U.S. patent application number 10/679708 was filed with the patent office on 2005-09-01 for novel compositions of saponin adjuvants and excipients.
This patent application is currently assigned to Antigenics, Inc.. Invention is credited to Beltz, Gerald A., Kensil, Charlotte A..
Application Number | 20050191310 10/679708 |
Document ID | / |
Family ID | 22009474 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191310 |
Kind Code |
A1 |
Kensil, Charlotte A. ; et
al. |
September 1, 2005 |
Novel compositions of saponin adjuvants and excipients
Abstract
Certain novel compositions of the adjuvant saponin QS-21 having
improved properties are disclosed. The compositions of the present
invention are designed (1) to minimize the lytic effects of QS-21,
(2) to improve the tolerance of QS-21 containing formulations in
humans or other animals, (3) to stabilize the QS-21 from alkaline
hydrolysis and/or (4) to maintain the high adjuvant potency of the
QS-21 product. These compositions may be employed with vaccines
comprising proteins or peptides, polysaccharides, lipids, or
nucleic acids.
Inventors: |
Kensil, Charlotte A.;
(Milford, MA) ; Beltz, Gerald A.; (Lexington,
MA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Antigenics, Inc.
|
Family ID: |
22009474 |
Appl. No.: |
10/679708 |
Filed: |
October 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10679708 |
Oct 3, 2003 |
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09143145 |
Aug 28, 1998 |
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6645495 |
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60057255 |
Aug 29, 1997 |
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Current U.S.
Class: |
424/185.1 ;
514/25; 514/44R; 514/54; 514/58 |
Current CPC
Class: |
A61K 47/26 20130101;
A61K 47/10 20130101; A61P 37/04 20180101; A61K 39/39 20130101; A61K
47/40 20130101; A61P 25/04 20180101; A61K 2039/55577 20130101; A61K
47/42 20130101; A61K 9/0019 20130101 |
Class at
Publication: |
424/185.1 ;
514/025; 514/058; 514/054; 514/044 |
International
Class: |
A61K 048/00; A61K
039/00; A61K 031/715; A61K 031/724 |
Claims
1-45. (canceled)
46. A method for enhancing an immune response to an antigen in an
individual to which said antigen is administered comprising
administering to said individual an amount, effective to enhance
said immune response, of a composition comprising a saponin
adjuvant and an excipient, said excipient being selected from the
group consisting of a .beta.-cyclodextrin, a human serum albumin, a
deacylsaponin, a Polysorbate, and Triton X-100.
47. The method of claim 46, wherein the saponin adjuvant is a
heterogeneous saponin adjuvant.
48. The method of claim 47, wherein the heterogenous saponin
adjuvant is Quil-A.
49. The method of claim 46, wherein the saponin adjuvant comprises
two or more substantially pure saponins selected from the group
consisting of QS-7, QS-17, QS-18, and QS-21.
50. The method of claim 46, wherein the saponin adjuvant is a
substantially pure saponin adjuvant.
51. The method of claim 50, wherein the substantially pure saponin
adjuvant is selected from the group consisting of QS-7, QS-17,
QS-18 and QS-21.
52. The method of claim 50, wherein the substantially pure saponin
adjuvant is QS-21.
53. The method of claim 50, wherein the substantially pure saponin
adjuvant is QS-7.
54. The method of claim 46, wherein the antigen is a peptide, a
protein, a polysaccharide, a lipid, or a nucleic acid.
55. The method according to claim 46, wherein the excipient is a
Polysorbate or Triton X-100.
56. The method according to claim 55, wherein the excipient is a
Polysorbate.
57. The method according to claim 56, wherein the Polysorbate is
Polysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate
80.
58. The method according to claim 46, wherein the excipient is
.beta.-cyclodextrin.
59. The method according to claim 58, wherein the
.beta.-cyclodextrin is hydroxypropyl-.beta.-cyclodextrin.
60. The method of claim 46, wherein the excipient is a human serum
albumin.
61. The method of claim 46, wherein the excipient is a
deacylsaponin ("DS").
62. The method of claim 61, wherein the excipient is DS-1.
63. The method of claim 46, wherein said antigen and said
composition are administered to said individual concurrently.
64. The method of claim 46, wherein said individual is a
mammal.
65. The method of any one of claims 46-53, 56, 58 and 63, wherein
said individual is a human.
Description
FIELD OF THE INVENTION
[0001] The present invention relate& to the field of immune
adjuvants and the use thereof as immune adjuvants in vaccines. The
compositions of the present invention exhibit significantly
improved properties relevant to the lytic effect, tolerance to
QS-21 associated pain, and product stability of QS-21, and maintain
full adjuvant activity.
BACKGROUND OF THE INVENTION
[0002] Adjuvant saponins have been identified and purified from an
aqueous extract of the bark of the South American tree, Quillaja
saponaria Molina. Among the 22 peaks which were separable and
displayed saponin activity, QS-21 was one of the more predominant
purified saponins. This saponin has been substantially purified by
high pressure liquid chromatography (HPLC), low pressure liquid
silica chromatography, and hydrophilic interactive chromatography
(HILIC). QS-21 has been found to be useful as an immune adjuvant
for enhancing immune responses in individuals at a much lower
concentration than the previously available heterogeneous saponin
preparations without the toxic effects associated with crude
saponin preparations.
