Note: Descriptions are shown in the official language in which they were submitted.
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VACCINE DELIVERY COMPOSITIONS
AND METHODS OF USE
FIELD OF THE INVENTION
[0001] This invention relates generally to immunogenic compositions and, in
particular to
vaccine delivery compositions that bind to MHC alleles.
BACKGROUND INFORMATION
[0002] Although significant progress in vaccine development and administration
has been made,
alternative approaches that enhance the efficacy and safety of vaccine
preparations remain under
investigation. Sub-unit vaccines such as recombinant proteins, synthetic
peptides, and
polysaccharide-peptide conjugates are emerging as novel vaccine candidates.
However, traditional
vaccines, consisting of attenuated pathogens and whole inactivated organisms,
contain impurities
and bacterial components capable of acting as adjuvants, an activity which
these subunit vaccines
lack. Therefore the efficacy of highly purified sub-unit vaccines delivered as
stand-alone
formulations will requize addition of potent adjuvants.
[0003] Currently, aluminum compounds remain the only FDA approved adjuvants
for use in
human vaccines in the United States. Despite their good safety record, they
are relatively weak
adjuvants and often require multiple dose regimens to elicit antibody levels
associated with
protective immunity. Aluminum compounds may therefore not be ideal adjuvants
for the induction
of protective immune responses to sub-unit vaccines. Although many candidate
adjuvants are
presently under investigation, they suffer from a number of disadvantages
including toxicity in
humans and requirements for sophisticated techniques to incorporate antigens.
100041 Use of peptidic antigens in vaccines is based on knowledge of operation
of the immune
system in mammals and other animals, especially the major histocompatibility
complexes (MHC).
MHC molecules are synthesized and displayed by most of the cells of the body.
The MHC works
coordinately with specialized types of T cell (for example, the cytotoxic T
cell) to rid the body of
"nonself' or foreign viral proteins. The antigen receptor on T-cells
recognizes an epitope that is a
mosaic of the bound peptide and portions of the alpha helices that make up the
groove flanking it.
Following generation of peptide fragments by cleavage of a foreign protein,
the presentation of
peptide fragments by the MHC molecule allows for antigen-restricted cytotoxic
T cells to survey
cells for the expression of "nonself' or foreign viral proteins. A functional
T-cell will exhibit a
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cytotoxic immune response upon recognition of an MHC molecule containing bound
peptidic
antigen for which the T-cell is specific.
10005] Exogenous antigens are those from outside cells of the body. Examples
include bacteria,
free viruses, yeasts, protozoa, and toxins. These exogenous antigens enter
antigen-presenting cells
or APCs (macrophages, dendritic cells, and B-lymphocytes) through
phagocytosis. The microbes
are engulfed and protein antigens are degraded by proteases into a series of
peptides. These
peptides eventually bind to grooves in MHC-II molecules and are transported to
the surface of the
APC. T4-lymphocytes are then able to recognize peptide/MHC-II complexes by
means of their T-
cell receptors (TCRs) and CD4 molecules. Peptides that are presented by APCs
in class II MHCs
are about 10 to about 30 amino acids, for example about 12 to about 24 amino
acids in length
(Marsh, S. G. E. et al. (2000) The HLA Facts Book, Academic Press, p. 58-59).
The effector
functions of the activated T4-lymphocytes include production of antibodies by
B cells and
microbiocidal activities of macrophages, which are the main mechanisms by
which extracellular or
phagocytosed microbes are destroyed.
[0006] One of the body's major defenses against viruses, intracellular
bacteria, and cancers is
destruction of endogenous infected cells and tumor cells by cytotoxic T-
lymphocytes or CTLs.
These CTLs are effector cells derived from T8-lymphocytes during cell-mediated
immunity.
However, in order to become CTLs, naive T8-lymphocytes must become activated
by cytokines
produced by APCs. This interaction between APCs and naive T8-lymphocytes
occurs primarily in
the lymph nodes, the lymph nodules, and the spleen. The process involves
dendritic cells and
macrophages engulfing and degrading infected cells, tumor cells, and the
remains of killed infected
and tumor cells. It is thought that in this manner, endogenous antigens from
diseased cells are able
to enter the APC, where proteases and peptidases chop the protein up into a
series of peptides, of
about 8 to about 10, possibly about 8 to about 11, or about 8 to about 12
amino acids in length. The
MHC class I molecules with bound peptide, which appear on the surface of the
APCs, can now be
recognized by naive T8-lymphocytes possessing TCRs and CD8 molecules with a
complernentary
shape. This recognition of the peptide epitope by the TCR serves as a first
signal for activating the
naive T8-lymphocyte for cell-mediated immunity function. A single cell may
have up to 250,000
molecules of MHC-I with bound epitope on its surface.
[0007] The past decade has seen development of interest in adjuvants that
function by triggering
or blocking operation of innate immunological pathways via Toll-like Receptors
(TLR). TLRs are
a family of proteins homologous to the Drosophila Toll receptor, which
recognize molecular
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patterns associated with pathogens and thus aid the body in distinguishing
between self and non-
self molecules. Substances common in viral pathogens are recognized by TLRs as
pathogen-
associated molecular patterns. For example, Toll-like receptor 3 (TLR-3)
recognizes patterns in
double-stranded RNA; Toll-like receptor 4 (TLR-4) recognizes pattems in LPS;
Toll-like receptors
7 and 8 (TLR-7/8) recognize patterns containing Adenosine in viral and
bacterial RNA and DNA;
and Toll-like receptor 9 (TLR-9) recognizes un-methylated CpG-containing
sequences of single-
stranded DNA, which are enriched in bacteria. When a TLR is triggered by such
pattern
recognition, a series of signaling events occurs that leads to inflammation
and activation of innate
and adaptive immune responses. Synthetic ligands containing the molecular
pattems recognized by
various TLRs have been used to activate immune responses at a level where the
molecular
mechanisms involved are better defined than for empirically derived adjuvants.
[0008] Despite these developments in the art, there is still a need for new
and better vaccine
delivery compositions utilizing peptidic antigens, rather than deactivated
pathogens, and for new
and better adjuvants, such as TLR agonists. Methods for production and use of
such compositions
to induce an immune response in individuals against pathogenic organisms that
are identified by
IvIHC class I and class II alleles are also needed.
SUMMARY OF TJ3E INVENTION
[0009] The present invention is based on the premise that biodegradable
polymers that contain
amino acids in the polymer chain, such as certain poly (ester amide) (PEA),
poly (ester urethane)
(PEUR), and poly (ester urea) (PEU) polymers, can be used to formulate
completely synthetic and,
hence, easy to produce vaccine delivery compositions for stimulating an immune
response to a
variety of pathogenic organisms in humans and other mammals.
[0010] In one embodiment the invention provides a vaccine delivery composition
formulated for
administration in the form of a liquid dispersion of a polymer in which is
dispersed an effective
amount of at least one MHC class I or cIass II peptidic antigen containing
from 5 to about 30 amino
acids, and an adjuvant. The polymer contains at least one or a blend of
biodegradable polymers
selected from a poly(ester amide) (PEA) having a structural formula described
by structural
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formula (1),
0 H O O H
8-R'-C-N-6-6-0-R4-0-6 -6-N
H R3 R3 H n
Formula (I)
wherein n ranges from about 5 to about 150; R' is independently selected from
residues of am-
bis(4-carboxyphenoxy)-(C,-C8) alkane, 3,3'-(alkanedioyidioxy)dicinnamic acid
or 4,4'-
(alkanedioyldioxy)dicinnamic acid, (C2 - C2o) alkylene, or (C2-C20)
alkenylene; the R3s in
individual n monomers are independently selected from the group consisting of
hydrogen, (CI-C6)
alkyl, (C7-C6) alkenyl, (C2-C6) alkynyl, (C6-Cio) aryl (Ci-C20) alkyl, and -
(CH2)2S(CH3); and R4 is
independently selected from the group consisting of (C2-C2o) alkylene, (C2-
C20) alkenylene, (C2-Cs)
alkyloxy, (C2-C20) alkylene, a residue of a saturated or unsaturated
therapeutic diol, bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of structural formula (II), and
combinations thereof, (C2 -
C20) alkylene, and (C2-C20) alkenylene;
\
CH O
H2C~ ~CH2
p CH
Formula (II)
or a PEA having a chemical formula described by structural formula III:
11p 1 0 H O O H 0 1 0 H 13
C-R -G-N-C-C-O-R4-O-C-C-N C-R -C-N-C-R -N
H R3 R3H m H C-O-R2 H p
o n
Formula (III)
[0011] wherein n ranges from about 5 to about 150, m ranges about 0.1 to 0.9:
p ranges from
about 0.9 to 0.1; wherein R' is independently selected from residues of a,co-
bis(4-
carboxyphenoxy)-(Cl-Ca) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4'-
(alkanedioyldioxy)dicinnamic acid, (C2 - CZo) alkylene, or (C2-C20)
alkenylene; each RZ is
independently hydrogen, (C1-C12) alkyl or (C6-Cio) aryl or a protecting group;
the R3s in individual
m monomers are independently selected from the group consisting of hydrogen,
(C1-C6) alkyl, (C2-
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C6) alkenyl, (CrC6) alkynyl, (C6-C1o) aryl (CI-Czo) alkyl, and -(CHZ)ZS(CH3);
and R4 is
independently selected from the group consisting of (C2-C20) alkylene, (C2-
C20) alkenylene, (C2-Cs)
alkyloxy, (C2-C20) alkylene, a residue of a saturated or unsaturated
therapeutic diol or bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of structural formula(II), and
combinations thereof; and R13
is independently (CI-C2o) alkyl or (C2-C2o) alkenyl;
or a poly(ester urethane) (PEUR) having a chemical formula described by
structural
formula (IV),
4 O 0 H O O H
11 11 98 1
C-O-RB-O-C"-N-C-C-O-Ra-O-C-C-N
H R3 R3 H
n
.Formula IV
wherein n ranges from about 5 to about 150; wherein R3s in independently
selected from the group
consisting of hydrogen, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-
Clo) aryl (CI-C20)
alkyl, and -(CH2)2S(CH3); R4 is selected from the group consisting of (C2-Cao)
alkylene, (CZ-Cao)
alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic
diol, bicyclic-fragments
of 1,4:3,6-dianhydrohexitols of structural formula (II); and combinations
thereof, and Rs is
independently selected from (C2-C20) alkylene, (C2-C2o) alkenylene or
alkyloxy, bicyclic-fragments
of 1,4:3,6-dianhydrohexitols of general formula (II), and combinations
thereof;
or a PEUR polymer having a chemical structure described by general structural
formula
(V)
110 s ~ H O 4 O H 0 s 0 H
13
C-O-R -O-C-N-C-C-O-R -O-C-C-N C-O-R -O-C-N-C-R -N
H R3 R3 H '
m C-O-R2 H p
O n
Formula (V)
[0012] wherein n ranges from about 5 to about 150, m ranges about 0.1 to about
0.9: p ranges
from about 0.9 to about 0.1; R2 is independently selected from hydrogen, (C6-
C1 o) aryl (Ct-C2o)
alkyl, or a protecting group; the R3s in an individual m monomer are
independently selected from
the group consisting of hydrogen, (Cl-C6) alkyl, (C2-C6) alkenyl, (C2-C6)
alkynyl, (C6-Clo) aryl (Cl-
C2o) alkyl and -(CH2)2S(CH3); W is selected from the group consisting of (C2-
CZO) alkylene, (C2-
C20) alkenylene or alkyloxy, a residue of a saturated or unsaturated
therapeutic diol and bicyclic-
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fragments of 1,4:3,6-dianhydrohexitols of structural formula (II) and
combinations thereof; and R6
is independently selected from (C2-C2o) alkylene, (C2-C2o) alkenylene or
alkyloxy, bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of general formula (II), an effective
amount of a residue of
a saturated or unsaturated therapeutic diol, and combinations thereof; and R13
is independently (Cl-
C20) alkyl or (C2-C2o) alkenyl
or a poly(ester urea) (PEU) having a chemical formula described by general
structural
formula (VI):
1O H O O H
C-N-6-6-O-R4-0-C-C-N
H R3 R3 H n
Formula (VI),
wherein n is about 10 to about 150; the R3s within an individual n monomer are
independently
selected from hydrogen, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6 -
CIo) aryl (C1-C20)
alkyl and -(CH2)2S(CH3); R4 is independently selected from (C2-C2o) alkylene,
(C2-C2o) alkenylene,
(C2-Cs) alkyloxy (C2-C2o) alkylene, a residue of a saturated or unsaturated
therapeutic diol; or a
bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II),
and combinations
thereof:
or a PEU having a chemical formula described by structural formula (VII)
lb O H O 4 Q H 0 H 13
C-N-C-C-O-R -O-C-C-N C-N-C-R -N
H R3 R3 H H C-O-Ra H
m 0 pn
Formula (VII),
wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about
10 to about 150; each R2
is independently hydrogen, (Ci-C1Z) alkyl or (Co-C1 o) aryl; the R3s within an
individual m monomer
are independently selected from hydrogen, (CI -C6) alkyl, (C2-C6) alkenyl, (C2-
C6) alkynyl, (C6 -
CIo) aryl (CI -C2o) alkyl and -(CH2)2S(CH3); each R4 is independently selected
from (C2-C20)
alkylene, (CZ-Cao) alkenylene, (C2-Cs) alkyloxy (C2-C20) alkylene, a residue
of a saturated or
unsaturated therapeutic diol; a bicyclic-fragment of a 1,4:3,6-
dianhydrohexitol of structural formula
(II), and combinations thereof and R13 is independently (CI -C2o) alkyl or (C2-
C20) alkenyl.
[0013] In another embodiment, the invention provides methods for inducing an
immune
response in a mammal by administering to the mammal an invention vaccine
delivery composition
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in the form of a liquid dispersion of particles or molecules of a polymer
described by structural
formulas I and III-VII, in which is dispersed an effective amount of class I
or class II peptidic
antigens and an adjuvant. The invention composition is taken up by antigen
presenting cells of the
mammal so as to induce an immune response in the mammal.
[0014] In yet another embodiment, the invention provides methods for
delivering a vaccine to a
mammal by administering to the mammal an invention vaccine delivery
composition in the form of
a liquid dispersion of particles or molecules of a polymer described by
structural formulas I and III-
VII. At least one class I or class II peptidic antigen and an
immunostimulatory adjuvant is
dispersed in the polymer of the composition, which is taken up by antigen
presenting cells of the
mammal to deliver the class I or class II peptidic antigen and the
immunostimulatory adjuvant to
the mammal.
A BRIEF DESCRIPTION OF THE DRAWINGS
100151 Fig. 1 is a schematic drawing illustrating the generation of particles
of PEA, PEUR or
PEU with various types of active agents, such as a peptidic antigen, dispersed
therein by double and
triple emulsion procedures described herein.
[0016] Fig. 2 is a schematic drawing illustrating invention micelles
containing dispersed
peptidic antigens, as described herein.
[0017] Fig. 3 is a flow chart of the process for making an invention vaccine
and testing the in
vitro human T-Cell response to the invention vaccine.
[0018] Figs. 4A-B are graphs showing T Cell activation in response to
dendritic cells exposed to
polymer-peptide conjugates. Fig. 4A shows T-Cell proliferation over 96 hours
in which PEA-
peptide conjugates stimulated significant proliferation over peptide or PEA
alone. Fig. 4B shows
T-Cell IL-2 secretion over 96 hours in which PEA-peptide (Formula III, Example
B1) stimulated
significant IL-2 secretion compared to peptide or PEA alone.
[0019] Figs. 5A-B are graphs showing T cell activation in response to
mononuclear cells
exposed to polymer peptide conjugates. Human mononuclear cells were exposed to
peptidic
antigens and then the cells were mixed with T cells from melanoma patients.
Killing of the
mononuclear cells by the T cells were measured 3 and 7 days after mixing. Fig.
5A shows CTL
killing of mononuclear cells presenting peptidic antigen delivered to those
cells via PEA-1VIART-1
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conjugates. Fig. SB shows CTL killing in response to PEA-gp 100 conjugates.
The CTLs are only
activated when the peptidic antigens are delivered via conjugation to PEA co-
polymer.
[0020] Fig. 6 is a graph showing a T cell response in vivo to PEA-HIV peptidic
antigen vaccine
compositions. Secretion of cytokines was used to enumerate epitope specific T
cells by ELISpot.
Peptide only (A), adjuvant only (B) and PEA only (C) did not induce cytokine
secretion. Two
different PEA-peptide fornnulations (E and F) are shown, which stimulated T
cell responses as
strongly without adjuvant as did peptide plus adjuvant (D).
(0021] Fig.7 is a graph showing protection against lethal Influenza A
challenge using the PEA-
hemagglutinin (HA) vaccine delivery composition. Mice were immunized with PEA-
HA (5 gg HA)
live PR8 strain of the H1N1 virus (i.p.), 5 g HA with or without alum or CpG
adjuvants, or not
immunized. At day 21 post-vaccination, the animals were challenged
intranasally with 10 LD50 of
the PRS strain of the H1N1 virus to produce a fatal influenza infection and
were monitored for
weight loss over 7 days. The protein antigen HA-PEA polymer invention vaccine
delivery
composition confers 100% protection against lethal infection.
[0022] Fig. 8 is a graph showing the prevention of human papilloma virus (HPV)
protein-
expressing tumor growth after immunization with PEA-E6E7 oncogene fusion
protein vaccine
delivery composition. The material was 10 g E6E7 protein + CpG, PEA-E6E7
(containing 10 g
protein antigen) + CpG invention vaccine composition, irradiated tumor cells,
or nothing. Five
weeks after immunization, mice were challenged with 5 x 10S C3-43 HPV-
transfected cells by
subcutaneous injection in the flank. Fifteen days after tumor challenge, the
mice were euthanized,
and tumors removed and weighed, showing that 4 of 5 mice immunized with PEA-
E6E7 had
negligible tumors; whereas 5 of 5 mice immunized with the fusion protein alone
had large tumors.
[0023] Figs. 9A-B are graphs showing the more potent activation of antigen
presenting cells by
adjuvant when the adjuvant is delivered via encapsulation in PEA copolymer. In
Fig 9A, traces are
shown of the FACS analysis intensity distribution of CDI lc-positive bone
marrow derived
dendritic cells (BMDC) from Balb/c mice stained for elevation of surface CD40.
PEA-,imiquimod
(IMQ) increased CD40 expression at both 10 and I uM IMQ whereas IMQ alone only
increased
CD40 expression at 10 uM. In Fig. 9B, supernatants from 5 x 104 BMDC cultured
overnight with
the substances indicated at the bottom of the panel are analyzed for IL- 12
cytokine secretion.
Again, PEA-IMQ was over 10-fold more potent at stimulating a response than IMQ
alone.
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DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention is based on the discovery that biodegradable polymers
that contain at least
one amino acid per monomer can be used to create a synthetic vaccine delivery
composition for
subcutaneous or intramuscular injection or mucosal administration that is
reproducible in large
quantities, safe (containing no attenuated virus), stable, and can be
lyophilized for transportation
and storage. Due to structural properties of the polymer used, the vaccine
delivery composition
provides high copy number and local density of antigen and adjuvant.
100251 In one embodiment, the polymer can be formulated into vaccine delivery
compositions
with different properties. For example, the polymer can act as a time-release
polymer depot
releasing the adjuvant and peptidic antigen or antigen-polymer fragments to be
taken up by APCs
and presented by 1VIHC class I or class II alleles as the polymer depot
biodegrades in vivo. In other
embodiments, the polymer carries the peptidic antigen and adjuvant into the
APC, and the peptidic
antigen and adj uvant are released for presentation intracellularly. The
polymer may actually
stimulate the APCs by inducing phagocytosis of polymer-antigen and polymer
adjuvant conjugates.
[0026] In yet another embodiment, the invention provides methods for inducing
an immune
response in a mammal by administering to the mammal an effective amount of an
invention vaccine
delivery composition, which is taken up by antigen presenting cells of the
mammal to induce an
immune response in the mammal.
[00271 In addition to treatment of humans, the invention vaccine delivery
compositions are also
intended for use in veterinary treatment of a variety of mammalian and avian
patients, such as pets
(for example, cats, dogs, rabbits, and ferrets), farm animals (for example,
chickens, ducks, swine,
horses, mules, dairy and meat cattle) and race horses.
[0028] Polymer particles or molecules delivered directly or released from an
in vivo polymer
depot are sized to be readily taken up by antigen presenting cells (APCs) and
contain peptidic or
protein antigens and adjuvants, dispersed within polymer particles or
conjugated to functional
groups on the polymer molecules. The APCs display the peptidic/protein antigen
via MHC
complexes and are recognized by T-cells, such as cytotoxic T-cells, to
generate and promote
endogenous immune responses leading to destruction of pathogenic cells bearing
matching or
similar antigens. The polymers used in the invention vaccine delivery
composition can be designed
to tailor the rate of biodegradation of the polymer depots, molecules and
particles to result in
continuous contact of the peptidic/protein antigen with antigen presenting
cells over a selected
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period of time. For instance, typically, the polymer depot will degrade over a
time selected from
about twenty-four hours, about seven days, about thirty days, or about ninety
days, or longer.
Longer time spans are particularly suitable for providing an implantable
vaccine delivery
composition that eliminates the need to repeatedly inject the vaccine to
obtain a suitable immune
response.
[0029] The present invention utilizes biodegradable polymer-mediated delivery
techniques to
elicit an immune response against a wide variety of pathogens, including
mucosally transmitted
pathogens. The composition affords a vigorous immune response, even when the
antigen is by
itself weakly immunogenic. Although the individual components of the vaccine
delivery
composition and methods described herein were known, it was unexpected and
surprising that such
combinations would enhance the efficiency of antigens beyond levels achieved
when the
components were used separately and, moreover, that the polymers used in
making the vaccine
delivery composition would obviate the need for additional adjuvants in some
cases.
[0030] Although the invention is broadly applicable for providing an immune
response against
any of the herein-described pathogens, the invention is exemplified herein by
reference to influenza
virus and HIV.
