U.S. patent application number 17/212051 was filed with the patent office on 2021-10-07 for stimulation of an immune response by enantiomers of cationic lipids.
The applicant listed for this patent is PDS Biotechnology Corporation. Invention is credited to Frank Bedu-Addo, Weihsu Claire Chen, Gregory Conn, Leaf Huang, Kenya N. Toney Johnson, Elizabeth Ann Vasievich.
Application Number | 20210308157 17/212051 |
Document ID | / |
Family ID | 1000005653104 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210308157 |
Kind Code |
A1 |
Vasievich; Elizabeth Ann ;
et al. |
October 7, 2021 |
STIMULATION OF AN IMMUNE RESPONSE BY ENANTIOMERS OF CATIONIC
LIPIDS
Abstract
A composition and method for activating immune cells ex-vivo, or
inducing an immune response in a subject including a composition
comprising at least one chiral cationic lipid. The chiral cationic
lipid in one embodiment comprises a nonsteroidal cationic lipid
having a structure represented by formula (I); wherein in R.sup.1
is a quaternary ammonium group, Y.sup.1 is a space chosen from a
hydrocarbon chain, an ester, a ketone, and a peptide, C* is a
chiral carbon, R.sup.2 and R.sup.3 are independently chosen from a
saturated fatty acid, an unsaturated fatty acid, an ester-linked
hydrocarbon, phosphor-diesters, and combination thereof.
Inventors: |
Vasievich; Elizabeth Ann;
(Chapel Hill, NC) ; Chen; Weihsu Claire; (Toronto,
CA) ; Toney Johnson; Kenya N.; (Mason, OH) ;
Conn; Gregory; (Lawrenceburg, IN) ; Bedu-Addo;
Frank; (Bethel, CT) ; Huang; Leaf; (Durham,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PDS Biotechnology Corporation |
North Brunswick |
NJ |
US |
|
|
Family ID: |
1000005653104 |
Appl. No.: |
17/212051 |
Filed: |
March 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16899763 |
Jun 12, 2020 |
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17212051 |
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|
15702063 |
Sep 12, 2017 |
10702541 |
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16899763 |
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12988236 |
Dec 23, 2010 |
9789129 |
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PCT/US2009/040500 |
Apr 14, 2009 |
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15702063 |
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61045837 |
Apr 17, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/685
20130101 |
International
Class: |
A61K 31/685 20060101
A61K031/685 |
Claims
1. A method of enhancing an immunostimulatory effect in an immune
system of a mammal to respond prophylactically or therapeutically
to an infection, said method comprising the step of administering a
immunogenic composition to the mammal, wherein the composition
comprises a cationic lipid and an antigen, wherein the antigen
consists of T-cell receptor gamma alternate reading frame protein
or (TARP) or Mucin 1 (MUC 1).
2. The method of claim 1, wherein the cationic lipid comprises a
chiral cationic lipid.
3. The method of claim 2, wherein the chiral cationic lipid
comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP),
N-1-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride
(DOTMA), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), and
combinations thereof.
4. The method of claim 3, wherein the composition further comprises
one or more therapeutic ingredients selected from the group
consisting of drug molecules, cytokines, chemokines, polymer
complexes and pharmaceutical carriers.
5. The method of claim 1, wherein the antigen consists of TARP and
antigenic fragments thereof.
6. The method of claim 5, wherein the composition further comprises
a nucleic acid molecule that encodes the amino acid sequence of
TARP and antigenic fragments thereof.
7. The method of claim 6 wherein the modified to increase its
hydrophobicity or wherein the protein domain of the antigen is
modified to facilitate purification by adding a C-terminal protein
tag that includes a poly-histidine tag and a proteolytic cleavage
sequence for removal of the tags.
8. The method of claim 1, wherein the antigens consists of MUC 1
and antigenic fragments thereof.
9. The method of claim 8, wherein the composition further comprises
a nucleic acid molecule that encodes an amino acid sequence of MUC
1 and antigenic fragments thereof.
10. The method of claim 9, wherein the antigen is modified to
increase its hydrophobicity or wherein the protein domain of the
antigen is modified to facilitate purification by adding a
C-terminal protein tag that includes a poly-histidine tag and a
proteolytic cleavage sequence for removal of the tags.
11. The method of claim 1, wherein the cationic lipid comprises
DOTAP.
12. The method of claim 11, wherein the cationic lipid consists of
R-DOTAP.
13. The method of claim 1, wherein the mammal is a human.
14. The method of claim 1, wherein the wherein the composition is
administered as an aerosol or as a liquid solution for
intratumoral, intraarterial, intravenous, intratracheal,
intraperitoneal, subcutaneous, or intramuscular administration.
15. The method of claim 1, wherein the immunostimulatory effect
includes production of immune system regulatory molecules.
16. A pharmaceutical composition comprising an immunologically
active enantiomer of a cationic lipid component, wherein the
cationic lipid component consists of R-DOTAP, and an antigen,
wherein the antigen consists of T-cell receptor gamma alternate
reading frame protein or (TARP) or Mucin 1 (MUC 1), and wherein the
composition is effective to activate an immune system in a
patient.
17. The pharmaceutical composition of claim 16, further comprising
additional lipids selected from the group consisting of lyso
lipids, lysophosphatidylcholine, 1-oleoyl lysophosphatidylcholine,
cholesterol, neutral phospholipids, dioleoyl phosphatidyl
ethanolamine (DOPE), dioleoyl phosphatidylcholine (DOPC),
lipophilic surfactants, Tween-80 and PEG-PE, and combinations
thereof.
18. The pharmaceutical composition of claim 16, wherein the antigen
consists of TARP, or antigenic fragments thereof.
19. The pharmaceutical composition of claim 18, wherein the
composition further comprises a nucleic acid molecule that encodes
an amino acid sequence of TARP, or antigenic fragments thereof.
20. The pharmaceutical composition of claim 16, wherein the antigen
consists of MUC 1, or antigenic fragments thereof.
21. The pharmaceutical composition of claim 20, wherein the
composition further comprises a nucleic acid molecule that encodes
an amino acid sequence of MUC 1, or antigenic fragments
thereof.
22. The pharmaceutical composition of claim 16, wherein the antigen
includes an antigen modified to increase its hydrophobicity.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/899,763 filed Jun. 12, 2020, which is a
continuation U.S. patent application Ser. No. 15/702,063 filed Sep.
12, 2017, now issued as U.S. Pat. No. 10,702,541 on Jul. 7, 2020,
which is a divisional of U.S. patent application Ser. No.
12/988,236 filed Dec. 23, 2010, now issued as U.S. Pat. No.
9,789,129 on Oct. 17, 2017, which is a 371 of International Patent
Application. No. PCT/US2009/040500 filed Apr. 14, 2009, which
claims benefit of U.S. Provisional Application No. 61/045,837 filed
Apr. 17, 2008, the disclosures of which are incorporated by
reference herein in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 16, 2021, is named PDS-16-1033WO-US-DC-CON_SL.txt and is
648 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention generally related to stimulating an
immune response, and more particularly to the use of the R and S
enantiomers of lipids in stimulating immune responses.
BACKGROUND OF THE INVENTION
[0004] This section is intended to introduce the reader to various
aspects of art that may be related to various aspects of the
present invention, which are described and/or claimed below. This
discussion is believed to be helpful in providing the reader with
background information to facilitate a better understanding of
various aspects of the present invention. Accordingly, it should be
understood that these statements are to be read in this light, and
not as admissions of prior art.
[0005] Development of safe and effective immunotherapies for human
use remains an urgent medical need for patients worldwide. In order
to elicit appropriate immune responses, immunologic modifiers
("immunomodifiers") that enhance, direct or promote an immune
response can be used in vaccine design or immunotherapy
[Gregoriadis, G., Immunological adjuvants: a role for liposomes,
Immunol Today 11:89 (1990)]. For example, vaccines may include
antigens to stimulate an immune response. However, some potential
vaccines that include antigens are weak stimulators of an immune
response because the vaccines do not efficiently deliver the
antigen to antigen presenting cells ("APC") of the immune system
and/or the antigen is weakly immunogenic. Thus, immunotherapies
that effectively deliver antigens to APC, and also stimulate the
immune system to respond to the antigen, are needed.
Immunomodifiers have the potential to function as such an
immunotherapy. Such immunotherapies may have these and other
benefits. For example, when included as part of a therapeutic
vaccine, an immunomodifier should at least (1) improve antigen
delivery and/or processing in the APC [Wang, R. F., and Wang, H. Y.
Enhancement of antitumor immunity by prolonging antigen
presentation on dendritic cells. Nat Biotechnol 20:149 (2002)1, (2)
induce the production of immunomodulatory cytokines that favor the
development of immune responses to the vaccine antigen, thus
promoting cell mediated immunity, including cytotoxic T-Lymphocytes
("CTL"), (3) reduce the number of immunizations or the amount of
antigen required for an effective vaccine [Vogel, F. R. Improving
vaccine performance with adjuvants. Clin Infect Dis 30 Suppl 3:S266
(2000)], (4) increase the biological or immunological half-life of
the vaccine antigen, and (5) overcome immune tolerance to antigen
by inhibiting immune suppressive factors [Baecher-Allan, C., and
Anderson, D. E. Immune regulation in tailor-bearing hosts. Curr
Opin Immunol 18:214 (2006)].
