U.S. patent application number 11/944912 was filed with the patent office on 2008-06-05 for antigen delivery compositions and methods of use.
This patent application is currently assigned to PDS BIOTECHNOLOGY CORPORATION. Invention is credited to Zhengrong Cui, John Dileo, Su-Ji Han, Leaf Huang, Dileep P. Vangasseri.
Application Number | 20080131455 11/944912 |
Document ID | / |
Family ID | 35541619 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131455 |
Kind Code |
A1 |
Huang; Leaf ; et
al. |
June 5, 2008 |
Antigen Delivery Compositions And Methods Of Use
Abstract
The present invention provides antigen delivery compositions and
methods of using same to prevent or to treat cancers and other
infectious diseases.
Inventors: |
Huang; Leaf; (Durham,
NC) ; Cui; Zhengrong; (Philomath, OR) ; Dileo;
John; (Stafford, VA) ; Han; Su-Ji; (Gwangju,
KR) ; Vangasseri; Dileep P.; (Thrissur, IN) |
Correspondence
Address: |
WOOD, HERRON & EVANS, LLP
2700 CAREW TOWER, 441 VINE STREET
CINCINNATI
OH
45202
US
|
Assignee: |
PDS BIOTECHNOLOGY
CORPORATION
Liberty Township
OH
|
Family ID: |
35541619 |
Appl. No.: |
11/944912 |
Filed: |
November 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11121840 |
May 2, 2005 |
7303881 |
|
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11944912 |
|
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60567291 |
Apr 30, 2004 |
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Current U.S.
Class: |
424/196.11 ;
424/193.1; 424/197.11; 562/512 |
Current CPC
Class: |
A61K 39/12 20130101;
A61P 37/04 20180101; A61K 39/02 20130101; A61K 2039/585 20130101;
Y10S 977/907 20130101; C12N 2710/20034 20130101; A61K 2039/53
20130101; A61K 39/0011 20130101; A61K 2039/6018 20130101; A61K
39/385 20130101; A61K 2039/55555 20130101; A61K 2039/6093
20130101 |
Class at
Publication: |
424/196.11 ;
424/193.1; 424/197.11; 562/512 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 39/00 20060101 A61K039/00; C07C 53/00 20060101
C07C053/00; A61P 37/04 20060101 A61P037/04; A61K 39/02 20060101
A61K039/02 |
Claims
1. A method of inducing an immune response in a subject comprising
administering to the subject an antigen/lipid complex comprising an
antigen and a non-steroidal cationic lipid having a structure
represented by the formula: ##STR00001## wherein in R.sup.1 is a
quaternary ammonium group, Y.sup.1 is chosen from a hydrocarbon
chain, an ester, a ketone, and a peptide, 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; to form an antigen/lipid complex to induce an
immune response to treat or prevent disease in the subject.
2. The method of claim 1 wherein said cationic lipid is selected
from the group consisting of 1,2-myristoyl-3-trimethylammonium
propane (DMTAP), 1,2-distearoyl-3-trimethylammonium propane
(DSTAP), 1,2-palmitoyl-3-trimethylammonium propane (DPTAP),
1,2-oleoyl-3-trimethylammonium propane (DOTAP),
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC),
1,2-distearoyl-sn glycerol-3-ethylphosphocholine (DSEPC),
1,2-dimyristoyl-sn-glycero-3-ethylphophocholine (DMEPC),
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC),
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),
O,O'-dimyristyl-N-lysyl-aspartate (DMKE),
O,O'-dimyristyl-N-lysyl-glutamate (DMKD),
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoracetate (DOSPA),
N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride
(DOTMA), their structural variants and derivatives, and
combinations thereof.
3. A method for stimulating an immune response to a tumor in a
subject comprising administering to the subject the complex of
claim 1 wherein said antigen is a tumor-associated antigen thereby
stimulating an immune response to a tumor in the subject.
4. A method for inhibiting the growth of a tumor cell in a subject,
said method comprising administering to a subject the complex of
claim 1 wherein said antigen is a tumor-associated antigen, or a
tumor-associated antigen modified to retain its antigenic activity
but to eliminate its tumorigenic activity, thereby preventing or
inhibiting the growth of a tumor cell in said subject.
5. The method of claim 1 wherein the antigen is derived from
pathogens associated with cancer risk wherein said pathogens are
selected from the group consisting of hepatitis B virus, hepatitis
C virus, Epstein Barr virus, HTLVL, oncogenic human papilloma
viruses types 16, 18, 33, 45 and the bacterium Helicobacter
pylori.
6. The method of claim 3, wherein the tumor associated antigen is
selected from the group consisting of mutated antigens,
overexpressed antigens found in tumors, melanocyte differentiation
antigens, prostate associated antigens, reactivated embryonic gene
products, human papilloma virus antigens, oncofetal antigens, self
antigens, cancer testis antigens, mucins, gangliosides, whole cell
and tumor cell lysates, immunogenic portions of whole cell and
tumor cell lysates, immunoglobulin idiotypes expressed on
monoclonal proliferations of B lymphocytes, and combinations
thereof.
7. The method of claim 6 wherein said human papilloma virus antigen
is an HPV 16 E7 protein or peptide.
8. The method of claim 7 wherein said HPV 16 E7 protein or peptide
has been modified to retain its antigenic activity but to eliminate
its tumorigenic activity.
9. The method of claim 1, wherein said antigen/lipid complex
includes at least one additional component selected from the group
consisting of a cationic salt, an adjuvant, an immunostimulant, a
cholesterol, a surfactant, a polymer, and combinations thereof.
10. A method of inducing an immune response to treat or prevent an
infectious disease in a subject comprising administering to the
subject an antigen/lipid complex comprising an antigen and a
non-steroidal cationic lipid having a structure represented by the
formula: ##STR00002## wherein in R.sup.1 is a quaternary ammonium
group, Y.sup.1 is chosen from a hydrocarbon chain, an ester, a
ketone, and a peptide, 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; to form an antigen/lipid complex to induce an immune
response to treat or prevent an infectious disease in the
subject.
11. The method of claim 10 wherein the cationic lipid selected from
the group consisting of 1,2-myristoyl-3-trimethylammonium propane
(DMTAP), 1,2-distearoyl-3-trimethylammonium propane (DSTAP),
1,2-palmitoyl-3-trimethylammonium propane (DPTAP),
1,2-oleoyl-3-trimethylammonium propane (DOTAP),
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC),
1,2-distearoyl-sn glycerol-3-ethylphosphocholine (DSEPC),
1,2-dimyristoyl-sn-glycero-3-ethylphophocholine (DMEPC),
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC),
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),
O,O'-dimyristyl-N-lysyl-aspartate (DMKE),
O,O'-dimyristyl-N-lysyl-glutamate (DMKD),
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoracetate (DOSPA),
N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride
(DOTMA), and combinations thereof.
12. The method of claim 10 wherein said antigen is a viral or
microbial antigen.
13. The method of claim 12 wherein said viral antigen is selected
from the group consisting of retroviridae, picornaviridae,
orthomyxoviridae, herpesviridae, hepatitis A, hepatitis B,
hepatitis C, Epstein Barr virus, HTLVL, oncogenic human papilloma
viruses types 16, 18, 33, and 45, and combinations thereof.
14. The method of claim 12 wherein said microbial antigen is
selected from the group consisting of Mycobacteria species,
Pasteurella species, Staphylocci species, Streptococcus species,
Pseudomonas species, Salmonella species, Helicobacter pylori, and
combinations thereof.
15. The method of claim 10 wherein said antigen is a protein or
peptide associated with a virus or microbe.
16. The method of claim 10 wherein said antigen is a protein or
peptide associated with a virus or microbe, and the protein or
peptide is modified to retain its antigenic activity but to
eliminate its pathogenic activity.
17. The method of claim 10, wherein the cationic lipid includes
structural variants and derivatives thereof.
18. An antigen/lipid complex comprising an antigen and a
non-steroidal cationic lipid having a structure represented by the
formula: ##STR00003## wherein in R.sup.1 is a quaternary ammonium
group, Y.sup.1 is chosen from a hydrocarbon chain, an ester, a
ketone, and a peptide, 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; to form an antigen/lipid complex.
19. The complex of claim 18 wherein the cationic lipid is selected
from the group consisting of 1,2-myristoyl-3-trimethylammonium
propane (DMTAP), 1,2-distearoyl-3-trimethylammonium propane
(DSTAP), 1,2-palmitoyl-3-trimethylammonium propane (DPTAP),
1,2-oleoyl-3-trimethylammonium propane (DOTAP),
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC),
1,2-distearoyl-sn glycerol-3-ethylphosphocholine (DSEPC),
1,2-dimyristoyl-sn-glycero-3-ethylphophocholine (DMEPC),
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC),
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),
O,O'-dimyristyl-N-lysyl-aspartate (DMKE),
O,O'-dimyristyl-N-lysyl-glutamate (DMKD),
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoracetate (DOSPA),
N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride
(DOTMA), and combinations thereof.
20. The complex of claim 18 further including an additive selected
from the group consisting of cationic salt, adjuvant,
immunostimulant, cholesterol, surfactant, polymer, and combinations
thereof, to improve the stability or activity of the
combination.
21. The complex of claim 18 wherein the cationic lipid includes
structural variants and derivatives thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation that claims the
benefit of currently pending U.S. patent application Ser. No.
11/121,840, filed May 2, 2005 by the same inventors and having the
same assignee as the present invention, and U.S. Provisional
Application Ser. No. 60/567,291, filed on Apr. 30, 2004 by the same
inventors and having the same assignee as the present invention,
each of which is incorporated by reference herein in their
entireties.
BACKGROUND OF THE INVENTION
[0002] To prevent and treat many of the deadly diseases and
cancers, effective vaccines are needed. The new generation vaccine
has many advantages over traditional vaccine. However, the potency
of the new generation vaccine needs to be enhanced. Unfortunately,
after 80 some years of development, Alum is still the only single
adjuvant approved by the Food and Drug Administration (FDA) in the
United States for use in humans. Therefore, novel vaccine delivery
systems and/or adjuvants are desperately needed and desired.