[0003] QS-21 is a membrane-lytic triterpene glycoside saponin. It
forms micelles of approximately the same radius as bovine serum
albumin (Kensil, U.S. Pat. No. 5,057,540) and has a critical
micellar concentration of approximately 50 .mu.g/ml in PBS
(Soltysik, S., et al., 1995, Vaccine 13:1403-1410).
[0004] The potency of an adjuvant formulation containing an antigen
plus QS-21 can be assessed in experiments that address the
relationship of adjuvant dose to immunological function
(dose-response experiments). A decrease in adjuvant potency is
expected to increase the minimum dose (threshold dose) required for
enhancement of immune response. A desirable composition is expected
to maintain an equivalent or better potency than the formulation
that is used as a reference. For QS-21, the reference formulation
is a simple solution in phosphate-buffered saline (PBS) or
saline.
[0005] The adjuvant activity of QS-21 is assessed in animal models
such as mice. The primary responses measured are increases in
antigen-specific antibody and antigen-specific cytotoxic T
lymphocytes (CTL). The threshold dose of QS-21 that will enhance
murine immune response (antibody or CTL) has been measured in
simple buffer solution such as PBS. A dose of 2.5 .mu.g has been
shown to be the threshold dose for antibody (Kensil, C. R., et al.,
1993, Vaccine Research 2:273-281) and for CTL (Newman, M. J., et
al., 1992, J. Immunology 148:2357-2362) to the antigen ovalbumin
(OVA) in C57BL/6 mice in PBS. Similar threshold doses were observed
when aluminum hydroxide was included in the PBS formulation
(Kensil, C. R., et al., 1993, Vaccine Research 2:273-281). However,
it is expected that there may be differences in potency between
different compositions of a given adjuvant.
[0006] Despite these beneficial qualities, QS-21 possesses some
unwelcome qualities as well. For instance, QS-21 associates with
phospholipid bilayers and causes a lytic effect on certain cell
membranes (i.e., erythrocytes). QS-21 will absorb to the
phospholipid bilayer of sheep erythrocytes and cause the red blood
cells to release hemoglobin This hemoglobin release, which is known
as hemolysis, occurs at a concentration of approximately 5-7
.mu.g/ml in a simple buffer such as saline or PBS (Kensil, C. R.,
et al., 1991, J. Immunology 146:431-437). At higher concentrations
(above the critical micellar concentration of QS-21), total lysis
of the red blood cell membrane occurs. The lytic effect of QS-21
is, therefore, an undesirable property for a composition.
[0007] In in vivo studies, hemolysis is not noted. However, after
intramuscular injection of QS-21/saline solutions into New Zealand
white rabbits, mild to moderate fibroblast damage or necrosis is
noted in some animals when the injection site is analyzed
histopathologically (Kensil, C. R., et al., 1995, In: Vaccine
Design: The Subunit and Adjuvant Approach Powell, M. F. and Newman,
M. J., Eds., Plenum Press, NY). Further, creatine kinase, a marker
for muscle damage is increased after injection with QS-21 in saline
or PBS. This rise is believed to be due to the lytic effect of
QS-21 on cell membranes.
[0008] Moreover, in clinical trials, some individuals have
experienced an immediate, transient pain after injection with QS-21
in simple buffer solutions (saline or PBS). This pain, described by
most individuals as a burning pain, may be a secondary reaction
correlated with the lytic effect of the QS-21 adjuvant. Patient
pain is likewise an objectionable property for a composition.
[0009] Product stability is another concern for QS-21 containing
compositions. The shelf life of a vaccine product is typically
defined by the extent of time to reach a defined and acceptable low
level of degradation (such as, the time to 10% degradation, also
known as tgo). Most commercial vaccine products have a shelf life
of at least 18 to 24 months when stored in the refrigerator at
4.degree. C. Adjuvants, which are essential components of vaccines,
therefore must also have equally long shelf lives. However, the
shelf life of a 50 .mu.g/ml solution of QS-21 at pH 7.0 at
4.degree. C. is reached in about 3 months. The reason for the short
shelf life is because the ester bond of QS-21 is increasingly
labile at increasing pH and because monomers of QS-21, as opposed
to micelles, are subject to hydrolysis. The need to stabilize
compositions of QS-21 adjuvant is significant.
SUMMARY OF THE INVENTION
[0010] A need exists for compositions of the saponin adjuvant QS-21
that may be used to boost the antigenic immune response in a
relatively low dose with low local reactions and side effects, but
also features a reduced lytic effect, improved tolerance to QS-21,
and an increased stability. Accordingly, the present invention
provides novel compositions of QS-21 that have these improved
characteristics compared to a simple solution of QS-21 in a buffer
such as saline or PBS. Surprisingly, the full adjuvant potency of
QS-21 in the disclosed compositions is not compromised compared to
a control formulation of QS-21 in PBS.
DESCRIPTION OF THE FIGURES
[0011] FIG. 1 depicts a graph showing the adjuvant potency of
various compositions. FIG. 1A shows the effect of Polysorbate 40,
Polysorbate 60, and Polysorbate 80 on the immune response of Balb/c
mice to ovalbumin at different concentrations of QS-21. FIG. 1B
shows the effect of methyl-.beta.-cyclodextrin on the immune
response of Balb/c mice to ovalbumin at different concentrations of
QS-21.
