DESCRIPTION
Immunogenic Composition
TECHNICAL FIELD
[0001]
The present invention relates to an immunogenic composition comprising as
an effective ingredient an antigen-adjuvant microparticle complex containing an
antigen encapsulated in an adjuvant microparticle composed of an amphiphilic
polymer(s).
BACKGROUND ART
[0002]
For enhancement of the immune-activating capacity of an antigen, an
adjuvant is used together with the antigen. Although complete Freund's adjuvant
(CFA) is known to have an excellent effect as an adjuvant, CFA is composed of
killed bacteria and an oil emulsion, and hence has strong side effects such as strong
inflammatory reaction and formation of ulcerative swelling (granuloma) at the
administration site. Therefore, use of CFA for human is not permitted in view of
safety. Adjuvants whose administration to human is permitted are limited.
Examples of the adjuvants whose administration to human is permitted include
aluminum hydroxide adjuvants, but their immune-activating capacities are not
necessarily sufficient and hence they need to be repeatedly administered in order to
allow acquisition of immunity. Therefore, development of an immunogenic
composition using an efficient and strong adjuvant, which composition can be used
for human, has been demanded.
[0003]
For development of a novel adjuvant aiming to attain a high immune-
activating capacity, a method wherein an antigen is encapsulated in a microparticle

has been attempted. It has been reported that administration of a microparticulated
antigen enhances immunological reactions such as antibody production compared to
the case of administration of an antigen alone, but the effect of its administration is
not necessarily high, and only an effect at almost the same level as in the case of the
above-mentioned aluminum hydroxide adjuvant has been reported. This is
considered to be due to difficulty in efficient encapsulation of hydrophilic antigen
molecules such as protein in microparticles studied so far, such as microparticles
comprising hydrophobic polylactic acid-polyglycolic acid copolymers, while
maintaining the structures of the antigen molecules (Non-patent Document 1).
[0004]
In recent years, a novel microparticle technology has been reported (Patent
Documents 1 and 2), which technology uses an amphiphilic polymer and enables
highly efficient encapsulation of a high molecular protein. Although this novel
microparticle has been studied for its sustained-release performance for drugs, its
adjuvant function in cases where an antigen is encapsulated therein has not been
studied at all. Further, in terms of the mechanism by which a microparticle
containing an antigen functions as an adjuvant, it is thought that the function for
sustained release of the antigen molecule as well as the mechanism by which the
microparticle containing an antigen is incorporated in its entirety into an immunocyte
and releases the antigen in the cell are important, and it is further thought that the
function of drug release from the particle and the performance as an adjuvant are not
necessarily correlated with each other. Therefore, it is difficult to infer the adjuvant
function from the sustained-release performance of the particle, and an effective
adjuvant having a much better performance than aluminum adjuvants has not been
realized so far by conventional technologies using microparticles in spite of the
demand for its development.
PRIOR ART DOCUMENTS

Patent Documents
[0005]
[Patent Document 1] WO2006/095668
[Patent Document 2] JP 2008-088158 A
[0006]
Non-patent Documents
[Non-patent Document 1] Advanced drug delivery reviews, 2005, vol. 57, pp.
391-410
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0007]
The present invention aims to provide an immunogenic composition which
shows a high immune-activating capacity even with a small antigen amount and/or a
small number of doses.
MEANS FOR SOLVING THE PROBLEMS
[0008]
In order to solve the above-described problems, the present inventors studied
a method by which a high level of immune activation can be induced using a small
amount of antigen and with a small number of doses thereof, and, as a result,
discovered that an antigen-adjuvant microparticle complex containing an antigen
encapsulated in an adjuvant microparticle has a high immune-activating capacity in
vivo. That is, the present invention has the following constitution.
[0009]
(1) An immunogenic composition comprising as an effective ingredient an
antigen-adjuvant microparticle complex containing an antigen encapsulated in an
adjuvant microparticle composed of an amphiphilic polymer(s) whose hydrophobic
segment is a poly(hydroxy acid).

[0010]
(2) The immunogenic composition according to (1), comprising as an
effective ingredient a particle composed of the antigen-adjuvant microparticle
complex associated together.
[0011]
(3) The immunogenic composition according to (1) or (2), wherein the
adjuvant microparticle has a hydrophilic portion in the inside thereof, the hydrophilic
portion being composed of a hydrophilic segment of the amphiphilic polymer, and
has an outer layer composed of a hydrophobic portion constituted by the hydrophobic
segment of the amphiphilic polymer.
[0012]
(4) The immunogenic composition according to any of (1) to (3), wherein the
hydrophilic segment of the amphiphilic polymer is a polysaccharide or a polyethylene
glycol.
[0013]
(5) The immunogenic composition according to any of (1) to (4), wherein the
amphiphilic polymer is a graft amphiphilic polymer composed of a polysaccharide
backbone and a poly(hydroxy acid) graft chain.
[0014]
(6) The immunogenic composition according to (4) or (5), wherein the
polysaccharide is dextran.
[0015]
(7) The immunogenic composition according to any of (1) to (4), wherein the
amphiphilic polymer is a block polymer composed of a poly(hydroxy acid) and a
polyethylene glycol.
[0016]
(8) The immunogenic composition according to any of (1) to (7), wherein the

poly(hydroxy acid) is a poly(lactic-co-glycolic acid).
[0017]
(9) The immunogenic composition according to any of (1) to (8), further
comprising a surface modifier bound to the poly(hydroxy acid) of the adjuvant
microparticle.
[0018]
(10) The immunogenic composition according to any of (1) to (9), wherein
the average particle size of the antigen-adjuvant microparticle complex or the particle
composed of the antigen-adjuvant microparticle complex associated together is 0.1 to
50 µm.
[0019]
(11) The immunogenic composition according to any of (1) to (10), further
comprising an immune-activating substance.
[0020]
(12) The immunogenic composition according to (11), wherein the immune-
activating substance is a nucleic acid.
[0021]
(13) The immunogenic composition according to (11) or (12), wherein the
immune-activating substance is CpG.
EFFECT OF THE INVENTION
[0022]
By the present invention, an immunogenic composition with which stronger
immune activation than before is possible in vivo is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023]
Fig. 1 shows immunological evaluation 1 of OVA-containing immunogenic
compositions.

Fig. 2 shows immunological evaluation (total IgG) of CEA-containing
immunogenic compositions.
Fig. 3 shows immunological evaluation (IgG2a) of CEA-containing
immunogenic compositions.
Fig. 4 shows immunological evaluation 2 of OVA-containing immunogenic
compositions.
Fig. 5 shows immunological evaluation 3 of OVA-containing immunogenic
compositions.
Fig. 6 shows immunological evaluation of HCV structural protein-containing
immunogenic compositions.
Fig. 7 shows immunological evaluation 2 of CEA-containing immunogenic
compositions.
Fig. 8 shows immunological evaluation 3 of CEA-containing immunogenic
compositions.
Fig. 9 shows immunological evaluation 4 of OVA-containing immunogenic
compositions.
Fig. 10 shows immunological evaluation 4 of CEA-containing immunogenic
compositions.
Fig. 11 shows immunological evaluation (IgG2a/IgGl ratio) of CEA-
containing immunogenic compositions.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024]
The present invention relates to an immunogenic composition comprising an
antigen-adjuvant microparticle complex containing an antigen encapsulated in an
adjuvant microparticle composed of an amphiphilic polymer(s) whose hydrophobic
segment is a poly(hydroxy acid).
[0025]

First, the amphiphilic polymer constituting the adjuvant microparticle is
described. Being "amphiphilic" means that properties of both hydrophilicity and
hydrophobicity are retained. When solubility of a certain portion in water is higher
than those of other portions, the portion is said to be hydrophilic The hydrophilic
portion is preferably soluble in water, but even in cases where the portion has poor
solubility in water, it is sufficient if the solubility in water is higher than those of
other portions. When solubility of a certain portion in water is lower than those of
other portions, the segment is said to be hydrophobic. The hydrophobic portion is
preferably insoluble in water, but even in cases where the portion is soluble in water,
it is sufficient if the solubility in water is lower than those of other portions
[0026]
The amphiphilic polymer means a polymer having the above-mentioned
amphiphilicity as the whole molecule. The polymer means that the molecule has a
molecular structure wherein the hydrophilic segment or the hydrophobic segment of
the amphiphilic polymer, or the both, is/are constituted by a structure(s) in which
minimum units (monomers) are repeated. The amphiphilic polymer of the present
invention may have a structure having a hydrophilic segment(s) and a hydrophobic
segment(s), and may be a linear block polymer having a hydrophilic segment(s) and a
hydrophobic segment(s) linked to each other; a branched polymer having a
branch(es) in which one or both of a hydrophilic segment(s) and a hydrophilic
segment(s) exist(s); or a graft polymer in which plural hydrophobic segments are
grafted to a hydrophilic segment or plural hydrophilic segments are grafted to a
hydrophobic segment. The amphiphilic polymer of the present invention is
preferably a polymer having one hydrophilic segment, most preferably a linear block
polymer having one each of a hydrophilic segment and a hydrophobic segment, or a
graft polymer having plural hydrophobic segments grafted on a hydrophilic segment
backbone.