[0031] The method of the invention provides for cell-mediated immunity, and/or
humoral
antibody responses. Accordingly, the methods of the present invention will
find use with any
antigen for which cellular and/or humoral immune responses are desired,
including antigens
derived from viral, bacterial, fungal and parasitic pathogens that may induce
antibodies, T- helper
cell activity and T-cell cytotoxic activity. Thus, "immune response" as used
herein means
production of antibodies, T-helper cell activity or T-cell cytotoxic activity
specific to the
peptidic/protein antigen used. Such antigens include, but are not limited to
those encoded by
human and animal pathogens and can correspond to either structural or non-
structural proteins,
polysaccharide-peptide conjugates, or DNA.
[0032] For example, the present invention will find use for stimulating an
immune response
against a wide variety of proteins from the herpes virus family, including
proteins derived from
herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2
glycoproteins gB, gD and
gH; antigens derived from varicella zoster virus (VZV), Epstein-Barr virus
(EBV) and
cytomegalovirus (CMV) including CMV gB and gH; and antigens derived from other
human
herpes viruses such as HHV6 and HHV7. (See, e.g. Chee et al.,
Cytomegaloviruses (J. K.
McDougall, ed., Springer-Verlag 1990) pp. 125-169, for a review of the protein
coding content of
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cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a
discussion of the
various HSV-1 encoded proteins; U.S. Pat. No. 5,171,568 for a discussion of
HSV-1 and HSV-2
gB and gD proteins and the genes encoding therefor; Baer et al., Nature (1984)
310:207-211, for
the identification of protein coding sequences in an EBV genome; and Davison
and Scott, J. Gen.
Virol. (1986) 67:1759-1816, for a review of VZV.)
[0033] Antigens from the hepatitis family of viruses, including hepatitis A
virus (HAV),
hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus
(HDV), hepatitis E.virus
(HEV) and hepatitis G virus (HGV), can also be conveniently used in the
techniques described
herein. By way of example, the viral genomic sequence of HCV is known, as are
methods for
obtaining the sequence. See, e.g., Intemational Publication Nos. WO 89/04669;
WO 90/11089; and
WO 90/14436. The HCV genome encodes several viral proteins, including El (also
known as E)
and E2 (also known as E2/NSI) and an N-terminal nucleocapsid protein (termed
"core") (see,
Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV
proteins, including El
and E2). Each of these proteins, as well as antigenic fragments thereof, will
find use in.the present
methods. Similarly, the sequence for the S-antigen from HDV is known (see,
e.g., U.S. Pat. No.
5,378,814) and this antigen can also be conveniently used in the present
methods. Additionally,
antigens derived from HBV, such as the core antigen, the surface antigen, sAg,
as well as the
presurface sequences, pre-S 1 and pre-S2 (formerly called pre-S), as well as
combinations of the
above, such as sAg/pre-S1, sAg/pre-S2, sAg/pre-Sl/pre-S2, and pre-Sl/pre-S2,
will find use herein.
See, e.g., "HBV Vaccines--from the labbratory to license: a case study" in
Mackett, M. and
Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176, for a
discussion of HBV
structure; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513, incorporated
herein by reference in
their entireties; Beames et al., J. Virol. (1995) 69:6833-6838, Bimbaum et
al., J. Virol. (1990)
64:3319-3330; and Zhou et al., J. Virol. (1991) 65:5457-5464.
[00341 Antigens derived from other viruses will also find use in the claimed
methods, such as
without limitation, proteins from members of the families Picornaviridae
(e.g., polioviruses, etc.);
Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);
Flaviviridae; Coronaviridae;
Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.);
Filoviridae; Paramyxoviridae
(e.g., mumps virus, measles virus, respiratory syncytial virus, etc.);
Orthomyxoviridae (e.g.,
influenza virus types A, B and C, etc.); Bunyaviddae; Arenaviridae;
Retroviradae (e.g., HTLV-I;
HTLV-II; HIV-1 (also known as HTLV-III LAV, ARV, hTLR, etc.)), including but
not limited to
antigens from the isolates HIVIIib, HIVsF2, HNLAV, HIVLAI, HIVmN); HIV-
ICNt235, HIV-1US4 ; HIV-
2; simian immunodeficiency virus (SIV) among others. Additionally, antigens
may also be derived
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12
from human papillomavirus (HPV) and the tick-borne encephalitis viruses. See,
e.g. Virology, 3rd
Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N.
Fields and D. M.
Knipe, eds. 1991), for a description of these and other viruses.
[0035] More particularly, the envelope proteins from any of the above HIV
isolates, including
members of the various genetic subtypes of HIV, are known and reported (see,
e.g., Myers et al.,
Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.Mex.
(1992); Myers et al.,
Human Retroviruses and Aids, 1990, Los Alamos, N.Mex.: Los Alamos National
Laboratory; and
Modrow et al., J. Virol. (1987) 61:570-578, for a comparison of the envelope
sequences of a variety
of HIV isolates) and antigens derived from any of these isolates will find use
in the present
methods. Specifically, the synthetic peptide, R15K (Nehete et al. Antiviral
Res. (2002) 56:233-
251), derived from the V3 loop of gp120 and having the sequence
RIQRGPGRAFVTIGK (SEQ
ID NO:1), will have use in the invention compositions and methods.
Furthermore, the invention is
equally applicable to other immunogenic proteins derived from any of the
various HIV isolates,
including any of the various envelope proteins such as gp 160 and gp41, gag
antigens such as
p24gag and p55gag, as well as proteins derived from the pol region.
Furthermore, multi-epitope
cocktails of the invention composition carrying various epitopes from HIV
proteins are envisioned.
For example, 6 conserved peptides from gp120 and gp4l have been shown to
reduce viral load and
prevent transmission in a rhesus/SHIV model : SVITQACSKVSFE (S13E) (SEQ ID
NO:2),
GTGPCTNVSTVQC (G13C) (SEQ ID NO:3), LWDQSLKPCVKLT (L13T) (SEQ ID NO:4),
VYYGVPVWKEA (V11A) (SEQ ID NO:5), YLRDQQLLGIWG (V12G) (SEQ ID NO:6), and
FLGFLGAAGSTMGAASLTLTVQARQ (F25Q) (SEQ ID NO:7) (Nehete et al. Vaccine (2001)
20:813-). The amino acid sequence of the antigen tested in the invention
compositions and
methods is IFPGKRTIVAGQRGR (SEQ ID NO:8), wherein all amino acids are natural,
L-amino
acids.
[0036] As explained above, influenza virus is another example of a virus for
which the present
invention will be particularly useful. Specifically, the envelope
glycoproteins HA and NA of
influenza A are of particular interest for generating an immune response, as
are the nuclear proteins
and matrix proteins. Numerous HA subtypes of influenza A have been identified
(Kawaoka et al.,
Virology (1990) 12:759-767; Webster et al., "Antigenic variation among type A
influenza viruses,"
p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza
viruses. Springer-Verlag,
New York). Thus, proteins derived from any of these isolates can also be used
in the immunization
techniques described herein. In particular, the conserved 13 amino acid
sequence of HA can be
used in the invention vaccine delivery composition and methods. In H3 strains
used in current
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13
vaccine formulations, this amino acid sequence is PRYVKQNTLKLAT (SEQ ID NO:9),
and in H5
strains it is predominantly PKYVKSNRLVLAT (SEQ ID NO:10). In addition, the
whole of HA in
its monomer or trimer form can be used in the invention vaccine delivery
composition and
methods. In particular, the H5N1 strain of avian influenza that is used in
vaccine formulations is
MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCD
LDGVKPLILRDCS VAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKH
LLSRINHFEKIQIIPKSSWSSHEASLGV SSACPYQGKSSFFRNVVWLIKKNSTYPTIKRS
YNNTNQEDLLVLW GIHHPNDAAEQTKLYQNPTTYIS V GTSTLNQRLVPRIATRSKVNGQS
GRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA
INS SMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQG
MV DGWYGYHHSNEQGS GYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAV GREFNNLER
RIENLNKKMEDGFLDV WTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKEL
GNGCFEFYHKCDNECMESVRNGTYDYPQYSEE (SEQ ID NO: 11). The nucleoprotein (NP)
sequence of the same H5N1 strain is
MASQGTKRSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQNSI
TIERMV LS AFDERRNRYLEEHP S AGKDPKKTGGPIYRRRDGKW VRELILYDKEEIRRIW RQ
ANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMC SLMQGSTLPRRSGAAG
AAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAM
MDQVRESRNPGNAEIEDLIFLARSALILRGS V AHKS CLPAC VYGLAVAS GYDFEREGYSLV
GIDPFRLLQNS QVF SLIRPNENPAHKSQLV W MACHSAAFEDLRV SSFIRGTRV VPRGQLSTR
GVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFER
ATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDV SFQGRGVFELSDEKATNPIVPSFDMNN
EGSYFFGDNAEETS (SEQ ID NO: 12).
A fusion of NP and the extracellular domain of the matrix protein (M2e) of the
same H5N1 strain
can also be used as the protein antigen in the invention vaccine compositions:
MASQGTKRSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLIQN SI
TIERM V LSAFDERRNRYLEEHP SAGKDPKKTGGPIYRRRDGKW VRELILYDKEEIRRIW RQ
ANNGEDATAGLTHLMIWHSNLNDATYQRTRALV RTGMDPRMC SLMQGSTLPRRSGAAG
AAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAM
MDQVRESRNPGNAEIEDLIFLARSALILRGS VAHKS CLPAC VYGLAVAS GYDFEREGYS LV
GIDPFRLLQNS QVFSLIRPNENPAHKSQLV W MACHSAAFEDLRV S SFIRGTRW PRGQLSTR
GVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFER
ATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDV SFQGRGVFELSDEKATNPIV P SFDMNN
EGSYFFGDNAEETSHMSLLTEVETPTRNEWECRCSDSSDKSR (SEQ ID NO: 13).
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[0037] The methods described herein will also find use with numerous bacterial
antigens, such
as those derived from organisms that cause diphtheria, cholera, tuberculosis,
tetanus, pertussis,
meningitis, and other pathogenic organism, including, without limitation,
Meningococcus A, B and
C, Hemophilus influenza type B (HIB), and Helicobacter pylori. Examples of
parasitic antigens
include those derived from organisms causing malaria and Lyme disease.
[0038] Furthermore, the methods described herein provide a means for treating
a variety of
malignant cancers. Although the invention is broadly applicable for providing
an immune response
against a range of cancers, the invention is exemplified herein by reference
to melanoma and
human papilloma virus-induced cervical cancer.
[0039] For example, the composition of the present invention can be used to
mount both
humoral and cell-mediated immune responses to particular proteins specific to
the cancer in
question, such as an activated oncogene, a fetal antigen, or an activation
marker. Such tumor
antigens include any of the various MAGEs (melanoma associated antigen E),
including MAGE 1,
2, 3, 4, etc. (Boon, T. Scientific American (March 1993):82-89); any of the
various tyrosinases;
MART 1 (melanoma antigen recognized by T cells), mutant ras; mutant p53; p97
melanoma
antigen; CEA (carcinoembryonic antigen), among others. Additional melanoma
peptidic antigens
useful in the invention compositions and compositions include the following:
DESIGNATION ANTIGEN SEQUENCE PROTEIN
Martl-27 AAGIGILTV MART1
(SEQ ID NO:14)
Gp100-209* ITDQVPFSV Melanocyte lineage-specific antigen GP100
(SEQ ID NO:15)
GplOO-154 KTWGQYWQV Melanocyte lineage-specific antigen GPi00
(SEQ ID NO:16)
Gp100-280 YLEPGPVTA Melanocyte lineage-specific antigen GP 100
(SEQ ID NO:17)
*GP100 is also called melanoma-associated ME20 antigen.
[0040J Malignant cancers that express foreign antigens, such as those from a
tumor-inducing '
virus, are additional targets for the invention vaccine delivery compositions.
Strains of the human
papilloma virus (HPV) can cause cervical cancer, and cytotoxic T cell immune
responses to the
peptidic or protein antigens are of particular interest. In particular, a
fusion protein of the E6 and E7
oncogenes with the sequence
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MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVGDFAFRDLCNYRDGNPYA
V CDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQ KPLCPEEKQRHLDKK
QRFHNIRGRWTGRCMSCCRS SRTRRETQLHGDTPTLHEYMLDLQPETTDLYGYGQLNDSS
EEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPIC
SQKP (SEQ ID NO: 18) can be used in the invention vaccine delivery
compositions.
[0041] It is readily apparent that the subject invention can be used to
prevent or treat a wide
variety of diseases.
[0042] The peptidic/protein antigens dispersed within the polymers in the
invention vaccine
delivery compositions can have any suitable length, but may incorporate a
peptidic antigen segment
of 8 to about 30 amino acids that is recognized by a peptide-restricted T-
lymphocyte. Specifically,
the peptidic antigen segment that is recognized by a corresponding class I
peptide-restricted
cytotoxic T-cell contains 8 to about 12 amino acids, for example 9 to about 11
amino acids and, the
peptidic antigen segment that is recognized by a corresponding class II
peptide-restricted T-helper
cell contains 8 to about 30 amino acids, for example about 12 to about 24
amino acids.
[0043] While natural T-cell mediated immunity works via presentation of
peptide epitopes by
MHC molecules (on the surface of APCs), MHCs can also present peptide adjunct--
in particular
glycol-peptides and lipo-peptides, in which the peptide portion is held by the
MHC so as to display
to the T-cell the sugar or lipid moiety. This consideration is particularly
relevant in cancer
vaccinology because several tumors over-express glyco-derivatized proteins or
lipo-derivatized
proteins, and the glyco- or lipo-derivatized peptide fragments of these can,
in some cases, be
powerful T-cell epitopes. Moreover, the lipid in such T-cell epitopes can be a
glyco-lipid.
[0044] Unlike the normal peptide-alone presentation, in these cases T-cell
recognition is
dominated by the sugar or lipid group on the peptide, so much so that short
synthetic peptides that
bind to MHCs with high affinity, but were not derived from the tumor proteins,
yet to which the
tumor-associated sugar or lipid molecule is covalently attached synthetically_
have been
successfully used as peptidic antigens. This approach to building an
artificial T-cell epitope
directed against a natural tumor cell line has recently been adopted by Franco
et al., J. Exp. Med
(2004) 199(5):707-716. Therefore, synthetic peptide derivatives and even
peptidomimetics can be
substituted for the peptidic antigen and are encompassed by the term "peptidic
antigen" as used in
the description of and claims to the invention vaccine delivery compositions
to act as high-affinity
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16
MHC-binding ligands that form a platform for the presentation to T-cells of
peptide branches and
non-peptide antigens.
[0045] Accordingly, the term "peptidic antigen", as used herein, refers to
peptides, wholly
peptide derivatives (such as branched peptides) and covalent hetero- (such as
glyco- and lipo- and
glycolipo-) derivatives of peptides. It also is intended to encompass
fragments of such materials
that are specifically bound by a specific antibody or specific T lymphocyte.
[0046] The peptidic antigens can be synthesized using any technique as is
known in the art. The
peptidic antigens can also include "peptide mimetics." Peptide analogs are
commonly used in the
pharmaceutical industry as non-peptide bioactive agents with properties
analogous to those of the
template peptide. These types of non-peptide compound are termed "peptide
mimetics" or
"peptidomimetics." Fauchere, J. (1986) Adv. Bioactive agent Res., 15:29; Veber
and Freidinger
(1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem., 30:1229; and are
usually developed
with the aid of computerized molecular modeling. Generally, peptidomimetics
are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical
property or
pharmacological activity), but have one or more peptide linkages optionally
replaced by a linkage
selected from the group consisting of.- - -CH2NH--, --CH2S--, CH2-CH2--, --
CH=CH-- (cis and
trans), --COCH2--, --CH(OH)CH2--, and --CH2SO--, by methods known in the art
and further
described in the following references: Spatola, A.F. in "Chemistry and
Biochemistry of Amino
Acids, Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New York,
p. 267 (1983);
Spatola, A.F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone
Modifications"
(general review); Morley, J.S., Trends. Pharm. Sci, (1980) pp. 463-468
(general review); Hudson,
D. et al., Int. J Pept. Prot. Res., (1979) 14:177-185 (--CH2 NH--, CH2CH2--);
Spatola, A.F. et al.,
Life Sci:, (1986) 38:1243-1249 (--CH2--S--); Harm, M. M., J. Chem. Soc. Perkin
Trans 1(1982)
307-314 (--CH=CH--, cis and trans); Almquist, R.G. et al., J. Med. Che z.,
(1980) 23:2533 (--
COCH2--); Jennings-Whie, C. et al., Tetrahedron Lett., (1982) 23:2533 (--COCH2-
-); Szelke, M. et
al., European Appln., EP 45665 (1982) CA: 97:39405 (1982) (--CH(OH)CH2--);
Holladay, M_ W.
et al., Tetrahedron Lett., (1983) 24:4401-4404 (--C(OH)CH2--); and Hruby,
V.J., Life Sci., (1982)
31:189-199
(-CH2-S-). Such peptide mimetics may have significant advantages over
polypeptide
embodiments, including, for example: more economical production, greater
chemical stability,
enhanced pharmacological properties (half-life, absorption, potency, efficacy,
etc.), altered
specificity (e.g., a broad-spectrum of biological activities), reduced
antigenicity, and others.
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17
[0047] Additionally, substitution of one or more amino acids within a peptide
(e.g., with a
D-Lysine in place of L-Lysine) may be used to generate more stable peptides
and peptides resistant
to endogenous proteases. Alternatively, the synthetic peptidic antigens, e.g.,
covalently bound to
the biodegradable polymer, can also be prepared from D-amino acids, referred
to as inverso
peptides. When a peptide is assembled in the opposite direction of the native
peptide sequence, it is
referred to as a retro peptide. In general, peptides prepared from D-amino
acids are very stable to
enzymatic hydrolysis. Many cases have been reported of preserved biological
activities for retro-
inverso or partial retro-inverso peptides (US patent, 6,261,569 B1 and
references therein; B.
Fromme et al., Endocrinology (2003)144:3262-3269.
[00481 In addition to peptidic antigens, whole proteins can be used in the
invention vaccine
delivery compositions. Peptidic antigens are defined as peptides generally
less than 10,000 daltons
molecular weight. Proteins are larger macromolecules composed of one or more
peptidic antigen
chains.
[0049] The selected peptidic/protein"antigen and adjuvant are combined with
the biodegradable
polymer for subsequent administration to a mammalian subject. The invention
vaccine delivery
composition can be prepared for intravenous, mucosal, intramuscular, or
subcutaneous delivery.
For example, useful polymers in the methods described herein include, but are
not limited to, the
PEA, PEUR and PEU polymers described herein. These polymers can be fabricated
in a variety of
molecular weights, and the appropriate molecular weight for use with a given
antigen is readily
determined by one of skill in the art. Thus, e.g., a suitable molecular weight
will be on the order of
about 5,000 to about 300,000, for example about 5,000 to about 250,000, or
about 75,000 to about
200,000, or about 100,000 to about 150,000.
[0050] The invention vaccine delivery composition includes an adjuvant that
can augment
immune responses, especially cellular immune responses to the peptidic/protein
antigen, by
increasing delivery of antigen, stimulating cytokine production, and/or
stimulating antigen
presenting cells. The adjuvants can be administered by dispersing the adjuvant
along with the
peptidic/protein antigen within the polymer matrix, for example by conjugating
the adjuvant to the
antigen. Alternatively, the adjuvants can be administered concurrently with
the vaccine delivery
composition of the invention, e.g., in the same composition or in separate
compositions. For
example, an adjuvant can be administered prior or subsequent to the vaccine
delivery composition
of the invention. Alternatively still, the adjuvant or an adjuvant/antigen can
be dispersed in (e.g.,
chemically bonded to) the polymer as described herein for simultaneous
delivery. Such adjuvants
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include, but are not limited to: (1) aluminum salts (alum), such as aluminum
hydroxide, aluminum
phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations
(with or without other
specific immunostimulatory agents such as muramyl peptides or bacterial cell
wall components),
such as for example (a) MF59 (International Publication No. WO 90/14837),
containing 5%
Squalene, 0.5% Tween 80TM, and 0.5% SpanTM 85, optionally containing various
amounts of MTP-
PB, formulated into submicron particles using a microfluidizer such as Model
110Y microfluidizer
(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween
80TM, 5%
pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a
submicron emulsion or
vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant
composition (RAS),
(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80TM,
and one or
more bacterial cell wall components from the group consisting of
monophosphorylipid A (MPL),
trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS
(DetoxTM); (3)
saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, Mass.)
may be used or
particle generated therefrom such as ISCOMs (immunostiniulating complexes);
(4) Complete
Freunds adjuvant (CFA) and Incomplete Freunds adjuvant (IFA); (5) cytokines,
such as
interleukins (IL-1, IL-2 etc.); macrophage colony stimulating factor (M-CSF),
tumor necrosis factor
(TNF), etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such
as a cholera toxin
(CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT),
particularly LT-K63 (where lysine
is substituted for the wild-type amino acid at position 63) LT-R72 (where
arginine is substituted for
the wild-type amino acid at position 72), CT-S109 (where serine is substituted
for the wild-type
amino acid at position 109), and PT-K9/Gl29 (where lysine is substituted for
the wild-type amino
acid at position 9 and glycine substituted at position 129) (see, e.g.,
International Publication Nos.
W093/13202 and W092/19265); and (7) QS21, a purified form of saponin and 3D-
monophosphoryl
lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS), to enhance
cellular and humoral
immune responses (Moore, et al., Vaccine. 1999 Jun 4;17(20-21):2517-27).
(0051] Particularly desirable immunostimulatory adjuvants to enhance the
effectiveness of the
invention vaccine delivery compositions are immunostimulatory drugs (i.e.,
small molecules),
polymers, lipids, lipid/sugars, lipid/salts, sugars, salts and biologics,
examples of which are
arranged by type in Table 1 below. A "biologic" as the term is used herein
includes oligo- and
poly-nucleotides (DNA, RNA, cDNA, and the like) polypeptides (i.e., peptide or
protein
adjuvants), and proteins.