SUMMARY OF THE INVENTION
[0003] The present invention provides antigen delivery
compositions/adjuvants and methods of using same to prevent or to
treat cancers and other infectious diseases. More particularly, the
present invention provides a lipid-protamine-DNA (LPD) vaccine
delivery system/adjuvant and methods of using such LPD vaccine
delivery system/adjuvant for the prevention and treatment of cancer
and other infectious diseases. In fact, it has surprisingly been
found that the LPD vaccines of the present invention can induce
strong cellular immunity to cause complete regression of
established tumors and to prevent the formation of new tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1. One time treatment of LPD/E7 nanoparticles
eradicates established tumors. C57BL/6 mice were s.c. inoculated
with 1.times.10.sup.5 TC-1 cells on day 4, 6, 8, or 12, mice were
s.c. injected with LPD nanoparticles containing 10 .mu.g of E7
peptide. Tumor growth was measured three times per week. Mean
.+-.SD, 5 mice per group. One representative of three experiments
showing similar tumor growth kinetics is shown.
[0005] FIG. 2. LPD/E7 vaccination prevents tumor establishment
after i.p. injection of TC-1 cells (A) and induces tumor specific
immune response (B). A. Mice were s.c. injected with LPD particles
with 10 .mu.g E7 peptide or 10 .mu.g E7 peptide in PBS on days 0
and 5 or left untreated. On day 10, the mice were i.p. challenged
with 1.times.10.sup.6 TC-1 cells and the survival rate was checked.
B. 50 days after, the survived mice were s.c. rechallenged with
1.times.10.sup.5 TC-1 or 24 JK cells. Tumor formation was monitored
three times per week by palpation.
[0006] FIG. 3. All E7 peptide containing LPD nanoparticles can
induce antigen specific anti tumor effect. Mice (n=5) were sc
injected with LPD nanoparticles on day 0 and 5. On day 10, the mice
were sc challenged with 1.times.10.sup.5 TC-1 cells. Tumor
formation was monitored three times per week.
[0007] FIG. 4. The role of TLR9 on the antigen specific immune
response induced by LPD/E7 nanoparticles. The splenocytes from wild
type or TLR9-/- mouse were re-stimulated with 1 .mu.g/ml of E7
peptide for 24 h. The culture supernatants were collected and
IFN-.alpha. levels were measured using ELISA.
[0008] FIG. 5. IFN-.gamma. ELISPOT assay. Mice were s.c. injected
with LPD nanoparticles containing 10 .mu.g of E7 peptide or LPD
nanoparticles without E7 peptide on day 0 and 5. Five days after
the last injection, lymph node cells were isolated, re-stimulated
with 1 .mu.g/ml of E7 peptide for 24 h for tumor specific T cell
expansion. NK cells were depleted by injection with 100 .mu.g
anti-NK1.1 starting 2 days before the first vaccination and ending
2 days after the last vaccination.
[0009] FIG. 6. The expression of co-stimulatory molecules, CD80 and
CD86, on DC2.4 cells after stimulation by LPD or the components of
LPD.
[0010] FIG. 7. Expression of co-stimulatory molecules, CD80 and
CD86, on DC2.4 cells after stimulation with different cationic
liposomes.
[0011] FIG. 8. Hydrocarbon chain length dependence of cationic
lipids on the expression of co-stimulatory molecule (CD 80) on DC
2.4 cells.
[0012] FIG. 9. Tumor growth kinetics on mice treated with LPD/E7
and other different formulations. The letters a, b, and c indicate
that the final mean tumor sizes were significantly different
between the groups, but not different within the group.
[0013] FIG. 10. Specific IgG level in serum after diluted for
1.000-fold. Mice (n=4-5) were immunized with E7 protein alone,
E7/LPD, and E7 adjuvanted with `Alum` (15 .mu.g/mouse) (E7/Alum).
The dose of E7 protein was 20 .mu.g/mouse. As control, one group of
mice was left untreated. Mice were immunized on day 0 and 14. On
day 28, mice were bled via tail vein. ANOVA analysis on the three
treatments showed a p value of 0.001. * indicates that the value
from E7/LPD is significantly different from that of the others.
[0014] FIG. 11. Immunization with E7/LPD prevented TC-1 tumor cell
growth in C57BL/6 mice (n=6). Mice were immunized on day 0 and 14
as in FIG. 1. On day 21, TC-1 cells (5.times.10.sup.5) were
injected subcutaneously. The growth of the tumors was monitored for
15 days. Showing is % of tumor free mice as a function of time.
Statistic analysis showed that, the line for E7/Alum is different
from that for E7/LPD (p=0.02).
[0015] FIG. 12. Treatment with E7/LPD caused regression of tumors.
Mice (n=5) were subcutaneously injected with TC-1 cells
(5.times.10.sup.5/mouse) on day 0. On day 4, they were treated with
E7 alone, E7/LPD, or E7/Alum. Dosage is same as in FIG. 1. Showed
is the tumor growth kinetics. On day 25, the tumor size from E7/LPD
treated mice is significantly smaller than that from other
treatments (p=5.times.10.sup.-6, ANOVA).
[0016] FIG. 13. Relative RLU in 293 cells transfected with a CMV
driven .beta.-galactosidase gene containing plasmid, cdc25A
Sac1-luc, and pCDNA3(+) with or without E7/E7m gene. * indicates
that the value for pCDNA-E7 is different from that of the others
(p=0.002, ANOVA). Also, the value for pCDNA3 is not different from
that for pCDNA-E7m (p=0.19).
[0017] FIG. 14. Specific IgG levels in mice immunized with E7m,
E7m/LPD, E7, and E7/LPD. Mice (n=5-6) were immunized on day 0 and
14. Shown are IgG levels in serum on day 28 (.smallcircle.) and 60
(.quadrature.). The IgG levels from E7 or E7m alone immunized mice
were significantly lower than those from E7/LPD or E7m/LPD
immunized mice. On day 28, the value for E7m/LPD is lower than that
for E7/LPD (p=0.02); on day 60, the values from these two
treatments are similar (p=0.53). For E7m/LPD, the value from day 28
is similar to that from day 60 (p=0.81); for E7/LPD, these two
values are different (p=0.004).
[0018] FIG. 15. E7m/LPD and E7/LPD are equally effective in
treating tumor. Mice (n=5 or 10, 10 for E7/LPD and E7m/LPD) were
injected with TC-1 cells (5.times.10.sup.5/mouse) on day 0. On day
4 and 10, they were treated with E7 in different formulations.
Tumor sizes were reported as a function of time. Statistical
analysis showed that the values for E7m/LPD and E7/LPD are not
different.
[0019] FIG. 16. Distribution of LPD/E7 particles in the spleen
following IV administration. Spleens from mice that were injected
with LPD/E7 particles containing Cy3 labeled ODN were visualized at
the indicated times after administration.
[0020] FIGS. 17A and 17B. Uptake of LPD/E7 particles by CD11b+ and
CD11c+ cells. Mice were injected with LPD particles containing 25
.mu.g pNGVL3, 0.1 .mu.g FITC labeled ODN and 10 .mu.g E7 peptide in
150 .mu.l 5% dextrose. (A) Splenocytes were collected 24 h later,
stained with PE labeled anti-CD11b or CD11c antibodies and analyzed
by flow cytometry. (B) Spleens were collected at the indicated
times post injection and the percent of all CD11b+ (.box-solid.)
and CD11c+ ( ) that were DNA positive was determined. Mean .+-.SD,
n=3. One representative of 3 experiments is shown.
[0021] FIGS. 18A and 18B. Tumor specific CTL activity induced by
LPD/E7 vaccination. Mice were IV (A) or SC (B) injected with either
LPD particles containing 0 (.diamond-solid.), 1 (.tangle-solidup.),
10 (.box-solid.), or 20 ( ) .mu.g of E7 peptide, SL liposomes
containing 20 .mu.g E7 peptide (.smallcircle..largecircle.), or 20
.mu.g E7 peptide in PBS (*) on days 0 and 5. Five days after the
last injection, splenocytes were isolated, restimulated for 4 days
and used as effectors in a chromium release assay. Non-specific
lysis was <8% in all groups. Mean .+-.SD, 2 mice/group. One
representative of 2 experiments is shown.
[0022] FIGS. 19A and 19B. LPD/E7 vaccination prevents tumor
establishment. Mice were IV (A) or SC (B) injected with LPD
particles with ( ) or without (.box-solid.) 20 .mu.g E7 peptide, or
20 .mu.g E7 peptide in PBS (.diamond-solid.) on days 0 and 5 or
left untreated (.tangle-solidup.). On day 10, the mice were SC
challenged with 0.5.times.10.sup.6 TC-1 cells. Tumor formation was
monitored twice per week by palpation. 5 mice per group. One
representative of 3 experiments showing similar tumor formation
kinetics is shown.
[0023] FIGS. 20A and 20B. LPD/E7 treatment eradicates established
tumors. Intact (closed symbols) or asplenic mice (open symbols)
were SC inoculated with 0.5.times.10.sup.6 TC-1 cells on day 0. On
days 3 and 6, mice were or IV (A) or SC (B) injected with LPD
particles with ( ) or without (.box-solid.) 10 .mu.g E7 peptide or
left untreated (.diamond-solid.). Tumor growth was measured 3 times
per week. Mean .+-.SD, 5 mice per group. One representative of 3
experiments showing similar tumor growth kinetics is shown.
[0024] FIGS. 21A and 21B. Tumor specific CTL activity induced by
LPD/E7 treatment. Tumor bearing mice were IV (A) or SC (B) treated
with LPD particles with (.tangle-solidup.) or without (.box-solid.)
10 .mu.g E7 peptide or E7 peptide in PBS ( ). Ten days after the
last treatment, splenocytes were isolated, restimulated for 4 days
and used as effectors in a chromium release assay. EL4 cells pulsed
with E7 peptide were used as specific targets. Non-specific lysis
was <7% in all groups. Mean .+-.SD, 2 mice per group. One
representative of 2 experiments is shown.