[0006] Presently, the primary class of agents used to enhance the
efficacy of antigens, such as peptide or protein antigens, in
eliciting an immune response are adjuvants such as water-in-oil
emulsions, alum, and other chemicals which enhance antigen
responses; however, these adjuvants are not immunomodifiers, as
described above, because they have no direct immunomodulatory
effects themselves [Vogel, F. R., and Powell, M. F. A compendium of
vaccine adjuvants and excipients, Pharm Biotechnol 6:141 (1995)1.
Several such adjuvants are available for use in animals and some of
them have been tested in clinical trials. In addition to
traditional adjuvants such as the aluminum salts, products such as
influenza virosomes [Gluck, R., and Walti, E. 2000. Biophysical
validation of Epaxal Berna, a hepatitis A vaccine adjuvanted with
immunopotentiating reconstituted influenza virosomes ORM. Dev Biol
(Basel) 103:189 (2000)1, and Chiron's MF59 [Kahn, J. 0., et al.
Clinical and immunologic responses to human immunodeficiency virus
(I.sup.-1.17) type ISF2 gp120 subunit vaccine combined with MF59
adjuvant with or without mummy! tripeptide
dipalinitoylphosphatidylethanolamine in non-I.sup.-IIV-ittfected
human volunteers. J Infect Dis 170:1288 (1994)], which have
intrinsic immune effects, are being marketed. For example, MF59,
which is a submicron emulsion based adjuvant, is internalized by
dendritic cells [Dupuis, M., et al., Dendritic cells internalize
vaccine adjuvant after intramuscular injection. Cell Immunol 186:18
(1998)]. However, according to clinical trial reports on HSV and
influenza vaccines [Jones, C. A., and Cunningham, A. L. Vaccination
strategies to prevent genital herpes and neonatal herpes simplex
virus (HSV) disease. Herpes 11:12 (2004); Minutello, M. et al.,
Safety and immunogenicity of an inactivated subunit influenza virus
vaccine combined with ilifF59 adjuvant emulsion in elderly
subjects, immunized for three consecutive influenza seasons.
Vaccine 17:99 (1999)1, evidence from animal models suggests that
the MF59 adjuvant enhances production of neutralizing antibodies
rather than enhancing responses. Thus, new methods of stimulating
cell mediated immune responses are needed.
[0007] Further, as mentioned above, some antigens are weak
stimulators of an immune response. Thus, in addition to
co-administering antigen with substances that stimulate immune
responses, as described above, a weakly immunogenic antigen can be
modified to increase its immunogenicity. For example, a weakly
immunogenic antigen can be coupled to immunogenic peptides,
polysaccharides, or lipids to increase its immunogenicity. However,
simply coupling weakly immunogenic antigens to these types of
compounds may not be sufficient to elicit an immune response. For
example, the resulting immune response may be directed to
immunogenic epitopes on the coupled compound and not the weak
antigen, or the coupled antigen may not be efficiently delivered to
APC of the immune system. Thus, additional methods are needed to
stimulate immune responses to antigens that are weakly
immunogenic.
SUMMARY OF THE INVENTION
[0008] Certain exemplary aspects of the invention are set forth
below. Tt should be understood that these aspects arc presented
merely to provide the reader with a brief summary of certain forms
the invention might take and that these aspects are not intended to
limit the scope of the invention. Indeed, the invention may
encompass a variety of aspects that may not be explicitly set forth
below.
[0009] This invention is directed to the chirality of cationic
lipids and the use of the R and S enantiomers of cationic lipids,
which under certain dose and composition conditions act as a novel
class of immune-stimulants, to (1) effectively present or deliver
an antigen to the immune system and (2) stimulate the immune system
to respond to the antigen.
[0010] Liposomes have been extensively used for delivering small
molecular weight drugs, plasmid DNA, oligonucleotides, proteins,
and peptides. Vaccines using liposomal vehicles as nonviral antigen
carriers are preferable compared to traditional immunizations using
live attenuated vaccines or viral vectors such as vaccinia or
influenza virus. U.S. patent application Ser. No. 12/049,957,
assigned to the assignee of the present application, discloses
simple yet effective lipid-based immunotherapies, including a
cationic lipid/antigen complex, which has two molecules, a cationic
lipid and an antigen, and the effects of the lipid dose on the
resulting immune response. The reported results demonstrate that
the cationic liposome complexed with an antigen serves to stimulate
immune responses and initiate dendritic cell (an APC) interaction
with T-cells.
[0011] In the present invention, additional studies performed with
the two enantiomers of a selected cationic lipid have led to the
discovery that differences exist in the ability of the R and S
enantiomers of the cationic lipids to act as potent immune
activators under various conditions. In combination with an
antigen, the cationic lipid/antigen complex containing the R
enantiomer, under various dose conditions (including low dose
conditions), induces strong immune responses specific to the
antigen formulated in the complex and results in tumor regression.
Complexes consisting of S-DOTAP and the antigen however were able
to induce only limited tumor regression, and not at all doses at
which R-DOTAP was effective. Both enantiomers of DOTAP are however
equally effective at inducing maturation and activation of
dendritic cells, which is the first step in inducing a cellular
immune response.
[0012] Thus, one aspect of the invention provides a composition of
at least one enantiomer of a cationic lipid in a dose sufficient to
induce an immune response in a subject.
[0013] Another aspect of the invention provides a method of
inducing an immune response in a subject by administering a
specific enantiomer or a mixture of enantiomers of a cationic lipid
to the subject.
[0014] Another aspect of the invention provides a composition of an
R or S enantiomer of a cationic lipid in a dose sufficient to
induce an immune response in a subject.
[0015] Additional aspects of the invention involve the addition of
at least one antigen to the R or S enantiomer to form a cationic
lipid/antigen complex in which case the immune response is
antigen-specific.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Various features, aspects, and advantages of the present
invention will become better understood when the following detailed
description is read with reference to the accompanying figures,
wherein:
[0017] FIGS. 1A and 1B depict chirality of
I,2-dioleoyl-3-trimethylammonium propane ("DOTAP").
[0018] FIG. 2 is a graph depicting activation of human dendritic
cells resulting in expression of the co-stimulatory molecule CD 80
by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
[0019] FIG. 3 is a graph depicting activation of human dendritic
cells resulting in expression of the co-stimulatory molecule CD 83
by R-DOTAP, S-DOTAP and the racernic mixture RS-DOTAP.
[0020] FIG. 4 is a graph depicting activation of human dendritic
cells resulting in expression of the co-stimulatory molecule CD 86
by R-DOTAP, S-DOTAP and the racemic mixture RS-DOTAP.
[0021] FIG. 5 is a graph depicting stimulation of human dendritic
cells resulting in production of the chemokine CCL-3 by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0022] FIG. 6 is a graph depicting stimulation of human dendritic
cells resulting in production of the chemokine CCL-4 by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0023] FIG. 7 is a graph depicting stimulation of human dendritic
cells resulting in production of the chemokine CCL-5 by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0024] FIG. 8 is a graph depicting stimulation of human dendritic
cells resulting in production of the chemokine CCL-19 by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0025] FIG. 9 is a graph depicting stimulation of human dendritic
cells resulting in production of the cytokine IL-2 by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0026] FIG. 10 is a graph depicting stimulation of human dendritic
cells resulting in production of the cytokine IL-S by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0027] FIG. 11 is a graph depicting stimulation of human dendritic
cells resulting in production of the cytokine 1L-12 by R-DOTAP,
S-DOTAP and the racemic mixture RS-DOTAP.
[0028] FIG. 12 is a graph demonstrating the in vivo antitumor
effect of various doses of a cationic lipid/antigen complex based
on tumor size and. time post-injection.
[0029] FIG. 13 is a graph demonstrating the effect of S-DOTAP dose
on the in vivo anti tumor efficacy of the cationic lipid/antigen
complex.
[0030] FIG. 14 is a graph demonstrating the effect of R-DOTAP dose
on the in vivo anti tumor efficacy of the cationic lipid/antigen
complex.
[0031] FIG. 15 is a graph depicting the lipid dose response effects
of the racemic mixture of DOTAP, R-DOTAP and S-DOTAP on the in vivo
anti-tumor immune response of the cationic lipid/antigen complex
with antigen dose of 201.1 g. The effect of antigen dose is also
demonstrated with the racemic mixture of DOTAP. R-DOTAP compared to
S-DOTAP: * p<0.05, ** p<0.01, n=5-6.
DETAILED DESCRIPTION
[0032] One or more specific embodiments of the present invention
will be described below. In an effort to provide a concise
description of these embodiments, all features of an actual
implementation may not be described in the specification. It should
be appreciated that in the development of any such actual
implementation numerous implementation-specific decisions must be
made to achieve the developers' specific goals, which may vary from
one implementation to another. Moreover, it. should be appreciated
that such a development effort might be complex and time consuming,
but would nevertheless be a routine undertaking for those of
ordinary skill having the benefit of this disclosure.
[0033] When introducing elements of the present invention (e.g.,
the exemplary embodiments(s) thereof), the articles "a", "an",
"the" and "said" are intended to mean that there are one or more of
the elements. The terms "comprising", "including" and "having" are
intended to be inclusive and mean that there may be additional
elements other than the listed elements.
