Note: Descriptions are shown in the official language in which they were submitted.
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CARBOHYDRATE VACCINES FGR VIRAL DISEASES
This application claims the benefit of U..S. Provisional
Application No. 60/102,657, filed October 1, 1998, the
contents of which are hereby incorporated by reference.
5 Throughout this application; various references are referred
to within parentheses. Disclosures of these publications in
their entireties are hereby incorporated by reference into
this application to more fully describe the state of the art
_ ,to which this invention pertains.
to
Backaround of the Invention
Dramatic changes in carbohydrate metabolism and cell surface
expression have been observed in a:~sociation with infectious
diseases, such as viral infection's. For example, in vivo and
15 in vitro infection of human lymphocytes with retroviruses
such as the human immunodeficiency virus (HIV) or human T
lymphotropic virus type-1 (HTLV-1) leads to increased cell
~~
surface expression of the Lewis y antigen (Adachi et al., J.
Exp. Med, 167:323, 1988) and of t:he gangliosides GM2, GD2,
20 GM1, GMIa, GDla, and GM3 (Sorice, et al., JAIDS, 12:112,
1996; Misasi et al., Clin. Immunol. Immunopathol., 67:216,
1993; Furukawa et al., PNAS, 90:1972, 1993; Matsuda et al.,
Biochem. Biophys. Acta, 1168:12?'., 1993; Auci et: al., J.
Leukoc. Biol. 52:282, 1992). Increased expression of GM2,
25 GD2 and GM3 was observed in cell lines transfected with
human adenovirus genes or infected with a hybrid adenovirus
(Sanai et al., J. Biochem. [Tokyo] 107:740, 1990, Jambrosic
et al., Int. J. Cancer, 44:1117, :1989).
30 GM2, GD2 and other gangliosides acre sialic acid containing
glycosphingolipids composed of a complex carbohydrate moiety
linked to a hydrophobic ceramide portion. Embedded within
the outer leaflet of the cell membrane, the carbohydrate
chain is exposed to the extracellular matrix. The
35 oligosaccharide portion of gangliosides such as GD2 may also
be linked to peptide moieties and activate cytotoxic T
lymphocytes when presented on the cell surface in
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-2-
association with major histocompatibility molecules {Zhao
and Cheung, J. Exp. Med. 182:67, 1.995). The Lewes y antigen
is a neutral oligosaccharide found on glycolipids and
glycoproteins. A growing number of glycolipids are known to
elicit cytotoxic T lymphocyte responses when presented on
the cell surface in association with members of the CD1
family of antigen presenting molecules.
As with virally infected cells, a variety of human tumors
are characterized by altered expression of carbohydrate
structures, including GM2, GiD2, and the Lewis y
antigen(reviewed in Ragupathi, Cancer Immunol. Immunother.
43:152, 1996). Melanoma patients with naturally occurring
antibodies to GM2 have prolonged! disease-free and overall
survival periods. Cell surface carbohydrate antigens have
thus been identified as target:a for active and passive
immunotherapy of cancers, and different approaches have been
adopted to induce immune responses against these structures.
These include whole or lysed. tumor cells, purified
carbohydrates, mimotopes and anti-idiotype antibodies. The
immunogenicity of purified carbohydrates can be improved via
their conjugation to immunogenic carrier proteins.
While each vaccine approach has shown promise in initial
experimentation, adsorption or covalent attachment of
purified carbohydrate antigens to immunogenic T-dependent
protein carriers is the concept that has been pursued most
vigorously, resulting in vaccines that have ~ in same
instances been shown to be effective in clinical trials.
3a
In studies aimed at inducing a humoral immune response
against gangliosides in melanoma patients by active
immunization, GM2/Bacillus Calmette-Guerin {BCG) vaccines
were shown to be effective (Livingston et al., Proc. Natl.
Acad. Sci. USA 84:2911, 1987; Livingston et al., Cancer Res.
49:4045, 1989). In a randomized study with 122 melanoma
patients, who were disease-free after surgery, that it was
shown that, out of 64 patients treated with BCG alone and 58
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-3-
patients with GM2/BCG, the majority of patients (860)
receiving the GM2 vaccine produced. antibodies. Patients who
produced anti-GM2 antibodies had a significantly longer
disease free interval and overall survival than antibody
5 negative patients. (Livingston et al., J. Clin. Oncol.
12:1036, 1994).