DETAILED DESCRIPTION OF THE INVENTION
A. LPD Complexes
[0025] As mentioned, the present invention provides
antigen/lipid/polycationic polypeptide salt complexes comprising an
antigen, at least one lipid species and at least one polycationic
polypeptide salt. LPD complexes are described in U.S. Pat. No.
6,008,202, which issued to Huang et al. on Dec. 28, 1999, the
teachings of which are incorporated by reference. The teachings in
U.S. Pat. No. 6,008,202 directed to LPD complexes are fully
applicable to the present invention, except that it has now been
surprisingly found that when an antigen is incorporated into the
LPD, the antigen-LPD complex can be used as an effective vaccine to
prevent and to treat cancers and other infectious diseases.
[0026] This invention relates to lipid-comprising antigen delivery
complexes having a net positive charge and/or a positively charged
surface at pH 6.0-8.0. These complexes comprise lipids, antigens
and optionally further comprise polycations. The invention further
relates to a method for producing these complexes where the method
may optionally include the step of purifying these formulations
from excess individual components. For the production of the
antigen/LPD complexes of this invention, inclusion of the
purification step is a preferred embodiment. The lipid-comprising
antigen delivery complexes of this invention are stable, capable of
being produced at relatively high concentrations, and retain
biological activity of the antigen component over time in
storage.
[0027] The "antigen" which is contained in the lipid-comprising
drug delivery complexes of the present invention may be nucleic
acids, polyanionic proteins, polysaccharides and other
macromolecules which can 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 cationic
lipid. The use of this process permits delivery of positive or
neutral charged drug to cells by the complexes of the present
invention.
[0028] The cationic liposomes mixed with antigen or with antigen
and polycation to form the complexes of the present invention may
contain a cationic lipid alone or a cationic lipid in combination
with a neutral lipid. Suitable cationic lipid species include, but
are not limited to: 3-.beta.[.sup.4N-(.sup.1N,.sup.8-diguanidino
spermidine)-carbamoyl] cholesterol (BGSC);
3-.beta.[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol
(BGTC); N,N.sup.1N.sup.2N.sup.3Tetra-methyltetrapalmitylspermine
(cellfectin);
N-t-butyl-N'-tetradecyl-3-tetradecyl-aminopropion-amidine
(CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB);
1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
(DMRIE);
2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-p-
ropanaminium trifluorocetate) (DOSPA);
1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER);
4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM)
N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3
dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); 1,2
bis(oleoyloxy)-3-(trimethylammonio) propane (DOTAP);
N-1-(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 trimethylammonium 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-p-hydroxyethylammonium) or
DORIE
(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-p-hydroxyethylammonium)
(DORIE) or analogs thereof as disclosed in WO 93/03709;
1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC);
cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as
dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl
phosphatidylethanolamylspermine (DPPES) or the cationic lipids
disclosed in U.S. Pat. No. 5,283,185,
cholesteryl-3.beta.-carboxylamido-ethylenetrimethylammonium iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propylcholesteryl
carboxylate iodide, cholesteryl-3-.beta.-carboxyamidoethyleneamine,
cholesteryl-3-.beta.-oxysuccinamido-ethylenetrimethylammonium
iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-.beta.-oxysu-
ccinate iodide, 2-(2-trimethylammonio)-ethylmethylamino
ethyl-cholesteryl-3-.beta.-oxysuccinate iodide,
3-.beta.-N--(N',N'-dimethylaminoethane) carbamoyl cholesterol
(DC-chol), and
3-.beta.-N-(polyethyleneimine)-carbamoylcholesterol.
[0029] Examples of preferred cationic lipids include
N-t-butyl-N'-tetradecyl-3-tetradecylaminopropion-amidine
(CLONfectin),
2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium trifluoroacetate (DOSPA),
1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), N-[1-(2,3,
dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) (DOTMA),
cholesteryl-3-.beta.-carboxyamidoethylenetrimethylammonium iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl
carboxylate iodide, cholesteryl-3-.beta.-carboxyamidoethyleneamine,
cholesteryl-3-.beta.-oxysuccinamidoethylenetrimethyl-ammonium
iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-.beta.-oxysu-
ccinate iodide, 2-(2-trimethylammonio)ethylmethylamino
ethyl-cholesteryl-3-.beta.-oxysuccinateiodide,
3-.beta.-N--(N',N'dimethyl-aminoethane)-carbamoyl-cholesterol
(DC-chol), and 3-.beta.-N-(polyethyleneimine)-carbamoyl
cholesterol.
[0030] In certain embodiments, it has been found that certain
cationic lipids have an increased immunostimulatory effect, i.e.,
increased immunostimulation activity. It has surprisingly been
found that cationic lipids having shorter acyl chains have
increased immunostimulation activity. In addition, it has
surprisingly been found that cationic lipids having cis-unsaturated
double bonds have increased immunostimulation activity. As such, in
preferred embodiments, the cationic lipids used in the antigen-LPD
complexes of the present invention are those that have shorter acyl
chains and, in addition, those that have cis-unsaturated double
bonds.
[0031] Since 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), it will be understood by those of ordinary skill
in the art that preferred cationic lipids are those lipids in which
bonds between the lipophilic group and the amino group are stable
in aqueous solution. While such bonds found in cationic lipids
include amide bonds, ester bonds, ether bonds and carbamoyl bonds,
preferred cationic lipids are those having a carbamoyl bond. An
example of a preferred cationic lipid having a carbamoyl bond is
DC-Chol. Those of skill in the art would readily understand that
liposomes containing more than one cationic lipid species may be
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 (Brunette, E. et al. (1992) Nucl. Acids Res.,
20:1151).
[0032] It is to be further understood that in considering cationic
liposomes suitable for use in mixing with antigen and optionally
with polycation, to form the complexes of this invention, 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.
[0033] Thus, in addition to cationic lipids, cationic liposomes
used to form the complexes of the invention may contain other
lipids in addition to the cationic lipids. These lipids include,
but are not limited to, lyso lipids of which
lysophosphatidylcholine (1-oleoyl lysophosphatidylcholine) is an
example, cholesterol, or neutral phospholipids including dioleoyl
phosphatidyl ethanolamine (DOPE) or dioleoyl phosphatidylcholine
(DOPC) as well as various lipophylic surfactants, containing
polyethylene glycol moieties, of which Tween-80 is one example. The
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.
[0034] It is further contemplated that in the cationic liposomes
utilized to form the complexes of the invention, the ratio of
lipids may be varied to include a majority of cationic lipids in
combination with cholesterol or with mixtures of lyso or neutral
lipids. When the cationic lipid of choice is to be combined with
another lipid, a preferred lipid is a neutral phospholipid, most
preferably DOPE.
[0035] 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 nm
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.
[0036] In the cationic liposomes utilized to produce the drug/lipid
complexes of this invention, the cationic lipid is present in the
liposome at from about 10 to about 100 mole % of total liposomal
lipid, preferably from about 20 to about 80 mole % and most
preferably about 20 to about 60 mole %. The neutral lipid, when
included in the liposome, may be present at a concentration of from
about 0 to about 90 mole % of the total liposomal lipid, preferably
from about 20 to about 80 mole %, and most preferably from 40 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, preferably
from about 0 mole % to about 40 mole %. In a preferred embodiment,
the liposomes contain a cationic and a neutral lipid, most
preferably DC-Chol and DOPE in ratios between about 2:8 to about
6:4. 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.
Potential targets include, but are not limited to, liver, blood
cells, endothelial cells and tumor cells. Furthermore, to enhance
the circulatory half-life of the complexes, the positive surface
charge can be sterically shielded by incorporating lipophilic
surfactants which contain polyethylene glycol moieties.
[0037] It is to be further understood that the positive charge of
the complexes of this invention may be affected not only by the
lipid composition of the complex, but also by the pH of the
solution in which the drug/lipid complexes are formed. For example,
increasing pH (more basic) will gradually neutralize the positive
charge of the tertiary amine of the cationic lipid DC-Chol. In a
preferred embodiment, the complexes of the present invention are
produced, and stored, at a pH such that the complexes have a net
positive charge and/or positively charged surface. A preferred pH
range is pH 6.0-8.0, most preferably pH 7.0-7.8.
[0038] When a polycation is to be mixed with nucleic acid and
cationic liposomes, the polycation may be selected from organic
polycations having a molecular weight of between about 300 and
about 200,000. These polycations also preferably have a valence of
between about 3 and about 1000 at pH 7.0. The polycations may be
natural or synthetic amino acids, peptides, proteins, polyamines,
carbohydrates and any synthetic cationic polymers. Non-limiting
examples of polycations include polyarginine, polyornithine,
protamines and polylysine, polybrene (hexadimethrine bromide),
histone, cationic dendrimer, spermine, spermidine and synthetic
polypeptides derived from SV40 large T antigen which has excess
positive charges and represents a nuclear localization signal. In
one embodiment, the polycation is poly-L-lysine (PLL).
[0039] In another more preferred embodiment, the polycation is a
polycationic polypeptide having an amino acid composition in which
arginine residues comprise at least 30% of the amino acid residues
of the polypeptide and lysine residues comprise less than 5% of the
amino acid residues of the polypeptide. In addition, preferably
histidine, lysine and arginine together make up from about 45% to
about 85% of the amino acid residues of the polypeptide and serine,
threonine and glycine make up from about 10% to about 25% of the
amino acid residues of the polypeptide. More preferably, arginine
residues constitute from about 65% to about 75% of the amino acid
residues of the polypeptide and lysine residues constitute from
about 0 to about 3% of the amino acid residues of the
polypeptide.
[0040] In addition to the above recited percentages of arginine and
lysine residues, the polycationic polypeptides of the invention may
also contain from about 20% to about 30% hydrophobic residues, more
preferably, about 25% hydrophobic residues. The polycationic
polypeptide to be used in producing drug/lipid/polycation complexes
may be up to 500 amino acids in length, preferably about 20 to
about 100 amino acids in length; more preferably, from about 25 to
about 50 amino acids in length, and most preferably from about 25
to about 35 amino acids in length.