[0012] FIG. 2 depicts a graph showing the effect of Polysorbate 80
and hydroxypropyl-.beta.-cyclodextrin on Type 14 IgG3 antibody
response to a T-independent polysaccharide antigen.
[0013] FIG. 3 shows a bar graph of patients' tolerance to pain for
various excipients in QS-21 adjuvant compositions from Trial 1.
This figure shows how the pain scores are classified as no pain,
mild pain, moderate pain, or severe pain, where 0=no pain, 1-3=mild
pain, 4-7=moderate pain, and 8-10=severe pain.
[0014] FIG. 4 shows the individual scores for the patients'
tolerance to pain in FIG. 3. This figure shows individual immediate
pain scores after injection of a given formulation on a scale of
0-10, where 0 is no pain and 10 is maximum pain.
[0015] FIG. 5 shows a bar graph of patients' tolerance to pain for
various excipients in QS-21 adjuvant compositions from Trial 2.
This figure shows how the pain scores are classified as no pain,
mild pain, moderate pain, or severe pain, where 0=no pain, 1-3=mild
pain, 4-7=moderate pain, and 8-10=severe pain.
[0016] FIG. 6 shows the individual scores for the patients'
tolerance to pain in FIG. 5. This figure shows individual immediate
pain scores after injection of a given formulation on a scale of
0-10, where 0 is no pain and 10 is maximum pain. Mean and median
scores for each formulation are listed below each formulation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] The saponins of the present invention may be obtained from
the tree Quillaja saponaria Molina.
[0018] The term "saponin" as used herein includes glycosidic
triterpenoid compounds which produce foam in aqueous solution, have
hemolytic activity in most cases, and possess immune adjuvant
activity. The invention encompasses the saponin per se, as well as
biologically active fragments thereof.
[0019] The invention also concerns compositions, such as
immunologic compositions, comprising one or more substantially pure
saponin fractions, and methods of using these compositions as
immune adjuvants.
[0020] More particularly, the compositions of the present invention
may reduce the in vitro lytic effects of a saponin adjuvant
containing formulation. Another preferred composition is one that
may maintain the maximum adjuvant activity of a saponin. Yet
another preferred composition may increase the stability of a
saponin adjuvant containing composition from alkaline hydrolysis.
Other compositions may preferably improve an individual's tolerance
to saponin adjuvant associated pain from a formulation containing a
saponin adjuvant.
[0021] As described in Kensil, et al., U.S. Pat. No. 5,057,540, the
contents of which are fully incorporated by reference herein, the
adjuvant activity of such saponins may be determined by any of a
number of methods known to those of ordinary skill in the art. The
increase in antibody titer of antibody against specific antigen
upon administration of an adjuvant may be used as a criteria for
adjuvant activity. (Dalsgaard, Acta Verterinia Scandinavica, 69:1
(1978); Bomford, Int. Archs. Allergy Appl. Immun. 77:409 (1985).)
Briefly, one such test involves injecting CD-1 mice intradermally
with an antigen (for instance, i.e., bovine serum albumin, BSA)
mixed with varying amounts of the potential adjuvant. Sera was
harvested from the mice two weeks later and tested by ELISA for
anti-BSA antibody.
[0022] "QS-21" designates the mixture of isomeric components
QS-21-V1 and QS-21-V2 which appear as a single peak on reverse
phase HPLC on Vydac C4 (5 .mu.m particle size, 300 .ANG. pore, 4.6
mm ID.times.25 cml) in 40 mM acetic acid in methanol/water (58/42,
v/v). The component fractions are referred to specifically as
QS-21-V1 and QS-21-V2 when describing experiments performed on the
further purified components.
[0023] The term "substantially pure" means substantially free from
compounds normally associated with the saponin in its natural state
and exhibiting constant and reproducible chromatographic response,
elution profiles, and biologic activity. The term "substantially
pure" is not meant to exclude artificial or synthetic mixtures of
the saponin with other compounds.
[0024] The substantially pure QS-7 saponin also referred to as QA-7
in U.S. Pat. No. 5,057,546) is characterized as having immune
adjuvant activity, containing about 35% carbohydrate (as assayed by
anthrone) per dry weight, having a UV absorption maxima of 205-210
nm, a retention time of approximately 9-10 minutes on RP-HPLC on a
Vydac C.sub.4 column having a 5 .mu.m particle size, 300 .ANG.
pore, 4.6 mm ID.times.25 cm L in a solvent of 40 mM acetic acid in
methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting
with 52-53% methanol form a Vydac C.sub.4 column having a 5 .mu.m
particle size, 300 .ANG. pore, 10 mM ID.times.25 cm L in a solvent
of 40 mM acetic acid with gradient elution from 50 to 80% methanol,
having a critical micellar concentration of approximately 0.06%
(w/v) in water and 0.07% (w/v) in phosphate buffered saline,
causing no detectable hemolysis of sheep red blood cells at
concentrations of 200 .mu.g/ml or less, and containing the
monosaccharide residues terminal rhamnose, terminal xylose,
terminal glucose, terminal galactose, 3-xylose, 3,4-rhamnose,
2,3-fucose, and 2,3-glucuronic acid, and apiose (linkage not
determined).