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TABLE 1
IMMUNOSTIMULATORY ADJUVANT TYPE
Calcitrol Drug
Loxoribine Drug
Poly rA: Poly rU Drug
S-28463 Drug
SM360320 Drug
SAF-1TM Polymer
SPT' Polymer
Avridine Lipid
Bay R1005 Lipid
DDA Lipid
DHEA Lipid
DMPC Lipid
DMPG Lipid
D-Murapalmitine Lipid
DOC/Alum Complex Lipid
ISCOM Lipid
IscoprepTM 7Ø3 Lipid
Liposomes Lipid
MF59 Lipid
MontanideTM ISA 51 Lipid
MontanideTM ISA 720 Lipid
MPL Lipid
MTP-PE Lipid
MTP-PE Liposomes Lipid
Murapalmitine Lipid
Non-Ionic Surfactant Vesicles Lipid
Polysorbate 80 Lipid
Protein Cochleates Lipid
Span 85 (sorbitan monostearate) Lipid
Stearyl Tyrosine Lipid
Theramide Lipid
Gerbu AdjuvantTM Lipid/Sugar
QS-21 Lipid/Sugar
Quil ATM Lipid/Sugar
Walter Reed Liposomes2 Lipid/Salt
Algal Glucan Sugar.
AlgammulinTM Sugar
Gamma Inulin Sugar
Glucosaminyl-muramyl dipeptide Sugar
ImmTherg Sugar
N-acetyl-muramyl dipeptide Sugar
Beta glucan from Pleurotus ostreatus Sugar
Threonyl-muramyl dipeptide Sugar
Adju-Phos Salt
AlhydrogelTM Salt
Calcium Phosphate Gel Salt
Rehydragel HPA Salt
Rehydragel LV Salt
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Cytosine-phosphate-guanosine oligodeoxynucleotide (TLR-9) TLR Agonist
Bacterial Flagellin (TLR 5) TLR Agonist
Imiquimod, R-848 (TLR-7) TLR Agonist
Lipopeptides (PAM3CSK4) (TLR-2) TLR Agonist
Lipopolysaccharide (TLR-4) TLR Agonist
Macrophage-activating lipopeptide-2 (TLR-2 AND TLR-6) TLR Agonist
Peptidoglycans (TLR-2) TLR Agonist
Polyriboinosinic:polyribocytidylic acid (TLR-3) TLR Agonist
Cholera Holotoxin; Cholera toxin B Subunit Biologic
Cholera toxin Al-subunit-Protein A D-fragment
fusion protein Biologic
Cytokine-containing liposomes Biologic
GM-CSF Biologic
Liposomes Containing Antibodies to Costimulatory Biologic
Molecules (DRV)
Interferon-y Biologic
Interleukin-12 Biologic
Interleukin-1 B Biologic
Interleukin-2 Biologic
Interleukin-7 Biologic
heat-labile cholera-like toxin of E. coli. LT-OA Biologic
Neuraminidase and galactose oxidase (NAGO) Biologic
Sclaro Peptide3 Biologic
Sendai Proteoliposomee Biologic
Sendai-containing Lipid Matrices Biologic
Ty Particles Biologic
Freund's Complete Adjuvant Oil
Freund's Incomplete Adjuvant Oil
SpecolTM Oil
Squalane Oil
Squalene Oil
Vaccine Design, The Subunit and t~L(IjWuu nJ Approach" edited by M. Powell
and,
M. Newman, Plenum Press, 1995, p 147.
2 Vogel, F. R., and M. F. Powell. 1995. Section on Walter Reed liposomes,
p. 226-227. In M. F. Powell and M. J. Neman (ed.), Vaccine design: the subunit
and adjuvant approach. Plenum Press, New York, N.Y.
3 Reimer G, et al. Arthritis Rheum (1988) 31:525-532; Reichlin M, et al., J
Clin
Immunol (1984) 4e40-44; and Oddis CV, et al., Arthritis Rheum (1992) 35:1211-
1217.
4 Ozawa, M and A Asano. J. Biol. Chem., (1981) 256: 5954-5956.
[0052] Among the immunostimulatory adjuvants listed in Table 1 are Toll-like
receptor (TLR)
agonists, which are among the specific immunostimulatory adjuvants. TLR
agonists are certain
adjuvant ligands, many synthetic, that contain a molecular pattern recognized
by a particular
member of the TLR family and activate a corresponding immune response. As
described herein,
the invention vaccine delivery compositions based on PEA, PEUR and PEU
molecules and
particles are efficiently taken up (phagocytosed) by antigen presenting cells
(APCs)( including
dendritic cells) and the peptidic/protein antigen incorporated therein is
processed within these cells,
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21
i.e., intracellularly. Accordingly, the preferred TLR agonists for use in the
invention compositions
and methods are those that target receptors that act intracellularly, such as
TLRs-7, -8, and -9. For
example, TLR agonists recognized by TLRs-7 and -8 include certain drugs or
small molecule
ligands based on Adenosine and 8-hydroxy-adenine prodrugs thereof, such as
Imiquimod and
SM360320 (J. Lee et al. PNAS (2006) 103(6):1828-1823 and A. Kurimoto et al.
Chem Pharm. Bull.
(2004) 53(3):466-469). Imiquimod, which is a TLR-7 agonist, is used in
treatment of superficial
basal cell carcinoma, actinic keratosis, genital warts and melanoma, among
others (A. Gupta, et al.
J. Cutan. Med. Surg. (2004) 8(5):338-352). TLR-9 agonists include
deoxyribonucleotides of about
20 residues that contain unmethylated CpG segments and which trigger a Th-1
response without
triggering a Th-2 immune response (G. Haker et al. Immunology (2002) 105:245-
251).
[0053] Complement domain-3 (C3d) or CD40-ligand (CD40L) are examples of
biologic
adjuvants that enhance adaptive immunity by binding to complement receptor 2
on B cells and
follicular dendritic cells, resulting in enhanced antigen-specific antibody
production. As will be
described below, a protein or polypeptide immunogenic adjuvant can be
incorporated into the
invention compositions using the invention one-step method for vaccine
preparation_
[0054] Polymers suitable for use in the practice of the invention bear
functionalities that allow
the peptidic/protein antigen, adjuvant, or antigen-adj uvant conjugate either
to be conjugated to the
polymer or dispersed therein. For example, a polymer bearing carboxyl groups
can readily react
with an amino moiety, thereby covalently bonding the peptide or protein or a
peptide or protein
adjuvant to the polymer via the resulting amide group. As will be described
herein, the
biodegradable polymer and the peptide or adjuvant may contain numerous
complementary
functional groups that can be used to covalently attach the peptidic/protein
antigen and/or the
adjuvant to the biodegradable polymer.
[0055] The polymer in the invention vaccine delivery composition plays an
active role in the
endogenous immune processes at the site of implant by holding the
peptidic/protein antigen and
adjuvant at the site of injection for a period of time sufficient to allow the
individual's immune cells
to interact with the peptidic/protein antigen and adjuvant to affect immune
processes, while slowly
releasing the particles or polymer molecules containing such agents during
biodegradation of the
polymer. The fragile biologic peptidic/protein antigen is protected by the
more slowly
biodegrading polymer to increase half-life and persistence of the antigen.
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22
[0056] The polymer itself may also have an active role in delivery of the
antigen into APCs by
stimulating phagocytosis of the polymer-antigen-adjuvant composition. In
addition, the polymers
disclosed herein (e.g., those having structural formulae (I and III-VIII),
upon enzymatic
degradation, provide essential amino acids while the other breakdown products
can be harmlessly
metabolized in the way that fatty acids and sugars are metabolized. Uptake of
the polymer with
antigen and adjuvant is safe: studies have shown that the APCs survive,
function normally, and can
metabolize/clear these polymer degradation products. The invention vaccine
delivery compositions
are, therefore, substantially non-inflammatory to the subject both at the site
of injection and
systemically, apart from the trauma caused by injection itself. Moreover, in
the case of active
uptake of polymer by APCs, the polymer may also act as an adjuvant for the
antigen.
[0057] The biodegradable polymers useful in forming the invention
biocompatible vaccine
delivery compositions include those comprising at least one amino acid
conjugated to at least one
non-amino acid moiety per monomer. The term "non-amino acid moiety" as used
herein includes
various chemical moieties, but specifically excludes amino acid derivatives
and peptidomimetics as
described herein. In addition, the polymers containing at least one amino acid
are not contemplated
to include polyamino acid segments, including naturally occurring
polypeptides, unless specifically
described as such. In one embodiment, the non-amino acid is placed between two
adjacent amino
acids in the monomer. In another embodiment, the non-amino acid moiety is
hydrophobic. The
polymer may also be a block co-polymer.
[0058] Preferred biodegradable polymers for use in the invention compositions
and methods are
polyester amides (PEAs) and polyester urethanes (PEURs), which have built-in
functional groups or
PEA or PEUR backbones, and these built-in functional groups can react with
other chemicals and le
to the incorporation of additional functional groups to expand the
functionality of PEA or PEUR
further. Therefore, for example, the polymers are ready for reaction with
peptidic/protein antigens,
adjuvants, and other agents, without the necessity of prior modification, or
with other molecules hav
a hydrophilic structure, such as PEG, to increase water solubility.
[0059] In addition, the polymers used in the invention vaccine delivery
compositions display no
hydrolytic degradation when tested in a saline (PBS) medium, but in an
enzymatic solution, such as
chymotrypsin or CT, a uniform erosive behavior has been observed.
[0060] In one embodiment the invention vaccine delivery composition comprises,
as the
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23
[0061] biodegradable polymer, at least one or a blend of the following:
a PEA having a chemical formula described by structural formula (I),
4 O 0 H O O H
C -R~ -C -N -C -C -O -R4 - O -C 11 -C -N
H R3 R3 H
n
Formula (I)
wherein n ranges from about 5 to about 150; R' is independently selected from
residues of a,w-
bis(4-carboxyphenoxy)-(C1-C$) alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid
or4,4'-
(alkanedioyldioxy)dicinnamic acid, (C2 - C20) alkylene, or (Ca-Cao)
alkenylene; the R3s in
individual n monomers are independently selected from the group consisting of
hydrogen, (CI-C6)
alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-C10) aryl (CI -C20) alkyl, and -
(CH2)2S(CH3); and R4 is
independently selected from the group consisting of (C2-C20) alkylene, (CZ-
C20) alkenylene, (C2-Cg)
alkyloxy, (C2-C20) alkylene, a residue of a saturated or unsaturated
therapeutic diol, bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of structural formula (II), and
combinations thereof, (C2
-
C20) alkylene, and (C2-C2o) alkenylene;
\
CH O
HZC; \CH2
O CH
Formula (II)
or a PEA having a chemical formula described by structural forrnula III:
lb O 0 H O O H 0 0 Fi
" 1 " ' " 4 " ' " 1 " 13
C-R -C-N-C-C-O-R -O-C-C-N C-R -C-N-C-R -N
H R3 R3 H m H C-O-Ra H p
O ' n
Formula (III)
wherein n ranges from about 5 to about 150, m ranges about 0.1 to 0.9: p
ranges from about 0.9 to
0.1; wherein R' is independently selected from residues of a,o)-bis(4-
carboxyphenoxy)-(Ci-C$)
alkane, 3,3'-(alkanedioyldioxy)dicinnamic acid or 4,4'-
(alkanedioyldioxy)dicinnamic acid, (C2 -
C20) alkylene, or (C2-C20) alkenylene; each RZ is independently hydrogen, (CI-
C1Z) alkyl or (C6-C10)
aryl or a protecting group; the R3s in individual m monomers are independently
selected from the
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group consisting of hydrogen, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl,
(C6-Clo)aryl (C1-
C20) alkyl, and -(CH2)2S(CH3); and R4 is independently selected from the group
consisting of (C2-
C20 alkylene, (C2-C20) alkenylene, (C2-C$) alkyloxy, (C2-Cao) alkylene, a
residue of a saturated or
unsaturated therapeutic diol or bicyclic-fragments of 1,4:3,6-
dianhydrohexitols of structural
formula (II), and combinations thereof; and R13 is independently (C1-C20)
alkyl or (C2-C20) alkenyl;
or a PEUR having a chemical formula described by structural formula (IV),
1 0 H O O H
L C-0-R6-0-C-N-6-C-O-R4-0-C-6-N
H R3 R3 !i
n
Formula IV
wherein n ranges from about 5 to about 150; wherein R3s in independently
selected from the group
consisting of hydrogen, (CI-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-
Cto) aryl (CI-CZO)
alkyl, and -(CH2)2S(CH3); R4 is selected from the group consisting of (C2-C20)
alkylene, (C2-C20)
alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic
diol, bicyclic-fragments
of 1,4:3,6-dianhydrohexitols of structural formula (II); and combinations
thereof, and R6 is
independently selected from (C2-C2o) aikylene, (C2-Cao) alkenylene or
alkyloxy, bicyclic-fragments
of 1,4:3,6-dianhydrohexitols of general formula (II), and combinations
thereof;
or a PEUR having a chemical structure described by general structural formula
(V)
IFO B O H O 4 O H U s O 0 H 13
C-O-R -O-C-N-C-G-O-R -O-C-C-N C-O-R -O-C-N-C-R -N
3 3
R R H m H C-O-R2 H p
U n
Formula (V)
wherein n ranges from about 5 to about 150, m ranges about 0.1 to about 0.9: p
ranges from about
0.9 to about 0.1; R2 is independently selected from hydrogen, (C6-C1o) aryl
(CI-C2o) alkyl, or a
protecting group; the R3s in an individual m monomer are independently
selected from the group
consisting of hydrogen, (Ci-C6) alkyl, (CZ-C(,) alkenyl, (C2-C6) alkynyl, (C6-
Clo) aryl (CI-CZO) alkyl
and -(CH2)2S(CH3); R4 is selected from the group consisting of (C2-C20)
alkylene, (C2-C20)
alkenylene or alkyloxy, a residue of a saturated or unsaturated therapeutic
diol and bicyclic-
fragments of 1,4:3,6-dianhydrohexitols of structural formtila (II) and
combinations thereof; and R6
is independently selected from (C2-Cao) alkylene, (C2-C2o) alkenylene or
alkyloxy, bicyclic-
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fragments of 1,4:3,6-dianhydrohexitols of general formula (II), an effective
amount of a residue of
a saturated or unsaturated therapeutic diol, and combinations thereof, and R13
is independently (CI -
C2o) alkyl or (C2-C20) alkenyl, for example, (C3-C6) alkyl or (C3-C6) alkenyl;
or a PEU having a chemical formula described by general structural formula
(VI):
O H O O H
C-N-6-8-O-R4-O-8-C-N
H R3 R3 H n
Formula (VI),
wherein n is about 10 to about 150; the R3s within an individual n monomer are
independently
selected from hydrogen, (CI-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6 -
CIo) aryl (Ct-C20)
alkyl and -(CH2)2S(CH3); R4 is independently selected from (C2-C20) alkylene,
(CZ-CZo) alkenylene,
*(C2-C8) alkyloxy (C2-C2o) alkylene, a residue of a saturated or unsaturated
therapeutic diol; or a
bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of structural formula (II);
or a PEU having a chemical formula described by structural formula (VII)
IFO H O O H 0 H
6-N-6-C-O-R4-O-C-6-N C-N-C-R13-N
i~ 1 3~ I ' e
H R R H }{ O-0-R2 H
m
O Fn
Formula (VII),
wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about
10 to about 150; each R2
is independently hydrogen, (CI-CiZ) alkyl or (C6-Clo) aryl; the R3s within an
individual m monomer
are independently selected from hydrogen, (Ci-C6) alkyl, (C2-C6) alkenyl, (C2-
C6) alkynyl, (C6 -
Clo) aryl (CI -C2o) alkyl and--(CH2)2S(CH3); eachR~ is independently selected
from (C2-C20)
alkylene, (C2-C2o) alkenylene, (C2-Cs) alkyloxy (C2-Czo) alkylene, a residue
of a saturated or
unsaturated therapeutic diol; a bicyclic-fragment of a 1,4:3,6-
dianhydrohexitol of structural formula
(II), and combinations thereof, and R13 is independently (CI-C2o) alkyl or (C2-
C20) alkenyl, for
example, (C3-C6) alkyl or (CJ-C6) alkenyl.
[0062] For example, in one alternative in the PEA polymer used in the
invention particle
delivery composition, at least one R' is a residue of a,cobis(4-
carboxyphenoxy) (C1-C8) alkane,
3,3'-(alkanedioyldioxy)dicinnamic acid, or 4,4'-(alkanedioyldioxy)dicinnamic
acid and R4 is a
bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of general formula(II). In
another alternative, R' in
the PEA polymer is either a residue of a,w-bis (4-carboxyphenoxy) (CI-Cs)
alkane, 3,3'--
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26
(alkanedioyldioxy)dicinnamic acid,or 4,4'-(alkanedioyldioxy)dicinnamic acid.
In yet another
alternative, in the PEA polymer R' is a residue a,w-bis (4-carboxyphenoxy) (CI-
Cs) alkane, such as
1,3-bis(4-carboxyphenoxy)propane (CPP), 3,3'-(alkanedioyldioxy)dicinnamic acid
or 4,4'-
(adipoyldioxy)dicinnamic acid and R4 is a bicyclic-fragment of a 1,4:3,6-
dianhydrohexitol of
general formula (iI), such as DAS.
[0063] In one altemative in the PEUR polymer, at least one of R~ is a bicyclic
fragment of
1,4:3,6-dianhydrohexitol (formula (II)), such as 1,4:3,6-dianhydrosorbitol
(DAS); or R6 is a
bicyclic fragment of 1,4:3,6-dianhydrohexitol, such as 1,4:3,6-
dianhydrosorbitol (DAS). In still
alternative in the PEUR polymer, R~ and/or R6 is a bicyclic fragment of
1,4:3,6-dianhydrohexitot,
such as 1,4:3,6-dianhydrosorbitol (DAS).
[0063] These PEU polymers can be fabricated as high molecular weight polymers
useful for
making the invention vaccine delivery compositions for delivery to humans and
other mammals of
a variety of pharmaceutical and biologically active agents. The invention PEUs
incorporate
hydrolytically cleavable ester groups and non-toxic, naturally occurring
monomers that contain a.-
arnino acids in the polymer chains. The ultimate biodegradation products of
PEUs will be a-amino
acids (whether biological or not), diols, and CO2. In contrast to the PEAs and
PEURs, the invention
PEUs are crystalline or semi-crystalline and possess advantageous mechanical,
chemical and
biodegradation properties that allow formulation of completely synthetic, and
hence easy to
produce, crystalline and semi-crystalline polymer particles, for example
nanoparticles.
[00641 For example, the PEU polymers used in the invention vaccine delivery
compositions
have high mechanical strength, and surface erosion of the PEU polymers can be
catalyzed by
enzymes present in physiological conditions, such as hydrolases.
[0065] In one alternative in the PEU polymer, at least one R' is a bicyclic
fragment of a 1,4:3,6-
dianhydrohexitol, such as 1,4:3,6-dianhydrosorbitol (DAS).
[0066] Suitable protecting groups for use in practice of the invention include
t-butyl and others
as are known in the art. Suitable bicyclic-fragments of 1,4:3,6-
dianhydrohexitols can be derived
from sugar alcohols, such as D-glucitol, D-mannitol, and L-iditol.
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27
[0067] For example, 1,4:3,6-dianhydrosorbitol (isosorbide, DAS) is
particularly suited for use
as a bicyclic-fragment of 1,4:3,6-dianhydrohexitol.
[0068] In one alternative, the R3s in at least one n monomer are CH2Ph and the
a-amino acid
used in synthesis is L-phenylalanine. In alternatives wherein the R3s within a
monomer are -CH2-
CH(CH3)2, the polymer contains the a-amino acid, leucine. By varying the R3s,
other a-amino
acids can also be used, e.g., glycine (when the R3s are -H), proline (when the
R3s are ethylene
amide); alanine (when the R3s are -CH3), valine (when the R3s are -CH(CH3)2),
isoleucine (when
the R3s are -CH(CH3)-CH2-CH3), phenylalanine (when the R3s are -CH2-C6H5);
lysine (when the
R3s are -(CH2)4NH2); or methionine (when the R3s are -(CHZ)2S(CH3).
[0069] In yet a further embodiment wherein the polymer is a PEA, PEUR or PEU
of formula I
or III-VII, at least one of the R3s further can be -(CH2)3- and the at least
one of the R3s cyclizes to
form the chemical structure described by structural formula (XVIII):
H O
N-C-C-O-
H2C,CCH2
H2
Formula (VIII)
When the R3s are -(CH2)3, an a-imino acid analogous to pyrrolidine-2-
carboxylic acid (proline) is
used.
[0070] The PEAs, PEURs and PEUs are biodegradable polymers that biodegrade
substantially
by enzymatic action so as to release the dispersed peptidic/protein antigen
and adjuvant over time.
Due to structural properties of these polymers, the invention vaccine delivery
compositions provide
for stable loading of the peptidic/protein antigens and adjuvants while
preserving the three
dimensional structure thereof and, hence, the bioactivity.
[0071] As used herein, the tenns "amino acid" and "a-amino acid" mean a
chemical compound
containing an amino group, a carboxyl group and a pendent R group, such as the
R3 groups defined
herein. As used herein, the term "biological a-amino acid" means the amino
acid(s) used in
synthesis are selected from phenylalanine, leucine, glycine, alanine, valine,
isoleucine, methionine,
proline, or a mixture thereof.
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28
[0072] In the PEA, PEUR and PEU polymers useful in practicing the invention,
multiple
different a-amino acids can be employed in a single polymer molecule. These
polymers may
comprise at least two different amino acids per repeat unit and a single
polymer molecule may
contain multiple different a-amino acids in the polymer molecule, depending
upon the size of the
molecule. In one alternative, at least one of the a-amino acids used in
fabrication of the invention
polymers is a biological a-amino acid.