Improved humoral immune responses were elicited in humans by
a vaccine containing GM2 covalently linked to the carrier
10 protein Keyhole Limpet ~Hem~cyanin (KLH) and administered
with the adjuvant QS-21. QS~21 is a carbohydrate extracted
from the bark of the South American tree Quillaja,saponaria
Molina. The monosaccharide composition, molecular weight,
adjuvant effect and toxicity for ~~ series of these saponins
15 have been described (Kensil, Crit. Rev. Ther. Drug Carrier
Syst. 13:1, 1996). Studies have s_dentified the 100 ug dose
of QS-2l as the optimal well tolerated dose for induction of
antibodies against GM2 and KLH in humans (Livingston et al.,
Vaccine 22:1275, 1994).
20
In the presence of QS-21 adjuvant, the GM2-KLH conjugate
vaccine consistently induced high-titer, long-lived IgM
responses against GM2 in melanoma patients (Livingston et
al., Cancer Immunol. Immunother" 43:324, 1997). Tn the
25 majority of treated patients, the 'vaccine also induced anti-
GM2 IgG antibodies,. which have the ability to mediate
antibody-dependent oe~Il-mediated cytotoxicity (ADCC). The
elicited antibodies were shown to specifically bind and kill
cancer cells via complement mediated lysis and ADCC.
30
In addition to gangliosides, other carbohydrate antigens
have been made immunogenic by conjugation to appropriate
carrier proteins. Coupling of capsular polysaccharides from
the bacterium Haemophilus influenzae type b to diphtheria
35 toxiod resulted in a significant increase in immune response
and protection (Eskola et al., New England J. Med. 323:1381,
1990). This product has been licensed by the U.S. Food and
Drug Administration for use in children and in adults with
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_4
splenic dysfunction. Similarly, robust humoral immune
responses were elicited in children by pneumococcal
polysaccharides conjugated to either tetanus toxoid or the
outer membrane protein complex of Neisseria meningitides
(Kayhty et al., J. Infect. Dis. L?2:1273, 1995; Anttila et
al., J. Infect. Dis., 177:1614, 1998). In addition,
vaccination of ovarian cancer patients with synthetic
Thompson Friedenreich tumor antic3en conjugated to. keyhole
limpet.hemocyanin elicited humoral IgM and IgG responses
(MacLean et al., J. Immunother<~py 11:292, 1992}. The
important finding common in theses studies a as the isotype
switch from a IgM response of short duration to a long
lasting, high affinity IgG ~ response indicating that
activation of T-cell dependent pathways against
carbohydrates is likely to occur.
This approach is now applied to the use of ganglioside and
other carbohydrate-based vaccines for the treatment or
prevention of viral infections wherein carbohydrate
metabolism and expression are altered. The goal is to
similarly induce immune responses to carbohydrate antigens
associated with diseased cells. Through well known
mechanisms, the immune system can destroy the infected cells
or viruses that display the carbohydrate antigens and
thereby prevent or favorably alter the course of the viral
infection.
Antibodies are known to be an important component of the
host defense against viruses (Klein; Immunology, Blackwell
Scientific Publications, Boston, 1HA, 1990). Antibodies can
bind to viruses and eliminate their infectivity by direct
neutralization, complement-mediated virolysis, or Fc
receptor-mediated phagocytosis. IrA addition, antibodies can
eliminate virally infected cells via either complement-
mediated cytolysis or antibody-dependent cell-mediated
cytotoxicity. Incorporation of host cell antigens into
virus particles is a well-established phenomenon, and it has
been further demonstrated that protection from viral
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infections can be afforded by the immunological response to
host cell antigens as well as virally encoded antigens
iSchultz and Stott, AIDS, 8:[suppl 1]:5203, 1994).
5 Similarly, cell-mediated immunity is known to play a major
role in controlling viral infections. Importantly,
cytotoxic T lymphocytes can eliminate virally infected cells
wherein antigen expression is altered. As described above,
cytotoxic T lymphocytes can recognize carbohydrate antigens
to that are either lipid-linked and. presented in association
with CD1 molecules or peptide-linked and 'presented in
association with major histocompatibility molecules.
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Summnarv of the Invention
This invention provides a method for treating or preventing
a viral disease in a subject comprising administering to the
subject a cellular carbohydrate antigen that is
5 overexpressed during the viral disease, or a molecular mimic
thereof, the amount of such. carbohydrate or such mimic being
effective to treat or prevent the viral disease. In an
embodiment, the viral disease is caused by the human
immunodeficiency virus type-1.
to
In an embodiment, the carbohydrate is the Le~niis y antigen,
a ganglioside, or the oligosaccharide portion of a
ganglioside. The gangliosides may include, but not to be
limited to, GM1, GMla, GM2, GM3, GDla, and GD2. In a
Z5 preferred embodiment, the gangl:ioside is GM2, ~GD2 or a
combination thereof.