[0025] The substantially pure QS-17 saponin (also referred to as
QA-17 in U.S. Pat. No. 5,057,540) is characterized as having immune
adjuvant activity, containing about 29% carbohydrate (as assayed by
anthrone) per dry weight, having a UV absorption maxima of 205-210
nm, a retention time of approximately 35 minutes on RP-HPLC on a
Vydac C.sub.4 column having a 5 .mu.m particle size, 300 .ANG.
pore, 4.6 mm ID.times.25 cm L in a solvent of 40 mM acetic acid in
methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting
with 63-64% methanol from a Vydac C.sub.4 column having a 5 .mu.m
particle size, 300 .ANG. pore, 10 mM ID.times.25 cm L in a solvent
of 40 mM acetic add with gradient elution from 50 to 80% methanol,
having a critical micellar concentration of approximately 0.06%
(w/v) in water and 0.03% (w/v) in phosphate buffered saline,
causing hemolysis of sheep red blood cells at 25 .mu.g/ml or
greater, and containing the monosaccharide residues terminal
rhamnose, terminal xylose, 2-fucose, is characterized as having
immune adjuvant activity, containing about 35% carbohydrate (as
assayed by anthrone) per dry weight, having a UW absorption maxima
of 205-210 nm, a retention time of approximately 9-10 minutes on
RP-HPLC on a Vydac C.sub.4 column having a 5 .mu.m particle size,
300 .ANG. pore, 4.6 mm ID.times.25 cm L in a solvent of 40 mM
acetic acid in methanol/water (58/42; v/v) at a flow rate of 1
ml/min, eluting with 52-53% methanol form a Vydac C.sub.4 column
having a 5 .mu.m particle size, 300 .ANG. pore, 10 mM ID.times.25
cm L in a solvent of 40 mM acetic acid with gradient elution from
50 to 80% methanol, having a critical micellar concentration of
approximately 0.06% in water and 0.07% in phosphate buffered
saline, causing no detectable hemolysis of sheep red blood cells at
concentrations of 200 .mu.g/ml or less, and containing the
monosaccharide residues terminal rhamnose, terminal xylose,
2-fucose, 3-xylose, 3,4-rhamnose, 2,3-glucuronic acid, terminal
glucose, 2-arabinose, terminal galactose and apiose (linkage not
determined).
[0026] The substantially pure QS-18 saponin (also referred to as
QA-18 in U.S. Pat. No. 5,057,540) is characterized as having immune
adjuvant activity, containing about 25-26% carbohydrate (as assayed
by anthrone) per dry weight, having a UV absorption maxima of
205-210 nm, a retention time of approximately 38 minutes on RP-HPLC
on a Vydac C.sub.4 column having a 5 .mu.m particle size, 300 .ANG.
pore, 4.6 mm ID.times.25 cm L in a solvent of 40 mM acetic add in
methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting
with 64-65% methanol from a Vydac C.sub.4 column having a 5 .mu.m
particle size, 300 .ANG. pore, 10 mM ID.times.25 cm L in a solvent
of 40 mM acetic acid with gradient elution from 50 to 80% methanol,
having a critical micellar concentration of approximately 0.04%
(w/v) in water and 0.02% (w/v) in phosphate buffered saline,
causing hemolysis of sheep red blood cells at 25 .mu.g/ml or
greater, and containing the monosaccharide residues terminal
arabinose, terminal apiose, terminal xylose, terminal glucose,
terminal galactose, 2-fucose, 3-xylose, 3,4-rhamnose, and
2,3-glucuronic acid.
[0027] The substantially pure QS-21 saponin (also referred to as
QA-21 in U.S. Pat. No. 5,057,540) is characterized as having immune
adjuvant activity, containing about 22% carbohydrate (as assayed by
anthrone) per dry weight, having a UV absorption maxima of 205-210
nm, a retention time of approximately 51 minutes on RP-HPLC on a
Vydac C.sub.4 column having a 5 .mu.m particle size, 300 .ANG.
pore, 4.6 mm ID.times.25 cm L in a solvent of 40 mM acetic acid in
methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting
with 69-70% methanol from a Vydac C.sub.4 column having a 5 um
partide size, 300 .ANG. pore, 10 mM ID.times.25 cm L in a solvent
of 40 mM acetic acid with gradient elution from 50 to 80% methanol,
having a critical micellar concentration of approximately 0.03%
(w/v) in water and 0.02% (w/v) in phosphate buffered saline,
causing hemolysis of sheep red blood cells at 25 .mu.g/ml or
greater. The component fractions, substantially pure QS-21-V1 and
QS-21-V2 saponins, have the same molecular weight and identical
spectrums by FA-BMS. They differ only in that QS-21-V1 has a
terminal apiose which is xylose in QS-21-V2 (which therefore has
two terminal xyloses and no apiose). The two components
additionally contain the monosaccharides terminal arabinose,
terminal apiose, terminal xylose, 4-rhamnose, terminal galactose,
2-fucose, 3-xylose, and 2,3-glucuronic acid.
[0028] The invention may also encompass impure forms of saponin
adjuvants. For example, one preferred embodiment is the heterogenic
saponin adjuvant known as "Quil A." Commercial preparations of Quil
A are available from Superfos (Vedbaek, Denmark) and have been
isolated from the bark of the South American tree, Quillaja
saponaria Molina. Quil A is characterized chemically as
carbohydrate moieties in glycosidic linkage to the triterpenoid
quillaic acid. Quil A possesses immune adjuvant activity and
separates into 20 discrete peaks by RP-HPLC on Vydac C.sub.4 column
having a 5 .mu.m particle size, 300 .ANG. pore, 4.6 mM ID.times.25
cm L in a solvent of 40 mM acetic acid in methanol water (U.S. Pat.