[0073] For example, when the R3s are CH2Ph, the biological a-amino acid used
in synthesis is
L-phenylalanine. In alternatives wherein the R3s are CH2-CH(CH3)2, the polymer
contains the
biological a-amino acid, L-leucine. By varying the R3s within co-monomers as
described herein,
other biological a-amino acids can also be used, e.g., glycine (when the R3s
are H), alanine (when
the R3s are CH3), valine (when the R3s are CH(CH3)2), isoleucine (when the R3s
are CH(CH3)-
CH2-CH3), phenylalanine (when the R3s are CH2-C6HS), or methionine (when the
R3s are -
(CH2)2S(CH3), and mixtures thereof. When the R3s are -(CH2)3- as in 2-
pyrrolidinecarboxylic acid
(proline), a biological a-imino acid can be used. ln yet another alternative
embodiment, all of the
various a-amino acids contained in the invention vaccine delivery compositions
are biological a-
amino acids, as described herein.
100741 The polymer molecules may have the peptidic/protein antigen conjugated
thereto via a
linker or incorporated into a crosslinker between molecules. For example, in
one embodiment, the
polymer is contained in a polymer-antigen conjugate having structural formula
IX:
11O 1 0 H O 4 O H 0 0 H
C-R -C-N-C-C-O-R-O-C-C-N C-R,-6-N-6-R13-N
H R3 R3 H tI H
m H C=O p n
R5
R7
Formula (IX)
wherein n, M. p, R1, R3, and R4 are as above, RS is selected from the group
consisting of-O-,
-S-, and -NR$-, wherein R8 is H or (Ci-C$)alkyl; and R7 is the
peptidic/protein antigen.
[0075] In yet another embodiment, two molecules of the polymer of structural
formula (IX) can
be crosslinked to provide an -RS-R7-RS-conjugate. In another embodiment, as
shown in structural
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formula X below, the peptidic/protein antigen is covalently linked to two
parts of a single polymer
molecule of structural formula IV through the -R5-R7-R5- conjugate and RS is
independently
selected from the group consisting of -0-, -S-, and -NR8-, wherein R8 is H or
(Ci-C8) alkyl; and R7
is the peptidic/protein antigen.
O 0 H O O H 0 0 H
" " 1 " " 1 11 '
C-R 1 -C-N-C-C-O-R 4 -O-C-C-NmC-R1-CI' -N-C-R 13-N
H R3 R3 H H C=0 H p
R7
RS
IF O~ 0 O H O O H 0
N-R 7a C-N-C-R 1-C N-C-C-O-R 4 =O-C-C-N-C-R C
6 HH p HR3 R3H m
Formula (X)
[00761 Alternatively still, as shown in structural formula (XI) below, a
linker, -X-Y-, can be
inserted between RS and peptidic/protein antigen R7, in the molecule of
structural formula (VIII),
wherein X is selected from the group consisting of (Ci-Cts) alkylene,
substituted alkylene, (C3-C$)
cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system
containing 1-3
heteroatoms =selected from the group 0, N, and S, substituted heterocyclic,
(C2-C 18) alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, C6 and CI aryl,
substituted aryl, heteroaryl,
substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl,
substituted arylalkynyl,
arylalkenyl,=substituted arylalkenyl, arylalkynyl, substituted arylalkynyl and
wherein the
substituents are selected from the group H, F, Cl, Br, I, (CI-C6) alkyl, -CN, -
NO2, -OH, -O(CI-Ca)
alkyl, -S(CI-C6) alkyl, -S[(=0)(CI-C6) alkyl], -S[(02)(Ci-C6) alkyl], -
C[(=O)(CI-C6) alkyl], CF3,-
O[(CO)-( C1-C6) alkyl], -S(02)[N(R?R 0)], -NH[(C=0)(Ct-C6) alkyl],
-NH(C=0)N(R9R1 ), -N(R9R'0); where R9 and Rl0 are independently H or (CI-C6)
alkyl; and Y is
selected from the group consisting of -0-, -S-, -S-S-, -S(O)-,-S(02)-, -NR$-, -
C(=O)-,
-OC(=O)-, -C(=0)O-, -OC(=0)NH-, -NRBC(=O)-, -C(=0)NRg-, -N RSC(=O)NR$-,
-N R$C(=0)NRS-, and -NR$C(=S)N R$-.
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11O, 0 H O 4 Q H 0 M1 0 H 13
C-R -C-N-C-C-O-R -O-C-C-N C-R -C-N-C-R -N
R3 R3 H I
m H C=0 H p
RS
I
X
Y
RT
Formula (XI)
[00771 In another embodiment, two parts of a single peptidic/protein antigen
are covalently
linked to the bioactive agent through an RS-IC-Y-X- RS-bridge (Formula XII):
O 0 H O O H 0 0 H
u ~ u i n u i n 1
C-R -C-N-C-C-O-R 4 -O-C-C-N C-R I-C-N-C-R 93-N
H R3 R3 H ~1 H
m H C=O p
R5
R7 Y X
R5
13 O~ O O H Q 4 O H O 10
N-R C-N-C-R1-C N-C-C-O-R =O-C-C-N-6-R C
H H N p FI R3 R3 H m
Formula (XII)
wherein, X is selected from the group consisting of (Ct-Ci$) alkylene,
substituted alkylene, (C3-C8)
cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system
containing 1-3
heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (CZ-
Ci$) alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, (C6 - Clo) aryl,
substituted aryl, heteroaryl,
substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl,
substituted arylalkynyl,
arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl,
wherein the substituents
are selected from the group consisting of H, F, Cl, Br, I, (C1-C6) alkyl, -CN,
-NO2,, -OH, -O(CI-C6) alkyl, -S(Ci-C6) alkyl, -S[(=0)(CI-C6) alkyl], -
S[(OZ)(CI-C6) alkyl],
-C[(=0)(C}-C6) alkyl], CF3,-O[(CO)-(Ci-C6) alkyl], -S(02)[N(R9Rl0)], -
NH[(C=O)(C1-C6) alkyl], -
NH(C=0)N(R9R10), wherein R9 and Rl0 are independently H or (CI-C6) alkyl, and -
N(R11R12),
wherein R' 1 and R12 are independently selected from (C2-C20) alkylene and (C2-
C20) alkenylene.
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31
[0078] In yet another embodiment, four molecules of the polymer are linked
together, except
that only two of the four molecules omit W and are crosslinked to provide a
single
-RS-X-R5- conjugate.
[0079] The term " aryl" is used with reference to structural formulas herein
to denote a phenyl
radical or an ortho-fused bicyclic carbocyclic radical having about nine to
ten ring atoms in which
at least one ring is aromatic. In certain embodiments, one or more of the ring
atoms can be
substituted with one or more of nitro, cyano, halo, trifluoromethyl, or
trifluoromethoxy. Examples
of aryl include, but are not limited to, phenyl, naphthyl, and nitrophenyl.
[0080] Theterm "alkenylene" is used with reference to structural formulas
herein to mean a
divalent branched or unbranched hydrocarbon chain containing at least one
unsaturated bond in the
main chain or in a side chain.
[0081] As used herein, a "therapeutic diol" means any diol molecule, whether
synthetically
produced, or naturally occurring (e.g., endogenously) that affects a
biological process in a*
mammalian individual, such as a human, in a therapeutic or palliative manner
when administered to
the mammal
[0082] As used herein, the term "residue of a therapeutic diol" means a
portion of a therapeutic
diol, as described herein, which portion excludes the two hydroxyl groups of
the diol. The
corresponding therapeutic diol containing the "residue" thereof is used in
synthesis of the polymer
compositions. The residue of the therapeutic diol is reconstituted in vivo (or
under similar
conditions of pH, aqueous media, and the like) to the corresponding diol upon
release from the
backbone of the polymer by biodegradation in a controlled manner that depends
upon the properties
of the PEA, PEUR or PEU polymer selected to fabricate the composition, which
properties are as
known in the art and as described herein.
100831 Due to the versatility of the PEA, PEUR and PEU polymers used in the
invention
compositions, the amount of the therapeutic diol incorporated in the polymer
backbone can be
controlled by varying the proportions of the building blocks of the polymer.
For example,
depending on the composition of the PEA, loading of up to 40% w/w of 17(3-
estradiol can be
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32
achieved. Three different regular, linear PEAs with various loading ratios of
170-estradiol are
illustrated in Scheme 1 below:
"homopoly"-bis-Leu-Estradiol-Adipate (40% w/w -estradiol on polymer)
CH(CH3)2 CH(CH3)Z
HZG O
CH2
O-C-C-NH-C-(CH2)4-6N-C-C-O q/ 0 H O
H H O
n
Copolymer: Leu(ED)3Lys(OEt)Adip4 with 38% w/w estradiol loading
CH(CH3)2 CH(CH3)Z
' H2C O
CH2 - O-C-C-NH-G-(CHZ)4-C
HH'CO' -O ~~ OH O
3n
O H H
C-(CHZ)4-C-N-(CH2)4 C-NH
~ COOEt
In
Scheme 1
Similarly, the loading of the therapeutic diol into PEUR and PEU polymer can
be varied by varying
the amount of two or more building blocks of the polymer.
[0084] In addition, synthetic steroid based diols based on testosterone or
cholesterol, such as 4-
androstene-3, 17 diol (4-Androstenediol), 5-androstene-3, 17 diol (5-
Androstenediol), 19-nor5-
androstene-3, 17 diol (19-Norandrostenediol) are suitable for incorporation
into the backbone of
PEA and PEUR polymers according to this invention. Moreover, therapeutic diol
compounds
suitable for use in preparation of the invention vaccine delivery compositions
include, for example,
amikacin; amphotericin B; apicycline; apramycin; arbekacin; azidamfenicol;
bambermycin(s);
butirosin; carbomycin; cefpiramide; chloramphenicol; chlortetracycline;
clindamycin;
clomocycline; demeclocycline; diathymosulfone; dibekacin, dihydrostreptomycin;
dirithromycin;
doxycycline; erythromycin; fortimicin(s); gentamycin(s); glucosulfone
solasulfone; guamecycline;
isepamicin; josamycin; kanamycin(s); leucomycin(s); lincomycin; lucensomycin;
lymecycline;
meclocycline; methacycline; micronomycin; midecamycin(s); minocycline;
mupirocin; natamycin;
neomycin; netilmicin; oleandomycin; oxytetracycline; paromycin; pipacycline;
podophyllinic acid
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33
2-ethylhydrazine; primycin; ribostamycin; rifamide; rifampin; rafamycin SV;
rifapentine;
rifaximin; ristocetin; rokitamycin; rolitetracycline; rasaramycin;
roxithromycin; sancycline;
sisomicin; spectinomycin; spiramycin; streptomycin; teicoplanin; tetracycline;
thiamphenicol;
theiostrepton; tobramycin; trospectomycin; tuberactinomycin; vancomycin;
candicidin(s);
chlorphenesin; dermostatin(s); filipin; fungichromin; kanamycin(s);
leucomycins(s); lincomycin;
lvicensomycin; lymecycline; meclocycline; methacycline; micronomycin;
midecamycin(s);
minocycline; mupirocin; natamycin; neomycin; netilmicin; oleandomycin;
oxytetracycline;
paramomycin; pipacycline; podophyllinic acid 2-ethylhydrazine; priycin;
ribostamydin; rifamide;
rifampin; rifamycin SV; rifapentine; rifaximin; ristocetin; rokitamycin;
rolitetracycline;
rosaramycin; roxithromycin; sancycline; sisomicin; spectinomycin; spiramycin;
strepton;
otbramycin; trospectomycin; tuberactinomycin; vancomycin; candicidin(s);
chlorphenesin;
dermostatin(s); filipin; fungichromin; meparticin; mystatin; oligomycin(s);
erimycin A; tubercidin;
6-azauridine; aclacinomycin(s); ancitabine; anthramycin; azacitadine;
bleomycin(s) carubicin;
carzinophillin A; chlorozotocin; chromomcin(s); doxifluridine; enocitabine;
epirubicin;
gemcitabine; mannomustine; menogaril; atorvasi pravastatin; clarithromycin;
leuproline; paclitaxel;
mitobronitol; mitolactol; mopidamol; nogalamycin; olivomycin(s); peplomycin;
pirarubicin;
prednimustine; puromycin; ranimustine; tubercidin; vinesine; zorubicin;
coumetarol; dicoumarol;
ethyl biscoumacetate; ethylidine dicoumarol; iloprost; taprostene;
tioclomarol; amiprilose;
romurtide; sirolimus (rapamycin); tacrolimus; salicyl alcohol; bromosaligenin;
ditazol; fepradinol;
gentisic acid; glucamethacin; olsalazine; S-adenosylmethionine; azithromycin;
salmeterol;
budesonide; albuteal; indinavir; fluvastatin; streptozocin; doxorubicin;
daunorubicin; plicamycin;
idarubicin; pentostatin; metoxantrone; cytarabine; fludarabine phosphate;
floxuridine; cladriine;
capecitabien; docetaxel; etoposide; topotecan; vinblastine; teniposide, and
the like. The therapeutic
diol can be selected to be either a saturated or an unsaturated diol.
[0085] The molecular weights and polydispersities herein are determined by gel
permeation
chromatography (GPC) using polystyrene standards. More particularly, number
and weight
average molecular weights (Mõ and M,,,) are determined, for example, using a
Model 510 gel
permeation chromatography (Water Associates, Inc., Milford, MA.) equipped with
a high-pressure
liquid chromatographic pump, a Waters 486 UV detector and a Waters 2410
differential refractive
index detector. Tetrahydrofuran (THF), N,N-dimethylformamide (DME) or N,N-
dimethylacetamide (DMAc) is used as the eluent (1.0 mL/rnin). Polystyrene or
poly(methyl
methacrylate) standards having narrow molecular weight distribution were used
for calibration.
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[0086] As used herein, the terms "amino acid" and "a-amino acid" mean a
chemical compound
containing an amino group, a carboxyl group and a pendent R group, such as the
R3 groups defined
herein. As used herein, the term "biological a-amino acid" means the amino
acid(s) used in
synthesis are selected from phenylalanine, leucine, glycine, alanine, valine,
isoleucine, methionine,
or a mixture thereof.
[0087] Methods for making the polymers of structural formulas (I) and (III-
VII), containing an
a-amino acid in the general formula are well known in the art. For example,
for the embodiment of
the polymer of structural formula (I) wherein R4 is incorporated into an a-
amino acid, for polymer
synthesis the a-amino acid with pendant R3 can be converted through
esterification into a bis-a,eo-
diamine, for example, by condensing the a-amino acid containing pendant R3
with a diol HO-R4-
OH. As a result, di-ester monomers with reactive a,co-amino groups are formed.
Then, the bis-
a,w-diamine is entered into a polycondensation reaction with a di-acid such as
sebacic acid, or its
bis-activated esters, or bis-acyl chlorides, to obtain the final polymer
having both ester and amide
bonds (PEA). Alternatively, for PEUR, instead of the di-acid, a di-carbonate
derivative, formula
(XIII), is used, where R6 is defined above and R14 is independently (C6-
Cjo)aryl, optionally
substituted with one or more of nitro, cyano, halo, trifluoromethyl or
trifluoromethoxy.
0 0
11 11 R 14-O-CO-R'-O-CO-R ~ 4
Formula (XIII)
[0088] More particularly, synthesis of the unsaturated poly(ester-amide)s
(UPEAs) useful as
biodegradable polymers of the structural formula (I) as disclosed above will
be described, wherein
0 O O H
(a) 11 Rl~-- -s Ae Clir
H o
and/or (b) R4 is -CHZ-CH=CH-CHZ- . In cases where (a) is present and (b) is
not present, R4 in (I)
is -C4118- or -C6H 12-. In cases where (a) is not present and (b) is present,
R1 in (I) is -C4H$- or -
C8H16-.
[0089] The UPEAs can be prepared by solution polycondensation of either (1) di-
p-toluene
sulfonic acid salt of bis (alpha-amino acid) diesters, comprising at least 1
double bond in R4, and di-
p-nitrophenyl esters of saturated dicarboxylic acid or (2) di-p-toluene
sulfonic acid salt of bis
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(alpha-amino acid) diesters, comprising no double bonds in R4, and di-
nitrophenyl ester of
unsaturated dicarboxylic acid or (3) di-p-toluene sulfonic acid salt of
bis(alpha-amino acid)
diesters, comprising at least one double bond in R4, and di-nitrophenyl esters
of unsaturated
dicarboxylic acids.
[0090] Salts of p-toluene sulfonic acid are known for use in synthesizing
polymers containing
amino acid residues. The aryl sulfonic acid salts are used instead of the free
base because the aryl
sulfonic salts of bis (alpha-amino acid) diesters are easily purified through
recrystallization and
render the amino groups as unreactive ammonium tosylates throughout workup. In
the
polycondensation reaction, the nucleophilic amino group is readily revealed
through the addition of
an organic base, such as triethylamine, so the polymer product is obtained in
high yield.
[0091] The di-p-nitrophenyl esters of unsaturated dicarboxylic acid can be
synthesized from p-
nitrophenol and unsaturated dicarboxylic acid chloride, e.g., by dissolving
triethylamine and p-
nitrophenol in acetone and adding unsaturated dicarboxylic acid chloride drop
wise with stirring at
-78 C and pouring into water to precipitate product. Suitable acid chlorides
useful for this purpose
include fumaric, maleic, mesaconic, citraconic, glutaconic, itaconic, ethenyl-
butane dioic and 2-
propenyl-butanedioic acid chlorides.
[0092] The di-aryl sulfonic acid salts of bis(alpha-amino acid) diesters can
be prepared by
admixing alpha-amino acid, p-aryl sulfonic acid (e.g. p-toluene sulfonic acid
monohydrate), and '
saturated or unsaturated diol in toluene, heating to reflux temperature, until
water evolution is
minimal, then cooling. The unsaturated diols useful for this purpose include,
for example,
2-butene-1,3-dioland 1,18-octadec-9-en-dio1.
[0093] Saturated di-p-nitrophenyl esters of dicarboxylic acids and saturated
di-p-toluene
sulfonic acid salts of bis(alpha-amino acid) di-esters can be prepared as
described in U. S. Patent
No. 6,503,538 B1.
[0094] Synthesis of the unsaturated poly(ester-amide)s (UPEAs) useful as
biodegradable
polymers of the structural formula (I) as disclosed above will now be
described. UPEAs having
the structural formula (I) can be made in similar fashion to the compound
(VII) of U. S. Patent No.
6,503,538 Bl, except that R4 of (III) of 6,503,538 and/or Rl of (V) of
6,503,538 is (C2-C20)
alkenylene as described above. The reaction is carried out, for example, by
adding dry
triethylamine to a mixture of said (III) and (IV) of 6,503,538 and said (V) of
6,503,538 in dry N,N-
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36
dimethylacetamide, at room temperature, then increasing the temperature to 80
C and stirring for
16 hours, then cooling the reaction solution to room temperature, diluting
with ethanol, pouring into
water, separating polymer, washing separated polymer with water, drying to
about 30 C under
reduced pressure and then purifying up to negative test on p-nitrophenol and p-
toluene sulfonate. A
preferred reactant (IV) is p-toluene sulfonic acid salt of Lysine benzyl
ester, the benzyl ester
protecting group is preferably removed from (II) to confer biodegradability,
but it should not be
removed by hydrogenolysis as in Example 22 of U.S. Patent No. 6,503,538
because hydrogenolysis
would saturate the desired double bonds; rather the benzyl ester group should
be converted to an
acid group by a method that would preserve unsaturation. Alternatively, the
lysine reactant (IV)
can be protected by a protecting group different from benzyl that can be
readily removed in the
finished product while preserving unsaturation, e.g., the lysine reactant can
be protected with t-
butyl (i.e., the reactant can be t-butyl ester of lysine) and the t-butyl can
be converted to H while
preserving unsaturation by treatment of the product (II) with acid.
100951 A working example of the compound having structural formula (I) is
provided by
substituting p-toluene sulfonic acid salt of bis(L-phenylalanine) 2-butene-1,4-
diester for (III) in
Example 1 of 6,503,538 or by substituting di-p-nitrophenyl fumarate for (V) in
Example 1 of
6,503,538 or by substituting p-toluene sulfonic acid salt of L-phenylalanine 2-
butene-l,3-diester for
III in Example 1 of 6,503,538 and also substituting de-p-nitrophenyl fumarate
for (V) in Example 1
of 6,503,538.
[0096] In unsaturated polymers having either structural formula (I) or (III),
the following hold:
Aminoxyl radical e.g., 4-amino TEMPO, can be attached using
carbonyldiimidazol, or suitable
carbodiimide, as a condensing agent. Peptidic/protein antigens, adjuvants and
peptidic
antigen/adjuvant conjugates, as described herein, can be attached via the
double bond functionality.
Hydrophilicity can be imparted by bonding to poly(ethylene glycol) diacrylate.
100971 In yet another aspect, polymers contemplated for use in forming the
invention vaccine
delivery systems include those set forth in U.S. Patent Nos. 5,516, 881;
6,476,204; 6,503,538; and
in U.S. Application Nos. I0/096,435; 10/101,408; 10/143,572; and 10/194,965;
the entire contents
of each of which is incorporated herein by reference.
[0098] The biodegradable PEA, PEUR and PEU polymers and copolymers may contain
up to
two amino acids per monomer, multiple amino acids per polymer molecule, and
preferably have
weight average molecular weights ranging from 10,000 to 125,000; these
polymers and copolymers
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37
typically have intrinsic viscosities at 25 C, determined by standard
viscosimetric methods, ranging
from 0.3 to 4.0, for example, ranging from 0.5 to 3.5,
[0099] PEA and PEUR polymers contemplated for use in the practice of the
invention can be
synthesized by a variety ofinethods well known in the art. For example,
tributyltin (IV) catalysts
are commonly used to form polyesters such as poly(E-caprolactone),
poly(glycolide), poly(lactide),
and the like. However, it is understood that a wide variety of catalysts can
be used to form
polymers suitable for use in the practice of the invention.