[0027]
The amphiphilic polymer constituting the immunogenic composition may be
a set of plural types of amphiphilic polymers composed of constituent polymers
having different hydrophilic portions and/or hydrophilic portions, or a set of
amphiphilic polymers having the same constituent polymers but having plural types
of linking patterns, as long as the amphiphilic polymer has properties as an adjuvant
microparticle. In view of achievement of stable performance and enhancement of
productivity, the amphiphilic polymer is preferably a set of a small number of types
of amphiphilic polymers, more preferably a set of mainly not more than 2 types of
amphiphilic polymers, and still more preferably constituted by mainly a single type of
amphiphilic polymer.
[0028]
In the present invention, the hydrophobic segment of the amphiphilic polymer
is a poly(hydroxy acid). The poly(hydroxy acid) is not restricted, and preferably a
biocompatible polymer which does not have a severely adverse effect upon
administration to a living body. The biocompatibility herein means that LD50 in
the case of oral administration of the polymer to rats is not less than 2,000 mg/kg.
Further, the polymer may be a copolymer of plural types of hydroxy acids, and is
preferably a polymer of not more than 2 types of hydroxy acids. Particular preferred
examples of the poly(hydroxy acid) include polyglycolic acid, polylactic acid, poly(2-
hydroxybutyric acid), poly(2-hydroxyvaleric acid), poly(2-hydroxycaproic acid),
poly(2-hydroxycapric acid) and poly(malic acid); and derivatives and copolymers of
these macromolecular compounds; among which polylactic acid, polyglycolic acid,
and poly(lactic-co-glycolic acid) copolymers are more preferred. Further, in cases
where the poly(hydroxy acid) is a poly(lactic-co-glycolic acid), the composition ratio
of the poly(lactic-co-glycolic acid) (lactic acid/glycolic acid) (mol/mol%) is not
restricted as long as the purpose of the present invention is achieved therewith, and

the ratio is preferably 100/0 to 30/70, more preferably 60/40-40/60.
[0029]
The hydrophilic segment of the amphiphilic polymer is not restricted, and
preferably a biocompatible polymer, as in the case of the hydrophobic segment.
Further, in order to give a persistent adjuvant capacity to the adjuvant microparticle
composed of an amphiphilic polymer, the segment is preferably a refractory polymer
which is not easily decomposed in a living body or cell of a mammal or bird.
Particular examples of the biocompatible and refractory polymer include
polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyethyleneimine,
polyacrylic acid, polymethacrylic acid, poly-l,3-dioxolane, 2-methacryloyloxyethyl
phosphoryl choline polymer, poly-l,3,6-trioxane, polyamino acid and refractory
polysaccharides (e.g., cellulose, chitin, chitosan, gellan gµm, alginic acid, hyaluronic
acid, pullulan and dextran). In cases where the hydrophilic segment is polyethylene
glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyethyleneimine, polyacrylic acid,
polymethacrylic acid, poly-l,3-dioxolane, 2-methacryloyloxyethyl phosphoryl
choline polymer, poly-l,3,6-trioxane or polyamino acid, the amphiphilic polymer is
preferably a linear block polymer having one each of a hydrophilic segment and a
hydrophobic segment, and, in cases where the hydrophilic segment is a
polysaccharide, the amphiphilic polymer is preferably a graft polymer having plural
hydrophobic segments grafted on a hydrophilic segment backbone. Further, the
hydrophilic segment of the amphiphilic polymer is preferably polyethylene glycol or
a refractory polysaccharide, and the polysaccharide is more preferably dextran.
[0030]
The amphiphilic polymer having a hydrophobic segment(s) composed of
poly(hydroxy acid) and a hydrophilic segment(s) preferably has water immiscibility
as the whole polymer, in view of antigen-encapsulation capacity and persistency
upon administration to a living body

[0031]
The average molecular weight of the hydrophilic segment of the amphiphilic
polymer is not restricted, and, in the case of a block polymer wherein a hydrophilic
segment(s) and a hydrophilic segment(s) are linearly linked to each other, the average
molecular weight is preferably 1,000 to 50,000, more preferably 2,000 to 15,000.
The term "block" herein means a portion in a polymer molecule, which portion is
composed of not less than 5 monomer units and different from the other adjacent
portion(s) in terms of the chemical structure or the configuration. A polymer
constituted by at least two blocks linearly linked to each other is called a block
polymer. Each block itself constituting the block polymer may be a random,
alternating or gradient polymer composed of not less than 2 types of monomer units.
In the present invention, the block polymer is preferably constituted by one each of a
polymer forming a hydrophilic segment and a polyhydroxy acid.
[0032]
In the case of a graft polymer having a hydrophobic segment(s) grafted on a
hydrophilic segment backbone, the average molecular weight of the hydrophilic
segment is preferably 1,000 to 100,000, more preferably 2,000 to 50,000, still more
preferably 10,000 to 40,000. The number of the graft chains is preferably 2 to 50.
The number of the graft chains can be calculated based on the ratio between the
hydrophilic segment backbone and the hydrophobic segment backbone; the average
molecular weight of the hydrophobic segment; and the average molecular weight of
the hydrophilic segment backbone used; which are obtained by 1H-NMR
measurement.
[0033]
The preferred average molecular weight ratio between the hydrophobic
segment and the hydrophilic segment varies depending on the amphiphilic polymer,
and, in the case of a block polymer wherein a hydrophobic segment(s) and a

hydrophilic segment(s) are linearly bound to each other, the average molecular
weight ratio between the hydrophilic segment(s) and the hydrophobic segment(s) is
preferably not less than 1:1, more preferably not less than 1:2, still more preferably
not less than 1:4, especially preferably not less than 1:4 and not more than 1:25.
[0034]
Preferably, in the case of a graft polymer having plural hydrophobic segments
grafted on a hydrophilic segment backbone, the average molecular weight ratio
between the hydrophilic segment backbone portion and the whole hydrophobic
segment graft chains is not less than 1:3 and the average molecular weight of each
graft chain is 2,500 to 40,000. More preferably, the overall average molecular
weight ratio is not less than 1:5 and the average molecular weight of each graft chain
is 5,000 to 40,000.
[0035]
It should be noted that the above-mentioned average molecular weight is a
number average molecular weight, unless otherwise specified. The number average
molecular weight is an average molecular weight calculated without weighting by the
molecular size, and the number average molecular weights of the amphiphilic
polymer and the polymers constituting the hydrophilic segment(s) of the amphiphilic
polymer can be calculated as molecular weights in terms of polystyrene and pullulan
measured by gel permeation chromatography (GPC). Further, the average
molecular weight of poly(hydroxy acid) can be calculated by measurement by nuclear
magnetic resonance (NMR), based on the ratio between the peak integration value for
terminal residues and the peak integration value for the others.
[0036]
The amphiphilic polymer used in the present invention may be synthesized by
a known method, and examples of the method include a method wherein a
poly(hydroxy acid) polymer is added to a polymer to be used as a hydrophilic

segment and condensation reaction is carried out with the resulting mixture to
produce an amphiphilic polymer; a method wherein hydroxy acid-activated
monomers are added to a polymer to be used as a hydrophilic segment and
polymerization reaction is carried out with the resulting mixture to produce an
amphiphilic polymer; and a method wherein, conversely, monomers for constituting
a hydrophilic segment are added to a hydrophobic segment which is a poly(hydroxy
acid) polymer and polymerization reaction is carried out with the resulting mixture to
produce an amphiphilic polymer
[0037]
For example, an amphiphilic polymer constituted by polyethylene glycol and
poly(hydroxy acid) can be produced by a method in which hydroxy acid-activated
monomers are added to polyethylene glycol in the presence of a tin catalyst, and
polymerization reaction is carried out with the resulting mixture for introduction of
the poly(hydroxy acid), thereby producing an amphiphilic block polymer [Joµm al of
Controlled Release, 71, p. 203-211 (2001)].
[0038]
Further, for example, a graft-type amphiphilic polymer constituted by a
polysaccharide and a poly(hydroxy acid) graft chain(s) can be produced as described
in (1), (2) or (3) below:
(1) a method wherein, in the presence of a tin catalyst, hydroxy acid-activated
monomers are added to a polysaccharide and polymerization reaction is carried out,
thereby introducing poly(hydroxy acid), to produce a graft-type amphiphilic polymer
[Macromolecules, 31, p. 1032-1039 (1998)];
(2) a method wherein unprotected hydroxyl groups in a part of a
polysaccharide in which most of its hydroxyl groups are protected by substituents are
activated by a base, and hydroxy acid-activated monomers are added thereto to
introduce a graft chain(s) composed of poly(hydroxy acid), followed by finally

removing the protecting groups, thereby producing a graft-type amphiphilic polymer
[Polymer, 44, p. 3927-3933, (2003)]; and
(3) a method wherein condensation reaction of a copolymer of poly(hydroxy
acid) with a polysaccharide is carried out using a dehydrating agent and/or a
functional-group-activating agent, thereby producing a graft-type amphiphilic
polymer [Macromolecules, 33, p. 3680-3685 (2000)].
[0039]
The adjuvant microparticle is described below. The adjuvant microparticle
is a microparticle having an adjuvant capacity, and the adjuvant capacity means a
capacity with which the immune response upon administration of an antigen to a
living body can be caused at a higher level than in the case of administration of the
antigen alone. Further, in the present invention, the adjuvant microparticle is a
microparticle composed of an amphiphilic polymer, and an antigen is encapsulated in
the adjuvant microparticle to form an antigen-adjuvant microparticle complex, which
is an effective ingredient of the immunogenic composition of the present invention.
[0040]
The structure of the adjuvant microparticle is not restricted, and a structure
wherein the hydrophilic segment of the amphiphilic polymer is included in the
adjuvant microparticle and the hydrophobic segment of the amphiphilic polymer is
contained as an outer layer is preferred in view of stable maintenance of the
encapsulated antigen. The method of production of an adjuvant microparticle
having such a structure is not restricted, and examples of the production method
include a method comprising: (a) the step of mixing an aqueous solvent A with a
water-immiscible organic solvent B in which an amphiphilic polymer is dissolved, to
form a reversed-phase emulsion; and (b) the step of removing the solvent from the
reversed-phase emulsion to obtain an adjuvant microparticle. In this process, by
inclusion of an antigen in the aqueous solvent A, an antigen-adjuvant microparticle

complex wherein the antigen is encapsulated can be constituted. The steps (a) and
(b) are described below.
[0041]
As the aqueous solvent A in the step (a), water, or an aqueous solution
containing a water-soluble component is used. Examples of the water-soluble
component include inorganic salts, sugars, organic salts and amino acids.
[0042]
The water-immiscible organic solvent B in the step (a) is preferably a solvent
in which the poly(hydroxy acid) of the amphiphilic polymer is soluble and the
polymer constituting the hydrophilic segment is poorly soluble or insoluble, and,
preferably, the solvent can be removed by vaporization by freeze drying. The
solubility of the water-immiscible organic solvent B in water is preferably not more
than 30 g (water-immiscible organic solvent B)/100 ml (water). Particular
examples of the water-immiscible organic solvent B include ethyl acetate, isopropyl
acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene chloride and
chloroform.
[0043]
The ratio between the water-immiscible organic solvent B and the aqueous
solvent A is 1,000:1 to 1:1, preferably 100:1 to 1:1. The concentration of the
amphiphilic polymer in the water-immiscible organic solvent B varies depending on
the types of the water-immiscible organic solvent B and the amphiphilic polymer,
and the concentration is 0.01 to 90% (w/w), preferably 0.1 to 50% (w/w), more
preferably 1 to 20% (w/w).
[0044]
In the step (a), in the process of formation of a reversed-phase emulsion with
an aqueous solvent A and a water-immiscible organic solvent B in which an
amphiphilic polymer is dissolved, the reversed-phase emulsion may be formed using,