[0034] One aspect of the present invention provides an enantiomer
of a cationic lipid to stimulate an immune response in a mammal to
prevent or treat disease The individual chiral lipids can function
independently as immunomodulators, in a dose dependent manner, such
as for production of chemokines and/or cytokines, by activating
various components of the MAP kinase signaling pathway. The dose
range that effectively induces an immune response is observed to
differ between the R and S enantiomers and also within various
mammalian species. For example, in the rodent species the
R-enantiomer of DOTAP effectively attenuates tumor growth over a
range of about 30 nmole to about 400 nmole. In contrast, the
S-enantiomer of DOTAP is effective over this same range of doses in
the same species of rodent, though less so than the R-enantiomer.
In another aspect, the chiral cationic lipid may be associated with
antigens or drugs for presentation to cells of the immune system
while simultaneously stimulating a strong antigen-specific immune
response. In some aspects of the invention, the antigen is a
lipopeptide.
[0035] U.S. Pat. No. 7,303,881, incorporated by reference herein in
its entirety, discloses that multiple cationic lipids complexed
with disease-associated antigens were shown to stimulate a
prophylactic immune response that prevented the specific disease
(e.g., HPV-positive cancer) and also a therapeutic immune response
that killed cells expressing the particular antigen and resulted in
an effective treatment of the disease. Presently, studies were
performed to further understand the effects of chirality on the
immunostimulatory capability of cationic lipids by using the R and
S enantiomers of DOTAP. (The R and S enantiomers of DOTAP are shown
in FIGS. I A and 113). These studies have led to the discovery that
individual enantiomers of cationic lipids can function
independently as immunodulators to stimulate an immune response
with (or without) antigens. Further, when enantiomers of cationic
lipids are complexed with an antigen, an antigen specific immune
response is generated. The extent of the disease specific immune
response differs significantly between the R and S enantiomers of
the cationic lipid.
[0036] In another aspect, the chiral cationic lipid, at a dose
sufficient to stimulate an immune response, is administered in
combination with an antigen or antigens. In this case the cationic
lipid/antigen combination is capable of generating an immune
response that is specific to the antigen(s) delivered in
combination with the cationic lipid. The response generated may
include production of specific cytotoxic T cells, memory T cells,
or B cells resulting in the prevention of, or therapeutic response
to, the specific disease associated with the antigen(s).
[0037] The chiral cationic lipids of the invention may be in the
form of cationic lipid complexes. The cationic lipid complex can
take the form of various vesicles such as liposomes, micelles, or
emulsions. The cationic lipid complexes may be unilaminar or
multilaminar. When an antigen is included, the antigen may be
encapsulated in the cationic lipid complex or may be
unencapsulated. Encapsulated is understood to mean that the antigen
may be contained within the internal space of the complex and/or
incorporated into the lipid walls of the complex.
[0038] Another aspect of the invention relates to a method for
producing these complexes, wherein the method may optionally
include the step of purifying these formulations from excess
individual components.
[0039] In certain embodiments, the cationic lipid complexes have a
net positive charge and/or a positively charged surface at pH
6.0-8.0.
[0040] The optional "antigen" which may be included with cationic
lipid complexes of the invention may be nucleic acids, peptides,
lipopeptides, proteins, lipoproteins, polysaccharides, and other
macromolecules which may be complexed directly with cationic
lipids. However, cationic drugs (e.g., large cationic protein) can
be directly complexed with an anionic lipid or sequentially
complexed first with anionic lipid or polymer followed by the
chiral cationic lipid. The use of this process permits delivery of
positive or neutral charged drugs to cells by the complexes of the
present invention.
[0041] One aspect of the present invention involves the use of the
chiral cationic lipid complexes to activate dendritic cells and
also to stimulate the production of chemokines and cytokines.
Chemokines and cytokines are important regulators of immune
responses. Chemokines were originally identified as potent
chemoattractants for inflammatory cells including neutrophils,
eosinophils, and in onocytes/macrophages. Subsequent studies have
revealed that chemokines have profound effects on immune reactions
by regulating the trafficking of dendritic cells and other
lymphocytes into lymphoid organs. Dendritic cells are migratory
cells that sample antigens in the tissue, migrate to the draining
lymph nodes and mature to stimulate the T cell response. CCL2, a
member of the CC chemolcines was originally identified as a
chemotactic and activating factor for monocytes/macrophages.
Subsequent studies showed that it can also affect the function of T
cells, natural killer cells, and neutrophils. Further exploration
found that CCL2 was the most potent activator of CD8+ cytotoxic T
lymphocytes ("CTL") activity, when in the presence of the Thl
cytokines, interleukin-12 ("IL-12") and interferon-y (IFN-y"). This
can be explained by a positive bidirectional interaction between
CCL2 and IFN-y systems. An absence of either the cytokine or
chemokine may interfere with Thl polarization and subsequent
specific tumor immunity generation. Another CC chemokine, CCL-4,
has also been shown to recruit and expand dendritic cells in vivo
and potentiate the immunogenicity of plasmid DNA vaccines.
Recently, it has been shown that chemokines enhance immunity by
guiding naive CDS+ T cells to sites of CD4+ T cell-dendritic cell
interaction and promote memory CD8+ T cell generation. A few
examples of chemokines that may be stimulated by the cationic lipid
complexes of the present invention are CCL-2, CCL-3, and CCL-4.
Examples of cytokines that may be stimulated by the cationic lipid
complexes of the present invention are IL-2, IL-8, IL-12 and IFN-y.
The inventors contemplate that the cationic lipid complexes of the
present invention may stimulate chemokines and cytokines in
addition to those disclosed in this specification.
[0042] Lipids
[0043] The chiral cationic lipid complexes of the present invention
may form liposomes that are optionally mixed with antigen and may
contain the chiral cationic lipids alone or chiral cationic lipids
in combination with neutral lipids. Suitable chiral cationic lipid
species include, but are not limited to the R and S enantiomers of:
3-.beta.[.sup.4N-(.sup.1N,.sup.8-diguanidino
spennidine)-carbaroyl]cholesterol (BGSC);
3-.beta.0[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol
(BGTC); N,N.sup.1N.sup.2N.sup.3Tetra-methyttetrapalmitylsperrnine
(cellfectin);
N-t-butyl-N'-tctraclecyl-3-tetradecyl-aininopropion-amidine
(CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB);
1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
(DMRIE);
2,3-dioleoyloxy-N-[2(spenninecarboxamido)ethyl]-N,N-dimethyl-l-p-
ropanaminium trifluoracetate) (DOSPA);
1,3-dioleoyloxy-2-(6-carboxyspennyl)-propyl amide (DOSPER);
4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM)
N,N,1\1.sup.1,Ni-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3
dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50);
N-I-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride
(DOTMA) or other N--(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted.
ammonium surfactants; 1,2 dioleoyl-3-(4'-trimethylammonio)
butanol-sn-glycerol (DOBT) or cholesteryl (4'trimethylammonia)
butanoate (ChOTB) where the trim ethylammonium group is connected
via a butanol spacer arm to either the double chain (for DOTB) or
cholesteryl group (for ChOTB); DORI
(DL-1,2-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammonium)
or DORIE
(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-(3-hydroxyethylammonium)
(DORIE) or analogs thereof as disclosed in WO 93/03709;
2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl
hemisuccinate ester (ChOSC); lipopolyamines such as
dioctadecylamidoglycylspermine (DOGS) and dipalinitoyl
phospliatidylethanolamylspennine (DPPES) or the cationic lipids
disclosed in U.S. Pat. No. 5,283,185, cholesteryl-3
.beta.-carboxyl-amido-ethylenetrimethylammonium iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl
carboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine,
cholesteryl-3-.beta.-oxysuccinamido-ethylenetrimethylammonium
iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-.beta.-oxosu-
ccinate iodide, 2-(2-trimethylammonio)-ethylmethylamino
ethyl-cholesteryl-3-.beta.-oxysuccinate iodide,
3-.beta.-N-(1\1',N'-dimethylaminoethane) carbamoyl cholesterol
(DC-chol), and 3-.beta.-N-(polyethyleneimine)-carbamoylcholesterol;
0,0'-dirnyristyl-N-lysyl aspartate (DMKE);
0,0'-dirnyristyl-N-lysyl-glutamate (DMKD);
1,2-climyristyloxypropyl-3-dintethyl-hydroxy ethyl ammonium bromide
(DMRIE); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DMEPC);
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC);
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC);
1,2-clipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC);
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DMEPC);
1,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyl dim
ethylaminopropane (DODAP); 1,2-pahnitoyl-3-trimethylammonium
propane (DSTAP); 1,2-distearoyl-3-trimetbylam onium propane
(DSTAP), 1,2-nlyristoyl-3-trimethylammonium propane (DMTAP); and
sodium dodecyl sulfate (SDS). The present invention contemplates
the use of structural variants and derivatives of the cationic
lipids disclosed in this application.
[0044] Certain aspects of the present invention include
nonsteroidal chiral cationic lipids having a structure represented
by the following formula:
##STR00001##
wherein in R.sup.3 is a quaternary ammonium group, Y.sup.1 is
chosen from a hydrocarbon chain, an ester, a ketone, and a peptide,
C* is a chiral carbon, R.sup.2 and R.sup.3 are independently chosen
from a saturated fatty acid, an unsaturated fatty acid, an
ester-linked hydrocarbon, phosphor-diesters, and combinations
thereof. DOTAP, DMTAP, DSTAP, DPTAP, DPEPC, DSEPC, DMEPC, DLEPC,
DOEPC, DMKE, DMKD, DOSPA, DOTMA, are examples of lipids having this
general structure.