[0041] In one embodiment, the arginine residues present in the
polycationic polypeptide are found in clusters of 3-8 contiguous
arginine residues and more preferably in clusters of 4-6 contiguous
arginine residues.
[0042] In another embodiment, the polycationic polypeptide is about
25 to about 35 amino acids in length and about 65 to about 70% of
its residues are arginine residues and 0 to 3% of its residues are
lysine residues.
[0043] The polycationic polypeptides to be used in formulating the
complexes of the invention may be provided as naturally occurring
proteins, particularly certain protamines having a high arginine to
lysine ratio as discussed above, as a chemically synthesized
polypeptide, as a recombinant polypeptide expressed from a nucleic
acid sequence which encodes the polypeptide, or as a salt of any of
the above polypeptides where such salts include, but are not
limited to, phosphate, chloride and sulfate salts.
[0044] The complexes formed by the methods of the present invention
are stable for up to about one year when stored at 4.degree. C. The
complexes may be stored in 10% sucrose or a 5% dextrose solution
upon collection from the sucrose gradient or they may be
lyophilized and then reconstituted in an isotonic solution prior to
use. In a preferred embodiment, the complexes are stored in
solution. The stability of the complexes of the present invention
is measured by specific assays to determine the physical stability
and biological activity of the complexes over time in storage. The
physical stability of the complexes is measured by determining the
diameter and charge of the 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 Examples. The physical
stability of the complex is "substantially unchanged" over storage
when the diameter of the stored complexes is not increased by more
than 100%, preferably by not more than 50%, and most preferably by
not more than 30%, over the diameter of the complexes as determined
at the time the complexes were purified.
[0045] Therapeutic formulations using the complexes of the
invention preferably comprise the complexes in a physiologically
compatible buffer such as, for example, phosphate buffered saline,
isotonic saline or low ionic strength buffer such as 10% sucrose in
H.sub.2O (pH 7.4-7.6) or in Hepes (pH 7-8, a more preferred pH
being 7.4-7.6). The complexes may be administered as aerosols or as
liquid solutions for intratumoral, intravenous, intratracheal,
intraperitoneal, and intramuscular administration.
[0046] Methods for preparing and purifying the antigen-LPD
complexes of the present invention are disclosed in U.S. Pat. No.
6,008,202, the teachings of which are incorporated by
reference.
B. Antigens
[0047] A "tumor-associated antigen," as used herein is a molecule
or compound (e.g., a protein, peptide, polypeptide, lipid,
glycolipid, carbohydrate and/or DNA) associated with a tumor or
cancer cell and which is capable of provoking an immune response
when expressed on the surface of an antigen presenting cell in the
context of an 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.
[0048] 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. A
non-limiting 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.
[0049] The term "antigen" is further intended to encompass peptide
or protein analogs of known or wild-type antigens such as those
described above. 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.
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.
[0050] In one embodiment, the antigen in the LPD complex comprises
an antigen associated with a tumor or cancer, i.e., a
tumor-associated antigen. As such, in a preferred 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 LPD complexes and methods of the present
invention can be inherently immunogenic, or non-immunogenic, or
slightly immunogenic. As demonstrated herein, even tumor-associated
self antigens may be 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, lipids, glycolipids, carbohydrates and DNA.
[0051] Tumor-associated antigens suitable for use in the subject
invention include both mutated and non-mutated 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 and
glycoproteins, tumor-specific patterns of expression of
carbohydrates, gangliosides, glycolipids and mucins have also been
documented. Exemplary tumor-associated antigens for use in the
subject 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.
[0052] Specific embodiments of tumor-associated antigens include,
e.g., mutated 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, PRAME, 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 1/Melan
A, gp100, gp75, Tyrosinase, TRP1 and TRP2; prostate associated
antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated
embryonic gene products such as MAGE 1, MAGE 3, MAGE 4, GAGE 1,
GAGE 2, BAGE, RAGE, and other cancer testis antigens such as
NY-ESO1, 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 Tn, 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.
[0053] 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,
MBr1, MBr8, Ber-EP4, 17-1A, C26 and T16 are also known carcinoma
antigens. Desmin and muscle-specific actin are antigens of myogenic
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. HMB-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 neuroendocrine 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.
[0054] In fact, in a preferred embodiment, the human papillomavirus
(HPV) subtype 16 E7 is used as the tumor-associated antigen. It has
been found that E7 antigen-LPD complexes of the present invention
are effective at preventing and treating cervical cancer. In
addition, the present invention provides a genetically engineered
E7 protein, i.e., E7m protein, having antigenic activity, but
without tumorigenic activity. It has been found that the E7m-LPD
complexes of the present invention induce cellular immunity to
cause complete regression of established tumors and, thus, can be
used as potent anti-cervical cancer vaccines.
[0055] 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.
[0056] 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 (EBV) (Burkitt lymphoma, nasopharynx cancer, PTLD in
immunosuppressed individuals), HTLVL (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, Bailliere Tindall, Great Britain 1983, the
entire contents of which is hereby incorporated by reference.
[0057] In another embodiment, the antigen in the LPD complex
comprises an antigen derived from or associated with a pathogen,
i.e., a microbial antigen. As such, in a preferred embodiment, the
pathogen vaccines of the present invention further comprise at
least one epitope of at least one microbial antigen. Pathogens
which may be targeted by the subject vaccines include, but are not
limited to, infectious virus, infectious bacteria, infectious
parasites and infectious fungi. In another preferred embodiment,
the pathogen vaccines of the present invention further comprise a
plurality of epitopes from one or more microbial antigens.
[0058] The microbial antigens finding use in the subject
compositions and methods may be inherently immunogenic, or
non-immunogenic, 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, lipids,
glycolipids, carbohydrates and DNA.
[0059] Exemplary viral pathogens include, but are not limited to,
infectious virus that infect mammals, and more particularly humans.
Examples of infectious 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-III/LAV, or HIV-III; and
other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,
hepatitis A virus; enteroviruses, human Coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause
gastroenteritis); Togaviridae (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 Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvovirida (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
Iridoviridae (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=internally transmitted; class
2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0060] Also, gram negative and gram positive bacteria may be
targeted by the subject compositions and methods in vertebrate
animals. Such gram positive bacteria include, but are not limited
to Pasteurella species, Staphylococci species, and Streptococcus
species. Gram negative bacteria include, but are not limited to,
Escherichia coli, Pseudomonas species, and Salmonella species.
Specific examples of infectious bacteria include but are not
limited to: Helicobacter pyloris, Borella burgdorferi, 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 pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
infuenzae, Bacillus antracis, corynebacterium diphtheriae,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, Rickettsia, and Actinomyces
israelli.
[0061] 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,
("IROMP"), an outer membrane protein ("OMP"), and an A-protein of
Aeromonis salmonicida which causes furunculosis, p57 protein of
Renibacterium salmoninarum 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 ("IROMP"), 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 Ichthyophthirius; and a
structural and regulatory protein of Cytophaga columnari; and a
structural and regulatory protein of Rickettsia. Such antigens can
be isolated or prepared recombinantly or by any other means known
in the art.
[0062] Examples of pathogens further include, but are not limited
to, infectious fungi that infect mammals, and more particularly
humans. Examples of infectious fungi include, but are not limited
to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. Examples of infectious parasites include Plasmodium such
as Plasmodium falciparum, Plasmodium malariae, 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
Ichthyophthirius.
[0063] 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, the entire
contents of which is hereby incorporated by reference. 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 non-human 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.
[0064] Exemplary non-human 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-1,
HIV-2, SIV, Visna virus, feline immunodeficiency virus ("FIV"), and
equine infectious anemia virus ("EIAV"), T-cell leukemia viruses
such as HTLV-1, HTLV-II, simian T-cell leukemia virus ("STLV"), and
bovine leukemia virus ("BLV"), and foamy viruses such as human
foamy virus ("HFV"), simian foamy virus ("SFV") and bovine foamy
virus ("BFV").
[0065] In preferred 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.
[0066] 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 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.
EXAMPLE I
LPD as a Vaccine Delivery System
[0067] 1. Subcutaneously Injected LPD/Peptide Complex Accumulates
in Local Lymphnode and Taken up by Various Immune Effector
Cells.
[0068] As delivery to organized lymph tissue is important for
successful vaccination, we wished to determine if LPD particles
would make their way into draining lymph nodes following
subcutaneous injection. C57BL/6 mice were footpad injected with
LPD/E7 particles containing trace amounts of Cy5-labeled DNA. At 16
h after injection, popliteal lymph nodes were collected, single
cell suspensions were prepared and LPD the percentage of cells that
took up LPD was determined by flow cytometry. Mice injected with
non-fluorescent LPD particles served as controls. After SC
administration, 2.4% of all lymph node cells took up LPD particles.
While only a small percentage of all lymph node cells take up LPD
particles, this can be sufficient to produces effective vaccination
of those cells are capable of effectively presenting the delivered
antigen. To determine what cell types were taking up LPD/E7
particles, C57BL/6 mice were footpad injected with LPD/E7 particles
containing trace amounts of Cy5-labeled DNA. At 16 h after
injection, popliteal LN cells were stained for the cell-type
specific surface markers CD11b (to identify macrophages and myeloid
lineage DCs), CD11c (to identify dendritic cells), CD19 (to
identify B cells), and NK1.1 (to identify NK cells) using
fluorescently labeled antibodies and subjected to flow cytometric
analysis. Phagocytic cells appear to be the main cell types that
take up LPD/antigen particles with high percentage of NK cells
(25%) and macrophages (25%) taking up particles while only 2.4% of
B cells took up LPD particles. Of particular interest, 16.8% of
dendritic cells took up LPD particles (Please see data in the table
in Dileo et al.).
[0069] 2. LPD/E7 Complexes Induce an E7 Specific Immune
Response.
[0070] To determine if LPD/E7 particles induce enhanced
immunization versus traditional liposome/peptide vaccines, mice
were SC or IV vaccinated with LPD particles containing 0, 1, 10, or
20 .mu.g of E7 peptide on days 0 and 5. For comparison, mice were
injected with 20 .mu.g E7 peptide in PBS or encapsulated in SL
liposomes. Five days after the final vaccination, splenocytes were
collected, and used as effector cells in a chromium release assay.