depending on the pharmaceutical purpose, a water-immiscible organic solvent B in
which two or more types of amphiphilic polymers are dissolved.
[0045]
In the step (a), in order to aid the formation of a reversed-phase emulsion and
to form a uniform and fine reversed-phase emulsion, an additive may be added. The
additive is preferably a compound selected from C3-C6 alkyl alcohols, C3-C6 alkyl
amines and C3-C6 alkyl carboxylic acids. The structure of each alkyl chain in these
additives is not restricted, and the alkyl chain may have either a linear structure or a
branched structure, and may be either a saturated alkyl or an unsaturated alkyl. In
the present invention, the additive is especially preferably tert-butanol, iso-propanol
or pentanol.
[0046]
In the step (b), the method of removal of the solvent from the reversed-phase
emulsion is not restricted, and examples thereof include heating, drying under
reduced pressure, dialysis, freeze drying, centrifugation, filtration and reprecipitation,
and combinations thereof. Among the methods of removal of the solvent from the
reversed-phase emulsion, freeze drying is preferred since it causes less structural
change due to fusion of particles in the reversed-phase emulsion, or the like. The
conditions and the apparatus for the freeze drying are those which allow inclusion of
a freezing process and a drying step under reduced pressure, and the process of freeze
drying especially preferably comprises prior freezing, primary drying under reduced
pressure at low temperature, and secondary drying under reduced pressure, which are
conventionally carried out in freeze drying. For example, in cases where a
dispersion of an antigen-adjuvant microparticle complex in a water-immiscible
solvent is to be obtained, the reversed-phase emulsion is cooled/frozen to not more
than the melting points of the aqueous solvent A and the water-immiscible organic
solvent B, and then dried under reduced pressure, to obtain freeze-dried adjuvant

microparticles. The temperature for the prior freezing may be experimentally
determined as appropriate depending on the solvent composition, and is preferably
not more than -20°C. The degree of reduction of the pressure during the drying
process may also be determined as appropriate depending on the solvent composition,
and is preferably not more than 3,000 Pa, more preferably not more than 500 Pa, in
view of shortening of the drying time. The freeze drying is preferably carried out
using a freeze dryer for laboratory use which has a cold trap and can be connected to
a vacuum pump, or a shelf-type vacuum freeze dryer used for production of
pharmaceuticals or the like. After the prior freezing with liquid nitrogen, a cooling
medium or the like, the drying under reduced pressure may be carried out with
cooling or at room temperature using a vacuum device such as a vacuum pump.
[0047]
The type of the antigen encapsulated in the adjuvant microparticle is not
restricted, and may be a peptide, protein, glycoprotein, glycolipid, lipid, carbohydrate,
nucleic acid or polysaccharide; or a virus, bacterial cell, allergenic substance, tissue
or cell comprising these. Particular examples thereof include pollen-derived
antigens, hepatitis A virus-derived antigens, hepatitis B virus-derived antigens,
hepatitis C virus-derived antigens, hepatitis D virus-derived antigens, hepatitis E
virus-derived antigens, hepatitis F virus-derived antigens, HIV virus-derived antigens,
influenza virus-derived antigens, herpes virus (HSV-1, HSV-2)-derived antigens,
anthrax-derived antigens, chlamydia-derived antigens, pneumococcus-derived
antigens, Japanese encephalitis virus-derived antigens, measles virus-derived
antigens, rubella virus-derived antigens, Clostridium tetani-derived antigens,
chickenpox virus-derived antigens, SARS virus-derived antigens, EB virus-derived
antigens, papilloma virus-derived antigens, Helicobacter pylori-derived antigens,
rabies virus-derived antigens, West Nile virus-derived antigens, hantavirus-derived
antigens, Streptococcus-derived antigens, Staphylococcus-derived antigens,

Bordetella pertussis-derived antigens, Mycobacterium tuberculosis-derived antigens,
Plasmodium-derived antigens, poliovirus-derived antigens, antigens derived from
various zoonotic infections, cancer antigens, and antigens derived from various food
allergies.
[0048]
The encapsulated antigen does not need to be a single antigen. In view of
application of the present invention, an immune response may be induced against
cancer cells, bacteria, viruses or the like which are constituted by plural constituents.
In such cases, the antigen may be plural types of proteins or the like which may cause
immune responses, or a mixture of substances whose types cannot be specified.
Further, inclusion of plural types of antigens for positively inducing immune
responses against the plural types of antigens is one of the modes of use of the
immunogenic composition of the present invention. Preferably not less than 3 types,
more preferably a single type of an antigen(s) is/are encapsulated in the adjuvant
microparticle.
[0049]
The antigen-adjuvant microparticle complex in the present invention may
change the retention capacity of the antigen depending on the type(s) of the
polymer(s) constituting the adjuvant microparticle and the preparation method. The
mechanism by which the immunogenicity is provided by the antigen-adjuvant
microparticle complex in the present invention may include plural processes, such as
a process wherein the antigen released from the adjuvant microparticle is recognized
by immunocompetent cells, and a process wherein the adjuvant microparticle itself is
recognized by immunocompetent cells. An excellent effect can be obtained also by
the synergistic effect of these processes.
[0050]
The type of the immune response induced by the process in which the

antigen-adjuvant microparticle complex makes immunocompetent cells recognize the
antigen varies depending on the type of the process, and a preferred process may be
selected depending on the type of the immune response to be induced and the site of
administration. That is, the antigen does not necessarily need to be released from
the antigen-adjuvant microparticle complex, and the mode with which the optimum
immunogenicity of interest is realized is attained by optimization depending on the
antigen and the type of the immune response to be activated in a preferred method of
use thereof. However, in cases where the antigen is extremely quickly released
from the antigen-adjuvant microparticle complex, a long-term continuous immune-
activating action, which is an excellent property of the present invention, cannot be
obtained, so that preferably not less than 10% of the antigen in the antigen-adjuvant
microparticle complex is still retained in the living body as the complex one week
after the administration, and more preferably not less than 50% of the antigen is still
encapsulated one week after the administration. These release behaviors can be
confirmed, as shown in Examples, by in vitro evaluation mimicking the in vivo
environment.
[0051]
The antigen-adjuvant microparticle complex attains a good effect as an
effective component of the immunogenic composition of the present invention even
in a particle state wherein the complex is associated. The term "association" herein
means that two or more particles are bound together by an interparticle force or via
another substance, to form an aggregate. The interparticle force is not restricted,
and examples thereof include the hydrophobic interaction, hydrogen bond, van der
Waals force and the like. The association is not restricted to the state wherein the
microparticles are in contact with each other, and a substance having an affinity to
the microparticles may exist between the microparticles, or the microparticles may be
dispersed in a matrix. As the substance having an affinity to the microparticles, or

the matrix, a polymer is preferred, and an amphiphilic polymer whose hydrophobic
portion is poly(hydroxy acid) and which has the same constituent as that of the
adjuvant microparticle is more preferred. Particular examples thereof include
amphiphilic polymers each composed of a polysaccharide backbone and a
poly(hydroxy acid) graft chain(s), block polymers each composed of polyethylene
glycol and poly(hydroxy acid), and poly(hydroxy acid).
[0052]
The association of the antigen-adjuvant microparticle complex may be either
in a state where the complexes are reisolated upon their use, or may be in a state
where they are not reisolated upon their use. It should be noted that, even in cases
where the shape of the particle formed by association of the antigen-adjuvant
microparticle complex is in a state from which the association of the complex cannot
be known, the particle is considered to have been formed by association of the
complex as long as the production process of the particle comprises a step of
associating the complex.
[0053]
The step of associating the complexes is not restricted, and particular
examples thereof include a step of introducing the antigen-adjuvant microparticle
complex or an antigen-adjuvant microparticle complex dispersion to a liquid phase C
containing a surface modifier to remove the disperse mediµm, thereby causing
association. This step is described below.
[0054]
In cases where the antigen-adjuvant microparticle complex is dispersed in a
disperse medium to prepare a dispersion of the complex, the disperse medium is not
restricted, and in cases where the adjuvant microparticle has a hydrophilic portion in
the inside thereof, the hydrophilic portion being composed of a hydrophilic segment
of an amphiphilic polymer, and has an outer layer comprising a hydrophobic portion

composed of a hydrophobic segment of an amphiphilic polymer, the disperse
medium is preferably a solvent in which poly(hydroxy acid) of the amphiphilic
polymer is soluble and the polymer constituting the hydrophilic segment is
substantially insoluble, for the purpose of protecting the structure of the adjuvant
microparticle. In this case, the solvent may be either a water-immiscible organic
solvent or a water-miscible organic solvent. Particular examples of the solvent in
which the poly(hydroxy acid) of the amphiphilic polymer is soluble and the polymer
constituting the hydrophilic segment is substantially insoluble include ethyl acetate,
isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene
chloride, chloroform, dioxane, toluene and xylene.
[0055]
Preferably, the liquid phase C is one in which a surface modifier is soluble,
and has a higher boiling point than the disperse mediµm The liquid phase C may
be any of an aqueous solvent, water-immiscible organic solvent and water-miscible
organic solvent. As the aqueous solvent, water or an aqueous solution containing a
water-soluble component is preferred, and examples of the water-soluble component
include inorganic salts, sugars, organic salts and amino acids. Examples of the
water-immiscible organic solvent include silicone oil, sesame oil, soybean oil, corn
oil, cottonseed oil, coconut oil, linseed oil, mineral oil, castor oil, hydrogenated
castor oil, liquid paraffin, n-hexane, n-heptane, glycerol and oleic acid. Examples
of the water-miscible organic solvent include glycerin, acetone, ethanol, acetic acid,
dipropylene glycol, triethanolamine and triethylene glycol. Among these, in the
present invention, the liquid phase C is preferably an aqueous solvent or a water-
miscible organic solvent. In cases where the liquid phase C is an aqueous solvent
and the disperse medium is a water-immiscible organic solvent, the obtained
suspension of an antigen-adjuvant microparticle complex is in the form of the so
called solid-in-oil-in-water (S/O/W) emulsion, and, in cases where the liquid phase C

immiscible in the disperse mediµm, the suspension is in the form of a solid-in-oil-in-
oil(S/01/02) emulsion.
[0056]
The surface modifier is preferably a compound which stabilizes the water-oil
interface of the S/O/W emulsion or the oil-oil interface of the S/Ol/02 emulsion,
which compound has a property to enhance the colloidal stability of the particle
formed by association of the antigen-adjuvant microparticle complex. The
enhancement of the colloidal stability herein means prevention or delaying of
aggregation, in the solvent, of the particles formed by association of the antigen-
adjuvant microparticle complex. The surface modifier may be a single agent or a
mixture of plural agents.
[0057]
The surface modifier used in the present invention is preferably a hydrophilic
polymer or an amphiphilic compound.
[0058]
The hydrophilic polymer as the surface modifier is preferably polyethylene
glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyethyleneimine, polyacrylic acid,
polymethacrylic acid, poly-l,3-dioxolane, 2-methacryloyloxyethyl phosphoryl
choline polymer, poly-l,3,6-trioxane, polyamino acid, peptide, protein or sugar
polysaccharide, or an analogue of any of these. Examples of the analogue of the
hydrophilic polymer include, but are not limited to, surfactants prepared from
hydrophilic polymers by, for example, partial modification of hydrophobic groups
such as long-chain alkyl.
[0059]
The polyethylene glycol analogue as the surface modifier is preferably
"Pluronic" (registered trademark of BASF) commercially available from BASF, or its