[0045] In one embodiment, chiral cationic lipids of the invention
are lipids in which bonds between the lipophilic group and the
amino group are stable in aqueous solution. Thus, an attribute of
the complexes of the invention is their stability during storage
(i.e., their ability to maintain a small diameter and retain
biological activity over time following their formation). Such
bonds used in the cationic lipids include amide bonds, ester bonds,
ether bonds and carbamoyl bonds. Those of skill in the art would
readily understand that liposomes containing more than one cationic
lipid species maybe used to produce the complexes of the present
invention. For example, liposomes comprising two cationic lipid
species, lysyl-phosphatidylethanolamine and .beta.-alanyl
cholesterol ester have been disclosed for certain drug delivery
applications [Brunette, E. et al., Nucl. Acids Res., 20:1151
(1992)].
[0046] It is to be further understood that in considering chiral
cationic liposomes suitable for use in the invention and optionally
mixing with antigen, the methods of the invention are not
restricted only to the use of the cationic lipids recited above but
rather, any lipid composition may be used so long as a cationic
liposome is produced and the resulting cationic charge density is
sufficient to activate and induce an immune response.
[0047] Thus, the complexes of the invention may contain other
lipids in addition to the chiral cationic lipids, These lipids
include, but are not limited to, lyso lipids of which
lysophosphatidylcholine (1-oleoyl lysophospliatidyleholine) is an
example, cholesterol, or neutral phospholipids including dioleoyl
phosphatidyl ethanolamine (DOPE) or dioleoyl phosphatidylcholine
(DOPC) as well as various lipophilic surfactants, containing
polyethylene glycol moieties, of which Tween-80 and PEG-PE are
examples.
[0048] The chiral cationic lipid complexes of the invention may
also contain negatively charged lipids as well as cationic lipids
so long as the net charge of the complexes formed is positive
and/or the surface of the complex is positively charged. Negatively
charged lipids of the invention are those comprising at least one
lipid species having a net negative charge at or near physiological
pH or combinations of these. Suitable negatively charged lipid
species include, but are not limited to, CHEMS (cholesteryl
hemisuccinate), NGPE (N-glutaryl phosphatidlylethanolanine),
phosphatidyl glycerol and phosphatidic acid or a similar
phospholipid analog.
[0049] Methods for producing the liposomes to be used in the
production of the lipid comprising drug delivery complexes of the
present invention are known to those of ordinary skill in the art.
A review of methodologies of liposome preparation may be found in
Liposome Technology (CFC Press New York 1984); Liposomes by Ostro
(Marcel Dekker, 1987); Methods Biochem Anal, 33:337-462 (1988) and
U.S. Pat. No. 5,283,185. Such methods include freeze-thaw extrusion
and sonication. Both unilamellar liposomes (less than about 200 mu
in average diameter) and multilamellar liposomes (greater than
about 300 nm in average diameter) may be used as starting
components to produce the complexes of this invention.
[0050] In the cationic liposomes utilized to produce the cationic
lipid complexes of this invention, the chiral cationic lipid is
present in the liposome at from about 10 mole % to about 100 mole %
of total liposomal lipid, or from about 20 mole % to about 80 mole
%. The neutral lipid, when included in the liposome, may be present
at a concentration of from about 0 mole % to about 90 mole % of the
total liposomal lipid, or from about 20 mole % to about 80 mole %,
or from 40 mole % to 80 mole %. The negatively charged lipid, when
included in the liposome, may be present at a concentration ranging
from about 0 mole % to about 49 mole % of the total liposomal
lipid, or from about 0 mole % to about 40 mole %. In one
embodiment, the liposomes contain a chiral cationic and a neutral
lipid, in ratios between about 2:8 to about 6:4.
[0051] It is further understood that the complexes of the present
invention may contain modified lipids, protein, polycations or
receptor ligands which function as a targeting factor directing the
complex to a particular tissue or cell type. Examples of targeting
factors include, but are not limited to, asialoglycoprotein,
insulin, low density lipoprotein (LDL), folate and monoclonal and
polyclonal antibodies directed against cell surface molecules.
Furthermore, to modify the circulatory half-life of the complexes,
the positive surface charge can be sterically shielded by
incorporating lipophilic surfactants which contain polyethylene
glycol moieties.
[0052] The cationic lipid complexes may be stored in isotonic
sucrose or dextrose solution upon collection from the sucrose
gradient or they may be lyophilized and then reconstituted in an
isotonic solution prior to use. In one embodiment, the cationic
lipid complexes are stored in solution. The stability of the
cationic lipid complexes of the present invention is measured by
specific assays to determine the physical stability and biological
activity of the cationic lipid complexes over time in storage. The
physical stability of the cationic lipid complexes is measured by
determining the diameter and charge of the cationic lipid complexes
by methods known to those of ordinary skill in the art, including
for example, electron microscopy, gel filtration chromatography or
by means of quasi-elastic light scattering using, for example, a
Coulter N4SD particle size analyzer as described in the Example,
The physical stability of the cationic lipid complex is
"substantially unchanged" over storage when the diameter of the
stored cationic lipid complexes is not increased by more than 100%,
or by not more than 50%, or by not more than 30%, over the diameter
of the cationic lipid complexes as determined at the time the
cationic lipid complexes were purified.
[0053] While it is possible for the chiral cationic lipid to be
administered in a pure or substantially pure form, it is preferable
to present it as a pharmaceutical composition, formulation or
preparation. Pharmaceutical formulations using the chiral cationic
lipid complexes of the invention may comprise the cationic lipid
complexes in a physiologically compatible sterile buffer such as,
for example, phosphate buffered saline, isotonic saline or low
ionic strength buffer such as acetate or Hepes (an exemplary pH
being in the range of about 3.0 to about 8.0). The chiral cationic
lipid complexes may be administered as aerosols or as liquid
solutions for intratumoral, intraarterial, intravenous,
intratracheal, intraperitoneal, subcutaneous, and intramuscular
administration.
[0054] The formulations of the present invention may incorporate
any stabilizer known in the art. Illustrative stabilizers arc
cholesterol and other sterols that may help rigidify the liposome
bilayer and prevent disintegration or destabilization of the
bilayer. Also agents such as polyethylene glycol, poly-, and
monosaccharides may be incorporated into the liposome to modify the
liposome surface and prevent it from being destabilized due to
interaction with blood-components. Other illustrative stabilizers
are proteins, saccharides, inorganic acids, or organic acids which
may be used either on their own or as admixtures.
[0055] A number of pharmaceutical methods may be employed to
control, modify, or prolong the duration of immune stimulation.
Controlled release preparations may be achieved through the use of
polymer complexes such as polyesters, polyamino acids,
methylcellulose, polyvinyl, poly(lactic acid), and hydrogels to
encapsulate or entrap the cationic lipids and slowly release them.
Similar polymers may also be used to adsorb the liposomes. The
liposomes may be contained in emulsion formulations in order to
alter the release profile of the stimulant. Alternatively, the
duration of the stimulant's presence in the blood circulation may
be enhanced by coating the surface of the liposome with compounds
such as polyethylene glycol or other polymers and other substances
such as saccharides which are capable of enhancing the circulation
time or half life of liposomes and emulsions.
[0056] When oral preparations are required, the chiral cationic
lipids may be combined with typical pharmaceutical carriers known
in the art such as, for example, sucrose, lactose, methylcellulose,
carboxymethyl cellulose, or gum Arabic, among others. The cationic
lipids may also be encapsulated in capsules or tablets for systemic
delivery.
[0057] Administration of the chiral cationic lipid of the present
invention may be for either a prophylactic or therapeutic purpose.
When provided prophylactically, the cationic lipid is provided in
advance of any evidence or symptoms of illness. When provided
therapeutically, the cationic lipid is provided at or after the
onset of disease. The therapeutic administration of the
immune-stimulant serves to attenuate or cure the disease. For both
purposes, the cationic lipid may be administered with an additional
therapeutic agent(s) or antigen(s). When the cationic lipids are
administered with an additional therapeutic agent or antigen, the
prophylactic or therapeutic effect may be generated against a
specific disease.
[0058] The formulations of the present invention, both for
veterinary and for human use, comprise a chiral cationic lipid
alone as described above, as a mixture of R and S enantiomers, or
also optionally, with one or more therapeutic ingredients such as
an antigen(s) or drug molecule(s). The formulations may
conveniently be presented in unit dosage form and may be prepared
by any method known in the pharmaceutical art.
[0059] Antigens
[0060] In one embodiment, the chiral cationic lipid is administered
without any additional agents in order to boost or lower various
immune responses, including production of other immune modulators,
and to boost the immune response to fighting disease. In another
embodiment, the chiral cationic lipid is administered in
combination with an antigen or antigens. In this case the objective
is to generate an immune response, which is specific to the
antigen(s) delivered in combination with the cationic lipid. The
response generated may include production of specific cytotoxic
T-cells, memory T-cells, or B-cells resulting in the prevention of
or therapeutic response to the specific disease associated with
those antigen(s). The antigen can be any tumor-associated antigen
or microbial antigen or any other antigen known to one skilled in
the art.