Consistent with previous reports, antigen containing SL liposomes
induced significant levels of CTL activity (61% specific lysis)
following SC injection only. Vaccination with 20 .mu.g LPD/E7
produced the highest levels of CTL activity by both routes.
Injection of 10 .mu.g LPD/E7 particles induced intermediate levels
of CTL activity in both cases, while 1 .mu.g LPD/E7 induced
significant CTL activity following IV administration only (Dileo et
al.).
[0071] 3. LPD/E7 Induces Both Protective and Therapeutic Immunities
Against HPV+ Tumor.
[0072] To determine if the induced immune response is adequate to
provide protective immunity, mice were IV or SC vaccinated with
either 20 .mu.g E7 peptide in PBS, empty LPD, or LPD containing 20
.mu.g of E7 peptide on days 0 and 5. Untreated mice served as
controls. Five days after the last vaccination, mice were SC
challenged with 0.5.times.10.sup.6 E7 expressing TC-1 cells. Mice
that received LPD/E7 particles by either route failed to develop
tumors, while control mice and mice that received LPD alone or free
E7 peptide developed tumors within 12 days (Dileo et al.). To
determine the potential of LPD/E7 complex for use as a therapeutic
strategy, subcutaneous tumors were established in C57BL/6 mice by
inoculation of 0.5.times.10.sup.6 TC-1 cells.
[0073] On days 3 and 6 following inoculation, mice were injected IV
or SC with LPD containing 10 .mu.g of E7 peptide. To determine the
importance of antigen delivery to the spleen in the generation of
the observed immune responses, a group of mice that had their
spleens surgically removed was included. Untreated mice and mice
receiving empty LPD served as controls. All mice that received IV
LPD/E7 peptide showed steady tumor shrinkage and complete
regression within 2 weeks. SC treatment also resulted in complete
regression but with slower kinetics. As expected, empty LPD
administration slowed tumor progression but failed to eradicate the
tumors. The anti-tumor effect in asplenic mice depended on the
route of administration. IV treatment showed tumor growth rates
similar to those observed in mice treated with LPD alone, while SC
delivery resulted in tumor regression. Untreated mice showed
unimpeded tumor progression (Dileo et al.). It has been shown that
LPD administration to tumor bearing mice induces non-specific
immune activation that can result in tumor regression. To confirm
that tumor regression was due to an E7 specific response, ten days
after the last treatment splenocytes were assayed for E7 specific
tumor lytic activity. Consistent with previously published results,
IV or SC injection of LPD without peptide induced a low level of
apparent CTL activity while treatment with LPD/E7 resulted in the
highest level of CTL activity in both cases (data not shown). Cells
from untreated mice showed no lytic activity (Dileo et al.,
Molecular Therapy, 7(5):640-648 (May 2003), a copy of which was
attached to the U.S. Provisional Patent Application No. 60/567,291
as Appendix A, which is incorporated herein by reference for all
purposes (herein after "Dileo, et al.").
[0074] 4. A Single s.c. Injection of LPD/E7 can Induce Regression
of Large Advanced Tumor.
[0075] To determine how late the LPD/E7 nanoparticle treatment can
still be effective, subcutaneous tumors were established in C57BL/6
mice by inoculation of 1.times.10.sup.5 TC-1 cells. On days 4, 6, 8
or 12 following inoculation, mice were s.c. injected with LPD
containing 10 .mu.g of E7 peptide. Untreated mice served as
control. Our data demonstrate that even a single injection of
LPD/E7 nanoparticles as late as 8 days after tumor inoculation
could still induce tumor regression in most of the treated animals
(FIG. 1). LPD treatment on 12 days after TC-1 cell inoculation
stopped tumor growth after 20 days. The experiment had to be
terminated at 25 days because the size of the tumors in the
untreated control group had grown to be too large. The average
diameter of tumor on day 12 was more than 7 mm and this time point
was at the middle of the entire course of the experiment. These
data indicate that LPD/E7 nanoparticles may not only provide potent
anti-tumor effects in vaccination protocols, but also be effective
for the treatment of advanced tumors.
[0076] 5. LPD/E7 is Effective in Treating Metastatic Tumor Model
and Induce Tumor Specific Memory Immune Response.
[0077] To identify the effect of LPD/E7 nanoparticle vaccination on
the metastatic cervical cancer, we established metastatic tumor
model with TC-1 cells. If TC-1 cells 1.times.10.sup.6 cells) were
injected intraperitoneally, numerous metastatic tumor nodules were
observed in the peritoneum in a few days later; many were also
found in organs such as liver, spleen, kidney and lymph nodes.
Inoculated mice died within 20 days after TC-1 cell injection (FIG.
2A). To determine if LPD/E7 vaccination induced anti-tumor immunity
against i.p. injected TC-1 cells, mice were vaccinated with LPD
particles containing 10 .mu.g E7 peptide or 10 .mu.g E7 peptide in
PBS on day 0 and 5. On day 10, mice received subcutaneous tumor
challenge at a dose of 1.times.10.sup.6 TC-1 cells per mouse and
the survival rate was measured. As shown in FIG. 2A, only 20% of
untreated and E7-alone injected mice survived beyond 20 days after
TC-1 cell injection, but the survival rate of LPD/E7 vaccinated
group was 100% for the entire course of the experiment (50 days).
To identify if survived mice from the LPD/E7 vaccination group
exhibited the antigen specific immune response, mice were
re-challenged subcutaneously with 1.times.10.sup.5 TC-1 cells or
24JK cells. Normal unvaccinated animals receiving an equal number
of TC-1 cells or 24JK cells were used as control groups. Mice that
survived from first challenge with TC-1 cells also failed to
develop tumors upon re-challenge with fresh TC-1 cells, while
normal animals injected with TC-1 cells and all mice injected with
24JK cells developed tumors (FIG. 2B). The data indicate that the
immune response induced by LPD/E7 not only protected mice against
the metastatic tumor challenge, but also provided protection for
subsequent challenge.
[0078] 6. Immunological Characterizations of LPD/E7 Peptide.
[0079] Previous experiments showed that LPD particles formulated
with ODN-1668 (containing unmethylated CpG motif), but not
ODN-1668GC (containing identical sequence as 1668 except the CpG in
the motif was replaced with GpC), could induce TH.sub.1 cytokines
and show anti-tumor activity (Whitmore et al, 2001). These
experiments were done with LPD particles without any specific tumor
antigen. Whether the same result would be true for an antigen
specific vaccine activity was not known. We prepared LPD/E7 complex
using either ODN-1668 or ODN-1668GC, but no plasmid DNA, and
injected them to animals and to see if any protective immunity was
developed in the animal against subsequent challenge of TC-1 cells.
As can be seen in FIG. 3, all formulations containing E7 peptide
had prevented the growth of tumor; whereas no formulations without
E7 peptide had any activity. The data indicate that the antitumor
activity was antigen dependent. Furthermore, since formulations
with or without the CpG motif had the same activity, it suggested
that the unmethylated CpG motif played little if any role in the
induction of antitumor immune responses. This was a somewhat
surprising result, because we had previously shown that CpG motifs
are necessary for an antigen independent antitumor activity of LPD
(Whitmore et al, 2001). Apparently, when the antigen is present,
the antigen dependent immune response dominates the independent
response. Moreover, the data also suggest that DNA or ODN is not
necessary for the antitumor immune response. As long as the
antigen, E7 peptide in this case, is carried by the cationic
lipids, it can elicit an antitumor response. This hypothesis was
independently verified by another experiment in which various LPD
components were tested for antitumor activity. Indeed, E7 peptide
complexed with DOTAP/chol liposomes could induce tumor regression
after a single s.c. injection.
[0080] TLR9 is responsible for the action of the immunostimulating
unmethylated CpG motifs, e.g., RRCGYY (Hemmi et al., 2000; Bauer et
al., 2001). It is a good guess that the adjuvant activity of LPD is
mediated by TLR9. To test this hypothesis, we have obtained TLR9
-/- mice (Hemmi et al., 2000) from Professor Shizuo Akira in Japan.
The mice are in the background of C57BL/6 and are syngeneic with
TC-1 cells. The splenocytes from wild type or TLR9-/- mouse
injected with LPD/E7 were re-stimulated with 1 .mu.g/ml E7 peptide
for 24 h. The culture supernatants were collected and IFN-.gamma.
levels were measured using ELISA. As can be seen in FIG. 4,
splenocytes from either wildtype or knockout mice could be
stimulated with LPD/E7 to produce IFN-.gamma. to the same extend,
indicating that TLR9 is not important in the process of immune
stimulation. Again, it suggests that cationic lipid, rather than
DNA, was the primary stimulant of the immune system.
[0081] According to our preliminary data, approximately 25% of
Lymphnode NK cells have taken up s.c. injected LPD. Our previous
work using LPD without E7 peptide also indicates that NK cells are
involved in the first phase of tumor killing, which is followed by
induction of tumor specific CTL. Thus, there is a real possibility
that NK cells may be activated by LPD and involves in tumor cell
killing. To identify whether the NK cells are necessary for the
antigen specific immune response, we have eliminated the NK cells
by treating the animals with anti-NK1.1 antibody. As can be seen in
FIG. 5, the antigen specific T-cell response, measured by
IFN-.gamma. ELISPOT assay, was approximately the same as the ones
without the NK elimination. This result indicates that NK cells are
only important for the antigen independent antitumor activity, but
not in the antigen specific activity.
[0082] 7. Cationic Lipids can Activate Dendritic Cells.