equivalent.
[0060]
The polyamino acid as the surface modifier is preferably polyaspartic acid or
polyglutamic acid, or its analogue. Analogues prepared by introducing long-chain
alkyl to a part of polyaspartic acid or polyglutamic acid are more preferred.
[0061]
Examples of the peptide as the surface modifier include basic peptides, and
the protein as the surface modifier is preferably gelatin, casein or albumin in view of
enhancement of dispersibility. Preferred examples of the protein also include
antibodies.
[0062]
The sugar as the surface modifier is preferably a monosaccharide,
oligosaccharide or polysaccharide. The polysaccharide is preferably cellulose,
chitin, chitosan, gellan gµm, alginic acid, hyaluronic acid, pullulan or dextran, and
cholesterol-bearing pullulan is especially preferred in view of enhancement of the
dispersibility of the particle. An analogue of any of cellulose, chitin, chitosan,
gellan gµm, alginic acid, hyaluronic acid, pullulan and dextran is preferred.
[0063]
The peptide, protein or sugar as the surface modifier is especially preferably
an analogue prepared by, for example, partial modification of hydrophobic groups
such as long-chain alkyl, or an analogue prepared by modifying the above-mentioned
hydrophilic polymer or amphiphilic compound.
[0064]
Examples of the amphiphilic compound as the surface modifier include lipids
and surfactants. Preferred examples of the surfactants include nonionic surfactants
such as polyoxyethylene polypropylene glycol copolymers, sucrose fatty acid esters,
polyethylene glycol fatty acid esters, polyoxyethylene sorbitan monofatty acid ester,

polyoxyethylene sorbitan difatty acid ester, polyoxyethylene glycerol monofatty acid
ester, polyoxyethylene glycerol difatty acid ester, polyglycerol fatty acid ester,
polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil; alkyl
sulfates such as sodium lauryl sulfate, ammonium lauryl sulfate and sodium stearyl
sulfate; and lecithin.
[0065]
The volume ratio between the disperse medium in which the antigen-adjuvant
microparticle complex is dispersed and the liquid phase C is 1,000:1 to 1:1,000,
preferably 100:1 to 1:100. The association number of the antigen-adjuvant
microparticle complex obtained varies depending on this volume ratio, and, as the
ratio of the lipid phase C increases, a dispersion, in water, of particles produced by
association of a larger number of the antigen-adjuvant microparticle complex is
obtained, while, as the ratio of the lipid phase C decreases, the association number
decreases. In cases where the ratio of the liquid phase C is smaller than a solution
ratio of 1:4, most of the particles in the dispersion in water are each constituted by a
single antigen-adjuvant microparticle complex. Thus, by controlling the volume
ratio of the liquid phase C in the series of processes for production of the particle
formed by association of the antigen-adjuvant microparticle complex, the antigen-
adjuvant microparticle complex and the particle formed by association of the
complex can be selectively prepared.
[0066]
When the disperse medium containing the antigen-adjuvant microparticle
complex is mixed with the liquid phase C, a stirring device such as a magnetic stirrer,
turbine stirrer, homogenizer, membrane emulsification apparatus equipped with a
porous membrane, or the like may be used as required.
[0067]
The liquid phase C may contain, in addition to the surface modifier, various

additives such as a buffer, antioxidant, salt, polymer and/or sugar depending on the
pharmaceutical purpose. Further, the disperse medium in which the antigen-
adjuvant microparticle complex is to be dispersed may contain various additives
soluble in the disperse mediµm, such as an acidic compound, basic compound,
amphiphilic polymer and/or biodegradable polymer, for the purpose of controlling
the release rate of the encapsulated antigen by degradation or disintegration of the
complex.
[0068]
Further, an emulsifying operation of the formed solid-in-oil-in-water (S/O/W)
emulsion or solid-in-oil-in-oil (S/Ol/02) emulsion may be carried out for the
purpose of producing a finer particle formed by association of the antigen-adjuvant
microparticle complexes. The method of emulsification is not restricted as long as a
stable emulsion can be prepared thereby, and examples thereof include methods by
stirring and methods using a high-pressure homogenizer, high-speed homomixer or
the like.
[0069]
In cases where the antigen-adjuvant microparticle complex is once dispersed
in the disperse mediµm, and the obtained dispersion is added to the liquid phase C
containing the surface modifier, a suspension of the particle formed by association of
the desired adjuvant microparticle can be obtained by removal of the disperse
mediµm The method of removal of the disperse medium is not restricted, and
examples thereof include solvent evaporation, dialysis, freeze drying, centrifugation,
filtration and reprecipitation, among which solvent evaporation and freeze drying are
especially preferred. In cases where an aqueous solvent was used as the liquid
phase C, an aqueous dispersion of the particle formed by association of antigen-
adjuvant microparticle complex can be obtained by this step.
[0070]

In one preferred embodiment, the surface modifier is bound to the antigen-
adjuvant microparticle complex or the particle formed by association of the antigen-
adjuvant microparticle complex. The binding herein may be either a non-covalent
bond or covalent bond. The non-covalent bond is preferably a hydrophobic
interaction, and may also be an electrostatic interaction, hydrogen bond or van der
Waals force, or a combination of these bonds. In the non-covalent bond,
poly(hydroxy acid) of the microparticle containing an amphiphilic polymer is
preferably bound to the hydrophobic portion of the surface modifier by a
hydrophobic interaction, and, in this case, the disperse medium for the antigen-
adjuvant microparticle complex in the microparticle dispersion is preferably water,
buffer, physiological saline, aqueous surface modifier solution or hydrophilic solvent.
[0071]
The average particle size of the antigen-adjuvant microparticle complex or the
particles formed by association of the complex is preferably 0.1 to 50 µm, more
preferably 0.1 to 10 µm. In particular, the average particle size of the antigen-
adjuvant microparticle complex is preferably 0.1 to 1 µm, more preferably 0.1 to 0.5
µm, and the average particle size of the antigen-adjuvant microparticle complex is
preferably 0.1 to 50 µm, more preferably 0.1 to 10 µm, still more preferably 1 to 10
µm The average particle size of the antigen-adjuvant microparticle complex or the
particle formed by association of the complex can be directly measured using image
analysis by a scanning electron microscope (SEM: e.g., S-4800 manufactured by
Hitachi, Ltd.).
[0072]
The immunogenic composition in the present invention is a composition
which may induce an immune response in a living body, and contains the antigen-
adjuvant microparticle complex as an immunogenic substance. The type of the
immune response which is induced by the immunogenic composition is not restricted.

Examples of the type of the immune response to be induced include the Thl immune
response and the Th2 immune response, and it is known that one of these immune
responses is predominantly induced depending on the antigen, the site of
administration, and the type of the administration method. The present invention
may induce both of the Thl and Th2 immune responses. The Thl immune response
can be effectively induced by the antigen-adjuvant microparticle complex of the
present invention having a small particle size, or the particle formed by association of
the complexes, as shown in Examples. The degrees of the Thl immune response
and the Th2 immune response can be evaluated by various known methods. For
example, in the case of mouse, the amount of production of the IgG2a antibody is
known to be an index for the Thl immune response. Further, as indices for the Th2
immune response, the IgG1 antibody and the total IgG antibody amount are known.
[0073]
The immunogenic composition of the present invention contains as an
effective ingredient the antigen-adjuvant microparticle complex or the particle
formed by association of the complex, and hence has an adjuvant capacity, but, by
further inclusion of an immune-activating substance, a higher immune-activating
capacity can be realized. The immune-activating substance may be either contained
in the outside of the adjuvant microparticle or encapsulated therein, and the substance
is preferably encapsulated in the adjuvant microparticle. The immune-activating
substance is not restricted as long as it may function as an immune-activating
substance, and examples thereof include oils, aluminum salts, calcium salts, gel-
forming polymers, immune-activating cytokines and TLR receptor ligands, among
which immune-activating cytokines and TLR receptor ligands are preferred.
[0074]
Examples of the immune-activating cytokines include interleukin 12,
interferon a, interleukin 18, TNFα, interleukin 6, NO, interferon γ and interferon β.

[0075]
Examples of the TLR receptor ligands include lipoproteins; double-stranded
RNAs such as poly I:C and poly I:CLC; flagellin; single-stranded RNAs; CpG;
profilin; MPL; QS21; and TDM, among which nucleic acids such as double-stranded
RNAs, single-stranded RNAs and CpG are preferred, and CpG is more preferred.
The CpG herein means unmethylated CpG (cytosine-guanine)-motif DNAs existing
in viruses, bacteria and the like (see Japanese Translated PCT Patent Application
Laid-open No. 2001-503254). Various effective sequences are reported as CpG
motifs, and the type of the sequence is not restricted as long as it has an immune-
activating capacity, and the sequence may be prepared using a base analogue or may
be selected from various types of modified products.
[0076]
In cases where the immunogenic composition of the present invention is used
as a pharmaceutical composition or a vaccine, various pharmaceutically usefµl additives may be contained in addition to the amphiphilic polymer, hydrophilic active
substance, surface modifier and disperse mediµm Examples of the additives which
may be added include buffers, antioxidants, salts, polymer and sugars.
[0077]
The method of induction of an immune response using the immunogenic
composition of the present invention is not restricted, and the immunogenic
composition may be either administered to a living body or brought into contact with
immunocompetent cells removed to the outside of a living body. The method of
administration of the immunogenic composition to a living body is not restricted, and
examples thereof include subcutaneous administration, intradermal administration,
intramuscular administration, transnasal administration, pulmonary administration,
oral administration, sublingual administration, intravaginal administration,
intraperitoneal administration and lymph node administration, among which

intradermal administration and subcutaneous administration are preferred.
[0078]
In terms of the amount of the immunogenic composition of the present
invention to be used upon induction of the immune response, the necessary amount
of the antigen required for induction of the immune reaction of interest is set
appropriately depending on the type of the antigen, administration method, and
number of doses. For example, in cases where the immunogenic composition of the
present invention is subcutaneously administered to human to induce the immune
response, 0.01 to 1,000 µg per dose of the antigen is administered, which antigen is
contained in the immunogenic composition. The number of doses may also be
appropriately set similarly to the dose, and the immune response can be induced by 1
to 10 times of administration since the immunogenic composition of the present
invention has an action to induce an immune response continuously.
[0079]
The living body to which the immunogenic composition is administered may
be either human or a non-human animal, and the living body is preferably human; or
pig, cow, bird, sheep, horse, donkey, goat or camel, dog, cat, ferret, rabbit, monkey,
rat, mouse or guinea pig, which is kept as a livestock, pet animal or experimental
animal.
EXAMPLES
[0080]
Examples are described below, but the present invention is not restricted to
these Examples.
[0081]
Example 1 Synthesis of Dextran-Poly(lactic-co-glycolic acid) (PLGA)
(1-1) Synthesis of TMS-Dextran
Dextran (Nacalai Tesque; special grade according to Nacalai standards;