In an embodiment, the molecular mimic is an anti-idiotype
antibody. In another embodiment, the molecular mimic is a
20 peptide mimotope..
In an embodiment, the cellular carbohydrate antigen or.a
molecular mimic thereof is administered to the subject
together with a suitable adjuvant. In a preferred
25 embodiment, the adjuvant is QS-21.
In a further embodiment, the cellular carbohydrate antigen
or a molecular mimic thereof is adsorbed, covalently linked
or otherwise conjugated to an imrriunogenic carrier protein.
30 In a preferred embodiment, the carrier protein is Keyhole
Limpet Hemocyanin.
This invention also provides a vaccine for treating or
preventing a viral disease in a subject comprising
35 administering to the subject a cellular carbohydrate antigen
that is overe~cpressed during the viral disease, or a
molecular mimic thereof, the amount of such carbohydrate or
such mimic being effective to treat or~prevent the viral
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disease, and a pharmaceutically acceptable carrier.
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_g.
Detailed Desariytion of the Invention
This invention provides a method for treating or preventing
a viral disease in a subject compr:i.sing administering to the
subject a cellular carbohydrate antigen that is
5 overexpressed during the viral disease, or a molecular mimic
thereof, the amount of such~carbohydrate or such mimic being
effective to treat or prevent the viral disease.
As used herein, the molecular mimics is defined as a compound
which when administered to an appropriate host, stimulates
or enhances an immune respon~~ that recognizes the cellular
carbohydrate. Such mimics include, but are not limited to,
mimotopes and anti-idiotype antibodies. Mimotopes are
peptides which mimic the zmmunogenicity of the cellular
15 carbohydrate. It is known that anti-idiotype antibodies can
mimic carbohydrate antigens for the purposes of inducing
anti- carbohydrate immune responses. For example, U.S.
Patent No. 5,792,455, entitled, "Anti-idiotype antibody
vaccine," describes an anti-idiotype antibody which mimics
20 the ganglioside GD3; and U.S. Patent No. 5,653,977,
entitled, "Anti-idiotypic antibady that mimics the GD2
antigen," describes an anti-idiotypic monoclonal antibody
which elicits an immune response reactive against the
ganglioside GD2 antigen. The content of these patents is
25 incorporated into this application by reference.
In an embodiment, the cellular carbohydrate antigen or a
molecular mimic thereof is administered to the subject in
the presence of a suitable adjuvant. Suitable adjuvants
30 include the precipitated aluminum salts collectively known
as alum, cytokines such as IL-2 and interferon-gamma, block
copolymer-based adjuvants such as titermax, Ribi Detox and
other monophosphoryl lipid A containing adjuvants, and
saponins such as QS-21. In a preferred embodiment, the
35 adjuvant is QS-21.
Im an embodiment, the viral disease is caused by human
immunodeficiency virus type-1
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_g_
In an embodiment, the cellular carbohydrate antigen is the
Lewis y antigen, a ganglioside, or the oligosaccharide
portion of a ganglioside. Thn gangliosides which are
applicable to this invention include, but are not limited
5 to, GM1, GMla, GM2, GM3, GDla, and GD2. In a preferred
embodiment, the ganglioside is GM2, GD2 or a combination
thereof .
In an embodiment, the cellular carbohydrate antigen or a
molecular mimic thereof~is adsorbed, covalently linked or
otherwise conjugated to an immunogenic carri.r protein.
Ganglioside conjugate vaccines have been described. See
e.g. Livingston and Helling, "Ganglioside-KLH Conjugate
I5 Vaccines with QS-21" Patent Cooperation Treaty (PCT)
Application No: PCT/US94/00757, International Publication
Number: WO/94/16731, the content of which is incorporated
into this application by reference.
20 In a preferred embodiment of this invention, the conjugated
ganglioside is GM2, GD2 or a combination thereof.
Different effective amounts of the conjugated ganglioside or
oligosaccharide portion thereof may be used according to
25 this invention. A person of ordinary skill in the.art can
perform simple titration experiments to determine what
amount is required for effective immunization. An example
of such titration experiment is to inject different amounts
of the conjugated ganglioside ar conjugated oligosaccharide
30 portion thereof to the subject with or without a suitable
adjuvant, and then examine the immune response.