[0083] Previously we have shown that LPD is a potent vaccine
delivery system/adjuvant. Following are some data we collected when
studying the mechanism of the strong immunostimulation activity
from LPD. We have measured by flow cytometry the expression of
co-stimulatory molecules, CD86 and CD80, on DC2.4 cells as the
result of stimulation by various components of LPD, including the
liposome prepared from cationic lipid
1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol
(Chol), protamine, and DNA. Plasmid DNA alone, protamine alone, and
the complex of DNA/protamine did not stimulate the expression of
CD80/CD86 (FIG. 6). LPD stimulated DC2.4 cells expressed the
highest amount of CD80/CD86. Lipoplex prepared from liposome
(DOTAP/Chol) and DNA was as effective as the LPD, indicating that
protamine is not required for DC cell stimulation. In addition,
cholesterol is not required for DC cell stimulation (FIG. 6) since
LPD prepared from liposomes comprised of DOTAP alone is as
effective as those prepared from liposomes comprised of DOTAP and
cholesterol. Finally, it was found that CD80/CD86 expressions from
liposome alone (DOTAP/Chol) stimulated DC2.4 cells were up to 70%
of that from LPD stimulated DC2.4 cells, strongly demonstrating
that the DOTAP-based cationic liposome alone can activate DCs and
is responsible for the some of the LPD adjuvant activity.
Therefore, a few other cationic liposomes were also tested for
their ability to stimulate the expression of CD80/CD86 by DC2.4
cells. To our surprise, as shown in FIG. 7, 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(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoroacetate (DOSPA) and the neutral lipid dioleoyl
phosphatidylethanolamine (DOPE), and liposomes prepared from
O,O'-dimyristyl-N-lysyl aspartate (DMKE) and
O,O'-dimyristyl-N-lysyl-glutamate (DMKD), two newly synthesized
cationic lipids by Dr. Yong-Serk Park, strongly stimulated the
expression of CD80/CD86 by CD2.4 cells, whereas Lipofectin.RTM., a
1:1 (w/w) liposome formulation of cationic lipid
N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride
(DOTMA) and DOPE, DMRIEC.RTM., a 1:1 (M/M) liposome formulation of
cationic lipid 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl
ammonium bromide (DMRIE) and cholesterol, and liposome prepared
from cationic lipid dimethyldioctadecylammonium bromide (DDAB) and
cholesterol did not show any significant stimulation on the
expression of CD80/CD86 by DC2.4 cells.
[0084] The ability of different cationic lipids to stimulate the
expression of CD 80 on DC 2.4 cells varied significantly. 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 are, in general,
more potent than the DXTAP lipids with trimethylammonium propane
(TAP) head group. The effect of the hydrocarbon tail region of the
lipids on the DC cell stimulation also was systematically
investigated (FIG. 8). Within the lipids bearing one particular
head group structure, lipids with shorter
(1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC-12:0),
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC-14:0)) or
unsaturated (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(DOEPC-18:1)) acyl chains are found to be more potent than those
with longer (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine
(DPEPC-16:0)) or saturated
(1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC-16:0)) acyl
chains. Similar trends were observed with lipids having TAP head
groups also with 1,2-distearoyl-3-trimethylammonium propane
(DSTAP-18:0) and 1,2-palmitoyl-3-trimethylammonium propane
(DPTAP-16:0) showing much lower expression of CD80 on DC 2.4 cells
compared to that of 1,2-myristoyl-3-trimethylammonium propane
(DMTAP-14:0) and 1,2-oleoyl-3-trimethylammonium propane
(DOTAP-18:1). Detergent sodium dodecyl sulfate (SDS) could not
activate DC 2.4 cells for the expression of CD 80 suggesting that
the DC 2.4 activation by cationic lipids with shorter hydrophobic
chains were not due the possible detergent effect of shorter chain
lipids on the cell membrane.
[0085] To further prove that both DNA and cationic liposome are
required for the full activity of LPD, an in vivo tumor therapy
study was carried out. As shown in FIG. 9, 20 days after treatment
with E7 peptide-incorporated LPD (LPD/E7), the tumor (injected 6
days before the onset of treatment) almost totally regressed.
Liposome/E7 and lipoplex/E7 (LD/E7) showed effect to some extent
but significantly weaker than that of the LPD/E7. Tumors in mice
treated with other formulations including DNA/E7, protamine/E7,
LP/E7, PD/E7, and E7 alone kept growing rapidly with the final
tumor size comparable to those on the untreated naive mice.
Therefore, the result of this tumor therapy study again
demonstrated that both DNA and cationic liposome are required for
the full immunostimulation activity of LPD. It is interesting to
note that in the tumor model, protamine becomes functionally
important, as the activity of LPE/E7 was significantly greater than
that of LD/E7. It is speculated that by condensing DNA, protamine
helped to bring DNA inside the liposomes. E7 peptide was thought to
bind to DNA via electrostatic interaction (Dileo et al., 2003). In
LPD/E7, the E7 peptide might locate inside the liposome and should
be protected from enzymatic degradation after injection. On the
other hand, E7 might only be loosely bound in the LD/E7 particles.
In fact, the peptide incorporation efficiency of LPD/E7 was about
80%, whereas for LD/E7 and L/E7, it was about 65%.
[0086] 8. Genetically Engineered HPV 16 E7 Protein as a Potential
Cervical Cancer Vaccine.
[0087] Previously, we showed that a seven amino acid peptide from
HPV 16 E7 protein, when incorporated into LPD nanoparticles,
induced strong anti-tumor response the can prevent and treat tumors
grafted on mice. In the present study, we hypothesized that the LPD
particles could also be used as a carrier for E7 protein to induce
strong antibody and anti-tumor responses. We also hypothesized that
by introducing mutations on E7 protein, we can delete the oncogenic
activity of E7 but keep its antigenic activity.
[0088] To investigate whether strong immune responses can be
elicited when E7 protein is incorporated into LPD nanoparticles as
a vaccine, E7 protein was expressed in E. coli, purified,
detoxified, and incorporated into LPD particles. E7/LPD was then
used to immunize C57BL/6 mice. The resulting antibody, tumor
prevention, and tumor treatment activity from E7/LPD were compared
to that of E7 alone or E7 adjuvanted with `Alum`. Data showed that
E7/LPD induced antibody level (specific IgG) significantly higher
than that of E7/Alum (FIG. 10). Also, E7/LPD immunization prevented
the growth of TC-1 tumor cells on mice (FIG. 11); visible tumor (4
day old) on mice totally regressed when the mice were treated with
E7/LPD (FIG. 12).
[0089] To engineer the E7 protein to delete its oncogenic activity,
and hopefully, without disrupting its immunogenicity. The following
experiments were carried out. For the engineering of mutated E7
protein, an overlapping PCR was used. Amino acids D21 and C94 on E7
protein were changed to G21 and G94 by changing their codons from
GAT to GGT and TGT to GGT, respectively. The four primers used were
P1, 5-TTGGGATCCACCATGCATGGAGATACACCTAC-3 [SEQ. ID. NO. 1]; P2,
5-CGGAATTCATTCTTATGGTTTCTGAGAACCGATGGGGCACACA-3 [SEQ. ID. NO. 2];
P3, 5-GAGACAACTGGTCTCTACTGTTAT-3 [SEQ. ID. NO. 3]; and P4,
5-ACAGTAGAGACCAGTTGTCTCTGG-3 [SEQ. ID. NO. 4]. Using pET-E7 as the
template, two separate PCRs were completed using primer P1/P4 and
P2/P3 as the primer pairs, respectively. The PCR products were
purified using QiaQuick PCR purification Kit (Valencia, Calif.).
Another PCR was completed using P1 and P2 as the primers and equal
molar of the products from the two previous PCRs as the templates.
The PCR conditions were 94.degree. C. for 5 min followed by 35
cycles of 94.degree. C., 0.5 min, 56.degree. C., 1 min, and
72.degree. C., 0.5 min. Another 5 min of incubation at 72.degree.
C. was included prior to the end of the PCR reaction. Taq DNA
polymerase and dNTP were from Promega (Madision, Wis.). After
purification, the PCR product was then ligated to the pGEM.RTM.-T
vector from Promega. The ligation reaction was then transferred
into E. coli DH5a strain. Positive colonies were selected using
LB/ampicillin/IPTG/X-Gal plates. After confirmation of mutations on
the E7 gene of the pGEM.RTM.-T-E7m, the plasmid was digested with
BamHI and EcoRI, whose restriction sites are on primer P1 and P2,
respectively. The resulting DNA fragment was gel purified and then
cloned into the BamHI and EcoRI site of pCDNA3.1(+) vector
(Invitrogen, Carlsbad, Calif.) and pET vector (Novagen, Madison,
Wis.), respectively. The plasmid constructs were transferred into
E. coli DH5a and selected against ampicillin and kanamycin,
respectively.
[0090] An indirect method was used to verify that E7m lost its
ability to bind to pRB protein and therefore is unable to activated
E2F driven genes. E7 and E7m genes were inserted into the BamH1 and
EcoRI sites of pCDNA3.1(+) vector. The resulting plasmids were
amplified in E. coli DH5a strain and purified. Plasmid cdc25A
Sac1-luc, in which luciferase gene is driven by a 1173 (-755 to
+418) bp SacI fragment of the cdc25A promoter, is a gift from Dr.
D. DiMaio from Yale University (New Haven, Conn.). The SacI
fragment has both HPV 16 E2 protein binding site and the E2F
binding site. Plasmid cdc25A SacI-luc, pCDNA3.1 (+) with or without
E7 or E7m insert, and a CMV driven .beta.-galactosidase gene
containing plasmid were cotransfected into confluent 293 cells
(5.times.10.sup.5/well, incubated at 37.degree. C. and 5% CO.sub.2
overnight, DMEM medium with 10% FBS) with Lipofectamine.RTM.
(Invitrogen). The plasmid ratio was 2:1:10 (w/w/w) with pCDNA3.1(+)
plasmid at 1 .mu.g/well. Four hours after the addition of the
plasmids, the medium was replaced with fresh medium. After another
44 h, the incubation was stopped. Cells were washed with cold PBS
(10 mM, pH 7.4) for twice and lysed. Luciferase activity,
.beta.-galactosidase expression, and total protein amount were then
determined. Relative light unit normalized to galactosidase protein
level and total protein amount was reported. As shown in FIG. 13,
when 293 cells were co-transfected with E2F responsive element
driven luciferase gene encoding plasmid (cdc25A Sac I-luc) and an
E7 protein encoding plasmid (pCDNA-E7), significantly higher
luciferase expression was observed than when cdc25A Sac I-luc was
co-transfected with an empty plasmid (pCDNA3) (p=0.02). However,
when pCDNA-E7m was cotransfected with cdc25A Sac I-luc, the
resulting luciferase level was comparable to that when cdc25A Sac
I-luc and pCDNA3 were co-transfected (p=0.19). Taken together,
these date suggest that the mutations on E7m may abolish its
ability to bind to pRB and thus the oncogenic activity.