[0100] Such poly(caprolactones) contemplated for use have an exemplary
structural formula
(XIV) as follows:
O
4o__(c H2)s
Formula (XIV)
[0101) Poly(glycolides) contemplated for use have an exemplary structural
formula (XV) as
follows:
O H
I+
O-C-C
H n
Formula (XV)
[0102] Poly(lactides) contemplated for use have an exemplary structural
formula (XVI) as
follows:
O Me
O-C-C
H n
Formula (XVI)
[0103] An exemplary synthesis of a suitable poly(lactide-co-c-caprolactone)
including an
aminoxyl moiety is set forth as follows. The first step involves the
copolymerization of lactide and
e-caprolactone in the presence of benzyl alcohol using stannous octoate as the
catalyst to form a
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polymer of structural formula (XVII).
O O
Me\O O
~ ~ CH2OH + n 0 n1 ----
Me+
O
O H O
O-CH20 C-C-O C-(CHZ)5-O H
Me n m
Formula (XVII)
[0104] The hydroxy terminated polymer chains can then be capped with maleic
anhydride to
form polymer chains having structural formula (XVIII):
O H 0 0 0
O-CH20 C-C-O C-(CH2)5 O C-C=C-C-OH
Me n m H H
Formula (XVIII)
[0105] At this point, 4-amino-2,2,6,6-tetramethylpiperidine- 1 -oxy can be
reacted with the
carboxylic end group to covalently attach the aminoxyl moiety to the copolymer
via the amide bond
which results from the reaction between the 4-amino group and the carboxylic
acid end group.
Alternatively, the maleic acid capped copolymer can be grafted with
polyacrylic acid to provide
additional carboxylic acid moieties for subsequent attachment of further
aminoxyl groups.
[0106] In unsaturated polymers having structural formula (VII) for PEU the
following hold:
An amino substituted aminoxyl (N-oxide) radical bearing group e.g., 4-amino
TEMPO, can be
attached using carbonyldiimidazole, or suitable carbodiimide, as a condensing
agent. Additional
bioactive agents, and the like, as described herein, optionally can be
attached via the double bond.
[0107] For example, the invention high molecular weight semi-crystalline PEUs
having
structural formula (VI) can be prepared inter-facially by using phosgene as a
bis-electrophilic
monomer in a chloroform/water system, as shown in the reaction scheme (2)
below:
1. NaZCO31 HZO
H 0 H 2. CICOCI / CHCI3
HOTos.H2N-C-C-O-R4-O-C-C-NH2.TosOH - (VI)
R I 3 R3
Scheme (2)
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Synthesis of copoly(ester ureas) (PEUs) containing L-Lysine esters and having
structural formula
(VII) can be carried out by a similar scheme (3):
H O O H H
mHOTos.H2N-C-C-O-R4-O-C-C-NH2.TosOH + p HOTos.H2N-C-(CH2)4-NH2.TosOH
R3 R3 C-O-R2
0
1. Na2CO3 / H20
2. CICOCI / CHCIg ( VIII )
Scheme (3)
A 20% solution of phosgene (C1COC1) (highly toxic) in toluene, for example
(commercially
available (Fluka Chemie, GMBH, Buchs, Switzerland), can be substituted either
by diphosgene
(trichloromethylchloroformate) or triphosgene (bis(trichloromethyl)carbonate).
Less toxic
carbonyldiimidazole can be also used as a bis-electrophilic monomer instead of
phosgene, di-
phosgene, or tri-phosgene.
General Procedure for Synthesis of PEUs
[01081 It is necessary to use cooled solutions of monomers to obtain PEUs of
high molecular
weight. For example, to a suspension of di-p-toluenesulfonic acid salt of
bis(a-amino acid)-a,to-
alkylene diester in 150 mL of water, anhydrous sodium carbonate is added,
stirred at room
temperature for about 30 minutes and cooled to about 2 - 0 C, forming a first
solution. In parallel,
a second solution of phosgene in chloroform is cooled to about 15 -10 C. The
first solution is
placed into a reactor for interfacial polycondensation and the second solution
is quickly added at
once and stirred briskly for about 15 min. Then chloroform layer can be
separated, dried over
anhydrous Na2SO4, and filtered. The obtained solution can be stored for
further use.
[01091 All the exemplary PEU polymers fabricated were obtained as solutions in
chloroform
and these solutions are stable during storage. However, some polymers, for
example, 1-Phe-4,
become insoluble in chloroform after separation. To overcome this problem,
polymers can be
separated from chloroform solution by casting onto a smooth hydrophobic
surface and allowing
chloroform to evaporate to dryness. No further purification of obtained PEUs
is needed. The yield
and characteristics of exemplary PEUs obtained by this procedure are
summarized in Table 2
herein.
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General Procedure for Preparation of porous PEUs.
[0110] Methods for making the PEU polymers containing a-amino acids in the
general formula
will now be described. For example, for the embodiment of the polymer of
formula (I) or (II), the
a-amino acid can be converted into a bis-(a-amino acid)-a,co-diol-diester
monomer, for example,
by condensing the a-amino acid with a diol HO-R'-OH. As a result, ester bonds
are formed.
Then, acid chloride of carbonic acid (phosgene, diphosgene, triphosgene) is
entered into a
polycondensation reaction with a di-p-toluenesulfonic acid salt of a bis-(a-
amino acid) -alkylene
diester to obtain the final polymer having both ester and urea bonds.
j01111 The unsaturated PEUs can be prepared by interfacial solution
condensation of di-p-
toluenesulfonate salts of bis-(a-amino acid)-alkylene diesters, comprising at
least one double bond
in Rl. Unsaturated diols useful for this purpose include, for example, 2-
butene-1,4-diol and 1,18-
octadec-9-en-diol. Unsaturated monomer can be dissolved prior to the reaction
in alkaline water
solution, e.g. sodium hydroxide solution. The water solution can then be
agitated intensely, under
extemal cooling, with an organic solvent layer, for example chloroform, which
contains an
equimolar amount of monomeric, dimeric or trimeric phosgene. An exothermic
reaction proceeds
rapidly, and yields a polymer that (in most cases) remains dissolved in the
organic solvent. The
organic layer can be washed several times with water, dried with anhydrous
sodium sulfate,
filtered, and evaporated. Unsaturated PEUs with a yield of about 75%-85% can
be dried in
vacuum, for example at about 45 C.
[0112] To obtain a porous, strong material, L-Leu based PEUs, such as 1-L-Leu-
4 and 1-L-
Leu-6, can be fabricated using the general procedure described below. Such
procedure is less
successful in formation of a porous, strong material when applied to L-Phe
based PEUs.
[0113] The reaction solution or emulsion (about 100 mL) of PEU in chloroform,
as obtained
just after interfacial polycondensation, is added dropwise with stirring to
1,000 mL of about 80 C -
85 C water in a glass beaker, preferably a beaker made hydrophobic with
dimethyldichlorsilane to
reduce the adhesion of PEU to the beaker's walls. The polymer solution is
broken in water into
small drops and chloroform evaporates rather vigorously. Gradually, as
chloroform is evaporated,
small drops combine into a compact tar-like mass that is transformed into a
sticky rubbery product.
This rubbery product is removed from the beaker and put into hydrophobized
cylindrical glass-test-
tube, which is thermostatically controlled at about 80 C for about 24 hours.
Then the test-tube is
removed from the thermostat, cooled to room temperature, and broken to obtain
the polymer. The
obtained porous bar is placed into a vacuum drier and dried under reduced
pressure at about 80 C
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41
for about 24 hours. In addition, any procedure known in the art for obtaining
porous polymeric
materials can also be used.
101141 Properties of high-molecular-weight porous PEUs made by the above
procedure yielded
results as summarized in Table 2.
Table 2 Properties of PEU Polymers of Formula (VI) and (VII)
PEU* Y[e1d lfred miv b) Mn b) M"jMn b) Tg c) Tm c)
[%j [dL/gl t C1 i C1
1-L-Leu-4 80 0.49 84000 45000 1.90 67 103
1-L-Leu-6 82 0.59 96700 50000 1.90 64 126
1-L-Phe-6 77 0.43 60400 34500 1.75 - 167
[1-L-Leu-6]0.75- [1-L- 84 0.31 64400 43000 1.47 34 114
Lys(O$n)]o.25
X-L-Leu-DAS 57 0.28 55700d) 27700a) 2.1a) 56 165
*PEUs of general formula (VI), where,
1-L-Leu-4: R4 = (CH2)4, R3 = i-C4H9
1-L-Leu-6: R4 = (CH2)6, R3 = 1-C4H9
1-L-Phe-6:.R4 = (CHa)6, R3 -CH2-C6Hs=
1-L-Leu-DAS: R4 = 1,4:3,6-dianhydrosorbitol, R' = i-CQH
a) Reduced viscosities were n3easured in DMF at 25 C and a concentration 0.5
g/dL
b) GPC Measurements were carried out in DMF, (PMMA)
) Tg taken from second heating curve from DSC Measurements (heating ratel0
Clmin).
d) GPC Measuren3ents were carried out in DMAc, (PS)
[0115] Tensile strength of illustrative synthesized PEUs was measured and
results are
summarized in Table 3. Tensile strength measurement was obtained using
dumbbell-shaped PEU
films (4 x 1.6 cm), which were cast from chloroform solution with average
thickness of 0.125 mm
and subjected to tensile testing on tensile strength machine (Chatillon
TDC200) integrated with a
PC using Nexygen FM software (Amtek, Largo, FL) at a crosshead speed of 60
mm/min.
Examples illustrated herein can be expected to have the following mechanical
properties:
100010] 1. A glass transition temperature in the range fronn about 30 C to
about 90 C ,
for example, in the range from about 35 C to about 70 C ;
2. A film of the polymer with average thickness of about 1.6 cm will have
tensile stress
at yield of about 20 Mpa to about 150 Mpa, for example, about 25 Mpa to about
60 Mpa;
3. A fiIm of the polymer with average thickness of about 1.6 cm will have a
percent
elongation of about 10 % to about 200%, for example about 50 % to about 150%;
and
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42
4. A film of the polymer with average thickness of about 1.6 cm will have a
Young's
modulus in the range from about 500 MPa to about 2000 MPa. Table 2 below
summarizes the
properties of exemplary PEUs of this type.
[00011] Table 3 Mechanical Properties of PEUs
aO Tensile Stress Young's
Polymer designation Tg at Yield Percent Modulus
( C) (MPa) Elongation (%) (MPa)
I-L-Leu-6 64 21 114 622
1-L-Leu-6 o.7s- 1-L-L s OBn o.2s 34 25 159 915
[0116] The various components of the invention vaccine delivery composition
can be present in
a wide range of ratios. For example, the polymer repeating unit:antigen are
typically used in a ratio
of 1:50 to 50:1, for example 1:10 to 10:1, about 1:3 to 3:1, or about 1:1.
However, other ratios may
be more appropriate for specific purposes, such as when a particular antigen
is both difficult to
incorporate into a particular polymer and has a low immunogenicity, in which
case a higher relative
amount of the peptidic/protein antigen is required.
[0117] In certain embodiments, the invention vaccine delivery composition
described herein can
be provided as particles, with peptidic/protein antigen-adjuvant conjugate, or
antigens and
adjuvants either physically incorporated (dispersed) within the particle or
attached to polymer
functional groups, optionally by use of a linker, using any of several
techniques well known in the
art and as described herein. The particles are sized for uptake by APCs,
having an average
diameter, for example, in the range from about 10 nanometers to about 1000
microns, or in the
range from about 10 nanometers to about 10 microns. Optionally, the particles
can further
comprise a thin covering of the polymer to aid in control of their
biodegradation. Typically such
particles include from about 5 to about 150 peptidic/protein antigens per
polymer molecule.
[0118] The PEA, PEUR and PEU polymers used in the invention vaccine delivery
compositions, biodegrade by enzymatic action at the surface. Therefore, the
polymers, for example
particles thereof, administer the -antigen and adjuvant to the subject at a
controlled release rate,
which is specific and constant over a prolonged period.
[0119] As used herein, "biodegradable" as used to describe a polymer in the
invention vaccine
delivery compositions means the polymer is capable of being broken down into
innocuous products
in the normal functioning of the body. In one embodiment, the entire vaccine
delivery composition
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43
is biodegradable. The preferred biodegradable polymers have hydrolyzable ester
linkages that
provide the biodegradability, and are typically chain terrninated
predominantly with amino groups.
101201 As used herein "dispersed" means a peptidic/protein antigen or adjuvant
as disclosed
herein is dispersed, mixed, dissolved, homogenized, and/or covalently bound
("dispersed" or
loaded) in the polymer, which may or may not be formed into particles.
101211 While the peptidic/protein antigens and adjuvants can be dispersed
within the polymer
matrix without chemical linkage to the polymer carrier, it is also
contemplated that the antigen
and/or antigen-adjuvant conjugate can be covalently bound to the biodegradable
polymers via a
wide variety of suitable functional groups. For example, when the
biodegradable polymer is a
polyester, the carboxyl group chain end can be used to react with a
complimentary moiety on the
antigen or adjuvant, such as hydroxy, amino, thio, and the like. A wide
variety of suitable reagents
and reaction conditions are disclosed, e.g., in March's Advanced Organic
Chemistry, Reactions,
Mechanisms, and Structure, Fifth Edition, (2001); and Comprehensive Organic
Transformations,
Second Edition, Larock (1.999).
[0122] In other embodiments, an antigen and/or adjuvant can be linked to any
of the polymers
of structures (I) or (III-VII) through an amide, ester, ether, amino, ketone,
thioether, sulfinyl,
sulfonyl, disulfide linkage. Such a linkage can be formed from suitably
funetionalized starting
materials using synthetic procedures that are known in the art.
[0123] For example, in one embodiment a polymer can be linked to the
peptidic/protein antigen
or adjuvant via an end or pendent carboxyl group (e.g., COOH) of the polymer.
Specifically, a
compound of structures III, V and VII can react with an amino functional group
or a hydroxyl
functional group of a peptidic/protein antigen to provide a biodegradable
polymer having the
peptidic/protein antigen attached via an amide linkage or carboxylic ester
linkage, respectively. In
another embodiment, the carboxyl group of the polymer can be transformed into
an acyl halide,
acyl anhydride/"mixed" anhydride, or active ester. In other embodiments, the
free -1VH2 ends of
the polymer molecule can be acylated to assure that the peptidic/protein
antigen will attach only via
a carboxyl group of the polymer and not to the free ends of the polymer. For
example, the
invention vaccine delivery composition described herein can be prepared from
PEA, PEUR, or
PEU where the N-terminal free amino groups are acylated, e.g., with anhydride
RCOOCOR, where
the R=(CI -CZ4) alkyl, to assure that the bioactive agent will attach only via
a carboxyl group of the
polymer and not to the free ends of the polymer.
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[0124] Alternatively, the peptidic/protein antigen or adjuvant may be attached
to the polymer
via a linker molecule, for example, as described in structural formulae (VIII -
XI). Indeed, to
improve surface hydrophobicity of the biodegradable polymer, to improve
accessibility of the
biodegradable polymer towards enzyme activation, and to improve the release
profile of the
biodegradable polymer, a linker may be utilized to indirectly attach the
peptidic/protein antigen
and/or adjuvant to the biodegradable polymer. In certain embodiments, the
linker compounds
include poly(ethylene glycol) having a molecular weight (Mw) of about 44 to
about 10,000,
preferably 44 to 2000; amino acids, such as serine; polypeptides with repeat
units from 1 to 100;
and any other suitable low molecular weight polymers. The linker typically
separates the
peptidic/protein antigen from the polymer by about 5 angstroms up to about 200
angstroms.
[0125] In still further embodiments, the linker is a divalent radical of
formula W-A-Q, wherein
A is (Cl-C24) alkyl, (C2-C24) alkenyl, (C2-C24) alkynyl, (C3-Cs) cycloalkyl,
or (C6-Cto) aryl, and W
and Q are each independently -N(R)C(=O)-, -C(=O)N(R)-, -OC(=O)-, -C(=O)O,
-0-, -S-, -S(O), -S(O)2-, -S-S-, -N(R)-, -C(=0)-, wherein each R is
independently H or (CI-
C6)alkyl.
[0126] As used to describe the above linkers, the term "alkyl" refers to a
straight or branched
chain hydrocarbon group including methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, tert-butyl,
n-hexyl, and the like.
[0127] As used herein, "alkenyl" as used to describe linkers refers to
straight or branched chain
hydrocarbon groups having one or more carbon-carbon double bonds.
[0128] As used herein, "alkynyl" as used to describe linkers refers to
straight or branched chain
hydrocarbon groups having at least one carbon-carbon triple bond.
[0129] As used herein, "aryl" as used to describe linkers refers to aromatic
groups having in the
range of 6 up to 14 carbon atoms.
[0130] In certain embodiments, the linker may be a polypeptide having from
about 2 up to about
25 amino acids. Suitable peptides contemplated for use include poly-L-lysine,
poly-L-glutamic
acid, poly-L-aspartic acid, poly-L-histidine, poly-L-ornithine, poly-L-
threonine, poly-L-tyrosine,
poly-L-leucine, poly-L-lysine-L-phenylalanine, poly-L-arginine, poly-L-lysine-
L-tyrosine, and the
like.
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[0131] In one embodiment, the peptidic/protein antigen can covalently
crosslink the polymer,
i.e. the antigen is bound to more than one polymer molecule. This covalent
crosslinking can be
done with or without additional polymer-antigen linker.
[0132] The peptidic/protein antigen molecule can also form an intramolecular
bridge by covalent
attachment between two parts of a single macromolecule.
[0133] A linear polymer peptide conjugate is made by protecting the potential
nucleophiles on
the antigen backbone and leaving only one reactive group to be bound to the
polymer or polymer
linker construct. Deprotection is performed according to well known in the art
deprotection of
peptides (Boc and Fmoc chemistry for example).
[01341 In one embodiment of the present invention, the peptidic antigen is
presented as retro-
inverso or partial retro-invers peptide.
[0135] In other embodiments the peptidic/protein antigen is mixed with a
photocrosslinkable
version of the polymer in a matrix, and after crosslinking the material is
dispersed (ground) to a
phagocytosable size, i.e. 0.1-l0 rn.
[0136] The linker can be attached first to the polymer or to the
peptidic/protein antigen or
adjuvant. During synthesis, the linker can be either in unprotected form or
protected from, using a
variety of protecting groups well known to those skilled in the art. In the
case of a protected linker,
the unprotected end of the linker can first be attached to the polymer or the
peptidic/protein antigen.
The protecting group can then be de-protected using Pd/Ha hydrogenolysis, mild
acid or base
hydrolysis, or any other common de-protection method that is known in the art.
The de-protected
linker can then be attached to the peptidic/protein antigen, adjuvant, or
adjuvant-antigen conjugate.
[0137] An exemplary synthesis of a biodegradable polymer according to the
invention (wherein
the molecule to be attached is an aminoxyl) is set forth as follows. A
polyester can be reacted with
an amino substituted N-oxide free radical (aminoxyl) bearing group, e.g., 4-
amino-2,2,6,6-
tetramethylpiperidine- I -oxy, in the presence ofN,N'-carbonyldiimidazole to
replace the carboxylic
acid moiety at the chain end of the polyester with an amide bond to the amino
substituted
aminoxyl-containing radical, so that the amino moiety covalently bonds to the
carbon of the
carbonyl residue of the carboxyl group of the polymer. The N,N'-carbonyl
diimidazole or suitable
carbodiimide converts the hydroxyl moiety in the carboxyl group at the chain
end of the polyester
into an intermediate product moiety that will react with the aminoxyl, e.g., 4-
amino-2,2,6,6-
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tetramethylpiperidine-l-oxy. The aminoxyl reactant is typically used in a mole
ratio of reactant to
polyester ranging from 1:1 to 100: l. The mole ratio of N,N'-carbonyl
diimidazole to aminoxyl is
preferably about 1:1.
[0138] A typical reaction is as follows. A polyester is dissolved in a
reaction solvent and
reaction is readily carried out at the temperature utilized for the
dissolving. The reaction solvent
may be any in which the polyester will dissolve. When the polyester is a
polyglycolic acid or a
poly(glycolide-L-lactide) (having a monomer mole ratio of glycolic acid to L-
lactic acid greater
than 50:50), highly refined (99.9+% pure) dimethyl sulfoxide at 115 C to 130
C or
dimethylsulfoxide (DMSO) at room temperature suitably dissolves the polyester.
When the
polyester is a poly-L-lactic acid, a poly-DL-lactic acid or a poly(glycolide-L-
lactide) (having a
monomer mole ratio of glycolic acid to L-lactic acid 50:50 or less than
50:50), tetrahydrofuran,
methylene chloride and chloroform at room temperature to 50 C suitably
dissolve the polyester.
Polymer / Antigen Linkage
[0139] In one embodiment, the polymers used to make the invention vaccine
delivery
compositions as described herein have at least one peptidic/protein antigen
directly linked to the
polymer. The residues of the polymer can be linked to the residues of the one
or more
peptidic/protein antigens. For example, one residue of the polymer can be
directly linked to one
residue of the peptidic/protein antigen. The polymer and the peptidic/protein
antigen can each have
one open valence. Alternatively, more than one peptidic/protein antigen,
multiple peptidic/protein
antigens, or a mixture of peptidic/protein antigens from different pathogenic
organisms can be
directly linked to the polymer. However, since the residue of each
peptidic/protein antigen can be
linked to a corresponding residue of the polymer, the number of residues of
the one or more
peptidic/protein antigens can correspond to the number of open valences on the
residue of the
polymer.
[0140] As used herein, a "residue of a polymer" refers to a radical of a
polymer having one or
more open valences. Any synthetically feasible atom, atoms, or functional
group of the polymer
(e.g., on the polymer backbone or pendant group) of the present invention can
be removed to
provide the open valence, provided bioactivity is substantially retained when
the radical is attached
to a residue of a peptidic/protein antigen. Additionally, any synthetically
feasible functional group
(e.g., carboxyl) can be created on the polymer (e.g., on the polyrner backbone
or pendant group) to
provide the open valence, provided bioactivity is substantially retained when
the radical is attached
to a residue of a peptidic/protein antigen. Based on the linkage that is
desired, those skilled in the
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art can select suitably functionalized starting materials that can be derived
from the polymer of the
present invention using procedures that are known in the art.