[0061] A "tumor-associated antigen," as used herein is a molecule
or compound (e.g., a protein, peptide, polypeptide, lipoprotein,
lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid,
carbohydrate, RNA, and/or DNA) associated with a tumor or cancer
cell and which is capable of provoking an immune response (humoral
and/or cellular) when expressed on the surface of an antigen
presenting cell in the context of a major histocompatibility
complex ("MHC") molecule. Tumor-associated antigens include self
antigens, as well as other antigens that may not be specifically
associated with a cancer, but nonetheless enhance an immune
response to and/or reduce the growth of a tumor or cancer cell when
administered to an animal. More specific embodiments are provided
herein.
[0062] A "microbial antigen," as used herein, is an antigen of a
microorganism and includes, but is not limited to, infectious
virus, infectious bacteria, infectious parasites and infectious
fungi. Microbial antigens may be intact microorganisms, and natural
isolates, fragments, or derivatives thereof, synthetic compounds
which are identical to or similar to naturally-occurring microbial
antigens and, preferably, induce an immune response specific for
the corresponding microorganism (from which the naturally-occurring
microbial antigen originated). In a preferred embodiment, a
compound is similar to a naturally-occurring microorganism antigen
if it induces an immune response (humoral and/or cellular) similar
to a naturally-occurring microorganism antigen. Compounds or
antigens that are similar to a naturally-occurring microorganism
antigen are well known to those of ordinary skill in the art such
as, for example, a protein, peptide, polypeptide, Lipoprotein,
lipopeptide, glycoprotein, glycopeptides, lipid, glycolipid,
carbohydrate, RNA, and/or DNA. Another nonlimiting example of a
compound that is similar to a naturally-occurring microorganism
antigen is a peptide mimic of a polysaccharide antigen. More
specific embodiments are provided herein.
[0063] The term "antigen" is further intended to encompass peptide
or protein analogs of known or wild-type antigens such as those
described in. this specification. The analogs may be more soluble
or more stable than wild type antigen, and may also contain
mutations or modifications rendering the antigen more
immunologically active. Antigen can be modified in any manner, such
as adding lipid or sugar moieties, mutating peptide or protein
amino acid sequences, mutating the DNA or RNA sequence, or any
other modification known to one skilled in the art. Antigens can be
modified using standard methods known by one skilled in the
art.
[0064] Also useful in the compositions and methods of the present
invention are peptides or proteins which have amino acid sequences
homologous with a desired antigen's amino acid sequence, where the
homologous antigen induces an immune response to the respective
tumor, microorganism or infected cell.
[0065] In one embodiment, the antigen in the cationic lipid complex
comprises an antigen associated with a tumor or cancer, i.e., a
tumor-associated antigen, to make a vaccine to prevent or treat a
tumor. As such, in one embodiment, the tumor or cancer vaccines of
the present invention further comprise at least one epitope of at
least one tumor-associated antigen. In another preferred
embodiment, the tumor or cancer vaccines of the present invention
further comprise a plurality of epitopes from one or more
tumor-associated antigens. The tumor-associated antigens finding
use in the cationic lipid complexes and methods of the present
invention can be inherently immunogenic, or nonimmunogenic, or
slightly immunogenic. As demonstrated herein, even tumor-associated
self antigens maybe advantageously employed in the subject vaccines
for therapeutic effect, since the subject compositions are capable
of breaking immune tolerance against such antigens. Exemplary
antigens include, but are not limited to, synthetic, recombinant,
foreign, or homologous antigens, and antigenic materials may
include but are not limited to proteins, peptides, polypeptides,
lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA
and DNA. Examples of such vaccines include, but are not limited to,
those for the treatment or prevention of breast cancer, head and
neck cancer, melanoma, cervical cancer, Lung cancer, prostate
cancer, gut carcinoma, or any other cancer known in the art using a
cationic lipid in a complex with a tumor-associated antigen(s), It
is also possible to formulate the antigen. with the cationic lipid
without encapsulating it in the liposome. Thus, the chiral cationic
lipid complexes of the present invention may be used in methods to
treat or prevent cancer. In. such a case, the mammal to be
immunized maybe injected with the pharmaceutical formulation
containing the liposome with the encapsulated antigen(s).
[0066] Tumor-associated antigens suitable for use in the present
invention include both naturally occurring and modified molecules
which may be indicative of single tumor type, shared among several
types of tumors, and/or exclusively expressed or overexpressed in
tumor cells in comparison with normal cells. In addition to
proteins, glycoproteins, lipoproteins, peptides, and lipopeptides,
tumor-specific patterns of expression of carbohydrates,
gangliosides, glycolipids, and mucins have also been documented.
Exemplary tumor-associated antigens for use in cancer vaccines
include protein products of oncogenes, tumor suppressor genes, and
other genes with mutations or rearrangements unique to tumor cells,
reactivated embryonic gene products, oncofetal antigens,
tissue-specific (but not tumor-specific) differentiation antigens,
growth factor receptors, cell surface carbohydrate residues,
foreign viral proteins, and a number of other self proteins.
[0067] Specific embodiments of tumor-associated antigens include,
e.g., mutated or modified antigens such as the protein products of
the Ras p21 protooncogenes, tumor suppressor p53 and HER-2/neu and
BCR-abl oncogenes, as well as CDK4, MUM1, Caspase 8, and Beta
catenin; overexpressed antigens such as galectin 4, galectin 9,
carbonic anhydrase, Aldolase A, PRAMS, Her2/neu, ErbB-2 and KSA,
oncofetal antigens such as alpha fetoprotein (AFP), human chorionic
gonadotropin (hCG); self antigens such as carcinoembryonic antigen
(CEA) and melanocyte differentiation antigens such as Mart I/Melan
A, 0)100, gp75, Tyrosinase, TRP I and TRP2; prostate associated
antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated
embryonic gene products such as MA.GE 1, MAGE 3, MACE 4, GAGE 1,
GAGE 2, BAGE, RAGE, and other cancer testis antigens such as
NY-ES01, SSX2 and SCP1; mucins such as Muc-1 and Muc-2;
gangliosides such as GM2, GD2 and GD3, neutral glycolipids and
glycoproteins such as Lewis (y) and globo-H; and glycoproteins such
as To, Thompson-Freidenreich antigen (TF) and sTn. Also included as
tumor-associated antigens herein are whole cell and tumor cell
lysates as well as immunogenic portions thereof, as well as
immunoglobulin idiotypes expressed on monoclonal proliferations of
B lymphocytes for use against B cell lymphomas.
[0068] Tumor-associated antigens and their respective tumor cell
targets include, e.g., cytokeratins, particularly cytokeratin 8, 18
and 19, as antigens for carcinoma. Epithelial membrane antigen
(EMA), human embryonic antigen (HEA-125), human milk fat globules,
MBrl, MBr8, Ber-EP4, 17-1 A, C26 and T16 are also known carcinoma
antigens. Desmin and muscle-specific actin are antigens of
cryogenic sarcomas. Placental alkaline phosphatase, beta-human
chorionic gonadotropin, and alpha-fetoprotein are antigens of
trophoblastic and germ cell tumors. Prostate specific antigen is an
antigen of prostatic carcinomas, carcinoembryonic antigen of colon
adenocarcinomas. EIMB-45 is an antigen of melanomas. In cervical
cancer, useful antigens could be encoded by human papilloma virus.
Chromagranin-A and synaptophysin are antigens of neu pendocrine and
neuroectodermal tumors. Of particular interest are aggressive
tumors that form solid tumor masses having necrotic areas. The
lysis of such necrotic cells is a rich source of antigens for
antigen-presenting cells, and thus the subject therapy may find
advantageous use in conjunction with conventional chemotherapy
and/or radiation therapy.
[0069] In one embodiment, the human papillomavirus HPV antigens are
used. A specific HPV antigen that used as a tumor-associated
antigen is HPV subtype 16 E7. HPV E7 antigen-cationic lipid
complexes are effective at preventing and treating cervical cancer.
In addition, a genetically engineered E7 protein, i.e., E7 in
protein, having antigenic activity, but without tumorigenic
activity, is an effective tumor-associated antigen. Elm-cationic
lipid complexes induce cellular immunity to cause complete
regression of established tumors and, thus, are useful as potent
anti-cervical cancer vaccines.
[0070] Tumor-associated antigens can be prepared by methods well
known in the art. For example, these antigens can be prepared from
cancer cells either by preparing crude extracts of cancer cells
(e.g., as described in Cohen et al., Cancer Res., 54:1055 (1994)),
by partially purifying the antigens, by recombinant technology, or
by de novo synthesis of known antigens. The antigen may also be in
the form of a nucleic acid encoding an antigenic peptide in a form
suitable for expression in a subject and presentation to the immune
system of the immunized subject. Further, the antigen may be a
complete antigen, or it may be a fragment of a complete antigen
comprising at least one epitope.
[0071] Antigens derived from pathogens known to predispose to
certain cancers may also be advantageously included in the cancer
vaccines of the present invention. It is estimated that close to
16% of the worldwide incidence of cancer can be attributed to
infectious pathogens; and a number of common malignancies are
characterized by the expression of specific viral gene products.