In an embodiment, the effective amount of conjugated
ganglioside or conjugated oligosaccharide portion thereof is
35 an amount between about 1 ~g and about 500 ,ug.
In another embodiment, the effective amount of conjugated
ganglioside or conjugated oligrisaccharide portion thereof is
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an amount between about 50 ~.g and about 90 fig. In an
embodiment, the effective amount of conjugated ganglioside
or conjugated oligosaccharide portion thereof is about 70
~g-
In another embodiment, the effeci~ive amount of conjugated
ganglioside or conjugated oligosaccharide portion thereof is
between about 1 ~g and about 10 ~.g. In amore specific
embodiment, the effective amount of conjugated ganglioside
to or conjugated oligosaccharide portion thereof is between
about 7 ~.g and about 10 fig. In an embodiment, the effective
amount of conjugated ganglioside or conjugated
oligosaccharide portion thereof is about 7 fig.
In addition, the effective amount of the adjuvant may also
be similarly determined, i.e. by administering different
amounts of the adjuvant with t:he cellular carbohydrate
antigen or a molecular mimic thereof and examining the
immune response so as to determine which amount is
effective. When using QS-21 as adjuvant, the effective
amount of QS-21 may also to be similarly determined.
In a preferred embodiment, the effective amount of.QS-21 is
an amount between about lO~Cg anal about 200 ~,g. In an
25.embodiment, the effective amount of QS-21 is about 100 ~.g.
In another embodiment, the effecaive amount of QS-21 is
about 2 0 0 ~.g .
In a preferred embodiment, the cellular carbohydrate antigen
or a molecular mimic thereof is conjugated to an immunogenic
protein. As used herein, an immunogenic protein is a
polypeptide that, when conjugated to a carbohydrate or a
molecular mimic thereof stimulates or enhances an immune
response to the carbohydrate in the subject. In a further
embodiment, the immunogenic protein is Keyhole Limpet
Hemocyanin~or a derivative thereof. This invention also
provides the above-described vaccine wherein the cellular
carbohydrate antigen or a molecular mimic thereof is
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conjugated to Keyhole Limpet Hemocyanin or a derivative of
Keyhole Limpet Hemocyanin.
Keyhole Limpet Hemocyanin is a well-known protein. A
derivative of Keyhole Limpet Hemc>cyanin may be generated by
direct linkage of at least one immunological adjuvant such
as monophospholipid A or non-ionic block copolymers or
cytokines to Keyhole Limpet Hemocyanin. Cytokines are well
known to an ordinary skilled practitioner. Example cytokines
10 with adjuvant properties include interleukin 2 and
interferon-gamma. There are other known cytokines in the
art which may to be linked to. Keyhole Limpet Hemocyanin,
forming a derivative of Keyhole Limpet Hemocyanin.
15 In an embodiment, a ganglioside is conjugated to the
immunogenic protein by the proceas of reductive amination
following oxidation of a ceramide alkene structure to an
aldehyde. In another embodiment, the conjugation of the
ganglioside occurs through an aminolysyl group of the
20 Keyhole Limpet Hemocyanin.
In addition to reductive amination, various other
conjugation techniques may to be: used for this invention.
The conjugation techniques may include chemical linker
25 groups and should not adversely affect the immunogenicity of
the carbohydrate.
As used herein, a suitable adjuvant is an adjuvant which
when administered together with the carbohydrate or a
30 molecular mimic thereof or a conjugated carbohydrate or a
conjugated molecular mimic thereof stimulates or enhances an
immune response to the carbohydrate in the subject. In an
embodiment, the adjuvant is QS-:Z1. There are other known
adjuvants which may to be applicable to this invention.
35 There may be classes of QS-21 or QS-21 like chemicals which
may be similarly used in accordance with this invention.
This invention also provides a vaccine for treating or
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preventing a viral disease comprising a cellular
carbohydrate antigen that is overexpressed during the viral
infection, or a~molecular mimic thereof, the amount of such
carbohydrate or such mimic being effective to treat or
5 prevent the viral disease, and a pharmaceutically acceptable
carrier.
In an embodiment of this invention, the subject is a human.
10 In an embodiment, the cellular carbohydrate antigen is the
Lewis y ar:igen, a ganglioside: or the oligosaccharide
portion of a ganglioside.