101411 As used herein, a "residue of a compound of structural formula (*)"
refers to a radical of
a compound of polymer of formulas (I) and (III-VII) as described herein having
one or more open
valences. Any synthetically feasible atom, atoms, or functional group of the
compound (e.g., on the
polymer backbone or pendant group) can be removed to provide the open valence,
provided
bioactivity is substantially retained when the radical is attached to a
residue of a peptidic/protein
antigen. Additionally, any synthetically feasible functional group (e.g.,
carboxyl) can be created on
the compound of formulas (I) and (III-VII) (e.g., on the polymer backbone or
pendant group) to
provide the open valance, provided bioactivity is substantially retained when
the radical is attached
to a residue of a peptidic/protein antigen. Based on the linkage that is
desired, those skilled in the
art can select suitably functionalized starting materials that can be derived
from the compound of
formula (I) and (III-VII) using procedures that are known in the art.
[0142] For example, the residue of a peptidic/protein antigen can be linked to
the residue of a
compound of structural formulas (I) and (III-VII) through an amide (e.g., -
N(R)C(=O)- or
-C(=0)N(R)-), ester (e.g., -OC(=0)- or-C(=O)O-), ether (e.g., -0-), amino
(e.g., -N(R)-), ketone
(e.g., -C(=O)-), thioether (e.g., -S-), sulfinyl (e.g., -S(O)-), sulfonyl
(e.g., -S(O)2-), disulfide (e.g., -
S-S-), or a direct (e.g., C-C bond) linkage, wherein each R is independently H
or P-C6) alkyl.
Such a linkage can be formed from suitably functionalized starting materials
using synthetic
procedures that are known in the art. Based on the linkage that is desired,
those skilled in the art
can select suitably functional starting material that can be derived from a
residue of a compound of
any one of structural formulas (I) and (III-VII) and from a given residue of a
peptidic/protein
antigen or adjuvant using procedures that are known in the art. The residue of
the peptidic/protein
antigen or adjuvant can be linked to any synthetically feasible position on
the residue of a
compound of any one of structural formulas (I) and (III-VII). Additionally,
the invention also
provides compounds having more than one residue of a peptidic/protein antigen
or adjuvant
bioactive agent directly linked to a compound of any one of structural
formulas (I) and (III-VII).
101431 The number of peptidic/protein antigens that can be linked to the
polymer molecule can
typically depend upon the molecular weight of the polymer. For example, for a
compound of
structural formulas (I) or (III), wherein n is about 5 to about 150,
preferably about 5 to about 70, up
to about 150 peptidic/protein antigens (i.e., residues thereof) can be
directly linked to the polymer
(i.e., residue thereof) by reacting the peptidic/protein antigen with end
groups of the polymer. In
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unsaturated polymers, the peptidic/protein antigens can also be reacted with
double (or triple)
bonds in the polymer.
[0144] The PEA, PEUR and PEU polymers described herein readily absorb water (5
to 25 %
w/w water up-take, on polymer film), allowing hydrophilic molecules to readily
diffuse through
them. This characteristic makes PEA, PEUR and PEU polymers suitable for use as
an over coating
on particles to control release rate. Water absorption also enhances
biocompatibility of the
polymers and the vaccine delivery composition based on such polymers. In
addition, due to the
hydrophilic properties of the PEA, PEUR and PEU polymers, when delivered in
vivo the particles
become sticky and agglomerate, particularly at in vivo temperatures. Thus the
polymer particles
spontaneously form polymer depots when injected subcutaneously or
intramuscularly for local
delivery, such as by subcutaneous needle or needle-less injection. Particles
with average diameter
range from about 1 micron to about 100 microns, of a size that will not permit
circulation in the
body, are suitable for forming such polymer depots in vivo. AlteYnatively, for
oral administration,
the GI tract can tolerate much larger particles, for example micro particles
of about 1 micron up to
about 1000 microns average diameter.
[0145] For instance, typically, the polymer depot will degrade over a time
selected from about
twenty-four hours, about seven days, about thirty days, or about ninety days,
or longer. Longer
time spans are particularly suitable for providing an implantable vaccine
delivery composition that
eliminates the need to repeatedly inject the vaccine to obtain a suitable
immune response.
Preparation of Recombinant Protein or Peptidic Antigen
[0146] Techniques for recombinant production of heterologous polypeptides,
including peptide
and protein antigens, in unicellular organisms are well known in the art and
do not bear extensive
description in this application. For example, the preparation of the peptidic
antigens, peptide or
protein adjuvants, and fusion proteins used in the practice of this invention
can be carried out using
standard recombinant DNA methods. Preferably, a nucleotide sequence coding for
the desired
affinity peptide is first synthesized and then linked to a nucleotide sequence
coding for the His tag.
[0147] The thus-obtained hybrid gene can be incorporated into expression
vectors such as
plasmid pDS8/R.BSII, Sphl; pDS5/RBSII,3A+5A; pDS78/RBSII; pDS56/RBSII, or
other
commercial or generally accessible plasmids, using standard methods. Most of
the requisite
methodology can be found in Maniatis et al., "Molecular Cloning", Cold Spring
Harbor Laboratory,
2005, which illustrates the state of the art.
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10148] Methods for the expression of the fusion proteins of this invention are
also described by
Maniatis et al., supra. They embrace the following procedures: (a)
Transformation of a suitable
host organism, for example E. coli, with an expression vector in which the
hybrid gene is
operatively linked to an expression control sequence; (b) Cultivation of the
transformed host
organism under suitable growth conditions; and (c) Extraction and isolation of
the desired fusion
protein from the host organism. Host organisms that can be used include but
are not limited to
gram-negative and gram-positive bacteria, such as E. coli and B. subtilis
strains. E. c li strain M15
is especially preferred. Other E. coli strains that can be used include, e.g.,
E. coli 294 (ATCC No.
3144), E. coli RRI (ATCC No. 31343) and E. coli W31 10 (ATCC No. 27325).
Three methods to selectively capture peptidic or protein antigens and
adjuvants from a
recombinant cell lysate
[0149] For production of large quantities of antigens and adjuvants by
recombinant gene
technologies, coding regions for the proteins are integrated into artificial
genes which are replicated
and expressed in bacteria, usually E. Coli, or in a virus, such as
baculovirus, which replicates in host
insect cells. The same of a different unicellular organism may be used for
expression of
peptidic/protein antigen and the peptide or protein adjuvant, as described
herein. Whichever
method is used, the final cell colonies, containing many copies of the
expressed proteins, have to be
lysed so as to release the cell contents. In another embodiment, a fraction of
a cell extract or cell
lysate that has been enriched in the peptidic/protein antigen or peptide or
protein adjuvant may be
formed using methods well known in the art. The over-expressed antigen and/or
adjuvant must
then be selectively removed from the cell lysate, extract or enriched fraction
thereof for subsequent
incorporation into a vaccine construct.
[0150] Three methods are described here for the selective capture of target
peptidic/protein
molecules from cell lysate according to the invention methods. PEA and PEUR
polymers of
structural formulas III and IV, respectively, have been used to both capture
the target
peptidic/protein molecules and, simultaneously, to form the core of the
vaccine preparation. The
polymer is mixed directly with fresh lysate, cell extract, or an enriched
fraction thereof, resulting in
formation of a antigen-polymer complex. The process involved requires
contacting together a
solution or dispersion comprising 1) at least one Class I or Class II peptidic
antigen or a whole
protein antigen, wherein the antigen has been expressed by a unicellular
organism containing at
least one recombinant vector comprising a DNA sequence encoding the antigen;
and 2) a synthetic
biodegradable polymer comprising at least one or a blend of polymers having a
chemical formula
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described by Formulas (I) and (III-VII) to which has been attached an affinity
ligand that binds
specifically to the peptidic/protein antigen. The contacting is conducted
under conditions, as
described herein, in which a complex is formed that incorporates the polymer,
the affinity ligand,
and the peptidic/protein antigen. Optionally, the solution or dispersion may
also contain a peptide
or protein adjuvant that has been expressed by the same or a different
unicellular organism from a
DNA sequence encoding the peptide or protein adjuvant.
[0151] Optionally, a peptide or protein adjuvant-polymer complex can be
similarly formed.
Because there is a protein-capture point on every repeat unit of these PEA and
PEUR polymers, the
peptidic antigen-polymer complex and/or adjuvant-polymer complex molecules are
of sufficiently
high molecular mass that they can be removed from the protein and other
compounds in the
remaining liquid medium (e.g. cell lysate) by size-filtration.
[0152] Oligomerization This method may be used to capture antigenic proteins
that naturally
form oligomers. Examples are the functional trimer of hemaglutinin (HA) and
the tetramer of
neuraminidase (NA) from influenza A virus.
(0153] Previously prepared target antigen protomer is conjugated to repeat
units of the polymer:
The protomer-polymer complex is mixed with lysate under batch conditions that
promote
oligomerization of the antigenic proteins. The resulting oligomer-polymer
complex is removed
from the remaining filtrate by size-filtration. A more complete description of
preparation of the
invention vaccine delivery compositions by the oligomerization technique is
contained in U.S.
Patent application Serial No. 11/345,021, filed January 31, 2006.
[0154] Antibody (Ab) recognition This method may be used to capture antigenic
proteins
against which humanized monoclonal antibody molecules or active fragments
thereof (MAbs or
FAbs) have been pre-prepared, for example, as described herein.
[0155] Previously prepared MAb or FAb molecules against target antigen are
conjugated to
repeat units of the polymer, either directly using amide bond or cysteine-
maleimide bond
formation, or indirectly via polymer-conjugated Ab-binding protein domains,
such as protein A or
protein G. The Ab-polymer complex is mixed with lysate under batch conditions
that promote
antibody binding. The resulting antigen-Ab-polymer complex is removed from the
remaining
filtrate by size-filtration.
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[0156] Metal agz'nit,y complex fornzation Pre-functionalization of repeat
units of the polymer
with suitable metal affinity ligands may be performed by (A) an imidazole
derivative, or (B) an
NTA derivative, such as nitrilotriacetic acid (NTA) or iminodiacetic acid
(IDA), as follows:
Polymer-Affinity Ligand Linkage
[0157] The affinity ligands are directly conjugated to the biodegradable
polymers via a wide
variety of suitable functional groups. For example, when the biodegradable
polymer is a polyester,
the carboxyl group chain end can be used to react with a complimentary moiety
on the affinity
ligand (e.g., the one or more free amino groups, on the metal affinity ligand
NTA or IDA). A wide
variety of suitable reagents and reaction conditions are disclosed, e.g., in
March's Advanced
Organic Chemistry, Reactions, Mechanisms, and Structure, Fifth Edition,
(2001); and
Comprehensive Organic Transformations, Second Edition, Larock (1999).
[0158] In other embodiments, the affinity ligand can be linked to any of the
polymers of
structures (I) or (III-VII) through a free amide, ester, ether, amino, ketone,
thioether, sulfinyl,
sulfonyl, disulfide linkage. Such a linkage can be formed from suitably
functionalized starting
materials using synthetic procedures that are known in the art. For example,
in one embodiment the
polymer can be linked to the metal affinity ligand via an end or pendent
carboxyl group (e.g.,
COOH) of the polymer. Specifically, the metal affinity ligand used in the
invention methods can
react with a polymer with an amino functional group or a hydroxyl functional
group of the polymer,
such as those described by structural formulas III, V and VII, while leaving
free binding sites for
forming a coordination complex with a metal transition ion and metal binding
amino acids of a
peptidic antigen to provide a biodegradable polymer having the peptidic
antigen non-covalently
attached to the polymer via a metal affinity complex. In another embodiment,
the carboxyl group
of the polymer can be transformed into an acyl halide, acyl anhydride/"mixed"
anhydride, or active
ester. In other embodiments, the free -NH2 ends of the polymer molecule can be
acylated to assure
that the affinity ligand will attach only via a carboxyl group of the polymer
and not to the free ends
of the polymer. For example, the invention vaccine delivery composition
described herein can be
prepared from PEA, PEUR, or PEU where the N-terminal free amino groups are
acylated, e.g., with
anhydride RCOOCOR, where the R=(CI-CZ4) alkyl, to assure that the antigenic
protein or peptidic
antigen will attach only via an affinity complex formed at a carboxyl group of
the polymer and not
to the free ends of the polymer.
[0159] For example, in one embodiment, side-chain protected lysine (e.g.OBu-
Lys) is
conjugated via an amide bond to the activated carboxylate on the repeat unit
of the PEA, PEUR or
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PEU polymer of structural formulas III, IV or VII. Following de-protection,
the free amino groups
of these lysine residues are modified by reacting with a metal affinity
ligand, such as 2-
imidazolecarboxaldehyde.
[0160] A transition metal (TM) selected from Fea+, Cu2+, or Niz+ is then bound
to the metal
affinity ligand, e.g., 2-imidazolecarboxaldehyde. The resulting TM-derivatized
polymer is bio-
functionalized via the bound TM(II) with a genetically expressed protein
bearing a hexa-histidine
extension.
[0161] The strength of the metal affinity complexes formed varies according to
the number of
His and Trp in the peptide and the ions used. The metal ions used in practice
of the invention are
nickel (Ni2+) copper (Cu2{) zinc (Zn2+) and cobalt (Co2+). In general, the
strength of binding of the
peptidic antigen or fusion protein incorporating the peptidic antigen to the
metal ion decreases in
the following order: Cu2+ > Ni2+ > Co2+ > Zn2+.
[0162] The metal affinity ligands suitable for use in the invention methods
for preparing a
vaccine delivery composition include nitrilotriacetic acid (NTA) and
iminodiacetic acid (IDA).
NTA is a tetra-dentate metal affinity ligand known to bind to a variety of
transition metals with
stability constants of 109 to 1014. The stability constant remains high due to
the presence of
multiple free metal coordination sites therein after the NTA is conjugated to
available functional
groups in the polymer. When iminodiacetic acid (IDA) is used as the metal
affinity ligand, a
bidentate chelating moiety, to which a metal ion can be coordinated, remains
free after binding to
the polymer. Various metal ions can be coordinated via these bound metal
affinity ligands so that
free coordination sites on the metal ions in turn are free to bind to metal
binding amino acids in the
peptidic antigen. Because free functional groups are located along the
flexible polymer chains used
in the invention methods, the metal ion can be arranged in the best position
relative to the binding
sites on the surface of the peptidic antigen. As a result, the peptidic
antigens can be bound tightly,
yet non-covalently, to the polymer via the metal affinity complex formed.
[0163] The existence of at least one histidine residue in the antigenic
protein, peptidic antigen,
or fusion peptide comprising the peptidic antigen and a His tag, is an
important factor for the
binding of the antigen to the polymer. However, with the short peptidic
antigens used in the
invention methods and compositions, the alpha amino groups present also play a
role so that in
some cases the peptidic antigens can also be attached if no histidine residues
are present, especially
if other metal binding amino acids, such as cysteine and tryptophane, are
present in the peptidic
antigen to contribute to the binding. Since the pK value of the histidine
groups, contributing to the
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binding, lies in the neutral range, the binding of the peptidic antigen to the
polymer might be
expected to occur at a pH value of about 7. However, the actual pK value of an
individual amino
acid can vary strongly depending on the influence of neighboring amino acid
residues. Various
experiments have shown that depending on the protein structure, the pK value
of an amino acid can
deviate from the theoretical pK value up to one pH unit. Therefore, a reaction
solution with a pH
value of about 8 often achieves an improved binding.
[0164] Despite these complexities in the interactions taking place during
formation of the metal
coordination complex, the number of Histidines or Tryptophanes in the peptidic
antigen or fusion
protein incorporating the peptidic antigen provide general guidelines for
selection of the metal ion
to be used are found in Table 4 below:
TABLE 4
Presence of metal binding AA Suitable metal ion
in peptidic antigen
No His or Trp no adsorption
One His Cu2}
More than one His CuZ+ or Ni2+ (stronger adsorption)
Clusters of 3 to 10 His Cu2+, Ni2+, Zna+, Co'+
Several Trp, no His Cu2+
pH, Buffers, and Ionic Strength
[0165] The conditions present in the reaction solution or dispersion affect
formation of the metal
affinity complex in the invention methods. In general, a pH value of about 8
results in stronger
binding than a lower pH of about 6. Buffering agents also affect binding, with
highest binding
occurring in acetate or phosphate, moderate binding occurring in ammonium or
Tris, and weakest
binding occurring in citrate. Control of ionic strength in the reaction
solution also affects complex
formation. NaCl in a concentration range of about 0.1M to aboutl.0 M, for
example between about
0.5M and about 0.9 M may be used to suppress undesirable protein-protein ionic
interactions.
[0166] The presence of other substances that also bind to the metal ions in
the reaction solution
or dispersion can prevent binding of the target protein. For example, high
imidazole concentrations
strongly influence the binding characteristics of the metal complex,
especially if the metal ion is
copper. At the same time, a decrease of the pH value of the reaction solution
results in adsorption
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of fewer of the available peptidic antigens from a complex mixture, such as a
cell lysate. In
addition, to prevent ionic interactions between proteins and polymer carboxy
groups that might
remain uncharged with the affinity complex, relatively high ionic strength
should be present. For
example, the presence of about 0.1 M to 1.0 M NaCl, for example 0.5 M to about
0.9 M NaCl in the
reaction solution or dispersion is sufficient to prevent undesirable protein
binding in the reaction
solution.
101671 Preferably, there is at least one His at the amino- or carboxyl-
terminus of the peptidic
antigen (i.e.., a His tag), which results in improved specificity of binding
of the peptidic antigen to
the metal ion in the metal affinity complex. Therefore, in one embodiment, at
least one to about 10
adjacent His residues, for example, about six His residues, are incorporated
at one or both the
amino- and carboxy termini as a tag to ensure binding efficiency. If a His tag
is added, the His tag
and the metal chelate, for example the Ni-NTA metal chelate, are allowed to
remain in the final
vaccine delivery composition.
[0168] Whether or not a His tag is added to the peptidic antigen used in the
invention methods,
the metal coordination complex and the polymer remain along with the peptidic
antigen in the
vaccine delivery composition so that the peptidic antigen is non-covalently
bound to the polymer
via the metal coordination complex in the final product. Thus, once the
coordination complex is
formed linking the polymer non-covalently to the peptidic antigen, with or
without the presence of
a His tag, all that is required to yield the vaccine product from the reaction
solution is separation of
the complex that constitutes the vaccine delivery composition from other
materials and proteins the
reaction solution or dispersion. A simple procedure such as size-exclusion
filtration, or
centrifugation and washing techniques, for example as is known in the art and
described herein can
be used for this purpose.
[0169] For example, the affinity ligand N-(5-Amino-l-
carboxypentyl)iminodiacetic acid
(Aminobutyl- , or AB- NTA),
~C02H
Hz~l N __,COzH
CO2H
can be conjugated directly, via an amide bond, to the activated carboxylate on
the repeat unit of the
polymer. A transition metal (TM) as above is then bound to the chelating -NTA.
The resulting TM-
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derivatized polymer is contacted with cell lysate for bio-functionalization
via the bound TM with a
genetically expressed peptidic antigen bearing a His-containing tag, e.g., a
hexa-His tag.
[0170] For example, a complex between hexa-His tagged peptidic antigen or full
length
antigenic protein and TM-functionalized polymer can, under suitable metal
affinity complex
forming conditions as described herein, create cross-linked protein-polymer
complexes, because
only two Histidines of each hexaHis tag bind preferentially to each chelation
point of the transition
metal ion. Relative to lysate macromolecules, the large size of these cross-
linked protein-polymer
complexes, within a range controlled by stoichiometry, facilitates filtration
by size-exclusion to
separate the complexes from other proteins and compounds in the reaction
mixture.
101711 Accordingly, in one embodiment, the invention provides high efficiency
one-step
methods for preparing a vaccine delivery composition based on interaction of a
metal affinity
ligand, which is pre-conjugated to the polymer, and a metal transition ion,
which binds specifically
to free sites on metal-binding amino acids, especially tryptophane (Trp) and
histidine (His), in the
peptidic/protein antigen, and optionally in a peptide or protein adjuvant.
Typically, the metal ion
used is NiZ+ and the peptidic target(s) to be separated from the liquid medium
is in the form of a
fusion protein in which a His-containing tag (e.g., a hexa-His tag) is
attached to the carboxy
terminus of each of the peptidic target(s).
[0172] For use in the invention one-step method in this embodiment, the
polymer is prepared in
advance by attaching the metal affinity ligand thereto as described herein and
using methods known
in the art. The metal affinity ligand is also preloaded with the metal ion
before the prepared
polymer is contacted with the solution or dispersion containing the peptidic
target(s) for separation
therefrom. The solution or dispersion can be a lysate or extract of one or
more unicellular
organisms which have been engineered to express a fusion protein containing
the peptidic antigen
or a peptidic adjuvant with a His-containing tag. Alternatively, the solution
or dispersion can be a
fraction of such a lysate or extract that has been enriched in the one or more
fusion proteins.
[0173] A peptide or protein adjuvant may be simultaneously or separately
incorporated into the
invention vaccine delivery composition using these techniques. For example, a
peptide or protein
adjuvant may be expressed by the same or a separate unicellular organism
transformed with a
vector containing a DNA sequence encoding a peptide or protein adjuvant or a
fusion protein
encoding a peptide or protein adjuvant with attached specifically binding tag
(e.g., a His-containing
tag. In this case, the solution or dispersion contacted may contain fractions
of cell lysate or extract
that have been enriched as to each of the peptidic target molecules. Upon
contact of the loaded
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polymer (to which has been attached the affinity ligand (e.g., a metal
affinity ligand loaded with an
appropriate metal ion as described herein), separate affinity complexes formi
for incorporation of
each of the two peptidic targets.