Thus, the inclusion of one or more antigens from pathogens
implicated in causing cancer may help broaden the host immune
response and enhance the prophylactic or therapeutic effect of the
cancer vaccine. Pathogens of particular interest for use in the
cancer vaccines provided herein include the, hepatitis B virus
(hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein
Barr virus (EMT) (Burkitt lymphoma, nasopharynx cancer, PTLD in
immunosuppressed individuals), I-ITLVL (adult T cell leukemia),
oncogenic human papilloma viruses types 16, 18, 33, 45 (adult
cervical cancer), and the bacterium Helicobacter pylori (B cell
gastric lymphoma). Other medically relevant microorganisms that may
serve as antigens in mammals and more particularly humans are
described extensively in the literature, e.g., C. G. A Thomas,
Medical Microbiology, Baiiliero Tindall, Great Britain 1983, the
entire contents of which is hereby incorporated by reference.
[0072] In another embodiment, the antigen in the cationic lipid
complex comprises an antigen derived from or associated with a
pathogen, i.e., a microbial antigen. As such, in one embodiment,
the pathogen vaccines of the present invention further comprise at
least one epitope of at least one microbial antigen. Pathogens that
may be targeted by the subject vaccines include, but are not
limited to, viruses, bacteria, parasites and fungi. In another
embodiment, the pathogen vaccines of the present invention further
comprise a plurality of epitopes from one or more microbial
antigens.
[0073] The microbial antigens finding use in the cationic lipid
complexes and methods may be inherently immunogenic, or
nonimmunogenic, or slightly immunogenic. Exemplary antigens
include, but are not limited to, synthetic, recombinant, foreign,
or homologous antigens, and antigenic materials may include but are
not limited to proteins, peptides, polypeptides, lipoproteins,
lipopeptides, lipids, glycolipids, carbohydrates, RNA, and DNA.
[0074] Exemplary viral pathogens include, but are not limited to,
viruses that infect mammals, and more particularly humans. Examples
of virus include, but are not limited to: Retroviridae (e.g., human
immunodeficiency viruses), such as HIV-1 (also referred to as
HTLV-III, LAV or HTLV-IHiLAV, or HIV-III; and other isolates, such
as HIV-LP; Picornaviridac (e.g. polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g. strains that cause gastroenteritis); Togavridae
(e.g. equine encephalitis viruses, rubella viruses); Flaviridae
(e.g. dengue viruses, encephalitis viruses, yellow fever viruses);
Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular
stomatitis viruses, rabies viruses); Coronaviridae (e.g.
coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae
(e.g. parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g. influenza
viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,
phleboviruses and Naito viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (e.g. reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses,
vaccinia viruses, pox viruses); and lridoviridae (e.g. African
swine fever virus); and unclassified viruses (e.g. the etiological
agents of Spongiform encephalopathies, the agent of delta hepatitis
(thought to be a defective satellite of hepatitis B virus), the
agents of non-A, non-B hepatitis (class 1..sup.--internally
transmitted; class 2=parenterally transmitted (i.e. Hepatitis C);
Norwalk and related viruses, and astroviruses).
[0075] Also, gram negative and grain positive bacteria may be
targeted by the subject compositions and methods in vertebrate
animals. Such grain positive bacteria include, but are not limited
to Pasteurelkt species, Staphylococci species, and Streptococcus
species. Gram negative bacteria include, but are not limited to,
Escherich fa coli, Pseudomonas species, and Salmonella species.
Specific examples of infectious bacteria include but are not
limited to: Helicobacter pyloris, Borella burgdoiferi, Legionella
pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.
intracellulare, M. kansaii, M., gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneuinoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
infitenzae, Bacillus antracis, Corynebacterium diphtheriae,
coiynebacteriuin sp., Erysipelothrix rhusiopathiae, Clostridium
perfringers, Clostridium letani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, Rickettsia, and Actinoinyces.
[0076] Polypeptides of bacterial pathogens which may find use as
sources of microbial antigens in the subject compositions include
but are not limited to an iron-regulated outer membrane protein,
("TROMP"), an outer membrane protein ("OMP"), and an A-protein of A
eromonis sahnonicida which causes furunculosis, p57 protein of
Renibacterium salmonittarum which causes bacterial kidney disease
("BKD"), major surface associated antigen ("msa"), a surface
expressed cytotoxin ("mpr"), a surface expressed hemolysin ("ish"),
and a flagellar antigen of Yersiniosis; an extracellular protein
("ECP"), an iron-regulated outer membrane protein ("TROMP"), and a
structural protein of Pasteurellosis; an OMP and a flagellar
protein of Vibrosis anguillarum and V ordalii; a flagellar protein,
an OMP protein, aroA, and purA of Edwardsiellosis ictaluri and E,
tarda; and surface antigen of chthyophthirius; and a structural and
regulatory protein of Cytophaga columnari; and a structural and
rectulatory protein of Rickettsia. Such antigens can be isolated or
prepared recombinantly or by any other means known in the art.
[0077] Examples of pathogens further include, but are not limited
to, fungi that infect mammals, and more particularly humans.
Examples of fungi include, but are not limited to: Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, and Candida
albicans. `Examples of infectious parasites include Plasmodium such
as Plasmodium. falciparum, Plasmodium malartae, Plasmodium ovale,
and Plasmodium vivax. Other infectious organisms (i.e. protists)
include Toxoplasma gondii. Polypeptides of a parasitic pathogen
include but are not limited to the surface antigens of
khthyophthirius.
[0078] Other medically relevant microorganisms that serve as
antigens in mammals and more particularly humans are described
extensively in the literature (e.g., see C. G. A Thomas, Medical
Microbiology, Bailliere Tindall, Great Britain 1983). In addition
to the treatment of infectious human diseases and human pathogens,
the compositions and methods of the present invention are useful
for treating infections of nonhuman mammals. Many vaccines for the
treatment of nonhuman mammals are disclosed in Bennett, K.
Compendium of Veterinary Products, 3rd ed. North American
Compendiums, Inc., 1995; see also WO 02/069369, the disclosure of
which is expressly incorporated by reference herein in its
entirety.
[0079] Exemplary nonhuman pathogens include, but are not limited
to, mouse mammary tumor virus ("MMTV"), Rous sarcoma virus ("RSV"),
avian leukemia virus ("ALV"), avian myeloblastosis virus ("AMV"),
murine leukemia virus ("MLV"), feline leukemia virus ("FeLV"),
murine sarcoma virus ("MSV"), gibbon ape leukemia virus ("GALV"),
spleen necrosis virus ("SNV"), reticuloendotheliosis virus ("RV"),
simian sarcoma virus ("SSV"), Mason-Pfizer monkey virus ("MPMV"),
simian retrovirus type 1 ("SRV-1"), lentiviruses such as HIV-I,
HIV-2, SIV, Visna virus, feline immunodeficiency virus ("Fly"), and
equine infectious anemia virus ("EIAY"), T-cell leukemia viruses
such as HTLV-1, HTLY-II, simian T-cell leukemia virus ("STLV"), and
bovine leukemia virus ("III,V"), and foamy viruses such as human
foamy virus ("HFV"), simian foamy virus ("SFV") and bovine foamy
virus ("B1.sup.7V").
[0080] In some embodiments, "treatment," "treat," and "treating,"
as used herein with reference to infectious pathogens, refer to a
prophylactic treatment which increases the resistance of a subject
to infection with a pathogen or decreases the likelihood that the
subject will become infected with the pathogen; and/or treatment
after the subject has become infected in order to fight the
infection, e.g., reduce or eliminate the infection or prevent it
from becoming worse.
[0081] Microbial antigens can be prepared by methods well known in
the art. For example, these antigens can be prepared directly from
viral and bacterial cells either by preparing crude extracts, by
partially purifying the antigens, or alternatively by recombinant
technology or by de MVO synthesis of known antigens. The antigen
may also be in the form of a nucleic acid encoding an antigenic
peptide in a form suitable for expression in a subject and
presentation to the immune system of the immunized subject.
Further, the antigen may be a complete antigen, or it may be a
fragment of a complete antigen comprising at least one epitope.
[0082] In order to improve incorporation of the antigen into the
chiral cationic lipid vesicles and also to improve delivery to the
cells of the immune system, the antigen may be conjugated to a
lipid chain in order to improve its solubility in the hydrophobic
acyl chains of the cationic lipid, while maintaining the antigenic
properties of the molecule. The lipidated antigen can be a
lipoprotein, or a lipopeptide, and combinations thereof. The
lapidated antigen may have a linker conjugated between the lipid
and the antigen such as, for example, an N-terminal a or
a-palmitoyl lysine may be connected to antigen via a dipeptide
Ser-Ser linker. U.S. application Ser. No. 12/049,957 discloses that
the DOTAP/E7-lipopeptide complex exhibited an enhanced functional
antigen-specific CD8.sup.- T lymphocyte responses in vivo compared
to the DOTAP/E7 formulation, and therefore provided superior
anti-tumor efficacy.
[0083] The present invention will be further appreciated in light
of the following example.