number average molecular weight, 13,000; 5.0 g) was added to formamide (100 ml),
and the resulting mixture was heated to 80°C. To this solution, 1,1,1,3,3,3-
hexamethyldisilazane (100 ml) was added dropwise for 20 minutes. Thereafter, the
resulting mixture was stirred at 80°C for 2 hours. After completion of the reaction,
the reaction solution was allowed to cool to room temperature, and two layers were
separated from each other with a separatory funnel. The upper layer was
concentrated under reduced pressure, and methanol (300 ml) was added thereto,
followed by filtering and drying the obtained solids, to obtain TMS-dextran
(Compound (1)) (11.4 g) as white solids.
[0082]
By the same method, Compounds (2) and (3) were prepared using dextran
(manufactured by Sigma; average molecular weight, not more than 1,500);
Compounds (4) and (5) were prepared using dextran (manufactured by SERVA;
average molecular weight, not more than 5,000); Compound (6) was prepared using
dextran (the same reagent as the one used for the preparation of Compound (1)); and
Compounds (7), (8) and (9) were prepared using dextran (manufactured by Nacalai
Tesque; average molecular weight, 40,000).
[0083]
(1-2) Synthesis of Dextran-PLGA (Compounds (12)-(23))
Compound (1) (0.5 g) and potassium tert-butoxide (35 mg) were dried under
heat under reduced pressure for 2 hours, and tetrahydrofuran (10 ml) was added
thereto, followed by stirring the resulting mixture for 1.5 hours at room temperature.
To this solution, a solution of (DL)-lactide (0.56 g) and glycolide (0.45 g) in
tetrahydrofuran (15 ml) was added dropwise, and the resulting mixture was stirred
for 5 minutes, followed by adding 2 drops of acetic acid to stop the reaction. After
completion of the reaction, the solvent was concentrated under reduced pressure, and
reprecipitation purification with the chloroform-methanol system and the chloroform-

diethyl ether system was carried out, to obtain white solids, which were then
dissolved in chloroform (9 ml). To the resulting solution, trifluoroacetic acid (1.0
ml) was added, and the resulting mixture was stirred at room temperature for 30
minutes. After completion of the reaction, the solvent was evaporated under
reduced pressure, and the residue was dissolved in chloroform (10 ml), followed by
adding the resulting solution to diethyl ether which had been preliminarily cooled to
0°C and filtering the obtained product, to obtain dextran-PLGA as white solids
(Compound (12)). By the same method, Compound (13) was synthesized with
(DL)-lactide (0.78g) and glycolide (0.63g); Compound (14) was synthesized with
(DL)-lactide (1.12g) and glycolide (0.9g); Compound (15) was synthesized with
(DL)-lactide (1.67g) and glycolide (1.35g).
[0084]
Further, Compound (16) was synthesized using Compound (2) with (DL)-
lactide (0.56g) and glycolide (0.45g); Compound (17) was synthesized using
Compound (3) with (DL)-lactide (0.67g) and glycolide (0.54g); Compound (18) was
synthesized using Compound (4) with (DL)-lactide (0.78g) and glycolide (0.63g);
Compound (19) was synthesized using Compound (5) with (DL)-lactide (0.89g) and
glycolide (0.72g); Compound (20) was synthesized using Compound (6) with (DL)-
lactide (0.78g) and glycolide (0.63g); Compound (21) was synthesized using
Compound (7) with (DL)-lactide (0.78g) and glycolide (0.63g); Compound (22) was
synthesized using Compound (8) with (DL)-lactide (1.12g) and glycolide (0.9g); and
Compound (23) was synthesized using Compound (9) with (DL)-lactide (1.12g) and
glycolide (0.9g).
[0085]
The weight average molecular weight and the number average molecular
weight of each of the polymers of Compounds (12) to (15) were determined by GPC
measurement (column: manufactured by Tosoh Corporation, TSK-gel a-5000x2,

DMF solvent; detector: RI; standard: pullulan). The number average molecular
weight and the number of graft chains of Compounds (12) to (23) were determined
by 1H-NMR measurement (Table 1).
[0086]
[Table 1]

Example 2 Synthesis of PEG-PLGA (Compounds (10), (11))
Polyethylene glycol monomethyl ether (NIPPON OIL & FATS CO., LTD.;
SUNBRIGHT MEH-20H; number average molecular weight, 5,128; Mw/Mn=1.02),
(DL)-lactide and glycolide were mixed together in the feeding amounts shown in
Table 2, and the resulting mixture was heated to 140°C. After stirring the mixture
for 20 minutes, tin octylate (II) (0.05 wt% with respect to polyethylene glycol
monomethyl ether) was added to the mixture, and the resulting mixture was stirred at
180°C for 3 hours. The reaction liquid was allowed to cool to room temperature,
and dissolved in chloroform (such that the concentration becomes about 100 mg/ml),
followed by reprecipitation purification with diethyl ether which had been
preliminarily cooled to 0°C. The obtained solids were filtered, and dried under
reduced pressure, to obtain a PEG-PLGA polymer as white or light-brown solids.

The number average molecular weight of the polymer was determined by 1H-NMR
(Table 2).
[0088]
[Table 2]

Example 3 Preparation of Antigen-adjuvant Microparticle Complexes Using Dex-g-
PLGA Polymers (Dex-g-PLGA Particles (l)-(28))
In 100 µl of dimethyl carbonate, 5 mg of dextran-poly(lactic-co-glycolic acid)
(PLGA) (Compounds (12)-(23)) in Example 1 was dissolved, to prepare a 50 mg/ml
polymer solution. To this polymer solution, 20 µl of tert-butanol was added, and 50
µl of the encapsulated antigen ((OVA (ovalbumin) (Sigma) or CEA
(carcinoembryonic antigen) (COSMO BIO Co., Ltd.)) and/or immune-activating
substance (CpG) shown in Table 3 was/were added to the concentration(s) described,
and the resulting mixture was stirred with a vortex mixer, to produce a reversed-
phase emulsion. As the Cpg, 5'-gggggggCGACGATCGTCAgG-3' (lowercase
letters represent phosphorothioate-modified bases) (contract synthesis by Sigma-
Genosys) was used.
[0090]
The reversed-phase emulsion was subjected to prior freezing with liquid
nitrogen, and freeze-dried using a freeze dryer (EYELA, FREEZE DRYER FD-1000)
at a trap cooling temperature of -45 °C at a degree of vacuum of 20 Pa for 24 hours.
The obtained solids were dispersed in the dispersion medium in the amount shown in
Table 3, to prepare an S/O suspension. The S/O suspension was added dropwise to

2 ml of an aqueous 10% Pluronic F-68-containing solution, and the resulting mixture
was stirred/emulsified by the stirring method described in Table 3, to prepare an
S/O/W emulsion. From the S/O/W emulsion, the water-immiscible organic solvent
was removed by solvent evaporation, to provide an antigen-adjuvant microparticle
complex dispersion. The dispersion was subjected to prior freezing with liquid
nitrogen, and freeze-dried using a freeze dryer (EYELA, FREEZE DRYER FD-1000)
at a trap cooling temperature of-45°C at a degree of vacuum of 20 Pa for 24 hours, to
obtain dry powder of an antigen-adjuvant microparticle complex (average particle
size, 0.4 µm ) and a particle (average particle size, 5 to 40 µm) formed by association
of the antigen-adjuvant microparticle complex. The result of calculation of the
average particle size of the obtained particle by observation with a scanning electron
microscope (SEM: S-4800 manufactured by Hitachi, Ltd.) is shown in Table 3.
[0091]
[Table 3]
Table 3: Recipes and average particle sizes of Dex-g-PLGA particles

[0092]
Example 4 Preparation of Antigen-adjuvant Microparticle Complexes Using
PEG-PLGA Polymers (PEG-PLGA particles (1) to (4))
In 100 µl of dimethyl carbonate, 5 mg of the PEG-PLGA polymer prepared in
Example 2 (Compound (10) or (11)) was dissolved, to prepare a 50 mg/ml polymer
solution. To this polymer solution, 20 µl of tert-butanol was added, and 50 µl of the
antigen-containing solution shown in Table 4 was added to the resulting mixture,
followed by stirring the mixture, to produce a reversed-phase emulsion solution.
The reversed-phase emulsion solution was subjected to prior freezing with liquid
nitrogen, and freeze-dried using a freeze dryer (EYELA, FREEZE DRYER FD-1000)
at a trap cooling temperature of -45 °C at a degree of vacuum of 20 Pa for 24 hours.
The obtained solids were dispersed in ethyl acetate in the amount shown in Table 4,
to prepare an S/O suspension. The S/O suspension was added dropwise to 2 ml of
an aqueous 10% Pluronic F-68-containing solution, and the resulting mixture was
stirred/emulsified with a vortex mixer, to prepare an S/O/W emulsion. From the
S/O/W emulsion, the water-immiscible organic solvent was removed by solvent
evaporation, to provide an antigen-adjuvant microparticle complex dispersion. The
dispersion was subjected to prior freezing with liquid nitrogen, and freeze-dried
using a freeze dryer (EYELA, FREEZE DRYER FD-1000) at a trap cooling
temperature of -45 °C at a degree of vacuum of 20 Pa for 24 hours, to obtain dry
powder of an antigen-adjuvant microparticle complex. The result of calculation of
the average particle size of the antigen-adjuvant microparticle complex by
observation with a scanning electron microscope (SEM: S-4800 manufactured by
Hitachi, Ltd.) is shown in Table 4.
[0093]
[Table 4]