No. 5,057,540).
[0029] The invention also relates to a composition which comprises
a saponin adjuvant of the present invention, an antigen, and an
excipient. Preferably, the adjuvant is QS-21. Preferably, the
excipients may be nonionic surfactants, polyvinylpyrollidone, human
serum albumin, aluminum hydroxide, agents with anesthetic action,
and various unmodified and devivatized cyclodextrins. More
preferably, the nonionic surfactants may include Polysorbate 20,
Polysorbate 40, Polysorbate 60, and Polysorbate 80. The
polyvinylpyrollidone may preferably be Plasdone C15, a
pharmaceutical grade of polyvinylpyrollidone. The agent having
anesthetic action preferably is benzyl alcohol. A preferred
cyclodextrin is a hydroxypropyl-.beta.-cyclodextrin, which reduces
QS-21 lysis of red blood cells in vitro.
[0030] The term "immune adjuvant," as used herein, refers to
compounds which, when administered to an individual or tested in
vitro, increase the immune response to an antigen in the individual
or test system to which said antigen is administered. Preferably,
such individuals are humans, however, the invention is not intended
to be so limiting. Any animal that may experience the beneficial
effects of the vaccines of the invention are within the scope of
animals which may be treated according to the claimed invention.
Some antigens are weakly immunogenic when administered alone or are
toxic to the individual at concentrations which evoke immune
responses in said individual. An immune adjuvant may enhance the
immune response of the individual to the antigen by making the
antigen more strongly immunogenic. The adjuvant effect may also
lower the dose of said antigen necessary to achieve an immune
response in said individual.
[0031] The saponins of the present invention may be utilized to
enhance the immune response to any antigen. Typical antigens
suitable for the immune-response provoking compositions of the
present invention include antigens derived from any of the
following: viruses, such as influenza, feline leukemia virus,
feline immunodeficiency virus, HIV-1, HIV-2, rabies, measles,
hepatitis B, or hoof and mouth disease, bacteria, such as anthrax,
diphtheria, Lyme disease or tuberculosis; or protozoans, such as
Babeosis bovis or Plasmodium. The antigens may be proteins,
peptides, polysaccharides, lipids, or nucleic acids encoding the
protein or peptide. The proteins, peptides, lipids, or nucleic
acids may be purified from a natural source, synthesized by means
of solid phase synthesis, or may be obtained means of recombinant
genetics.
[0032] Administration of the compounds useful in the method of the
present invention may be by parenteral, intravenous, intramuscular,
subcutaneous, intranasal, oral or any other suitable means. The
dosage administered may be dependent upon the age, weight, species,
kind of concurrent treatment, if any, route of administration, and
nature of the antigen administered. In general, the saponin and
antigen may be administered at a dosage of about 0.001 to about 1.0
mg/kg of saponin adjuvant or antigen per weight of the individual.
The initial dose may be followed up with a booster dosage after a
period of about four weeks to enhance the immunogenic response.
Further booster dosages may also be administered.
[0033] The effective compound useful in the method of the present
invention may be employed in such forms as capsules, liquid
solutions, suspensions or elixirs for oral administration, or
sterile liquid forms such as solutions or suspensions. The vaccine
of the present invention may be administered parenterally,
intranasally, or orally.
[0034] Another preferred embodiment is a method for reducing the in
vitro lytic effect of an immune adjuvant composition comprising
administering to an individual an effective amount of QS-21 and an
excipient. Preferably, the excipients may be nonionic surfactants,
polyvinylpyrollidone, human serum albumin, aluminum hydroxide,
agents with anesthetic action, and various unmodified and
devivatized cyclodextrins. More preferably, the nonionic
surfactants may include Polysorbate 20, Polysorbate 40, Polysorbate
60, and Polysorbate 80. The polyvinylpyrollidone may preferably be
Plasdone C15, a pharmaceutical grade of polyvinylpyrollidone. The
agent having anesthetic action preferably is benzyl alcohol. A
preferred cyclodextrin is Encapsin, a
hydroxypropyl-.beta.-cyclodextrin, which reduces QS-21 lysis of red
blood cells in vitro.
[0035] Other preferred methods falling within the scope of the
invention include a method for maintaining the maximum adjuvant
activity of QS-21 comprising administering to an individual an
effective amount of QS-21 and an excipient and a method for
improving the tolerance to saponin adjuvant associated pain in an
individual to whom it is administered comprising administering an
effective amount of QS-21 and an excipient.
EXAMPLES
[0036] A variety of excipients were evaluated in combination with
QS-21 as novel compositions. These included various nonionic
surfactants (Triton X-100, Polysorbate 20, Polysorbate 40,
Polysorbate 60, and Polysorbate 80), polyvinylpyrollidone (Plasdone
C15), human serum albumin, aluminum hydroxide, agents with
anesthetic action (benzyl alcohol), and various unmodified and
derivatized cyclodextrins (hydroxypropyl-.beta.-cyclodextr- in,
hydroxypropyl-.gamma.-cyclodextrin, methyl-.beta.-cyclodextrin).