Example
[0084] Effective Stimulation of the Immune System by Enantiomers of
Cationic Lipids
[0085] I. Cell Lines and Peptides
[0086] TC-1 cells are C57BL/6 mouse lung endothelial cells that
have been transformed with the HPV16 E6 and E7 oncogenes and
activated H-ras. Cells were grown in RPMI medium (commercially
available from Invitrogen of Carlsbad, Calif.) supplemented with
10% fetal bovine serum and 100 U/mI penicillin, and 100 mg/ml
streptomycin. The MHC class I restricted peptide from the HPV 16 E7
protein (amino acid 11 to 20, YMLDLQPETT [SEQ. ID. NO. I]) was
synthesized by the University of Pittsburgh Peptide Synthesis
Facility by solid state synthesis using an Advanced ChernTech model
200 peptide synthesizer and purified by HPLC.
[0087] 2. Preparation of Lipid/Antigen Complexes and Determination
of Physical Properties
[0088] The enantiomers of DOTAP were supplied by Merck AG (EPROVA),
Switzerland. All other lipids were purchased from Avanti Polar
Lipids (Alabaster, Ala.). Small unilamellar DOTAP liposomes were
prepared by thin film hydration followed by extrusion. The lipid,
in chloroform, was dried as a thin layer under a stream of nitrogen
in a glass tube. The thin film was vacuum desiccated for 2-3 h and
then re-hydrated in cell culture grade water (commercially
available from Cambrex of Walkersville, Md.) or buffer (such
buffers are well known to those skilled in the art) containing E7
peptide to a final concentration of 0.7 mg lipids and 0.1 mg E7 per
mL (molar ratio=11:1). The lipid dispersion was sequentially
extruded through polycarbonate membranes with pore size of 0.4,
0.2, and 0.1 pan. The un-entrapped peptide was not removed. The
liposomes were stored at 4.degree. C. until use. E7 peptide
association with the liposome was determined by measuring the
percentage of liposome-bound peptide. In brief, unbound E7 peptide
from R-DOTAP/E7, S-DOTAP/E7 or RS-DOTAP/E7 complexes was separated
by a Microcon.RTM. centrifugal filtrate device (Millipore, Bedford,
Mass.) and the concentration of unbound peptide was measured by
Micro BCA.TM. Protein Assay Kit (Pierce, Rockford, Ill.). The
efficiency of peptide association was determined as percent unbound
peptide. Other methods used in general liposome preparation that
are well known to those skilled in the art may also be used.
[0089] 3. Statistical Analysis
[0090] Data are presented as mean.+-.SD of at least 3 independent
experiments. Two-tailed Student's t tests were used to assess
statistical significance for differences in means. Significance was
set at p<0.05.
[0091] 4. Individual R and S Enantiorners of Cationic Lipid/E7
Complexes Activate Human Dendritic Cells Similarly to the DOATP
Racemic Mixture.
[0092] Cationic liposomes were prepared as described above. The E7
antigen used in the formulation is the identified human E7 peptide
restricted by HLA-A*0201 [HPV-16 E7, amino acids 11-20, YMLDLQPETT
(SEQ. ID. NO. 1)1 The peptide was synthesized by the University of
Pittsburgh, Molecular Medicine Institute, Pittsburgh, Pa. Human
1-ILA-A2 human dendritic cells were obtained from Lonza (of
Walkersville, Md.). Frozen cryovials were thawed and the dendritic
cells were cultured in LGM-3 medium (commercially available from
Lonza of Walkersville, Md.) supplemented with 50 microgram/mI IL-4
and GM-CSF at 37.degree. C. and 5% CO.sub.2 at an initial plating
density of 125,000 cells/cm.sup.2 in 2 ml of medium in 12-well
tissue culture dishes. The cells were grown for 3 days in culture
and appeared as a mixture of adherent and rounded cells by
microscopic examination.
[0093] The cells were treated on day 3 with a fresh dose of 50
microgram/ml of IL-4 and GM-CSF (all wells) and test wells were
treated with either a mixture of interleukin 1-beta ("IL-.beta."),
interleukin 6 ("IL-6") and TNF-a at 10 ng/ml, and prostaglandin E2
("PGE-2") at 10 .mu.g/ml (positive control for activation), no
treatment (negative activation control), and S-DOTAP/E7 at 2.5, 10
and 40 micromolar final concentrations, and R-DOTAP/E7 at 2.5, 10
and 40 micromolar final concentrations. The treated dendritic cells
were maintained in culture for 24 hours and harvested for cell
surface marker staining and flow cytornetry analysis. The harvested
cells were counted by hernacytometer and 10 pi of the following
antibody conjugates were added sequentially to each sample for
labeling surface markers: CD80-FITC, CD83-APC, and CD86-PE (BD
Biosciences). The surface labeled cells were subsequently analyzed
by flow cytometry using a BD FACxcaliber flow cytometer, and the
co-stimulatory dendritic cell marker molecules CD80, CD83, and CD86
which are produced upon activation, were monitored. As seen in
FIGS. 2, 3 and 4 primary human dendritic cells treated with the
both enantiomers of the cationic lipid/E7 complex up-regulated the
expression of all three co-stimulatory markers of dendritic cell
activation evaluated and required for successful antigen
presentation to T-cells, similarly to what was observed with the
racemic mixture (RS-DOTAP) of the cationic lipid and reported in
U.S. application Ser. No. 12/049,957, assigned to the assignee of
the present application.
[0094] 5. Cationic Lipid/E7 Complexes Consisting of Individual R
and S Enantiomers Exhibit Different Potencies in Activating Human
Dendritic Cells to Induce Chemokine and Cytokine Production
[0095] Human HLA-A 2 dendritic cells (Lonza, Walkersville, Md.),
were treated and grown in culture as described above. On day 3 the
cells were treated with 40 micromolar DOTAP/E7 complex or the
potent immunostimulator lipopolysaccharide (LPS) at 50 micromolar
concentrations (positive control). Medium from assay wells was
removed and centrifuged at 1300 rpm in a microfuge for 5 minutes to
pellet unattached dendritic cells. The supernatants were removed
and treated with 10 microliters per ml of Calbiochem (La Jolla,
Calif.) protease inhibitor cocktail set I (Cat. No. 539131) and
stored frozen prior to analysis. Samples were analyzed for
chemokine and cytokine expression by Searchlight Protein Array
Multiplex ELISA assay [Pierce Biotechnology (Woburn, Mass.)].
[0096] Production of selected chemokines known to be essential in
the cellular immune response, CCL3, CCL4, CCL5, and CCL19 was
evaluated, and production of IL-2, 11-8 and IL-12 was evaluated
(FIGS. 5-11, which illustrate the ability of R-DOTAP/E7 and
S-DOTAP/E7 to induce production of CCL3, CCL4, CCL5, CCL-19, IL-2,
IL-8 and 1L-12), The figures illustrate that the DOTAP/E7 complex
containing the individual enantiomers of DOTAP induce cytokine and
chemokine production by human dendritic cells. Both enantiorners
however activate the immune system to different extents with the
R-enantiomer exhibiting higher potency.
[0097] 6. Kinetics of TC-.1 HPV-Positive Tumor Growth in Mice
Treated with DOTAP/E7 Compositions at Varying Doses of Racemic
Mixtures of DOTAP.
[0098] In FIG. 12, mice were subcutaneously injected with TC-1
cells on day 0 in order to induce the growth of HPV-positive
tumors, The DOTAP/E7 compositions were comprised of racemic
mixtures of DOTAP. The mice received DOTAP/E7 compositions
containing 10 11.g E7 peptide subcutaneously on the opposite side
of the abdomen on. day 6. DOTAP lipid concentration in the complex
varied from 3 to 600 nmole (3, 15, 30, 75, 150, 300, and 600
nmole), Low dose of DOTAP (15 nmole) showed partial tumor
inhibition effect (P<0.05) compared to the untreated control on
day 23, while DOTAP at 30, 150 or 300 nmole exhibited an enhanced
efficacy (P<0.01). DOTAP at 75 nmole showed the most significant
tumor regression effect (P<0.001). Again, mice given a high dose
of DOTAP (600 nmole) did not show anti-tumor activity, confirming
that DOTAP liposomes at a high dose might have induced a negative
regulation to the immune response. In addition, DOTAP liposomes at
the 100 nmole dose, but without E7 peptide, did not show
significant inhibition of tumor growth, indicating that the
anti-tumor effect was antigen specific. Further, liposomes of
1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), an anionic lipid,
administered at the 150 nmole dose with antigen failed to
significantly inhibit tumor growth.
[0099] 7. Kinetics of TC-1 HPV-Positive Tumor Growth in Mice
Treated with R-DOTAP/E7 and S-DOTAP Compositions at Varying Doses
of R and S DOTAP.
[0100] In FIGS. 13 and 14, mice were subcutaneously injected with
TC-1 cells on day 0 in order to induce the growth of HPV-positive
tumors. The mice received R and S-DOTAP/E7 compositions containing
20 E7 peptide subcutaneously on the opposite side of the abdomen on
day 6. R or S-DOTAP lipid concentrations in the complex varied from
3 to 600 nmole (3, 15, 30, 75, 100, 125, 150, 300, and 600 nmole).
Unlike the racemic mixture of DOTAP (FIG. 12), S-DOTAP complexes
did not exhibit the ability to inhibit tumor growth and no tumor
regression was observed (FIG. 13). A dose response effect was
however observed, and S-DOTAP closes of 600 nmole induced the
slowest tumor growth (P<0.05) compared to untreated control on
day 23. Referring to FIG. 14 the anti-tumor effect of complexes
containing R-DOTAP and antigen were similar to the effect observed
in the race/tile mixture (FIG. 12). 75-150 nmole doses of R-DOTAP
showed partial tumor inhibition effect (P<0.001) compared to the
untreated control on day 23, while R-DOTAP at 300 nmole exhibited
the most significant tumor regression efficacy (P<0.0001).