In another embodiment, the immunostimulatory adjuvant, whether drug, polymer,
biologic, or the
like, is not expressed into the liquid medium, but is preattached to the
polymer, either by a
functional group of the polymer or by a linker as described herein (no
affinity tag is necessary in
this embodiment for the adjuvant). Then, upon contact of the preloaded polymer
with an
expression cell lysate, extract, or enriched fraction thereof containing the
expressed target peptidic
antigen, the invention vaccine delivery composition forms in one step, i.e.,
by formation of a
complex that incorporates the polymer (e.g., with attached immunostimulatory
adjuvants), the
affinity ligand, the metal ion and the peptidic antigen. Alternatively still,
an immunostimulatory
adjuvant can be loaded or matrixed into the polymer carrier (without direct
attachment or inclusion
into an affinity complex), preferably after formation of the antigen-
containing affinity complex and
separation of the composition from other components in the liquid medium.
Alternatively still, an
immunostimulatory adjuvant can be incorporated into the invention vaccine
delivery composition
when the composition is formulated in polymer particles, as described below.
[0174] Using the same one-step method discussed above, a target peptide or
protein (other than
a synthetic peptidic antigen) that contains free metal-binding amino acids can
be separated from
any liquid solution or dispersion in which the target peptide or protein has
been sufficiently
enriched. Preferably, the target peptide or protein will be a fusion protein
containing a His-
containing tag and the target peptide or protein will be separated from the
other contents in the
liquid solution or dispersion by formation of an affinity complex containing
the target peptide or
protein, the affinity ligand and a polymer as described herein, which has been
prepared by
preattachment of an affinity ligand that binds specifically with the free
metal-binding amino acids
in His-containing tag. This one-step method of selectively binding a target
peptide or protein can
be used to separate the target from an expression cell lysate, extract, or
fraction thereof that has
been =enriched in the target protein, using methods that are well known in the
art (e.g., in Methods in
Enzymology. Guide to Protein Purification, Vol. 182 (1990) and Protein
Purification Principles
and Practice, Third Edition (1994)). The target peptide or protein will be
thus obtained by a
method using conditions gentle enough to prevent destruction of the biological
activity of the
peptide or protein.
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[0175] The invention vaccine delivery compositions, whether made by the one-
step method
from expressed peptidic/protein antigens and adjuvants, or not, can be
formulated as polymer
particles. Particulate formulations of the invention vaccine delivery
compositions can be made
using immiscible solvent techniques. Generally, these methods entail the
preparation of an
emulsion of two immiscible liquids. A single emulsion method can be used to
make polymer
particles that incorporate hydrophobic adjuvantand peptidic antigens, or
conjugates thereof. In the
single emulsion method, molecules to be incorporated into the particles are
mixed with polymer in
solvent first, and then emulsified in water solution with a surface
stabilizer, such as a surfactant. In
this way, polymer particles with hydrophobic adjuvant, peptidic antigen, or
adjuvant/peptidic
antigen conjugates are formed and suspended in the water solution, in which
hydrophobic
conjugates in the particles will be stable without significant elution into
the aqueous solution, but
such molecules will elute into body tissue, such as muscle tissue.
[0176] Most biologics, including synthetic peptidic antigens, are hydrophilic.
A double
emulsion method can be used to make polymer particles with liquid or
hydrophilic adjuvant and/or
antigens dispersed within. In the double emulsion method, liquid or
hydrophilic adjuvant and/or
antigens dissolved in water are emulsified in polymer solution first, and the
whole emulsion is put
into water to emulsify again to form particles with an external polymer
coating and liquid
adjuvant/peptidic antigens in the interior of the particles. Surfactant can be
used in both methods of
emulsification to prevent particle aggregation. Chloroform or dichloromethane
(DCM), which are
not miscible in water, are used as solvents for PEA and PEUR polymers, but
later in the preparation
the solvent is removed, using methods known in the art.
[0177] For certain peptidic antigens or adjuvants with low water solubility,
however, these two
emulsion methods have limitations. In this context, "low water solubility"
means an active agent
that is less hydrophobic than truly lipophilic drugs, such as Taxol, but which
is less hydrophilic than
truly aqueous-soluble drugs, such as many biologics. These types of
intermediate compounds are
too hydrophilic for high loading and stable matrixing into single emulsion
particles, yet are too
hydrophobic for high loading and stability within double emulsions. In such
cases, a polymer layer
is coated on to particles made of polymer and drugs with low water solubility,
by three
emulsification process. This method provides relatively low drug loading (-10%
w/w), but
provides structure stability and controlled drug release rate.
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[0178] The first emulsion is made by mixing the active agents into a polymer
solution and
emulsifying the mixture in a water solution with surfactant or lipid, such as
di-
(hexadecanoyl)phosphatidylcholine (DHPC; a short-chain derivative of a natural
lipid). In this
way, particles containing the active agents are formed and suspended in water
to form the first
emulsion. The second emulsion is formed by putting the first emulsion into a
polymer solution, and
emulsifying the mixture, so that water drops with the polymer/drug particles
inside are formed
within the polymer solution. Water and surfactant or lipid will separate the
particles and dissolve
the particles in the polymer solution. The third emulsion is then formed by
putting the second
emulsion into water with surfactant or lipid, and emulsifying the mixture to
form the final particles
in water. The resulting particle structure, as illustrated in Fig. 1 will have
one or more particles
made with polymer plus peptidic antigen and adjuvant at the center, surrounded
by water and
surface stabilizer, such as surfactant or lipid, and covered with a pure
polymer shell. Surface
stabilizer and water will prevent solvent in the polymer coating from
contacting the particles inside
the coating and dissolving them.
[0179] To increase loading of active agents, such as the peptidic antigen or
adjuvant, by the
triple emulsion method, active agents with low water solubility can be coated
with surface
stabilizer in the first emulsion, without polymer coating and without
dissolving the active agent
in water. In this first emulsion, water, surface stabilizer and active agent
have similar volume or
in the volume ratio range of (1 to 3):(0.2 to about 2):1, respectively. In
this case, water is used,
not for dissolving the active agent, but rather for protecting the active
agent with help of surface
stabilizer. Then the double and triple emulsions are prepared as described
above (Fig. 1)
[0180] Many emulsification techniques will work in making the emulsions used
in manufacture
of the particles. However, the presently preferred method of making the
emulsion is by using a
solvent that is not miscible in water. The emulsifying procedure consists of
dissolving polymer
with the solvent, mixing with adjuvant/peptidic antigen molecule(s), putting
into water, and then
stirring with a mixer and/or ultra-sonicator. Particle size can be controlled
by controlling stir speed
and/or the concentration of polymer, adjuvant/peptidic antigen molecule(s),
and surface stabilizer.
Coating thickness can be controlled by adjusting the ratio of the second to
the third emulsion. In
any of the methods of particle formation described above, the antigenic
peptide and adjuvant can
form a coating on the surface of the particles by conjugation to the polymers
in the particles after
particle formation.
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[01811 Suitable emulsion stabilizers may include nonionic surface active
agents, such as
mannide monooleate, dextran 70,000, polyoxyethylene ethers, polyglycol ethers,
and the like, all
readily commercially available from, e.g., Sigma Chemical Co., St. Louis, Mo.
The surface active
agent will be present at a concentration of about 0.3% to about 10%,
preferably about 0.5% to about
8%, and more preferably about 1% to about 5%.
[0182] Rate of release of the adjuvant/peptidic antigen from the compositions
can be controlled
by adjusting the coating thickness, number of antigens covering the exterior
of the particle, particle
size, structure, and density of the coating. Density of the coating can be
adjusted by adjusting
loading of the adjuvantlpeptidic antigen in the coating. When the coating
contains no
adjuvant/peptidic antigen, the polymer coating is densest, and the
adjuvantlpeptidic antigen elutes
through the coating most slowly. By contrast, when adjuvant/peptidic antigen
is loaded into the
coating, the coating becomes porous once the adjuvant/peptidic antigen has
eluted out, starting from
the outer surface of the coating and, therefore, the adjuvant/peptidic antigen
at the center of the
particle can elute at an increased rate. The higher the drug loading, the
lower the density of the
coating layer and the higher the elution rate. The loading of
adjuvant/peptidic antigen in the
coating can be lower than that in the interior of the particles beneath the
exterior coating. Release
rate of adjuvant/peptidic antigen from the particles can also be controlled by
mixing particles with
different release rates prepared as described above.
[0183] A detailed description of methods of making double and triple emulsion
polymers may
be found in Pierre Autant et al, Medicinal and/or nutritional microcapsules
for oral administration,
U.S. patent No. 6,022,562; losif Daniel Rosca et al., Microparticle formation
and its mechanism in
single and double emulsion solvent evaporation, Journal of Controlled
Release(2004) 99:271-280;
L. Mu and S.S. Feng, A novel controlled release formulation for the anticancer
drug paclitaxel
(Taxol): PLGA nanoparticles containing vitamin E(TPGS, J. Control. Release
(2003) 86:33- 48;
Somatosin containing biodegradable microspheres prepared by a modified solvent
evaporation
method based on W/O/W-multiple emulsions, Int. J. Pharm. (1995) 126:129- 138
and F. Gabor et
al., Ketoprofenpoly(d,1-lactic-co-glycolic acid) microspheres: influence of
manufacturing
parameters and type of polymer on the release characteristics, J.
Microencapsul. (1999) 16 (1):1-
12, each of which is incorporated herein in its entirety.
[0184] In yet further embodiments for delivery of aqueous-soluble peptidic
antigens and/or
adjuvant, the particles can be made into nanoparticles having an average
diameter of about 20nm to
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about 200 nm for delivery to the circulation. The nanoparticles can be made by
the single emulsion
method with the peptidic antigen dispersed therein, i.e., mixed into the
emulsion or conjugated to
polymer as described herein. The nanoparticles can also be provided as
micelles containing the
PEA or PEUR polymers described herein. The micelles are formed in water and
the water soluble
antigens with adjuvant protein are loaded into micelles at the same time
without solvent.
[0185] More particularly, the biodegradable micelles, which are illustrated in
Fig. 2, are formed
of a water soluble ionized polymer chain conjugated to a hydrophobic polymer
chain. Whereas, the
outer portion of the micelle mainly consists of the water soluble ionized
section of the polymer, the
hydrophobic section of the polymer mainly partitions to the interior of the
micelles and holds the
polymer molecules together.
[01861 The biodegradable hydrophobic section of the polymer used to make
micelles is made of
PEA, PEUR or PEU polymers, as described herein. For strongly hydrophobic PEA,
PEUR or PEU
polymers, components such as di- L-leucine ester of 1,4:3,6-dianhydro-D-
sorbitol or a rigid
aromatic di-acid like a,w-bis (4-carboxyphenoxy) (C1-C$) alkane may be
included in the polymer,
repeat unit. By contrast, the water soluble section of the polymer comprises
repeating alternating
units of polyethylene glycol, polyglycosaminoglycan or polysaccharide and at
least one ionizable or
polar amino acid, wherein the repeating alternating units have substantially
similar molecular
weights and wherein the molecular weight of the polymer is in the range from
about lOkD to about
300kD. The higher the molecular weight of the water soluble section, the
greater the porosity of the
micelle, with the longer chains enabling high loading of the water soluble
antigens and adjuvants.
[0187] The repeating alternating units may have substantially similar
molecular weights in the
range from about 300D to about 700D. In one embodiment wherein the molecular
weight of the
polymer is over lOkD, at least one of the amino acid units is an ionizable or
polar amino acid
selected from serine, glutamic acid, aspartic acid, lysine and arginine. In
one embodiment, the units
of ionizable amino acids comprise at least one block of ionizable poly(amino
acids), such as
glutamate or aspartate, can be included in the polymer. The invention micellar
composition may
further comprise a pharmaceutically acceptable aqueous media with a pH value
at which at least a
portion of the ionizable amino acids in the water soluble sections of the
polymer are ionized.
[0188] Charged moieties within the micelles partially separate from each other
in water, and
create space for absorption of water soluble agents, such as the peptidic
antigen and optional protein
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adjuvant. Ionized chains with the same type of charge will repel each other
and create more space..
The ionized polymer also attracts the peptidic antigen, providing stability to
the matrix. In addition,
the water soluble exterior of the micelle prevents adhesion of the micelles to
proteins in body fluids
after ionized sites are taken by the therapeutic agent. This type of micelle
has very high porosity,
up to 95% of the micelle volume, allowing for high loading of aqueous-soluble
biologics, such as
peptidic/protein antigen and peptide or protein adjuvant. Particle size range
of the micelles is about
20 nm to about 200 nm, with about 20 nm to about 100 nm being preferred for
circulation in the
blood.
[0189] Rate of release of the adjuvant/peptidic antigen from the compositions
can be controlled
by adjusting the coating thickness, particle size, structure, and density of
the coating. Density of the
coating can be adjusted by varying the loading of the adjuvant/peptidic
antigen in the coating.
When the coating contains no peptidic antigen or adjuvant, the polymer coating
is densest, and the
elution of the peptidic antigen and adjuvant through the coating is slowest.
By contrast, when
peptidic antigen or adjuvant is loaded into the coating, the coating becomes
porous once the
peptidic antigen or adjuvant has eluted out, starting from the outer surface
of the coating and,
therefore, the active agent(s) at the center of the particle can elute at an
increased rate. The higher
the drug loading in the coating layer, the lower the density and the higher
the elution rate. The
loading of adjuvant/peptidic antigen in the coating can be lower than that in
the interior of the
particles beneath the exterior coating. Release rate of adjuvant/peptidic
antigen from the particles
can also be controlled by mixing particles with different release rates
prepared as described above.
[0190] Particle size can be determined by, e.g., laser light scattering, using
for example, a
spectrometer incorporating a helium-neon laser. Generally, particle size is
determined at room
temperature and involves multiple analyses of the sample in question (e.g., 5-
10 times) to yield an
average value for the particle diameter. Particle size is also readily
determined using scanning
electron microscopy (SEM). In order to do so, dry particles are sputter-coated
with a
gold/palladium mixture to a thickness of approximately 100 Angstroms, and then
examined using a
scanning electron microscope. Alternatively, the polymer, either in the form
of particles or not,
can be covalently attached directly to the peptidic antigen, rather than
incorporating peptidic
antigen therein ("loading" or "matrixing") without chemical attachment, using
any of several
methods well known in the art and as described hereinbelow. The peptidic
antigen content is
generally in an amount that represents approximately 0.1% to about 40% (w/w)
peptidic antigen to
polymer, more preferably about 1% to about 25% (w/w) peptidic antigen, and
even more preferably
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about 2% to about 20% (w/w) peptidic antigen. The percentage of peptidic
antigen will depend on
the desired dose and the condition being treated, as discussed in more detail
below. Following
preparation of the particles or polymer molecules loaded with peptidic antigen
and adj uvant, the
composition can be lyophilized and the dried composition suspended in an
appropriate vehicle prior
to immunization.
[0191] Any suitable and effective amount of immunogenic particles or polymer
fragments
containing the peptidic antigen and any adjuvant included in the vaccine
deiivery composition can
be released with time from the polymer particles (including those in a polymer
depot formed in
vivo) and will typically depend, e.g., on the specific polymer, peptidic
antigen, adjuvant or
polymer/peptidic antigen linkage, if present. Typically, up to about 100% of
the polymer particles
or molecules can be released from the polymer depot. Specifically, up to about
90%, up to 75%, up
to 50%, or up to 25% thereof can be released from the polymer depot. Factors
that typically affect
the release rate from the polymer are the nature and amount of the polymer,
the types of
polymer/peptidic antigen linkage and/or polymer/bioactive agent linkage, and
the nature and
amount of additional substances present in the formulation.
[0192] Once the invention vaccine delivery composition is made, as above, the
compositions
are formulated for subsequent mucosal or subcutaneous delivery. The
compositions will generally
include one or more "pharmaceutically acceptable excipients or vehicles"
appropriate for mucosal
or subcutaneous delivery, such as water, saline, glycerol, polyethyleneglycol,
hyaluronic acid,
ethanol, etc. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH
buffering substances, and the like, may be present in such vehicles.
[0193] - For example, intranasal and pulmonary formulations will usually
include vehicles that
neither cause irritation to the nasal mucosa nor significantly disturb ciliary
function. Diluents such
as water, aqueous saline or other known substances can be employed with the
subject invention.
The nasal formulations may also contain preservatives such as, but not limited
to, chlorobutanol and
benzalkonium chloride. A surfactant may be present to enhance absorption by
the nasal mucosa.
[0194] For rectal and urethral suppositories, the vehicle will include
traditional binders and
carriers, such as, cocoa butter (theobroma oil) or other triglycerides,
vegetable oils modified by
esterification, hydrogenation and/or fractionation, glycerinated gelatin,
polyalkaline glycols,
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mixtures of polyethylene glycols of various molecular weights and fatty acid
esters of polyethylene
glycol.
[0195] For vaginal delivery, the formulations of the present invention can be
incorporated in
pessary bases, such as those including mixtures of polyethylene triglycerides,
or suspended in oils
such as corn oil or sesame oil, optionally containing colloidal silica. See,
e.g., Richardson et al., Int.
J. Pharm. (1995) 115:9-15.
[0196] For a further discussion of appropriate vehicles to use for particular
modes of delivery,
see, e.g., Remington: The Science and Practice ofPharmacy, Mack Publishing
Company, Easton,
Pa., 19th edition, 1995. One of skill in the art can readily determine the
proper vehicle to use for
the particular antigen and site of delivery.
[0197] The compositions used in the invention methods may comprise an
"effective arnount" of
the peptidic antigen of interest. That is, an amount of antigen will be
included in the compositions
that will cause the subject to produce a sufficient immunological response in
order to prevent,
reduce or eliminate symptoms. The exact amount necessary will vary, depending
on the subject
being treated; the age and general condition of the subject to be treated; the
capacity of the subject's
immune system to synthesize antibodies; the degree of protection desired; the
severity of the
condition being treated; the particular antigen selected and its mode of
administration, among other
factors. An appropriate effective amount can be readily determined by one of
skill in the art. Thus,
an "effective amount" will fall in a relatively broad range that can be
determined through routine
trials. For example, for purposes of the present invention, an effective dose
will typically range
from about 1 g to about 100 mg, for example from about 5 g to about 1 mg, or
about 10 g to
about 500 g of the antigen delivered per dose.
[0198] Once formulated, the compositions of the invention are administered
mucosally or
subcutaneously by injection, using standard techniques. See, e.g., Remington:
The Science and
Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition,
1995, for mucosal
delivery techniques, including intranasal, pulmonary, vaginal and rectal
techniques, as well as
European Publication No. 517,565 and Illum et al., J. Controlled Rel. (1994)
29:133-141, for
techniques of intranasal administration.
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[0199] Dosage treatment may be a single dose of the invention time release
vaccine delivery
composition, or a multiple dose schedule as is known in the art. A booster may
be with the same
formulation given for the primary immune response, or may be with a different
formulation that
contains the antigen. The dosage regimen will also be determined, at least in
part, by the needs of
the subject and be dependent on the judgment of the practitioner. Furthermore,
if prevention of
disease is desired, the vaccine delivery composition is generally administered
prior to primary
infection with the pathogen of interest. If treatment is desired, e.g., the
reduction of symptoms or
recurrences, the vaccine delivery compositions are generally administered
subsequent to primary
infection.
[02001 The invention compositions can be tested in vivo in a number of animal
models
developed for the study of subcutaneous or mucosal delivery. For example, the
conscious sheep
model is an art-recognized model for testing nasal delivery of substances See,
e.g., Longenecker et
al., J. Pharm. Sci. (1987) 76:351-355 and Illum et al., J. Controlled Rel.
(1994) 29:133-141. The
vaccine delivery composition, generally in powdered, lyophilized form, is
blown into the nasal
cavity. Blood samples can be assayed for antibody titers using standard
techniques, known in the
art, as described above. Cellular immune responses can also be monitored as
described above.
[0201J There are currently a series of in vitro assays for cell-mediated
immune response that use
cells from the donor. The assays include situations where the cells are from
the donor, however,
many assays provide a source of antigen presenting cells from other sources,
e.g., B cell lines.
These in vitro assays include the cytotoxic T lymphocyte assay;
lymphoproliferative assays, e.g.,
tritiated thymidine incorporation; the protein kinase assays, the ion
transport assay and the
lymphocyte migration inhibition function assay (Hickling, J. K. et al. (1987)
J. Virol., 61: 3463;
Hengel, H. et al. (1987) J. Immunol., 139: 4196; Thorley-Lawson, D. A. et al.
(1987) Proc. Natl.
Acad. Sci. USA, 84: 5384; Kadival, G. J. et al. (1987) J. Immunol.,139:2447;
Samuelson, L. E. et
al. (1987) J. Immunol.,139:2708; Cason, J. et al. (1987) J. Immunol.
Meth.,102:109; and Tsein, R.
J. et al. (1982) Nature, 293: 68. These assays are disadvantageous in that
they may lack true
specificity for cell mediated immunity activity, they require antigen
processing and presentation by
an APC of the same MHC type, they are slow (sometimes lasting several days),
and some are
subjective and/or require the use of radioisotopes.
[0202] To test whether a peptide recognized by a T-cell will activate the T-
cell to generate an
immune response, a so-called "functional test" is used. The enzyme-linked
immunospot (ELISpot)
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assay has been adapted for the detection of individual cells secreting
specific cytokines or other
effector molecules by attachment of a monoclonal antibody specific for a
cytokine or effector
molecule on a microplate. Cells stimulated by an antigen are contacted with
the immobilized
antibody. After washing away cells and any unbound substances, a tagged
polyclonal antibody or
more often, a monoclonal antibody, specific for the same cytokine or other
effector molecule is
added to the wells. Following a wash, a colorant that binds to the tagged
antibody is added such
that a blue-black colored precipitate (or spot) forms at the sites of cytokine
localization. The spots
can be counted manually or with automated ELISpot reader composition to
quantitate the response.
A final confirmation of T-cell activation by the test peptide may require in
vivo testing, for example
in a mouse or other animal model.