The final formulations were assessed for their capacity to reduce
the lytic effect of QS-21, to improve tolerance to QS-21 adjuvant
associated pain in humans, to stabilize QS-21 in aqueous solution,
and/or to maintain maximum adjuvant potency relative to a control
formulation of QS-21 in PBS.
Example 1
Compositions that Reduce the Lytic Effect of QS-21
[0037] A simple in vitro assay was used to screen excipients for
reducing the lytic effect of QS-21. The lytic effect of QS-21 can
be determined in an assay of hemolysis of sheep erythrocytes.
Briefly, various two fold serial dilutions of QS-21 in a given
excipient are prepared in a round bottom microtiter plate (100
.mu.l/well). All plates contain control wells containing excipient,
but no QS-21. The concentration of QS-21 ranges from 1.56 to 200
.mu.g/ml. A total volume of 25 .mu.l of sheep erythrocytes (washed
with PBS) is added to each 20 well, mixed with the QS-21/excipient
solution, and incubated at ambient temperature for 30 minutes.
After the end of the incubation, the round bottom plate is
centrifuged at 2000 rpm for 5 minutes to sediment any unlysed
cells. A total volume of 75 .mu.l of supernatant (containing
released hemoglobin) is transferred to the equivalent well of a
flat-bottom 96 well plate. The flat-bottom plate is centrifuged at
2000 rpm for 5 minutes to break any air bubbles. The absorbance at
570 nm is read in a microtiter plate reader. Absorbance at 570 nm
is plotted on the y-axis against QS-21 concentration plotted on the
x-axis. The absorbance of hemoglobin in the supernatant of a well
where no intact cell pellet was observed is defined as maximum
hemolysis. The hemolytic index of QS-21 is defined as the
concentration of QS-21 that yields an absorbance equivalent to 50%
of the maximum absorbance. An excipient that reduces the lytic
effect of QS-21 is expected to increase the hemolytic index.
[0038] Table 1 lists the hemolytic indices of QS-21 in various
excipients. All excipients were tested in the absence of QS-21. In
the absence of QS-21, no hemolysis was noted, indicating that the
excipient formulations were isotonic. Excipients that were shown to
be effective in minimizing the lytic effect (increase hemolytic
index) of QS-21 were hydroxypropyl-.beta.-cyclodextrin, aluminum
hydroxide, and Polysorbate 80 in saline.
1TABLE 1 Excipient Hemolytic Index (.mu.g/ml) PBS 5
.alpha.-cyclodextrin (2 mg/ml) 1.5 .beta.-cyclodextrin (2 mg/ml) 10
methyl- .beta.-cyclodextrin (2 mg/ml) 36
hydroxypropyl-.gamma.-cyclodextrin (2 mg/ml) 5
hydroxypropyl-.beta.-cyclodextrin (1 mg/ml) 9
hydroxypropyl-.beta.-cyclodextrin (2 mg/ml) 11
hydroxypropyl-.beta.-cyclodextrin (4 mg/ml) 18
hydroxypropyl-.beta.-cyclodextrin (8 mg/ml) 32
hydroxypropyl-.beta.-cyclodextrin (16 mg/ml) 51
hydroxypropyl-.beta.-cyclodextrin (32 mg/ml) 93 human serum albumin
(40 mg/ml) 9 QS-7 (250 .mu.g/ml) 30 aluminum hydroxide (2 mg/ml) in
PBS 5 aluminum hydroxide (2 mg/ml) in saline 13 Monophosphoryl
lipid A (25 .mu.g/ml) 4.9 Monophosphoryl lipid A (50 .mu.g/ml) 7.7
Monophosphoryl lipid A (100 .mu.g/ml) 6.5 Triton X-100 (50
.mu.g/ml) 1 Triton X-100 (100 .mu.g/ml) 1 Polysorbate 80 (2 mg/ml)
9 Polysorbate 80 (4 mg/ml) 18 Polysorbate 80 (10 mg/ml) 38
Example 2
Compositions that Reduce the Lytic Effects of Other Saponins
[0039] Other saponin adjuvants are also known to be hemolytic,
although to different extent than QS-21. These saponins include
substantially pure QS-7, QS-17, and QS-18. In addition,
heterogeneous adjuvant saponins such as Quil A are hemolytic. An
example of the effect of Polysorbate 80 and
hydroxypropyl-.beta.-cyclodextrin on the hemolytic indices of the
substantially pure QS-7 and heterogeneous Quil A is shown in Table
2. Hydroxypropyl-.beta.-cyclodextrin was shown to be effective in
reducing the lytic effect (increasing the hemolytic index) of QS-7.
Polysorbate 80 and hydroxypropyl-.beta.-cyclodextrin were shown to
be effective in minimizing the lytic effect (increasing the
hemolytic index) of Quil A.
2TABLE 2 Hemolytic Saponin Excipient Index (.mu.g/ml) QS-7 PBS 650
QS-7 Polysorbate 80 (8 mg/ml) 60 QS-7
Hydroxypropyl-.beta.-cyclodextrin >1000 (32 mg/ml) Quil A PBS 18
Quil A Polysorbate 80 (8 mg/ml) 43 Quil A
Hydroxypropyl-.beta.-cyclodextrin 200 (32 mg/ml)
Example 3
Compositions that Stabilize QS-21
[0040] QS-21 is an acylated bidesmodic triterpene saponin. It has a
fatty acid ester linked to the hydroxyl residues of fucose. In
aqueous solution, this fatty acid ester migrates between two
adjacent vicinal hydroxyl groups (fucose 3, 4) to form two
equilibrium isomers Jacobsen, N. E., Fairbrother, W. J., et al.,
1996, Carbohydrate Research 280:1-14). The predominant isomer is
acylated at fucose 4 and the minor isomer is acylated at fucose 3.