[0094]
Comparative Example 1 Preparation of PLGA Homopolymer Particles
Containing Antigen (PLGA particles (1) and (2))
PLGA particles containing an antigen were prepared using a known
technology (International Joµm al of Pharmaceutics, 2007, vol. 334, pp. 137-148).
In 15 ml of methylene chloride, 200 mg of PLGA (manufactured by SIGMA; average
molecular weight, 40,000-75,000) was dissolved, to prepare 13.3 mg/ml PLGA
solution. To 2 ml of the polymer solution, 100 µl of 5 mg/ml aqueous OVA
solution was added with stirring at 19,000 rpm (with a homogenizer manufactured by
Polytron), and the stirring was further carried out in the same manner for 5 minutes,
to produce a W/O solution. The W/O solution was added to 20 ml of 1% aqueous
polyvinyl alcohol solution under stirring at 19,000 rpm, and the stirring was further
carried out in the same manner for 5 minutes, to produce a W/O/W solution. The
W/O/W solution was stirred at 200 rpm for 12 hours and then subjected to prior
freezing with liquid nitrogen, followed by freeze-drying using a freeze dryer (EYELA,
FREEZE DRYER FD-1000) at a trap cooling temperature of -45 °C at a degree of
vacuum of 20 Pa for 12 hours, to obtain a PLGA particle containing an antigen.
The obtained particle was observed with a scanning electron microscope (SEM: S-
4800 manufactured by Hitachi, Ltd.) to calculate the average particle size, and the
average particle size was revealed to be 2 µm. Further, the PLGA particle (2) was

prepared using an aqueous CEA solution (0.25 mg/ml) and the average particle size
was calculated in the same manner with SEM. The average particle size was
revealed to be 2 µm .
[0095]
Example 5 Measurement of Antigen Encapsulation Rates and Antigen Retention
Capacities of CEA-adjuvant Microparticle Complexes and Associated Particles
Thereof

In a 1.5-ml Eppendorf tube, 20 mg of each of the adjuvant microparticle
complexes and their associated particles (hereinafter referred to as CEA-
encapsulating particles) prepared by the methods of Examples 3 and 4 was placed,
and dissolved in 1 ml of buffer A (PBS supplemented with 0.1% bovine serum
albumin, 0.1% Pluronic F-68 and 0.02 % sodium azide), followed by separation into
particles (precipitate) and the supernatant by centrifugation at 18,000xg for 10
minutes. The supernatant was collected in another tube, and the particles were
resuspended in 1 ml of a buffer, followed by carrying out the separation again into
particles and the supernatant by centrifugation under the above conditions. This
washing operation was repeated once more (a total of three times of centrifugation),
and the CEA concentration of the supernatant collected by each time of
centrifugation was measured using an ELISA kit (manufactured by Hope
Laboratories, TM-201).
[0096]
From the amount of CEA fed upon preparation of the particle (per a weight of
the CEA-encapsulating particle of 20 mg), the total of the amounts of CEA in the
supernatants obtained by the three times of centrifugation was subtracted, and the
encapsulation rate was calculated according to the following equation.
[0097]


In terms of measurement of the release capacity of the antigen, the particle
after the three times of washing was suspended/dispersed in 1.2 ml of buffer A. A
part of this liquid (40 µl) was transferred to another tube, and centrifugation was
carried out at 18,000xg for 10 minutes to precipitate the particle, followed by
collecting 30 ju.1 of the supernatant in another tube (0-hour sample). The remaining
particle suspension was gently mixed by inversion in a 1.5-ml Eppendorf tube placed
in a 37°C incubator using a rotator at a rate of 6 rpm. From this liquid, aliquots of a
small amount (40 ul) were collected with time, and the supernatant was separated by
centrifugation in the same manner as described above. The CEA concentration in
the supernatant sample collected at each time point was measured by the above-
described ELISA method, and the release rate (%) was calculated according to the
following equation.
[0099]

The antigen encapsulation rates of the CEA-encapsulating particles were as
shown in Table 5, and it was revealed that the antigen was encapsulated in any of the
CEA-encapsulating particles at a high rate. The retention capacity for the antigen
was low in the Dex-g-PLGA particle (7) having short hydrophobic graft chains,
wherein 67.3% of the encapsulated antigen was released in one week. On the other
hand, as the length of the hydrophobic graft chain increased, the retention capacity
for the antigen increased. In Dex-g-PLGA particles having long hydrophobic graft

chains, about 90% of the fed antigen was still encapsulated even after one week.
Also in the PEG-PLGA particle, only about 4% of the antigen was released in one
week, showing a high antigen retention capacity.
[0101]
[Table 5]
Table 5: Encapsulation rates and antigen retention capacities of CEA-encapsulating

[0102]
Example 6 Subcutaneous Administration of OVA-containing Immunogenic
Composition to Mice (1)

Among the OVA-adjuvant microparticle complex-associated particles
(hereinafter referred to as OVA-encapsulating associated particles) and OVA-
adjuvant microparticle complexes (hereinafter referred to as OVA-encapsulating
particles) prepared in Examples 3 and 4, 40 mg (50 µg in terms of the fed amount of

antigen) each of
OVA-encapsulating associated particles having hydrophobic chains having
different lengths (Dex-g-PLGA particles (2), (3) and (4));
an OVA-encapsulating associated particle and an OVA-encapsulating particle
having particle sizes different from that of the Dex-g-PLGA particle (3) (Dex-g-
PLGA particles (5) and (6)); and
a PEG-PLGA particle (3);
was suspended/dispersed in 3 ml of phosphate buffered saline (PBS), followed by
centrifugation at 80xg for 5 minutes to precipitate the particle and transferring the
supernatant to another tube. The supernatant was centrifuged again at 80xg for 5
minutes to precipitate the remaining particle, and the resulting supernatant was
removed. The precipitates obtained in the first centrifugation and the second
centrifugation were combined and redispersed in 1 ml of PBS, followed by repeating
3 times of the same centrifugation operation, thereby removing the antigen that was
not encapsulated in the particle. The precipitate was finally redispersed in 150 µl of
PBS, to provide a liquid for administration. This liquid was subcutaneously
administered by single injection to the back of male Balb/C mice (Japan SLC, Inc.)
of 9 weeks old. Administration, by single injection, of the PLGA particle produced
in Comparative Example 1 or the antigen solution (50 µl) alone, as a Comparative
Example; or a solution prepared by mixing 50 µl of the antigen solution with 50 µl of
the adjuvant "Imject Alum" (manufactured by Thermo Scientific, hereinafter also
referred to as Alum), as a Reference Example; was carried out. Under each
condition, the administration was carried out for 4 individuals of mice, and the
average values of the antibody titers are shown in Fig. 1.
[0103]
The mice after the administration were kept in an environment in which the
mice can freely take food and water, while collecting blood from the tail vein with

time. To the collected blood, heparin was added to a final concentration of 3.3
IU/ml, and centrifugation was carried out at 5,000 rpm for 5 minutes to collect blood
plasma, followed by measuring the antibody titer against OVA in the blood plasma.
[0104]
The antibody titer was measured by the following method. In a 96-well
microplate (MaxiSorp, manufactured by Nunc), 100 µl of a PBS solution containing
1 ug/ml OVA was placed, and the plate was left to stand at 4°C overnight. The
solution was discarded, and 400 µl of PBS supplemented with 0.5% BSA was placed
in the plate, followed by carrying out blocking at room temperature for 2 hours.
The wells were washed once with 400 µl of a washing liquid (PBS supplemented
with 0.05% Tween 20), and 100 µl of a blood plasma sample which had been 1,000-
to 100,000-fold diluted with a dilution liquid (PBS supplemented with 0.25% BSA
and 0.05% Tween 20) was placed in each well, followed by allowing the reaction to
proceed at room temperature for 40 minutes with shaking. The wells were washed
three times with the washing liquid, and 100 µl of HRP (horse radish peroxidase)-
labeled anti-mouse IgG antibody (Zymed) (10,000-fold diluted with the dilution
liquid) was placed in each well, followed by allowing the reaction to proceed at room
temperature for 20 minutes with shaking. The wells were washed three times with
the washing liquid, and 100 µl of a coloring liquid (0.1 M sodium acetate/citrate
buffer (pH 4.5) containing 0.006% hydrogen peroxide and 0.2 mg/ml
tetramethylbenzidine) was placed in each well, followed by allowing the reaction to
proceed at room temperature for 10 minutes with shaking. The reaction was
stopped by addition of 100 µl of 1 N sulfuric acid, and the absorbance at 450 nm was
measured using a microplate reader. As a standard sample, a serially diluted anti-
OVA monoclonal antibody (HYB 094-05, manufactured by Antibody Shop) was
measured at the same time to provide a calibration curve, and the amount of the
antibody in each sample was calculated as a concentration by weight (ng/ml).

[0105]

Change in the average value of the anti-OVA antibody titer in blood plasma
with time is shown in Fig. 1. The OVA-encapsulating particle and the OVA-
encapsulating associated particles using Dex-g-PLGA (Dex-g-PLGA particles (2), (3),
(4), (5) and (6)), and the OVA-encapsulating associated particle using PEG-PLGA
(PEG-PLGA particle (3)) showed continuous antibody titer-increasing effect for not
less than 6 weeks, showing much higher values than the cases of administration of
the PLGA particle or the antigen alone in the Comparative Examples and the case of
administration of the antigen+Alum in the Reference Example. The Dex-g-PLGA
particle (6), which has a small particle size, showed a tendency to have the highest
antibody titer-increasing effect.
[0106]
Example 7 Subcutaneous Administration of CEA-containing Immunogenic
Composition to Mice

The CEA-adjuvant microparticle complexes (hereinafter referred to as CEA-
encapsulating particles) and associated particles thereof (hereinafter referred to as
CEA-encapsulating associated particles) prepared by the methods of Examples 3 and
4 were evaluated by the same method as in Example 6. The dose per individual was
1 mg (5 ug in terms of the antigen), and this dose was administered by single
injection. As the CEA-encapsulating particles and the CEA-encapsulating
associated particles, the Dex-g-PLGA particles (8), (9) and (10), which have
hydrophobic graft chains having different lengths; the Dex-g-PLGA particles (11)
and (12), which were prepared using Compound (4) as in the case of the Dex-g-
PLGA particle (9) and have different particle sizes; the Dex-g-PLGA particle (13),
which was prepared by incorporating the antigen and 25 ug of CpG into the Dex-g-