[02031 As is readily apparent, the invention vaccine delivery compositions are
useful for
eliciting an immune response against viruses, bacteria, parasites and fungi,
for treating and/or
preventing a wide variety of diseases and infections caused by such pathogens,
as well as for
stimulating an immune response against a variety of tumor antigens. Not only
can the
compositions be used therapeutically or prophylactically, as described above,
the compositions may
also be used in order to prepare antibodies, both polyclonal and monoclonal,
for, e.g., diagnostic
purposes, as well as for immunopurification of the antigen of interest. If
polyclonal antibodies are
desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is
immunized with the
compositions of the present invention. The animal is optionally boosted 2-6
weeks later with one
or more administrations of the antigen. Polyclonal antisera is then obtained
from the immunized
animal and treated according to known procedures. See, e.g., Jurgens et al.
(1985) J. Chrom.
348:363-370.
[0204J Monoclonal antibodies are generally prepared using the method of Kohler
and Milstein,
Nature (1975) 256:495-96, or a modification thereof. Typically, a mouse or rat
is immunized as
described above. However, rather than bleeding the animal to extract serum,
the spleen (and
optionally several large lymph nodes) is removed and dissociated into single
cells. If desired, the
spleen cells may be screened (after removal of nonspecifically adherent cells)
by applying a cell
suspension to a plate or well coated with the protein antigen. B cells,
expressing membrane-bound
immunoglobulin specific for the antigen, will bind to the plate, and are not
rinsed away with the
rest of the suspension. Resulting B cells, or all dissociated spleen cells,
are then induced to fuse
with myeloma cells to form hybridomas, and are cultured in a selective medium
(e.g.,
hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas
are plated by
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limiting dilution, and are assayed for the production of antibodies which bind
specifically to the
immunizing antigen (and which do not bind to unrelated antigens). The selected
monoclonal
antibody-secreting hybridomas are then cultured either in vitro (e.g., in
tissue culture bottles or
hollow fiber reactors), or in vivo (as ascites in mice). See, e.g., M.
Schreier et al., Hybridoma
Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell
Hybridomas (1981);
Kennett et al., Monoclonal Antibodies (1980); see also U.S. Pat. Nos.
4,341,761; 4,399,121;
4,427,783; 4,444,887; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels
of monoclonal
antibodies produced against the polypeptide of interest can be screened for
various properties; i.e.,
for isotype, epitope, affinity, etc.
102051 The following example is meant to illustrate, and not to limit, the
invention.
EXAMPLE 1
Synthesis of PEA-Antigen Conjugate
[0206] Synthesis of PEA succinimidyl ester (PEA-OSu). All examples are from N-
acetylated
polymer (A). PEA 1.392g, 754 M, calculated for MW=1845 per repeating unit
(Formula I, R' _
(CHZ)8i RZ = H; R3 =(CH3)ZCHCH2; R4 =(CHZ)6; n= 70; m/m+p=0.75 and p/m+p=0.25)
was
dissolved in 7ml anhydrous DMF while stirring. To the slightly viscous
solution of PEA was
added N-Hydroxysuccinimide (NHS), 0.110g, 955 M as a solid. 1-Ethyl-3-(3'-
dimethylaminopropyl)carbodiimide hydrochloride, 146mg, 759.8 M, was
transferred as a
suspension in DMF. The total volume of DMF for the reaction was l Oml. The
reaction was carried
out at room temperature under nitrogen atmosphere for 24 hrs.
Synthesis of PEA-Influenza Peptide Conjugate:
102071 111) The synthesis of PEA-Peptide conjugate (Fonnula IV, R' =(CH2)8i;
R3 =
(CH3)2CHCH2; R4 = (CH2)6; R5 =. NH; n = 70; m/m+p=0.75 and p/m+p=0.25 and R7 =
PKYVKQNTLKLAT (SEQ ID NO: 19)) was performed with 49.5pM aliquot of the
activated ester
(A) in DMF and 96 mg (49:5 M) H-PKYVKQNTLKLAT (SEQ ID NO: 19) -OH, as a
trifluoroacetic acid salt. The peptide was dissolved and transferred to the
activated ester in 5ml
DMSO. One equivalent, i.e. 49.5 M ethyl-diisopropylamine was added and the
reaction was
continued for 24hrs under nitrogen. Distilled water, 30 1 in 300 1 DMSO was
added and stirring
was continued at room temperature for another 4hrs.
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102081 The reaction mixture was precipitated in diethyl ether (60 ml) and,
after centrifugation,
the obtained material was washed three times with 15ml of diethyl ether. After
being air- dried, the
obtained product was treated with 3x5m1 distilled water under sonication for a
minute. After
centrifugation, the obtained material was lyophilized. Yield 86mg, 47%.
102091 B2) The synthesis of PEA-Peptide conjugate (Formula IX, cross-linked
through R5-R7-
R5, wherein R' =(CHZ)8; R3 =(CH3)2CHCHZ; R4= (CHZ)6; R5 = NH; n = 70;
m/m+p=0.75 and
p/m+p=0.25 and R7= PKYVKQNTLKLAT (SEQ ID NO: 19)) was performed with 37.7 M
aliquot
of the activated ester (A) in DMF (600 1) and 74mg (37.7 M) H-PKYVKQNTLKLAT
(SEQ ID
NO: 19) -OH, as trifluoroacetic acid salt. The peptide was dissolved and
transferred to the activated
ester in 0.8m] DMSO (dimethylsulfoxide). Four equivalents, i.e. 198 M ethyl-
diisopropylamine
were added and the reaction was continued for 48hrs under nitrogen. The
transparent, gel like
material was separated from the organic solvents by decantation. After being
cut into 2-3mm large
pieces, the product was treated with 17 ml distilled water at +4 C for 18
hrs. After centrifugation
and decantation, the material was treated two times with 17m1 distilled water
(3hrs each time) and
after the last centrifugation the product was lyophilized. Yield: 75mg, 53%
102101 B3) The synthesis of PEA-Peptide conjugate (Formula IX cross-linked
through R5-R7-
R5, wherein R' = (CH2)8; R3 =(CH3)ZCHCHZ; R4 =(CHZ)6; R5 = NH; n = 8;
m/m+p=0.75 and
p/m+p=0.25 and R' = PKYVKQNTLKLAT (SEQ ID NO: 19)) was performed with 41.2 M
of the
activated ester, which was synthesized in a way similar to (A) in DMF (600 l)
and 40mg (20.6 M)
H-PKYVKQNTLKLAT (SEQ ID NO: 19) -OH, as trifluoroacetic acid salt. The peptide
was
dissolved and transferred to the activated ester in Sml DMSO. Four
equivalents, i.e. 80 M ethyl-
diisopropylamine were added and the reaction continued for 72hrs under
nitrogen. Distilled water,
75 1, (4.2mM) in 300111 DMSO was added and stirring continued for another
24hrs. Then the
reaction mixture was precipitated in 24m1 water/acetone (1:1 v/v). The
resulting precipitate was
treated with distilled water (4x 12m1) for about an hour each time at +4 C
followed by
centrifugation. After the last centrifugation, the product was lyophilized.
Yield 50mg, 45%.
Summary of In ritro Human T Cell Response Protocol
CD4+ T cells and monocytes are isolated from the peripheral blood of human
donors. The
monocytes are cultured for 48 hours in a cytokine-rich medium to induce
differentiation into
dendritic cells (antigen presenting cells). 24 hours into that culture period,
PEA or PEA-
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hemagglutinin peptide (307-319) conjugates are added to the medium. Two hours
prior to starting
the co-culture of dendritic cells and T cells, free peptide is added to
control wells. T cells cultured
together with dendritic cells are measured for activation by proliferation and
cytokine secretion at
48h, 72h, and 96h. A schematic diagram of the T-cell response protocol is
illustrated in Fig. 3
herein.
[0212] T-cell activation in response to dendritic cells exposed to polymer-
peptide conjugates
were tested using the above protocol. Fig. 4A shows T-cell proliferation over
96 hours in which
PEA-peptide conjugates stimulated significant proliferation over peptide or
PEA alone. Fig. 4B
shows secretion of IL-2 by T-cells over 96 hours in which PEA-peptide (Formula
III, Example B 1)
stimulated significant IL-2 secretion compared to peptide or PEA alone.
EXAMPLE 2
Cytotoxic T Cell Response from PEA-Melanoma Peptidic Antigen Delivery to APCs
[0213] We examined the ability of PEA-melanoma peptides to induce a cytotoxic
T lymphocyte
killing response. MHC I restricted peptides from 2 melanoma-associated
proteins, gplOO and
MART-1, were used as peptidic antigens and conjucated to PEA as described in
Example 1.
Peripheral blood was collected from healthy human donors who expressed the
MIIC I allele, HLA-
A2. Peripheral blood mononuclear cells (PBMC) were isolated from the blood and
exposed to the
MART-1 peptide, the gp100 peptide, PEA-MART-1 conjugates, or PEA-gp 100
conjugates. Tumor
infiltrating lymphocytes were isolated from HLA-A2 melanoma patients, and the
ability of these
cells to kill the peptide- or construct-treated peripheral blood mononuclear
cells was measured by
release of lactose dehydrogenase into the culture media by killed cells.
Polymer only, peptide only,
or a mixture of polymer and peptide did not induce the tumor infiltrating
lymphocytes to kill the
PBMC. Only conjugates of the peptides with the polymer induced killing.
Importantly, the killing
activity of the T lymphocytes was sustained over 7 days, suggesting
persistence to the processing of
PEA-peptide conjugates and persistent presentation of the MHCI-antigen complex
on the surface of
the PBMC. (Fig. 5 (A) MART-1 peptide (B) gplOO peptide.). Melanoma antigens
delivered by
PEA stimulated a strong and sustained cytotoxic T lymphocyte response from
cancer patient tumor
cells, a result that demonstrates the use of invention compositions for
peptide-based cancer
vaccines.
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EXAMPLE 3
In Vivo T Cell Response from PEA-HIV Peptidic Antigen Delivery
[0214] A peptide antigen, longer than the actual epitope, was used to
demonstrate proper
processing and an MHC-I restricted T cell response in vivo. BlockAide/CR (H-
RINRGPGRRAFVTIGK (SEQ ID NO:20) -NH2) (Adventrx Phanmaceuticals, Inc.) is a
synthetic
peptide based upon the structure of the V3 loop of the gp 120 coat protein
from human
immunodeficiency virus (HIV). The virus uses this structure to bind to the
cell surface before viral
fusion takes place. The peptidic antigen was conjugated to PEA as described in
Example 1 herein.
Mice were immunized with peptide, peptide-adjuvant mixtures, or PEA-peptide
conjugates
containing 20 g BlockAide/CR by up to 4 weekly subdermal injections into the
tail. By surgical
excision, spleens were collected from three mice per group I week following
the 2"d immunization
and I week following the 4`h immunization. Peptide-specific T cell responses
were analyzed by
IFN-y and IL-2 specific ELISpot assays to quantify the relative number and
activation of antigen
specific T cells in an animal. The results of the ELISpot assays (Fig. 6) show
that BlockAide/CR
was delivered to and processed by local APCs that in turn evoked a systemic
immune response as
measured by subsequent T cell activation from the spleen. It is noted that,
PEA-BlockAide/CR
conjugates without adjuvant stimulated as strong a response as did
BlockAide/CR plus adjuvant.
Secretion of cytokines was used to enumerate epitope specific T cells by
ELISpot. Peptide only
(A), adjuvant only (B) and PEA only (C) did not induce cytokine secretion. Two
PEA-peptide
formulations (E and F) are shown that, without adjuvant, stimulate T cell
responses as strongly as
does peptide plus adjuvant (D). In particular, formulation E induced a
sustained release of IL-2
compared to peptide plus adjuvant (D).
EXAMPLE 4
Protection from Lethal Influenza A Viral Challenge with PEA-Protein Antigen
Delivery
[0215] As show by the results presented in Fig. 7, protection from influenza A
viral challenge
has been demonstrated using the invention composition and methods. PEA
microspheres were
loaded with purified baculovirus-produced hemagglutinin (HA) from an HIN1
influenza A strain to
deliver 5 g protein antigen via a single subcutaneous immunization in a
liquid dispersion with, or
without, a CpG adjuvant. Alternatively, mice were immunized with live PR8
strain of the HINI
virus (i.p.), 5 g HA, with or without, alum or CpG adjuvants. Unimmunized
mice were used as a
G'IN6532937.I
330142-170
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control. At day 21 post-vaccination, the animals were challenged intranasally
with 10 LD50 of the
PR8 strain of the HIN1 virus to produce a fatal influenza infection and were
monitored for weight
loss over 7 days. The number of mice surviving per group is shown in Fig. 7,
which shows that the
protein antigen HA-PEA polymer invention vaccine delivery composition confers
100% protection
against lethal infection.
[01011 EXAMPLE 5
Prevention of Tumor Growth by Immunization with PEA-HPV Protein Antigen
[0216] The vaccine delivery compositions are also used as prophylactic and
therapeutic vaccines
against malignant cancers. Proof-of-concept of this application is
demonstrated by the preventing
establishment of HPV-transfected tumor cells upon injection into mice by
immunization with PEA
or PEUR microspheres loaded with E6E7 oncogene fusion protein.
Formulation of PEA/PEUR-Antigen Microspheres
[0217] Preparation of aqueous in organic primary emulsion: The desired polymer
type, PEA or
PEUR- lysine-nitrilotriacetic acid conjugate (PEA-NTA or PEUR-NTA) was
dissolved in an
organic solvent system (named phase Al) of 1,1,1,3,3,3-Hexafluoro-2-propanol
(HFIP) at room
temperature and 1 atmosphere pressure (RT, 1 Atm.). The polymer concentration
in phase Al was
5% or 50 mg of polymer per 1 ml of organic solvent. An aqueous in organic
emulsion was
generated by adding to phase Al a 1.16 molar equivalent of NiSO4 (0.1 M in
aqueous) to lysine-
nitrilotriacetic acid (NTA conjugated to the polymer) at room temperature
(RT), 1 Atm. More
particularly, 1.4 ml, 0.1 M NiSO4, is added to 285 mg of PEA-NTA dissolved in
5.7 ml HFIP (140
mol NiSO4 to 120 umol NTA). The emulsion (named phase A2) was rendered
homogeneous by
vortex stirring and placement in a sonication bath for 120 seconds each at RT,
1 Atm. An
additiona10.5 volume equivalent of de-ionized H20 to HFIP (2.35 ml de-ionized
H20 was added to
285 mg of PEA-NTA dissolved in 5.7 ml HFIP) was added to the homogenous A2,
and the solution
was subjected to vortex stirring and sonication again for 120 seconds each at
RT, 1 Atm.
[02181 Preparation of aqueous in organic in aqueous secondary emulsion: A
secondary
emulsion was generated when phase A2 was added into an aqueous solution (named
phase B 1)
formed by ultra sonication of 0.2% or 2 mg per 1 ml poly(vinyl) alcohol [80%
hydrolyzed, 20%
acetate] at 25 W power, 4 C, 1 Atm. (Fisher Scientific Sonic Dismembrator,
model 100). The
volume ratio of phase A2 to phase B 1 is 1:11; for example, 9.1 ml of phase A2
was added into 100
ml of phase B I. The secondary emulsion (called phase B2) underwent
rotoevaporation at 760 mHg
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vacuum for 600 seconds at 30 C to remove HFIP. Large microspheres were removed
from the
emulsion using a 20 m mesh filter. The filtrate then underwent dialysis in a
10,000 molecular
weight cut-off, mixed cellulose membrane for 24 hours to remove excess solvent
and counter ions
(SO42-)= The dialysis was carried out in de-ionized H2O at room temperature,
at a volume ratio of
40:1, outer volume to inner volume. 100 ml of phase B2 was dialyzed against 4
liters of de-ionized
H20. The de-ionized H20 was replaced with fresh H20 at 4, 8, and 20 hours of
dialysis time. Phase
B2 was removed from dialysis, filtered again through a 20 m mesh filter
(collecting aggregates),
frozen in liquid nitrogen, and lyophilized overnight. Following
lyophilization, spheres were a dry,
green powder, and could be reconstituted in de-ionized water, or a desired
buffer.
[02191 Preparation ofPEA/PEUR antigen particles: Lyophilized spheres from the
preparation
above are reconstituted in a desired protein solution, 4 C, at a loading rate
of 4 times the amount of
protein in solution. In this example, 20 mg of spheres were reconstituted in 5
mg of protein
solution (10 ml at 0.2 mg/ml). The suspension of particles in protein solution
was gently shaken
via a rocking-plate at 4 C for 1.1 hours. Excess free protein was removed
following centrifugation
of the suspension at 4,000 times g, 4 C, for 5 minutes and the resulting
supernatant was decanted.
The pellet was re-suspended in PBS 7.4 buffer, as a washing step, at an equal
volume as the protein
solution (10 ml in our example). This washing step is repeated twice more
(with analogous
centrifugation intermittently), and finally re-suspended in de-ionized H2O, at
an equal volume as
the starting protein solution. This solution was frozen in liquid nitrogen,
and lyophilized overnight.
Final product was a faint green powder, and could be reconstituted in a
desired physiological
buffer.
[0220] Mice were injected subcutaneously in the base of the tail with (1) 10
g E6E7 protein +
CpG, PEA-E6E7 (containing 10 gg protein antigen) + CpG invention vaccine
composition, (2)
irradiated tumor cells, or (3) nothing. Five weeks after immunization, mice
were challenged with 5
x 105 C3-43 HPV-transfected cells by subcutaneous injection in the flank.
Fifteen days after tumor
challenge, the mice were cuthanized, and tumors removed and weighed (Fig. 8).
Four of five mice
immunized with PEA-E6E7 had negligible tumors; whereas five of five mice
immunized with the
fusion protein alone had large tumors.
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EXAMPLE 6
Activation of Antigen Presenting Cells by Polymer-adjuvant Composition
[0221] The PEA antigen delivery platform was modified to rapidly couple whole
antigen in a
directed manner, such as encapsulating or covalently coupling different types
of adjuvants, such as
TLR agonists to PEA spheres. This provides a means for tailoring the invention
compositions to
potentiate the desired immune response of each vaccine candidate. Toll-like
receptors (TLRs) are a
family of molecules that mediate the innate immune response to pathogens, such
as bacteria or viral
DNA. They are the receptors for several known immunostimulatory adjuvants.
Since TLR-7 is an
intracellular receptor, it desirable to provide direct intracellular
stimulation using a TLR agonist,
such as imiquimod, for this receptor.
Synthesis of PEA Succinimidyl ester
[0222] PEA (I).Ac.H (1), 1.116g (606 mol, 1.0 eq), calculated for MW=1845 per
repeating unit
(Formula, R' =(CHz)s; RZ = H; R3 =(CH3)zCHCHZ; W =(CH2)6; n= 35; was dissolved
in 6.0 ml of
anhydrous DMF and stirred to dissolve the polymer completely. To the slightly
viscous solution of
PEA were added N-Hydroxysuccinimide (NHS), 0.137g (667 mol, 1.1 eq) and
Dicyclohexyl
carbodiimide (DCC) 0.076g (667 mo1, 1.1 eq) as solids. The reaction was
carried out at room
temperature under nitrogen atmosphere for 24 hrs. DCC urea was precipitated as
white solid. The
reaction mixture was filtered through a 0.2 pore filter.
Synthesis of PEA-Imiquimod conjugate
[0223] Synthesis of PEA-Imiquimod conjugate (Formula, Rl =(CH2)$; RZ = H; R3 =
(CH3)2CHCH2; R~ = (CH2)6; RS = NH; R6 = Imiquimod) was performed with
activated ester (2) in
DMF and DMSO, 0.16g (6671irnol, 1.1 eq) of imiquimod. The imiquimod was
dissolved in 25.0
ml of DMSO and transferred to the activated ester. 115 1 of ethyl-
diisopropylamine (6671imo1, 1.1
eq) was added and the reaction was continued for six days under nitrogen at 48-
50 C. The
reaction mixture was precipitated in DI water (30 ml). After centrifugation
and decantation, the
obtained material was washed two times with DI water (30 ml) and two times
0.1N HCI (25 ml) to
remove unreacted imiquimod and finally again washed two times with DI water
(25 ml). After
centrifugation and decantation, the obtained material was lyophilized. Yield
1.133g, 89.84%.
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[0224] As shown in Fig 9, the polymer can provide intracellular delivery of
one TLR-7 agonist,
imiquimod (IMQ). In this experiment, intracellular delivery produces a 10-100
fold better
stimulation than free IMQ. In Fig 9A, traces are shown of the FACS analysis
intensity distribution
of CD 11 c-positive bone marrow derived dendritic cells (BMDC) from Balb/c
mice incubated with
the indicated substances and stained for elevation of surface CD40, a marker
of activation. The
calculated geometric mean of the intensity distribution is tabulated for each
condition in the column
labeled "Geo Mean." PEA-IMQ increases CD40 expression at both 10 and 1 M IMQ;
whereas
IMQ alone only increases CD40 expression at 10 uM. In Fig. 9B, supernatants
from 5 x 104
BMDC cultured overnight with the substances indicated at the bottom of the
panel are analyzed for
cytokine secretion. The amount of IL-12, a cytokine that indicates BMDC
activation, was detected
by ELISA (BD Biosciences, kit Cat #555165) following the manufacturer's
instructions. Again,
PEA-IMQ was over 10-fold more potent at stimulating a response compared to IMQ
alone.
LipopoIysaccharide (LPS) is used as a positive control, and dimethylsulfoxide
(DMSO) as a vehicle
negative control.
[0225] All publications, patents, and patent documents are incorporated by
reference herein, as
though individually incorporated by reference. The invention has been
described with reference to
various specific and preferred embodiments and techniques. However, it should
be understood that
many variations and modifications might be made while remaining within the
spirit and scope of
the invention.
[0226] Although the invention has been described with reference to the above
examples, it will
be understood that modifications and variations are encompassed within the
spirit and scope of the
invention. Accordingly, the invention in this application is limited only by
the following claims.