This ester bond is the most labile bond in QS-21 and will hydrolize
under alkaline conditions to form a deacylated saponin and a fatty
acid-arabinose domain. The deacylated saponin and the fatty add
domain are both inactive as immunological adjuvants (Kensil, C. R.,
et al., 1996, In: Saponins Used in Traditional and Modern Medicine
Waller and Yamaski, Eds., Plenum Press, NY, 165-172). Various
conditions affect the stability of this ester bond (Cleland, J. L.,
et al., 1996, J. Pharmaceutical Sciences 85:22-28). Furthermore,
the monomer form of QS-21 is more susceptible to hydrolysis than
the micellar form.
[0041] Examples of the shelf life of QS-21 are shown in Table 3.
The aqueous shelf life for a 50 .mu.g/ml QS-21 solution at pH 7.0
at 4.degree. C. was shown to be only 94 days or approximately 3
months. This is representative of a typical clinical vaccine
formulation containing QS-21 adjuvant (which consists of QS-21 at a
concentration of 50-200 .mu.g/ml in a physiological pH buffer (pH
7.0-7.5)). Hence, in simple buffer and salt solutions at low
concentration, the QS-21 product does not maintain a desirable
stability profile. Some improvement in stability, however, can be
achieved by an increased concentration of the QS-21 product. For
instance, the shelf life of a 500 .mu.g/ml QS-21 solution at pH 7.0
at 4.degree. C. was shown to be 717 days, or 23.9 months. But a
concentrated QS-21 solution is not necessarily a practical method
of administering a low dose of adjuvant. For example,
administration of 25 .mu.g from a 500 .mu.g/ml solution would
require the syringe withdrawal of 0.05 ml of dose. Additionally,
some improved stability can be achieved by the use of a lower pH,
i.e., at pH 6.0. However, a pH substantially lower than the
physiological pH range may not be tolerated well or be compatible
with the antigen.
3TABLE 3 QS-21 Concentration pH t.sub.90 (days) 50 .mu.g/ml pH 7.0
94 50 .mu.g/ml pH 6.0 679 500 .mu.g/ml pH 7.0 717
[0042] Another way to evaluate the stability of QS-21 in aqueous
solution was to assay the solution by HPLC in an accelerated
stability assay at 37.degree. C. Although this is not the
temperature used for storage of vaccines (4.degree. C.), it was
expected that this assay at 37.degree. C. would show the relative
stabilizing power of a given excipient. For example, an excipient
that extended the t.sub.90 value by two fold at 37.degree. C. would
also be expected to extend the t.sub.90 value by two fold at
4.degree. C.
[0043] Specifically, QS-21 (100 .mu.g/ml) was prepared in various
excipients in PBS at pH 7.0. The solutions were incubated at
37.degree. C. for 7 days. At the end of 7 days, the solutions were
assayed by reversed phase-HPLC to determine the extent of
degradation. The data was plotted as log (fraction QS-21 t=7/QS-21
t=0 days) against time on the x-axis. The time to 10% degradation
(40) was extrapolated from this plot.
[0044] Table 4 shows the .sub.90 values of QS-21 in various
excipients. Stabilization of QS-21 is shown by an increase in
t.sub.90. Excipients that stabilized QS-21 by at least two fold are
Polysorbate 20, Polysorbate 80, native Quillaja saponin QS-7, and
the deacylsaponin resulting from alkaline hydrolysis of QS-21
(DS1).
4 TABLE 4 Excipient t.sub.90 (days) at 37.degree. C. PBS (pH 7.0)
1.2 Polysorbate 20 (720 .mu.g/ml) 2.9 Polysorbate 80 (250 .mu.g/ml)
3.2 Polysorbate 80 (500 .mu.g/ml) 4.3 Polysorbate 80 (1.0 mg/ml)
5.2 Polysorbate 80 (2.0 mg/ml) 7.2 Phenol (2.5 mg/ml) 2.3 Pluronic
F68 (1.0 mg/ml) 1.4 QS-7 (100 .mu.g/ml) 1.8 QS-7 (250 .mu.g/ml) 2.6
QS-7 (500 .mu.g/ml) 9.0 QS-7 (1.0 mg/ml) 16.0 DS-1 (100 .mu.g/ml)
2.2 DS-1 (250 .mu.g/ml) 3.3 DS-1 (500 .mu.g/ml) 7.2 DS-1 (1.0
mg/ml) 6.2 Monocaproyl-rac-glycerol (1.0 mg/ml) 1.7
.alpha.-cyclodextrin (5 mg/ml) 0.8 .beta.-cyclodextrin (5 mg/ml)
0.7 Methyl-.beta.-cyclodextrin (5 mg/ml) 1.5
hydroxypropyl-.gamma.-cyclodextrin (5 mg/ml) 1.0
hydroxypropyl-.beta.-cyclodextrin (5 mg/ml) 1.0
[0045] In addition, 0.9% benzyl alcohol, and Plasdone C15 were
evaluated for its ability to stabilize QS-21 (Table 5). AR QS-21
concentrations and incubation conditions were equivalent in this
experiment except that the QS-21 formulation was prepared in
Dulbecco's PBS (without calcium or magnesium) at pH 7.5. As
expected, the higher pH resulted in a faster degradation of QS-21
in PBS. However, Plasdone C15 stabilized QS-21.