[0091] To investigate whether the mutations introduced on E7 affect
the mutated protein's ability to induce antibody and antitumor
responses, E7 and E7m proteins were purified and used to immunize
mice. The resulting specific antibody levels and tumor treatment
abilities were compared. Shown in FIG. 14 are the specific antibody
levels in serum on day 27 and day 60. Again, both E7/LPD and
E7m/LPD induced significantly higher IgG level than E7 and E7m
alone without LPD. Interestingly, on day 27, the IgG level from
E7/LPD was higher than that from E7m/LPD (p=0.019); whereas on day
60, the IgG level from E7/LPD was comparable to that for E7m/LPD
(p=0.53). This is due to the significant decreased IgG level in
E7/LPD immunized mice as time passed (p=0.004). The above data are
the antibody levels when measured against E7 protein as an antigen.
Similar results were obtained when measured against E7m
protein.
[0092] Treatment of tumor bearing mice with E7m/LPD is as effective
as with E7/LPD in causing tumor regression (FIG. 15).
[0093] In summary, mutations in the E7 protein have been
successfully introduced. The resulting mutated E7m protein losses
its oncogenic property, but is as immunogenic as the original E7
protein.
EXAMPLE II
LPD Mediated Antigen Delivery to Antigen Presenting Cells Results
in Enhanced Anti-Tumor Immune Response
[0094] A. Materials and Methods
[0095] 1. DNA, Cell Lines and Peptides
[0096] TC-1 cells were provided by TC Wu (Johns Hopkins University,
Baltimore, Md.). These cells are C57BL6 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 (Invitrogen,
Carlsbad, Calif.) supplemented with 10% fetal bovine serum and 100
U/ml penicillin, and 100 mg/ml streptomycin. Cy3 labeled
phophodiester oligodeoxynucleotides were purchased form Invitrogen
(Carlsbad, Calif.). pNGVL3 was obtained from the National Gene
Vector Laboratory (University of Michigan) and contains the CMVie
promoter and no coding region. Plasmids were prepared using Qiagen
EndoFree Giga-Prep kits (Qiagen, Valencia, Calif.). The MHC class I
restricted peptide from the HPV 16 E7 protein (aa 49 to 57,
RAHYNIVTF [SEQ. ID. NO. 5]) was synthesized by the University of
Pittsburgh Peptide Synthesis Facility by solid state synthesis
using an Advanced ChemTech model 200 peptide synthesizer and
purified by HPLC. (Feltkamp, et al. Eur J Immunol 23, 2242-2249
(1993)).
[0097] 2. Liposome and LPD Preparation
[0098] All lipids were purchased from Avanti Polar Lipids
(Alabaster Ala.). Small unilamellar DOTAP
(1,2-Dioleoyl-3-Trimethylammonium-Propane):cholesterol (1:1 molar
ratio) liposomes were prepared by thin film hydration followed by
extrusion. LPD complexes were prepared using the procedure
described by Li with modifications (Li, et al. Gene Ther 5, 930-937
(1998)). Briefly, complexes contained an 8.5:0.6:1.0 weight ratio
of DOTAP/cholesterol liposomes:protamine:DNA. Liposomes and
protamine (Sigma, St Louis Mo.) were mixed in 75 .mu.l of 5.2%
dextrose solution. 75 .mu.l of a solution containing plasmid DNA
and E7 peptide was slowly added by dropwise addition with constant
mixing. Complexes were incubated for 20 min at room temperature
prior to injection. LPD/E7 particle size was determined by dynamic
light scattering using a Coulter N4 plus (Beckman Coulter, San
Francisco) and found to be indistinguishable from LPD particles
(110.+-.30 nm and 123.+-.40 nm, respectively n=3). Peptide
encapsulation efficiency was determined using FITC-labeled E7
peptide and was found to be 83.+-.4% (n=3).
[0099] Sterically stabilized liposomes (SL liposomes) were prepared
according to Ignatius et al (29). Briefly, small unilamellar
cholesterol:Palmitoyl-Oleoyl Phosphatidylcholine (POPC):PEG-PE
(2:3:0.3 molar ratio) liposomes were prepared by thin film
hydration with a 1 mg/ml E7 peptide containing solution followed by
extrusion.
[0100] 3. Immunizations/Treatments
[0101] Six-week-old female C57BL/6 mice (Charles River Labs,
Wilmington, Mass.) were used in all experiments. For vaccinations,
mice were injected through the tail vein or subcutaneously with LPD
particles containing 25 .mu.g of pNGVL3 (University of Michigan)
and 20 .mu.g of E7 peptide on days 0 and 5. On day 10, mice were
challenged by SC injection of 0.5.times.10.sup.6 TC-1 cells and
mice were observed for the formation of tumors by palpation twice
per week.
[0102] For therapy experiments, SC tumors were established by
injecting 0.5.times.10.sup.6 TC-1 cells on day 0. Mice were IV
injected with LPD complexes 3 and 6 days later as described in the
text. Tumor size was monitored twice per week and size was
determined by multiplying the two largest dimensions of the tumor.
In some cases, mice were anesthetized and spleens were surgically
removed 10 days prior to tumor inoculation.
[0103] 4. CTL Assays
[0104] Cytolytic lymphocyte activity was measured using standard
.sup.51Cr-release assays. Splenocytes were collected and cultured
in RPMI supplemented with 10% FBS, 50 U/ml penicillin/streptomycin,
2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino
acids, 40 U/ml IL2, and 200 ng/ml MHC class I restricted E7 peptide
for 4 days. Effector cells were plated into 96-well plates at
various effector-to-target (E:T) cell ratios. Targets used were
either EL4 cells pulsed with E7 peptide or non-pulsed EL4 cells.
Targets were labeled with 200 .mu.Ci .sup.51Cr (NEN Life Sciences,
Boston Mass.) for 18 h at 37.degree. C. Before mixing with
effectors, the targets were washed two times with medium, and
resuspended at 2.times.10.sup.5 cells/ml. The lysis reaction was
carried out for 4 h at 37.degree. C., after which the plates were
centrifuged, and 100 .mu.l of medium from each well were assayed
for .sup.51Cr content in a scintillation counter. Specific lysis
was calculated using the following equation: % specific
lysis=(experimental release-spontaneous release)/(maximum
release-spontaneous release).times.100.
[0105] 5. Histology
[0106] To visualize LPD distribution, mice were injected with LPD
particles containing 25 .mu.g pNGVL3, 0.1 .mu.g Cy3 labeled
oligodeoxynucleotides (ODN) and 10 .mu.g E7 peptide in 150 .mu.l 5%
dextrose. 12, 24, and 48 h later, spleens were collected, embedded
in OCT medium, frozen, and 6 .mu.m sections were prepared and
observed at 200.times. magnification using a Nikon Eclipse TE300
inverted fluorescent microscope and SPOT image analysis
software.
[0107] 6. Flow Cytometry
[0108] Phycoerythrin (PE) conjugated antibodies were purchased from
BD Pharmingen (San Diego Calif.). Spleens were collected 12, 24,
and 48 h after administration of LPD containing fluorescein (FITC)
labeled ODN. Single cell suspensions were prepared and stained for
CD11b or CD11c using the M1/70 and HL3 antibodies respectively.
Samples were run on an EPICS-XL benchtop cytometer
(Beckman-Coulter, San Francisco) and analyzed using EXPO 32
software.
[0109] B. Results
[0110] 1. LPD/Peptide Complexes Accumulate in the Spleen and are
Taken Up by APCs
[0111] As delivery to organized lymph tissue is important for
successful vaccination, we wished to visualize the distribution of
LPD particles within the spleen. To this end, C57BL6 mice were IV
injected with LPD/E7 particles containing trace amounts of
Cy3-labeled DNA. At 12, 24, and 48 h after injection, spleens were
collected, sectioned and the distribution of fluorescence was
observed. Mice injected with non-fluorescent LPD particles served
as controls. After IV administration, LPD particles rapidly
accumulate in the marginal zones of the spleen. (FIG. 16) At 24 h,
the majority of the fluorescence was still located in the marginal
zones but began to be seen in the white pulp. By 48 h, fluorescence
was less intense and was found in a diffuse pattern. Control mice
showed no fluorescence.
[0112] To determine if LPD/E7 particles are taken up by antigen
presenting cells, C57BL6 mice were IV injected with LPD/E7
particles containing trace amounts of FITC-labeled DNA. At 12, 24,
and 48 h after injection, splenocytes were collected, stained for
CD11c (to identify dendritic cells) and CD11b (to identify
macrophages and myeloid lineage DCs), using PE-labeled antibodies
and subjected to flow cytometric analysis (FIG. 17). Mice injected
with non-fluorescent LPD particles served as control. At 24 h, 3.9%
of total splenocytes were CD11b/DNA double positive and 1.7% were
CD11c/DNA positive (FIG. 17A). However, these numbers represent
approximately 18% (3.9% of 21.6%) and 30% (1.7% of 5.7%) of all
CD11b and CD11c positive cells respectively (FIG. 17B). Similar
percentages were observed at 12 and 48 h. Control mice and
splenocytes stained with isotype control antibodies showed <0.5%
positive cells for both CD11b and CD11c.