PLGA particle (9); and the PEG-PLGA particle (4); were evaluated. Further, 50 µl of an aqueous solution containing 5 ug of the antigen, as a Comparative Example;
and a mixture of 50 µl of an aqueous solution containing 5 µg of the antigen and 50
µl of Alum, as a Reference Example; were administered by single injection. Under
each condition, the administration was carried out for 4 individuals of mice, and the
average values for the respective groups are shown in Fig. 2 and Fig. 3.
[0107]
The antibody titer against CEA was measured by the following method. In a
96-well microplate (MaxiSorp, manufactured by Nunc), 100 µl of a PBS solution
containing 1 ug/ml CEA protein was placed, and the plate was left to stand at 4°C
overnight. The solution was discarded, and 400 µl of PBS supplemented with 0.5%
BSA was placed in the plate, followed by carrying out blocking at room temperature
for 2 hours. The wells were washed once with 400 µl of a washing liquid (PBS
supplemented with 0.05% Tween 20), and 100 µl of a blood plasma sample which
had been 1,000- to 100,000-fold diluted with a dilution liquid (PBS supplemented
with 0.25% BSA and 0.05% Tween 20) was placed in each well, followed by
allowing the reaction to proceed at room temperature for 40 minutes with shaking.
The wells were washed three times with the washing liquid, and 100 µl of HRP
(horse radish peroxidase)-labeled anti-mouse IgG antibody (Zymed) (10,000-fold
diluted with the dilution liquid) was placed in each well, followed by allowing the
reaction to proceed at room temperature for 20 minutes with shaking. The wells
were washed three times with the washing liquid, and 100 µl of a coloring liquid (0.1
M sodium acetate/citrate buffer (pH 4.5) containing 0.006% hydrogen peroxide and
0.2 mg/ml tetramethylbenzidine) was placed in each well, followed by allowing the
reaction to proceed at room temperature for 10 minutes with shaking. The reaction
was stopped by addition of 100 µl of 1 N sulfuric acid, and the absorbance at 450 nm
was measured using a microplate reader. As a standard sample, a serially diluted

anti-CEA monoclonal antibody (MAI-5308, manufactured by Affinity Bioreagents)
was measured at the same time to provide a calibration curve, and the amount of the
antibody in each sample was calculated as a concentration by weight (ng/ml). For
measurement of the IgG2a antibody titer, an HRP-labeled anti-mouse IgG2a antibody
(A90-107P, manufactured by Bethyl) was used instead of the HRP-labeled anti-
mouse IgG antibody, and a blood plasma sample from a single individual of mouse
whose antibody titer had increased was used as a reference sample. Samples
prepared by serial dilution of this sample were used as a standard to prepare a
calibration curve. The antibody titer corresponding to the 64,000-fold diluted
sample was represented as 1 U.
[0108]

The CEA-encapsulating particles and the CEA-encapsulating associated
particles using Dex-g-PLGA (the Dex-g-PLGA particles (8), (9), (10), (11) and (12),
and the PEG-PLGA particle (4)) showed continuous increase in the antibody titer for
about 6 weeks. Among these, the CEA-encapsulating particle having a small
particle size (Dex-g-PLGA particle (12)) showed the highest antibody titer-increasing
effect. Further, the Dex-g-PLGA particle (13) containing CpG together with the
antigen showed a higher antibody titer-increasing effect than the Dex-g-PLGA
particle (9) (Fig. 2).
[0109]
Continuous increase in the anti-IgG2a antibody titer was confirmed for the
Dex-g-PLGA particles (8), (9), (10), (11) and (12), and the PEG-PLGA particle (4).
The Dex-g-PLGA particle (13) prepared by incorporating the antigen and CpG into
the Dex-g-PLGA particle (9) showed a higher antibody titer-increasing effect than
the Dex-g-PLGA particle (9). On the other hand, in the Reference Example
wherein the mixture of the antigen and Alum was administered, continuous increase

in the antibody titer was observed, but the effect was weaker than in the cases of the
other Dex-g-PLGA particles. It was confirmed that the immunogenic composition
of the present invention activated cell-mediated immunity, for which increase in the
mouse IgG2a titer is known to be an index, continuously for a long time (Fig. 3).
[0110]
Example 8 Subcutaneous Administration of OVA-containing Immunogenic
Composition to Mice (2)

Using a Dex-g-PLGA polymer prepared by the same method as in the case of
Compound (4) in Example 1, a particle prepared by the same method as in the case of
the Dex-g-PLGA particle (3) in Example 3 (Dex-g-PLGA particle (A)) was evaluated
by the method described in Example 6. The dose per individual was 16 mg (20 fig
in terms of the amount of encapsulated OVA), and this dose was administered by
single injection. Further, as a Reference Example, a solution prepared by mixing 50
µl of an aqueous solution containing 20 µg of the OVA antigen and 50 µl of Alum
was administered by injection, once, or three times at intervals of 1 week. Under
each condition, the administration was carried out for 2 individuals of mice, and the
average values of the antibody titers are represented in Fig. 4 as measured values of
the absorbance at 450 nm.
[0111]

Change in the anti-OVA antibody in blood plasma with time is shown in Fig.
4. In the Reference Example in which the mixture of Alum and the antigen was
administered once, increase in the antibody titer was hardly observed. In the
Reference Example in which Alum and the antigen were administered three times,
sharp increase in the antibody titer was observed after the third administration, but
the increase was transient, and no increase was observed on Day 35 and later. In the

mice to which the immunogenic composition of the present invention (Dex-g-PLGA
particle (A)) was administered by single injection, continuous increase was observed
from two weeks after the administration, and continuous increase in the antibody titer
was confirmed until Day 56.
[0112]
Example 9 Subcutaneous Administration of OVA-containing Immunogenic
Composition to Mice (3)

The evaluation was carried out by the same method as in Example 6. The
dose of the OVA-encapsulating associated particle per individual was 10 mg (12.5 µg
in terms of the fed amount of the antigen). A particle prepared using a Dex-g-
PLGA polymer prepared by the same method as in the case of Compound (4) in
Example 1, which particle was prepared by the same method as in the case of the
Dex-g-PLGA particle (3) in Example 3 (Dex-g-PLGA particle (B)); a particle
prepared using a Dex-g-PLGA polymer prepared by the same method as in the case
of Compound (4) in Example 1, which particle was prepared by incorporation of
CpG together with OVA during preparation of the Dex-g-PLGA particle (3) in
Example 3 such that the dose of the CpG per individual is 6.25 µg (Dex-g-PLGA
particle (C)); or the PEG-PLGA particle (1) or (2); was administered by single
injection. As a Comparative Example, 12.5 µg of the antigen was administered, and,
as a Reference Example, a mixture of 12.5 µg of the antigen and 6.25 µg of CpG was
administered, by single injection. Blood was collected from 4 weeks after the
administration at intervals of 1 week, and the antibody titer was measured by the
same method as in Example 6. Under each condition, the administration was
carried out for 2 individuals of mice, and the average values of the antibody titers are
shown in Fig. 5.
[0113]

Any of the OVA-encapsulating associated particles (the PEG-PLGA particles
(1) and (2), and the Dex-g-PLGA particles (B) and (C)) caused increase in the
antibody titer of the animal to which the particle was administered, for not less than 6
weeks after the administration. The Dex-g-PLGA particles showed higher immune-
activating capacities than the PEG-PLGA particles. Further, the OVA-
encapsulating particle in which CpG is encapsulated (Dex-g-PLGA particle (C))
showed a higher antibody titer-increasing effect than the OVA-encapsulating particle
which does not contain CpG (Dex-g-PLGA particle (B)).
[0114]
Example 10 Subcutaneous Administration of HCV Structural Protein-containing
Immunogenic Composition to Mice

Using a Dex-g-PLGA polymer prepared by the same method as in the case of
Compound (4) in Example 1, and using the same preparation method as in the case of
the Dex-g-PLGA particle (3) in Example 3, a Dex-g-PLGA particle (D) (hereinafter
referred to as HCV-E2-encapsulating associated particle) was prepared, which Dex-
g-PLGA particle (D) was formed by association of an HCV structural protein-
adjuvant microparticle complex containing an HCV structural protein. This particle
was administered by single injection by the same method as in Example 6. As the
HCV structural protein, a chimeric protein composed of the E2 protein derived from
the J6CF strain and the Fc protein of human IgG, which chimeric protein was
prepared according to the method described in Japanese patent application No. 2008-
254338, was used. The dose per individual was 80 mg (1.5 ug in terms of the
antigen). Further, a mixture of 25 ug of CpG and the Dex-g-PLGA particle (D), and
a mixture of 25 µg of CpG, 50 µl of Alum and the Dex-g-PLGA particle (D) were
administered by single injection, respectively. As a Comparative Example, 1.5 µg

of the antigen alone was administered, and, as Reference Examples, 100 µl of an
aqueous solution containing 1.5 µg of the antigen and 25 µg of CpG, 100 µl of an
aqueous solution containing 1.5 ug of the antigen and 50 µl of Alum, and 100 µl of
an aqueous solution containing 1.5 ug of the antigen, 50 µl of Alum and 25 µg of
CpG were administered by single injection, respectively. Under each condition, the
administration was carried out for 2 individuals of mice.
[0115]
The antibody titer against the HCV structural protein was measured by the
following method. In a 96-well microplate (MaxiSorp, manufactured by Nunc), 100
µl of a PBS solution containing 0.5 µg/ml HCV structural protein was placed, and the
plate was left to stand at 4°C overnight. The solution was discarded, and 400 µl of
PBS supplemented with 0.5% BSA was placed in each well, followed by carrying out
blocking at room temperature for 2 hours. The wells were washed once with 400 µl
of a washing liquid (PBS supplemented with 0.05% Tween 20), and 100 µl of a
blood plasma sample which had been 1,000- to 100,000-fold diluted with a dilution
liquid (PBS supplemented with 0.25% BSA and 0.05% Tween 20) was placed in
each well, followed by allowing the reaction to proceed at room temperature for 40
minutes with shaking. The wells were washed three times with the washing liquid,
and 100 µl of HRP (horse radish peroxidase)-labeled anti-mouse IgG antibody
(Zymed) (10,000-fold diluted with the dilution liquid) was placed in each well,
followed by allowing the reaction to proceed at room temperature for 20 minutes
with shaking. The wells were washed three times with the washing liquid, and 100
µl of a coloring liquid (0.1 M sodium acetate/citrate buffer (pH 4.5) containing
0.006% hydrogen peroxide and 0.2 mg/ml tetramethylbenzidine) was placed in each
well, followed by allowing the reaction to proceed at room temperature for 10
minutes with shaking. The reaction was stopped by addition of 100 µl of 1 N
sulfuric acid, and the absorbance at 450 nm was measured using a microplate reader.

