocytes from mice
immunized with either empty vector CLDC (EV) or IL-2 CLDC (IL-2)
produced very little IFN*. These data further substantiate the
effectiveness of antigen-specific immunization using CLDC. It is
believed that this effectiveness stems in large part from the
innate immune response that is triggered by systemic administration
of any CLDC. This strong induction of innate immune responses
undoubtedly serves as a powerful adjuvant for inducing strong
immune responses to the antigen-encoding DNA.
Example 7
[0218] The following experiments a-b and FIGS. 24 and 25
demonstrate that administration of CLDC having RNA encoding a tumor
antigen induces strong antitumor immunity and tumor-specific CTL
responses in vivo.
[0219] (a) The following experiment shows that CLDC-mediated
immunization with tumor RNA plus a cytokine induces strong
antitumor immunity. The ability to immunize mice using
polyA-enriched RNA from tumor cells was evaluated by complexing the
RNA to a cationic lipid to form cationic lipid RNA complexes (CLRC)
(Sections A and B). The antitumor effects were evaluated in BALB/c
mice (4 per treatment group) with day 3 established CT26 lung tumor
metastases (Section I). RNA was prepared from the autologous tumor
cells (CT26 RNA) or from an irrelevant control tumor cell line
(C57B1/6 RNA), complexed to a cationic lipid, then injected i.v. to
deliver approximately 501g RNA per mouse (Section C). One group of
mice was treated with CLDC containing DNA encoding the IL-2 gene
alone (IL-2), and a final group was treated with CLRC containing
both CT26 RNA and DNA encoding the IL-2 gene (CT26+IL-2). The lung
tumor burden was quantitated 7 days after the second injection of
CLDC. FIG. 24 shows that RNA can be effectively used to immunize
mice against a tumor when combined into CLRC and delivered
systemically, and that this antitumor effect can be enhanced by
co-administering the RNA with the DNA encoding IL-2.
[0220] (b) This experiment demonstrates that immunization with
tumor-specific RNA induces tumor-specific CTL responses. Mice with
established CT26 tumors were immunized twice with CLRC containing
either irrelevant RNA (B16), DNA encoding the IL-2 gene (IL-2),
total CT26 RNA (CT26), or total CT26 RNA plus DNA encoding the IL-2
gene (CT26/IL-2) (Sections A, B, and I). One week after the second
immunization, spleen cells were harvested and assayed for their
ability to lyse CT26 target cells in vitro (Section F). FIG. 25
shows that immunization with either CT26 RNA or CT26 RNA plus 1L-2
induced the highest levels of anti-tumor CTL activity. Thus,
CLDC-mediated immunization with a broad range (library) of
unselected tumor antigens can induce tumor-specific immunity, and
this immunity can be augmented by co-administration of a cytokine
gene.
Example 8
[0221] The following experiment and FIG. 26 demonstrate that
intraperitoneal administration of CLDC containing DNA encoding IL-2
induces a reduction in FeLV viral titer. A cat chronically infected
with the feline leukemia virus (FeLV) was treated with weekly (for
4 weeks), and then twice monthly intraperitoneal injections of 250
.mu.g CLDC prepared (as described above) using plasmid DNA encoding
the feline IL-2 gene. At various time points after treatment was
initiated, blood was collected and the serum levels of FeLV p27
determined using an ELISA (assays performed by Dr. Ed Hoover,
Colorado State University). Over the course of 3 months of
treatment, the FeLV p27 levels declined by 50%, and the cat's
clinical signs improved (e.g., weight gain, increased hematocrit).
In contrast, for 2 months prior to IL-2 CLDC treatment, the FeLV
p27 levels had remained relatively constant (data not shown).
Example 9
[0222] The following experiments a-b and FIGS. 27-29 demonstrate
that the composition and method of the present invention abrogates
airway hyperresponsiveness and reduces airway eosinophil influx in
a murine model of allergic asthma.