5TABLE 5 Excipient t.sub.90 (days) at 37.degree. C., pH 7.5
Dulbecco's PBS 0.6 0.9% benzyl alcohol in Dulbecco's PBS 0.7
Plasdone C15 in Dulbecco's PBS (25 mg/ml) 1.6 Plasdone C15 in
Dulbecco's PBS (50 mg/ml) 7.7
Example 4
Adjuvant Potency of Compositions
[0046] FIGS. 1A and 1B show the effect of Polysorbate 40,
Polysorbate 60, Polysorbate 80, and methyl-.beta.-cyclodextrin on
the immune response of Balb/c mice to OVA plus various doses of
QS-21. Female mice (10/group, 8-10 weeks of age at the first
immunization) were immunized subcutaneously with 5 .mu.g of OVA and
the indicated dose of QS-21 in either PBS alone or in 2 mg/ml
excipient in PBS. A booster immunization was given by the same
route at week 2. Sera was collected at week 4 for EIA analysis of
the anti-VA response. Mice were analyzed for OVA-specific IgG2a by
a standard EIA analysis (Kensil, C. R., et al., 1993, Vaccine
Research 2:273-281) QS-21 was active in all excipients within two
fold of the threshold value determined in PBS. The same maximum
level of antibody response was reached at the optimum adjuvant dose
(typically 10 .mu.g and above).
[0047] FIG. 2 shows the effect of excipients on antibody response
to a T-independent polysaccharide antigen. Balb/c mice were
immunized subcutaneously with a commercial 23-valent S. pneumonia
polysaccharide vaccine (Pnu-Imune, 0.5 .mu.g/serotype) and
different doses of QS-21 in PBS, in 4 mg/ml Polysorbate 80 in PBS,
or in 16 mg/ml hydroxypropyl-.beta.-cyclodextrin in PBS. Anti-Type
14 IgG was determined by EIA on sera collected at day 7 after a
single immunization. Neither Polysorbate 80 or
hydroxypropyl-.beta.-cyclodextrin in the formulation reduced the
potency of the vaccine for stimulating an IgG3 response specific
for Type 14 polysaccharide serotype.
Example 5
Clinical Studies of Compositions--Trial 1
[0048] Various QS-21 compositions were administered to patients in
order to test for the compositions' pain tolerance. Fifteen
volunteers were recruited to receive four intramuscular injections,
with each injection given at one week intervals. The study was
carried out as a randomized, double-blind study. Three of the
formulations contained 50 .mu.g QS-21 in either Dulbecco's PBS
(without calcium or magnesium), in 4 mg/ml Polysorbate 80 in PBS,
or in 1 mg/ml aluminum hydroxide in saline. The fourth formulation
was a PBS control without QS-21. Volunteers were asked to rate the
immediate pain in the first five minutes after injection on a 0 to
10 scale (0=no pain, 1-3=mild, 4-7=moderate, 8-10=severe). The
results are shown in FIG. 3. The cumulative scores represented in
FIG. 3 of the patients' tolerance to pain is represented in FIG. 4
as individual scores. The QS-21 formulation containing 4 mg/ml
Polysorbate 80 resulted in an improved pain tolerance compared to
QS-21 in PBS. The highest score for this particular formulation was
rated as a 5.
Example 6
Clinical Studies of Compositions--Trial 2
[0049] Various other QS-21 compositions-were administered to
patients in order to test for the compositions' pain tolerance.
Fifteen volunteers were recruited to receive four intramuscular
injections, with each injection given at one week intervals. The
study was carried out as a randomized, double-blind study. The
excipients evaluated were benzyl alcohol,
hydroxypropyl-beta-cyclodextrin, and a higher dose of Polysorbate
80, which had been shown to be more effective than 4 mg/ml
Polysorbate 80 at reducing QS-21 lysis of red blood cells in vitro.
The five formulations tested were (1) 1 mg/ml aluminum hydroxide,
which served as the placebo control; (2) 50 .mu.g QS-21 in 0.72%
benzyl alcohol in saline; (3) 50 .mu.g QS-21 in 30 mg/ml
hydroxypropyl-.beta.-cyclodextr- in; (Encapsin, Janssen Biotech
N.V., Olen, Belgium) (4) 50 .mu.g QS-21 in 8 mg/ml Polysorbate 80;
and (5) 50 .mu.g QS-21 in PBS (Dulbecco's PBS without calcium or
magnesium), which served as a positive control formulation.
Volunteers were asked to rate the immediate pain in the first five
minutes after injection on a 0 to 10 scale (0=no pain, 1-3=mild,
4-7=moderate, 8-10=severe). The results are shown in FIG. 5. The
cumulative scores represented in FIG. 5 of the patients' tolerance
to pain is represented in FIG. 6 as individual scores. All
excipients were shown to reduce the mean and median pain scores
associated with QS-21 in PBS. The highest single score for the
QS-21/Encapsin formulation was rated as a 5, which compared more
favorably with the QS-21/Polysorbate 80 formulation that was rated
with a single 6 and two 5's.
[0050] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention as set forth below.
* * * * *