Again, mice given a high dose of R-DOTAP (600 nmole) did not show
significant anti-tumor activity, confirming that R-DOTAP liposomes
at a high dose might have induced a negative regulation to the
immune response. E7 peptide alone, did not show any inhibition of
tumor growth (not shown). FIG. 15 shows the lipid dose-response
curves for the tumor regression efficacy of the various cationic
lipid/E7 antigen complexes DOTAP, S-DOTAP, and R-DOTAP at 20 .mu.g
of the antigen and DOTAP at 10 .mu.g of the antigen.
[0101] 8. Induction of T Cell Proliferation by S-DOTAP and R-DOT.AP
Compositions.
[0102] We have previously demonstrated that in U.S. Provisional
Application No. 60/983,799, assigned to the assignee of the present
application, that DOTAP/E7 interacts directly with human T
lymphocytes, leading to clonal expansion and T cell activation.
Those studies examined the ability of racemic mixtures of DOTAP to
stimulate clonal expansion of T cells. In those studies, enriched
human lymphocytes obtained from an I-ILA-A2.sup.t healthy donor
were directly stimulated by medium, DOTAP alone, peptide alone or
DOTAP/hE7. The stimulation was repeated three times with a 7-day
interval. Three days after the third stimulation, lymphocytes
treated with DOTAP or DOTAP/E7 showed extensive expansion of T cell
colonies in culture in contrast to no clonal expansion in medium
control. The expanded T-cells also demonstrated significant CTL
activity.
[0103] In those studies, the DOTAP-mediated T cell activation was
further confirmed by ERK phosphorylation in T cells. DOTAP-induced
expression of the costimulatory molecule, CD86 on human T
lymphocytes was also observed. Those results suggested that DOTAP
has a direct impact on T cell activation via a MAP kinase mediated
cell proliferation.
[0104] In the present studies, the induction of human T-cell
proliferation by the R and S enantiomers of DOTAP was investigated
and confirmed using purified T-cells obtained from Lonza, Mass.
R-DOTAP induced more T-cell proliferation than S-DOTAP and was
similar in activity to the DOTAP racemic mixture.
[0105] Discussion
[0106] As described in U.S. Pat. No. 7,303,881, a broad class of
cationic lipids can act as potent immunostimulators together with
an antigen to generate antigen specific immune responses in the
treatment of disease. For example, U.S. Pat. No. 7,303,881
discloses that liposomes comprised of cationic lipids activate
dendritic cells as demonstrated by the stimulation by cationic
lipids of the expression of costimulatory molecules CD80/CD86 on
DC2A dendritic cells (FIGS. 14A and 14B). As shown in FIG. 14A of
U.S. Pat. No. 7,303,881, the ability to stimulate the expression of
CD80/CD86 on DC2.4 cells by different cationic liposomes varies
greatly. Lipofectamine.RTM., a 3:1 (w/w) liposome formulation of
the polycationic lipid
2,3-dioleyloxy-N-[2(spen-ninecarboxamido)ethyll-N,N-dimethyl-1-propanarni-
nium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl
phosphatidylethanolamine (DOPE), and liposomes prepared from
0,0.sup.1-dirnyristyl-N-lysyl aspartate (DMKE) and
0,0'-dimyristyl-N-lysyl-glutamate (DMKD), strongly stimulated the
expression of CD80/CD86 by CD2.4 cells.
[0107] As further disclosed in U.S. Pat. No. 7,303,881, the ability
of different cationic lipids to stimulate the expression of CD 80
on DC 2.4 cells varied. Both hydrophilic head and the lipophilic
tail of the lipids have significant effect on this ability. For
example, the DXEPC lipids with the ethyl phosphocholine (EPC) head
groups appear, in general, to be more potent than the DXTAP lipids
with trimethylammonium propane (TAP) head group. Within the lipids
bearing one particular head group structure, lipids with shorter
(1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DXEPC-12:0),
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC-14:0)) or
unsaturated (1,2-dioleoyl-sn-glycero-3-ethylphosphocholinc
(DOEPC-18:1)) acyl chains appear to be more potent than those with
longer (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine
(DXEPC-16:0)) or saturated
(1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC-16:0)) acyl
chains, These data however, demonstrated that multiple cationic
lipids were capable of stimulating the activation of dendritic
cells. Studies reported in U.S. application Ser. No. 12/049,957
highlight the mechanism by which cationic lipids act as
immunostimulators.
[0108] Data from the abovementioned studies have led to the
observation that the cationic lipids are not only efficient
targeting and delivery vehicles for antigens to APC of the immune
system, but also function as potent adjuvants under low dose
composition ranges to directly influence immune system function
through activation of MAP kinase dependent signaling pathways with
resultant production of immune system regulatory molecules
including cytokines and chemokines. A clear dose-response effect of
cationic lipid on the immunostimulatory capabilities of the
formulations have been demonstrated. It was demonstrated that upon
receiving the lipid/antigen complex, the particles were mainly
taken up by dendritic cells, the major professional antigen
presenting cells. The initiation of dendritic cell activation and
migration to the draining lymph node facilitates immune responses
against antigen specific TC-1 tumors as demonstrated. Functional
CD8.sup.+ T lymphocytes were generated in mice upon receiving a
DOTAP/E7 injection and tumor sizes decreased and exhibited enhanced
apoptosis, owing to the increasing number of infiltrating T cells
in the tumor microenvironment. The resulting bell-shaped (activity
decreases above and below the optimal dose) cationic lipid dose
response curve demonstrated activity at very low doses, indicating
that the activity of the cationic lipids as adjuvants or
immunostimulators is so potent that the EC.sub.50 is as low as
about 15 nmole per injection. High doses of cationic lipids
eliminate the immunostimulatory activity. We have also demonstrated
that when an antigen such as, for example, ovalbumin, is
incorporated into the cationic liposomes and administered in a
single subcutaneous injection, effective antibodies against the
antigen are produced. Cationic liposomes can also induce expression
of the co-stimulatory molecules CD80 and CD83 and activate human
dendritic cells. It is clear that at optimal dose compositions, the
cationic lipids and cationic lipid/antigen complexes in addition to
effective delivery to the dendritic cells are potent activators of
the immune system and provide simple, safe, and very efficient
immunotherapies useful in preventing and treating diseases.
[0109] Based on an understanding of the mechanism of
immunostimulation, further studies were performed to evaluate the
effect of chirality in cationic lipids and the immunostimulatory
capability of cationic lipids, To this effect pure synthesized R
and S enantiomers of DOTAP were utilized and compared with the
commonly utilized racemic mixture. Both R and S enantiomers of
DOTAP were demonstrated to possess similar ability to the racemic
DOTAP with regards to activation and maturation of dendritic cells.
All three lipids induced dendritic cells to express the
co-stimulatory molecules CD 80 CD 83 and CD 86.
[0110] An important characteristic of an immunostimulator capable
of inducing cellular immune responses to disease is its ability to
induce the production of critical chemokines and cytokines. As
reported in the Example, significant differences were observed
between the R and S enantiomers of DOTAP in their ability to induce
chetnokine and cytokine production. R-DOTAP was observed to be a
more potent immune activator than S-DOTAP. In all cases the potency
of R-DOTAP was equivalent to or higher than that of the DOATP
racemic mixture.
[0111] In order to determine if the in-vitro potency in cytokine
induction would translate to in-vivo therapeutic efficacy, three
formulations, R-DOTAP/E7, S-DOTAP/E7 and DOTAP/E7 (racemic mixture)
were evaluated for their ability to eradicate HPV-E7 positive
tumors in tumor-bearing mice. Each formulation was evaluated at
multiple lipid doses. As demonstrated in FIGS. 12-15, both R-DOTAP
and DOTAP containing formulations exhibited a bell-shaped
lipid-dose response with strong E7 specific activity leading to
tumor regression within specific optimal dose ranges. S-DOTAP
containing formulations did not induce tumor regression under any
condition observed, although high lipid formulations slowed tumor
growth.
[0112] It is therefore evident that the R enantiomer of DOTAP is
responsible for the majority of the observed adjuvant effect of
DOTAP. However, both enantiomers are potent activators of dendritic
cells leading to maturation.
[0113] The studies reported above identify specific unique
compositions and applications of cationic lipids consisting of
chiral lipid or mixtures of chiral lipids, which can be exploited
to develop simple, cost effective, and much needed immunotherapies
for several debilitating diseases.
[0114] As various changes could be made in the above-described
aspects and exemplary embodiments without departing from the scope
of the invention, it is intended that all matter contained in the
above description shall be interpreted as illustrative and not in a
limiting sense.
[0115] To that end, while the examples primarily discuss
enantiomers of the cationic lipid DOTAP, those skilled in the art
will recognize that this cationic lipids is merely exemplary and
that the methods and mechanisms are applicable to other cationic
lipids.
Sequence CWU 1
1
1110PRTAlphapapillomavirus 9 1Tyr Met Leu Asp Leu Gln Pro Glu Thr
Thr1 5 10
* * * * *