[0223] (a) BALB/c mice (at least 8 per treatment group) were
sensitized to ovalbumin as follows. Briefly, mice were sensitized
by intraperitoneal (i.p.) injection of 20 .mu.g ovalbumin (OVA)
(Grade V, Sigma Chemical Co., St. Louis, Mo.) together with 20 mg
alum (Al(OH).sup.3) (Inject Alum; Pierce, Rockford, Ill.) in 100 *1
PBS (phosphate-buffered saline), or with PBS alone. 72 hours before
the mice were airway challenged with ovalbumin, the mice were
treated with intravenous administration of IFN* CLDC (IFN-g) or
empty vector CLDC (EV). Controls included OVA-sensitized mice that
were not treated (IPN) as well as untreated mice that did not
receive airway sensitization (IP). Mice received subsequent OVA
aerosol challenge for 20 minutes with a 1% OVA/PBS solution.
Airways responsiveness (Penh) following increasing doses of
methacholine was assessed using whole body plethysmography (Buxco,
Troy, N.Y.) (asthma is known to increase the sensitivity of the
airways to contractile agonists such as methacholine). In this
system, an unrestrained spontaneously breathing mouse is placed
into the main chamber of the plethysmograph, and pressure
differences between this chamber and a reference chamber are
recorded. The resulting box pressure signal is caused by volume and
resultant pressure changes during the respiratory cycle of the
animal. From these box pressure signals, the phases of the
respiratory cycle, tidal volume, and the enhanced pause (Penh) can
be calculated. Penh represents a function of the proportion of
maximal expiratory to maximal inspiratory box pressure signals and
of the timing of expiration. It correlates closely with pulmonary
resistance measured by conventional two-chambered plethysmography
in ventilated animals. FIG. 27 shows that allergen sensitized and
challenged mice which received intravenous administration of IFN*
CLDC had significantly reduced airway hyperresponsiveness to
methacholine challenge (i.e., almost equal to that of control (IP)
mice), whereas airways responsiveness remained high in untreated
animals (IPN). Animals treated with empty vector (CLDC) showed
reduced hyperresponsiveness to methacholine at lower methacholine
challenge doses. Additionally, both intravenous administration of
IFN* CLDC and empty vector CLDC reduced airway hyperresponsiveness
to methacholine significantly better than administration of
recombinant IFN * protein (data not shown).
[0224] (b) In this experiment, BALB/c mice were sensitized to
ovalbumin as described in section (a) above, then treated with CLDC
delivered either intravenously (IV) or intratracheally (IT). The
degree of eosinophil infiltration into the airways (a measure of
airways allergen sensitization) was quantitated in bronchoalveolar
lavage fluid (BALF). The mean number of eosinophils per ml BALF
fluid was plotted for each group of mice (unsensitized control
{IP}; sensitized, untreated control {IPN}; and sensitized mice
treated with either intratracheal IFN* CLDC, intratracheal EV CLDC,
intravenous IFN* CLDC, or intravenous EV CLDC). FIG. 28
demonstrates that treatment with intravenous CLDC (both EV and IFN*
CLDC) significantly reduced eosinophil infiltration compared to
control (IPN) animals.
Example 10
[0225] The following example demonstrates that spleen and lung
cells from mice receiving intravenous, but not intratracheal,
administration of CLDC produce significant amounts of IFN*.
[0226] BALB/c mice were administered CLDC containing 10 .mu.g of
DNA either intravenously or intratracheally as described in
experiments above. 24 hours post-administration, IFN* production
was measured from isolated spleen (FIG. 29A) and lung (FIG. 29B)
cells of the animals. FIGS. 29A and 29B show that mice receiving
intravenous administration of CLDC produced significant amounts of
IFN* in contrast to mice receiving intratracheal administration of
CLDC.
Example 11
[0227] The following example demonstrates that intravenous
administration of CLDC containing DNA encoding IL-2 eradicates
metastatic lung tumors in a dog.
[0228] A canine patient had a rear limb amputation for
osteosarcoma, followed by adjuvant chemotherapy for prevention of
tumor metastasis. Osteosarcoma is a highly malignant tumor of dogs
that metastasizes readily to the lungs, even after complete removal
(amputation) of the primary tumor. The median survival time for
dogs following amputation is 4 months, with death due to tumor
metastases. Canine osteosarcoma is thus a highly relevant and
useful animal model of osteosarcoma in humans.