In Fig. 6, the average values of the antibody titers are represented as measured values
of the absorbance at 450 nm.
[0116]

The HCV-E2-encapsulating associated particle (Dex-g-PLGA particle (D))
showed continuous antibody titer-increasing effect for 7 weeks. Further, in the case
where the HCV-E2-encapsulating associated particle was mixed with CpG, and in
the case where the HCV-E2-encapsulating associated particle was mixed with CpG
and Alµm, a higher antibody titer-increasing effect was obtained compared to the
case where the HCV-E2-encapsulating associated particle alone was administered.
In the Comparative Example wherein the antigen alone was administered, increase in
the antibody titer was hardly observed. In the Reference Examples wherein the
mixture of the antigen and Alum; mixture of the antigen and CpG; or mixture of the
antigen, CpG and Alum; was administered, the antibody titer-increasing effect was
higher than in the case of administration of the antigen alone, but the titer-increasing
effect was much lower than in the case of administration of the HCV-E2-
encapsulating associated particle.
[0117]
Example 11 Subcutaneous Administration of CEA-containing Immunogenic
Composition to Mice (2)

The CEA-adjuvant microparticle complex-associated particles prepared by
the methods in Examples 3 and 4 (hereinafter referred to as CEA-encapsulating
associated particles) were evaluated by the same method as in Example 7. The dose
per individual was 400 ug (1 ug in terms of the antigen), and this dose was
administered at Week 0 and Week 4. As the CEA-encapsulating associated
particles, the Dex-g-PLGA particles (14) and (15) having hydrophilic chains of

dextran having a molecular weight of 1,500, Dex-g-PLGA particles (16) and (17)
having hydrophilic chains of dextran having a molecular weight of 5,000, Dex-g-
PLGA particle (18) having hydrophilic chains of dextran having a molecular weight
of 175,000, and Dex-g-PLGA particle (19) having hydrophilic chains of dextran
having a molecular weight of 40,000 were evaluated. Under each condition, the
administration was carried out for 5 individuals of mice, and the average value in
each group is shown in Fig. 7. The antibody titer against CEA was measured by the
same method as in Example 7.
[0118]

The CEA-encapsulating associated particles using Dex-g-PLGA (Dex-g-
PLGA particles (14), (15), (16), (17), (18) and (19)) showed continuous increase in
the antibody titer. Among these, the CEA-encapsulating associated particles
constituted by dextran having a molecular weight of 175,000 and a molecular weight
of 40,000 (Dex-g-PLGA particles (18) and (19)) showed higher antibody titer-
increasing effects than the CEA-encapsulating associated particles constituted by
dextran hydrophilic chains having a molecular weight of 1,500 and a molecular
weight of 5,000 (Dex-g-PLGA particles (14), (15), (16) and (17)) (Fig. 7).
[0119]
Example 12 Subcutaneous Administration of CEA-containing Immunogenic
Composition to Mice (3)

The CEA-adjuvant microparticle complex-associated particles (hereinafter
referred to as CEA-encapsulating associated particles) prepared by the methods of
Examples 3 and 4 were evaluated by the same method as in Example 7. The dose
per individual was 400 µg (1 µg in terms of the antigen), and this dose was
administered at Week 0 and Week 4. As the CEA-encapsulating associated

particles, 3 types of particles which were prepared using the same polymer but have
different particle sizes (Dex-g-PLGA particle (20) (particle size, 0.4 µm ), Dex-g-
PLGA particle (21) (particle size, 5 µm) and Dex-g-PLGA particle (particle size, 40
µm)) were evaluated. Under each condition, the administration was carried out for
5 individuals of mice, and the average value in each group is shown in Fig. 8. The
antibody titer against CEA was measured by the same method as in Example 7.
[0120]

The CEA-encapsulating particle and CEA-encapsulating associated particles
using Dex-g-PLGA (Dex-g-PLGA particles (20), (21) and (22)) showed continuous
increase in the antibody titer. Among these, the Dex-g-PLGA particle (20) having
an average particle size of 0.4 µm showed the highest antibody titer-increasing effect;
the Dex-g-PLGA particle (21) having an average particle size of 5 µm showed the
second highest antibody titer-increasing effect; and the Dex-g-PLGA particle (22)
having an average particle size of 40 µm showed the lowest antibody titer-increasing
effect (Fig. 8).
[0121]
Example 13 Subcutaneous Administration of OVA-containing Immunogenic
Composition to Mice (4)

The evaluation was carried out by the same method as in Example 9. The
dose per administration was 20 ug in terms of the amount of the antigen in all the
cases, and the antigen was administered a total of 3 times at Week 0, Week 2 and
Week 4. The evaluation was carried out by comparison among the case where a
mixture of 20 µg of OVA and the Dex-g-PLGA particle (24) containing no antigen
(16 mg in terms of the polymer amount) was administered, the case where the Dex-g-
PLGA particle (23) containing 20 ug of OVA (16 mg in terms of the polymer

amount) was administered, the case where a mixture of 20 µg of OVA and 50 µl of
Alum was administered, and the case where 20 µg of OVA and the Dex-g-PLGA
particle (24) containing no antigen were administered at different sites. The
antibody titer in blood was measured by the same method as in Example 9. Under
each condition, the administration was carried out for 2 individuals of mice. Fig. 9
shows the average value of the antibody titer.
[0122]

All the particles showed the antibody titer-increasing effect. The OVA-
encapsulating associated particle (Dex-g-PLGA particle (23)) showed a higher
antibody titer-increasing effect compared to the case where the mixture of OVA and
the particle (Dex-g-PLGA particle (24)) containing no antigen was administered, and
the case where OVA and the Dex-g-PLGA particle (24) were administered at
different sites.
[0123]
Example 14 Subcutaneous Administration of CEA-containing Immunogenic
Composition to Mice (4)

The evaluation was carried out by the same method as in Example 7. Only
in the case where a mixture of CEA and Alum was administered, a total of 3 times of
administration was carried out at Week 0, Week 2 and Week 4, and in the other cases
where the CEA-encapsulating particles and CEA-encapsulating associated particles
were administered, single administration was carried out at Week 0. The evaluation
was carried out by comparison among particles which were prepared using the same
polymer but have different particle sizes: Dex-g-PLGA particle (25) (particle size,
0.4 µm; polymer content, 4 mg; amount of administration of antigen, 10 µg), Dex-g-
PLGA particle (26) (particle size, 5 µm; polymer content, 4 mg; amount of

administration of antigen, 10 µg), Dex-g-PLGA particle (27) (particle size, 40 µm ;
polymer content, 4 mg; amount of administration of antigen, 10 µg) , and Dex-g-
PLGA particle (28) (particle size, 0.4 µm ; polymer content, 4 mg; amount of
administration of antigen, 1 µg) . Further, in terms of the Dex-g-PLGA particle (25),
comparisons were made with the cases where the amount of administration was
reduced to 1/10 (polymer content, 400 ug; amount of administration of antigen, 1 µg)
or 1/100 (polymer content, 40 ug; amount of administration of antigen, 0.1 µg) . As
a Comparative Example, the PLGA particle (2) prepared by encapsulation of CEA
was evaluated. Under each condition, the administration was carried out for 6
individuals of mice, and the antibody titer, IgGl and IgG2a in blood were measured
by the same method as in Example 7. Fig. 10 and Fig. 11 show the average values.
[0124]

The CEA-encapsulating particles (Dex-g-PLGA particles (25) and (26))
having an average particle size of 0.4 µm and an average particle size of 5 µm
showed higher antibody titer-increasing effect compared to the CEA-encapsulating
particle (Dex-g-PLGA particle (27)) having an average particle size of 40 µm
Although the Dex-g-PLGA particle (25) showed a high antibody titer-increasing
effect, reduction of it amount of administration to 1/10 or 1/100 resulted in decrease
in the antibody titer-increasing effect, and in the case where the amount of
administration of the particle was 1/100, only a low antibody titer-increasing effect
could be obtained. Comparison between the administration of the Dex-g-PLGA
particle (25) in an amount of 1/10 and the administration of the Dex-g-PLGA particle
(28), in both of which the amount of the antigen administered was 1 ug, showed that
administration of a larger amount of the polymer results in a higher antibody titer-
increasing effect (Fig. 10).
[0125]

Further, for blood at Week 6, the IgG2a antibody titer was measured by the
same method as in Example 7, and the IgGl antibody titer was measured by the same
method as in the measurement of the IgG2a antibody titer using an IgGl antibody.
According to measurement of the ratio between these, administration of a mixture of
Alum and the antibody resulted in a low IgG2a/IgGl value, while administration of
the Dex-g-PLGA particle (25), (26) or (27) resulted in a high IgG2a/IgGl value,
showing a tendency that a smaller particle size results in a higher IgG2a/IgGl value
(Fig. 11).
INDUSTRIAL APPLICABILITY
[0126]
The immunogenic composition of the present invention can be used as a
vaccine for therapy and/or prophylaxis of infectious diseases, cancer and the like.

we claim:
1. An immunogenic composition comprising as an effective ingredient an
antigen-adjuvant microparticle complex containing an antigen encapsulated in an
adjuvant microparticle composed of an amphiphilic polymer(s) whose hydrophobic
segment is a poly(hydroxy acid).
2. The immunogenic composition according to claim 1, comprising as an
effective ingredient a particle composed of said antigen-adjuvant microparticle
complex associated together.
3. The immunogenic composition according to claim 1 or 2, wherein said
adjuvant microparticle has a hydrophilic portion in the inside thereof, said
hydrophilic portion being composed of a hydrophilic segment of said amphiphilic
polymer, and has an outer layer composed of a hydrophobic portion constituted by
said hydrophobic segment of said amphiphilic polymer.
4. The immunogenic composition according to any of claims 1 to 3, wherein
said hydrophilic segment of said amphiphilic polymer is a polysaccharide or a
polyethylene glycol.
5. The immunogenic composition according to any of claims 1 to 4, wherein
said amphiphilic polymer is a graft amphiphilic polymer composed of a
polysaccharide backbone and a poly(hydroxy acid) graft chain.
6. The immunogenic composition according to claim 4 or 5, wherein said
polysaccharide is dextran.
7. The immunogenic composition according to any of claims 1 to 4, wherein
said amphiphilic polymer is a block polymer composed of a poly(hydroxy acid) and a
polyethylene glycol.
8. The immunogenic composition according to any of claims 1 to 7, wherein
said poly(hydroxy acid) is a poly(lactic-co-glycolic acid).

9. The immunogenic composition according to any of claims 1 to 8, further
comprising a surface modifier bound to said poly(hydroxy acid) of said adjuvant
microparticle.
10. The immunogenic composition according to any of claims 1 to 9, wherein the
average particle size of said antigen-adjuvant microparticle complex or said particle
composed of said antigen-adjuvant microparticle complex associated together is 0.1
to 50 µm
11. The immunogenic composition according to any of claims 1 to 10, further
comprising an immune-activating substance as an effective ingredient.
12. The immunogenic composition according to claim 11, wherein said immune-
activating substance is a nucleic acid.
13. The immunogenic composition according to claim 11 or 12, wherein said
immune-activating substance is CpG.

An immunogenic composition comprising as an effective ingredient an
antigen-adjuvant microparticle complex containing an antigen encapsulated in an
adjuvant microparticle composed of an amphiphilic polymer(s) whose hydrophobic
segment is a poly(hydroxy acid), or a particle composed of the antigen-adjuvant
microparticle complex associated together, can induce a high immune response
against the antigen even with a small amount of the antigen and a small number of
doses, so that the immunogenic composition is usefµl as a vaccine effective for
therapy and prophylaxis of infectious diseases, cancer and the like.