[0113] 2. LPD/E7 Complexes Induce an E7 Specific Immune
Response
[0114] Previous work has shown that the induction of E7 specific
CTL activity is important for tumor control (Cheng, et al. J Clin
Invest 108, 669-678 (2001); and Cheng, et al. J Virol 75, 2368-2376
(2001)). To determine if LPD/E7 particles induces significant CTL
activity, mice were SC or IV vaccinated with LPD particles
containing 0, 1, 10, or 20 .mu.g of E7 peptide on days 0 and 5. For
comparison, mice were injected with 20 .mu.g E7 peptide in PBS or
encapsulated in stabilized liposomes (SL liposomes). Five days
after the final vaccination, splenocytes were collected, and used
as effector cells in a chromium release assay. Consistent with
previous reports, antigen containing SL liposomes induced
significant levels of CTL activity (61% specific lysis) following
SC injection only (FIG. 18) (Ignatius, et al. Blood 96, 3505-3513
(2000)). Vaccination with 20 .mu.g LPD/E7 produced the highest
levels of CTL activity by both routes (92% and 72% specific lysis
by IV and SC respectively). Injection of 10 .mu.g LPD/E7 particles
induced intermediate levels of CTL activity in both cases, while 1
.mu.g LPD/E7 induced significant CTL activity following IV
administration only. Free E7 peptide showed no CTL induction by
either route.
[0115] 3. LPD/E7 Vaccination Protects Mice from HPV+ Tumor
Formation
[0116] To determine if the induce immune response is adequate to
provide protective immunity, mice were IV or SC vaccinated with
either 20 .mu.g E7 peptide in PBS, empty LPD, or LPD containing 20
.mu.g of E7 peptide on day days 0 and 5. Untreated mice served as
controls. Five days after the last vaccination, mice were SC
challenged with 0.5.times.10.sup.5 E7 expressing TC-1 cells. Mice
that received LPD/E7 particles by either route failed to develop
tumors, while control mice and mice that received LPD alone or free
E7 peptide developed tumors within 12 days. (FIG. 19)
[0117] 4. LPD/E7 Complexes can be Used to Eradicated Established
HPV+ Tumors
[0118] To determine the potential of LPD/E7 complexes for use as a
therapeutic strategy, subcutaneous tumors were established in
C57BL6 mice by inoculation of 0.5.times.10.sup.6 TC-1 cells. On
days 3 and 6 following inoculation, mice were injected IV or SC
with LPD containing 10 .mu.g of E7 peptide. To determine the
importance of antigen delivery to the spleen in the generation of
the observed immune responses, a group of mice that had their
spleens surgically removed was included. Untreated mice and mice
receiving empty LPD served as controls. All mice that received IV
LPD/E7 peptide showed steady tumor shrinkage and complete
regression within 2 weeks (FIG. 20). SC treatment also resulted in
complete regression but with slower kinetics. As expected, empty
LPD administration slowed tumor progression but failed to eradicate
the tumors. The anti-tumor effect in asplenic mice depended on the
route of administration. IV treatment showed tumor growth rates
similar to those observed in mice treated with LPD alone, while SC
delivery resulted in tumor regression. Untreated mice showed
unimpeded tumor progression.
[0119] It has been shown that LPD administration to tumor bearing
mice induces non-specific immune activation that can result in
tumor regression (Whitmore, et al., Gene Ther. 6, 1867-1875 (1999);
and Whitmore, et al., Cancer Immunol Immunother. 50, 503-514
(2001)). To confirm that tumor regression was due to an E7 specific
response, ten days after the last treatment splenocytes were
assayed for E7 specific tumor lytic activity. Consistent with
previously published results, IV or SC injection of LPD without
peptide induced a low level of apparent CTL activity while
treatment with LPD/E7 resulted in the highest level of CTL activity
in both cases (FIG. 21). Cells from untreated mice showed no lytic
activity.
[0120] C. Discussion
[0121] Following IV administration, peptide containing LPD
particles traffic to the spleen where they accumulate in the APC
rich marginal zones (Basak, et al., Blood 99, 2869-2879 (2002); and
McIlroy, et al., Blood 97, 3470-3477 (2001)). Flow cytometry
identified the cells that take up LPD/E7 complexes as mainly CD11b
and CD11c positive cells. Approximately 27% of all CD11c and 16% of
CD11b positive cells show LPD uptake as soon as 12 h after
injection and remained positive for at least 48 h. Over time these
particles or the cells that have internalized them appear to
traffic into the white pulp where immune activation can occur.
These observations are consistent with recently published studies
by Moron, et al. which suggest that CD11c+CD11b+ cells in the
marginal zone internalize VLPs and subsequently traffic to T-cell
areas of the spleen (Moron, et al., J Exp Med 195, 1233-1245
(2002))
[0122] While only a small number of APCs presenting an antigen are
needed to initiate an immune response, most tumor antigens
represent self antigens and are inherently less immunogenic than
virally encoded tumor antigens (Porgador, et al., J Exp Med 188,
1075-1082 (1998)). The ability of LPD particles to deliver antigen
to a large number of APCs should be beneficial when delivering
epitopes from these antigens.
[0123] Intravenous vaccination with as little as 1 .mu.g of
encapsulated peptide produces measurable antigen specific CTL
activity and vaccination with 20 .mu.g of peptide showed greater
immune induction than other commonly used liposome/peptide delivery
systems (SL liposomes). The level of immune induction is sufficient
to protect mice against tumor formation and caused the complete
regression of established tumors. IV Treatment of asplenic mice
with LPD/E7 particles showed therapeutic effects similar to
delivery of empty LPD particles to intact mice while the removal of
the spleen showed no effect on SC administration. The differential
effects of spleen removal are most likely due to the differences in
the site of T cell activation depending on the route of
administration. Following IV administration, T cells are activated
in the spleen and its removal prevents effective priming. The fact
that removal of the spleen does not completely remove the IV
therapeutic effect could be the result of LPD uptake by APCs in
other lymphatic tissues, such as lung or hepatic lymph nodes, or to
the anti-tumor effect of LPD mediated by their ability to induce
the production of T.sub.h1 cytokines (IL-12, IFN.gamma., and
TNF.alpha.) that possess direct anti-tumor activities (Whitmore, et
al., Gene Ther 6, 1867-1875 (1999)). While the spleen plays a role
in the production of these cytokines, a large amount of this
production occurs in the liver and lung (Whitmore, et al. Cancer
Immunol Immunother 50, 503-514 (2001)). However, when LPD/E7 is
given SC the particles will drain into the local lymph nodes where
successful priming can occur and removal of the spleen has no
effect. The fact that IV vaccination consistently induced higher
CTL activities than SC injection in combination with the
observations in asplenic mice shows that antigen delivery to the
spleen is important for the enhanced vaccination observed here.
[0124] These results seem to be in contrast with those obtained by
others using liposome/peptide complexes for vaccination (Ignatius,
et al. Blood 96, 3505-3513 (2000); and Ludewig, et al., Vaccine 19,
23-32 (2000)). In these studies, no immune response was observed
upon IV administration. This discrepancy may be due to differences
in liposome formulation used. LPD lipopolyplexes are more stable
than non-polycation containing lipoplexes (Li, et al. Gene Ther 5,
930-937 (1998)). It has been reported that prolonged stability
results in liposome accumulation in the spleen (Tam, et al. Gene
Ther 7, 1867-1874 (2000)). In the absence of extended circulation,
other formulations may be rapidly degraded and fail to deliver
sufficient amounts of antigen to the spleen.
[0125] The use of LPD particles for vaccination has some
significant advantages. The inclusion of plasmid DNA in the
particles allows for great flexibility in vaccine design. While we
used empty plasmid DNA to form our LPD particles, DNA encoding any
gene of interest may be substituted. For example, while LPD has a
built in adjuvant effect, this can be modified by using DNA
encoding cytokines designed to skew the immune response in a
particular direction, chemotactic factors, or costimulatory
molecules.
[0126] The biophysical properties of the LPD formulation are also
an advantage. Others have reported successful vaccination using
liposome based peptide delivery strategies, however these systems
are more complex and usually require higher doses of peptide or the
inclusion of helper peptides or other stimulatory molecules
(Ludewig, et al., Vaccine 19, 23-32 (2000)). From a pharmaceutical
point of view, LPD particles can be lyophilized, stored for
extended periods (at least 1 year), rehydrated, and used without
any loss of efficacy (Li, et al. J Pharm Sci 89, 355-364. [pii]
(2000)). These unique properties make large scale "off the shelf"
applications possible for cancers with known immunodominant
epitopes such as cervical cancer, prostate cancer and various
her2/neu expressing cancers (Terasawa, et al. Clin Cancer Res 8,
41-53 (2002); Knutson, et al., Clin Breast Cancer 2, 73-79 (2001);
Munger, et al., Oncogene 20, 7888-7898 (2001); and Zeng, G., J
Immunother 24, 195-204 (2001)).
[0127] In compliance with 37 C.F.R. .sctn. 1.821(e), the applicants
request that the compliant computer readable "Sequence Listing"
that is already on file for corresponding U.S. patent application
Ser. No. 11/121,840 be used with the present application. The paper
copy of the "Sequence Listing" in the present application is
identical to the computer readable copy filed with corresponding
U.S. patent application Ser. No. 11/121,840.
[0128] While the present invention has been disclosed by reference
to the details of preferred embodiments of the invention, it is to
be understood that the disclosure is intended as an illustrative
rather than in a limiting sense, as it is contemplated that
modifications will readily occur to those skilled in the art,
within the spirit of the invention and the scope of the amended
claims.
Sequence CWU 1
1
5132DNAArtificial SequenceDescription of Artificial
Sequenceoverlapping PCR primer P1 1ttgggatcca ccatgcatgg agatacacct
ac 32243DNAArtificial SequenceDescription of Artificial
Sequenceoverlapping PCR primer P2 2cggaattcat tcttatggtt tctgagaacc
gatggggcac aca 43324DNAArtificial SequenceDescription of Artificial
Sequenceoverlapping PCR primer P3 3gagacaactg gtctctactg ttat
24424DNAArtificial SequenceDescription of Artificial
Sequenceoverlapping PCR primer P4 4acagtagaga ccagttgtct ctgg
2459PRTArtificial SequenceDescription of Artificial Sequencehuman
papillomavirus subtype 16 E7 oncogene tumor-associated antigen MHC
class I restricted peptide 5Arg Ala His Tyr Asn Ile Val Thr Phe 1
5
* * * * *