[0229] Six months after this patient underwent amputation and
adjuvant chemotherapy, the dog was re-evaluated and metastatic
tumors were found in the lung on thoracic radiographs. The dog was
then entered into a cancer immunotherapy trial, using intravenously
administered CLDC encoding the canine IL-2 gene. The dog was
treated weekly for 12 weeks with increasing doses of CLDC, up to a
maximum dose of 500 .mu.g (10 .mu.g/kg body weight). After 6 weeks
of treatment, partial tumor regression was observed on thoracic
radiographs, and by 12 treatments, 90% regression of lung tumor
nodules was observed. Additional treatments have been given at once
monthly intervals and the dog remains in remission at 1.2 years
after entering into the study.
[0230] This example demonstrates the potential efficacy of
systemically administered CLDC as a cancer treatment in animals in
addition to mice. Thus, efficacy was demonstrated in a large,
outbred animal (dog) with a spontaneous, highly malignant
metastatic tumor (osteosarcoma), with minimal toxicity at the doses
employed here.
[0231] In summary, the above-described experiments have
demonstrated the following:
[0232] 1. Systemic injection of CLDC containing empty vector
(non-coding) plasmid DNA induces intense immune activation, as
assessed by upregulation of an early activation marker (CD69), by
induction of NK cell cytotoxic activity, increase in NK cell
numbers and by induction of cytokine release in vivo.
[0233] 2. Immune stimulation in vitro or in vivo (at the doses
evaluated here) is induced by the complex of DNA and cationic lipid
(CLDC), and not by either DNA or lipid alone.
[0234] 3. Immune activation induced by CLDC is quantitatively more
potent than that induced by either LPS (endotoxin) or poly I/C (a
classical inducer of antiviral immune responses). Furthermore, the
type of immune stimulation induced (e.g., the pattern of cytokines
induced) also differs qualitatively from that induced by LPS.
[0235] 4. Immune activation by CLDC can be induced by eukaryotic as
well as prokaryotic DNA, indicating that there is some property of
the CLDC that is inherently immune activating, regardless of the
source of the DNA.
[0236] 5. Immune activation is induced by complexes of CLDC
containing RNA.
[0237] 6. Although any complex of DNA and lipid can conceivably
induce some immune activation, CLDC prepared using MLV liposomes
induce the maximal and optimal immune stimulation which induces
effective antitumor responses.
[0238] 7. Systemic administration of tumor antigen genes using CLDC
is more effective than some more conventional routes of DNA
immunization (e.g., intramuscular), and equivalent to others (e.g.,
intradermal at higher doses of DNA), for inducing antigen-specific
humoral immunity. Intradermal administration, however, does not
provide the anti-tumor effect observed with systemic
administration.
[0239] 8. Systemic administration of one-tenth of the amount of DNA
using CLDC by intravenous administration induces equivalent levels
of antigen-specific CTL activity observed with intramuscular
injection.
[0240] 9. Intravenously administered, CLDC-mediated immunization
against a tumor antigen induces effective antitumor immunity,
whereas intramuscular (IM) or intradermal (ID) immunization does
not.
[0241] 10. Combined administration of an antigen-encoding (i.e.,
immunogen-encoding) gene with a cytokine-encoding gene induces
greater immune responsiveness to the antigen gene, and greater
antitumor activity.
[0242] 11. Systemic i.v. administration of CLDC prepared using MLV
liposomes induces preferential transfection of pulmonary tissues.
Furthermore, i.v. administration of CLDC encoding certain cytokine
genes (e.g., those that stimulate NK cells) induce greater
antitumor effects (against established lung tumors) than
administration of empty vector DNA.
[0243] 12. The primary anti-tumor effector cell induced by systemic
administration of CLDC is the NK cell.
[0244] 13. The cytokine response to administration of CLDC is
characteristic of the response to acute viral infections, and is
dominated by release of IFN* from macrophages, NK cells, and other
cell types throughout the body. This pattern of response is ideally
suited for treatment of cancer, viral infections, and to serve as
an adjuvant for certain types of vaccines.
[0245] 14. Systemic administration of CLDC containing DNA encoding
a cytokine induces a reduction in viral titer.
[0246] 15. Systemic administration of CLDC containing DNA encoding
a cytokine abrogates airway hyperresponsiveness and reduces airway
eosinophil influx in an allergic asthma model.
[0247] 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.
[0248] The words "comprise," "comprising," "include," "including,"
and "includes" when used in this specification and in the following
claims are intended to specify the presence of stated features,
integers, components, or steps, but they do not preclude the
presence or addition of one or more other features, integers,
components, steps, or groups thereof.
* * * * *