This invention further provides a vaccine for stimulating or
15 enhancing in a subject to which the=_ vaccine is administered,
an immune response which'recognizes a cellular carbohydrate
antigen that is overexpressed during a viral disease
comprising an amount of such carbohydrate or a molecular
mimic thereof effective to stimulate or enhance an anti-
20 carbohydrate immune response in the subject, and a
pharmaceutically acceptable vehicle, wherein the subject is
afflicted with the viral disease and the immune response
produced in the subject upon administration of the vaccine
effectively treats the viral disease.
This invention also provides a vaccine for stimulating or
enhancing in a subject to which they vaccine is administered,
an immune response which recognizes a cellular. carbohydrate
antigen that is overexpressed during a viral disease
30 comprising an amount of such carbohydrate or molecular mimic
thereof effective to stimulate: or enhance an anti-
carbohydrate immune response a.rl the subjects and a
pharmaceutically acceptable vehicle, wherein the subject is
susceptible to the viral disease and the immune response
35 produced in the subject upon administration of the vaccine
effectively. prevents the viral disease.
This invention further provides a vaccine for a viral
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_13_ _
disease, wherein the virus or the virus-infected cells have
gangliosides on their surface.
This invention further provides a method for treating viral
diseases in a subject afflicted with a viral disease
comprising administering to the subject an effective dose of
a vaccine for stimulating or enhancing in a subject to which
the vaccine is administered, production of an antibody which
recognizes a ganglioside, comprising an amount of
ganglioside or oligosaccharide portion thereof conjugated to
an immunogenic protein effective to stimulate or enhance
antibody production in the subject, an effective amount of
adjuvant and a pharmaceutically acceptable vehicle, wherein
the subject is afflicted with a viral disease and the
antibody produced in the subject.upon administration of the
vaccine effectively treats the viral disease.
This invention further provides a method for preventing a
viral disease in a subject susceptible to a viral disease
comprising administering to the subject an effective dose of
a vaccine for stimulating or enhancing in a subject to which
the vaccine is administered, production of an antibody which
recognizes a ganglioside, comprising an amount of
ganglioside or oligosaccharide portion thereof conjugated to
an immunogenic protein effective to stimulate or enhance
antibody production in the subject, an effective amount of
adjuvant and a pharmaceutically acceptable vehicle, wherein
the subject is susceptible to a viral disease and the
antibody produced in the subject upon administration of the
vaccine effectively prevents the viral disease.
This invention. also provides a method of using the above-
described vaccine, wherein the ganglioside or
oligosaccharide portion thereof is conjugated to Keyhole
Limpet Hemocyanin or a derivat-ive of Keyhole Limpet
Hemocyanin.. This invention further provides a method of
using the above-described vaccine wherein the adjuvant is
QS-21.
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This invention further provides a method of using the above-
described vaccine for treating or preventing viral disease,
wherein the virus or virus-infected cells have gangliosides
on their surface.
For the purposes of this invention "pharmaceutically
acceptable vehicles" means any of the standard
pharmaceutical vehicles. Examples of suitable vehicles are
well known in the art and may include, but not limited to,
any of the standard pharmaceutical vehicles such as a
phosphate buffered saline solutions, pho:~hate buffered
saline containing Polysorb 80, water, emulsions such as
oil/water emulsion, and various t.~rpes of wetting agents.
The vaccine of this invention may be administered
intradermally, subcutaneously and intramuscularly. Other
methods well known by a person of ordinary skill~in the art
may also be used.
In a preferred embodiment this invention provides a method
for stimulating or enhancing in a subject production of
antibodies which recognize a ganglioside comprising
administering to the subject an effective dose of a vaccine
for stimulating or enhancing in a subject to which the
vaccine is administered, production of an antibody which
recognizes a ganglioside, comprising an amount of
ganglioside or oligosaccharide portion thereof conjugated to
an immunogenic protein effective to stimulate or enhance
antibody production in the subject, an effective amount of
adjuvant and a pharmaceutically acceptable vehicle, wherein
the administering comprises adminie~tering the effective dose
at two or more~sites. °Administering the effective dose at
two or more sites" means that the effective dose is divided
into two or more portions and each portion is administered
at a different site of the subject. In a specific
embodiment,. the administering comprises administering at
three sites.
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This invention will to be better understood from the
Experimental Details which follow. However, one skilled in
the art will readily appreciate i~hat the specific methods
and results discussed are merely illustrative of the
invention as described more fully i.n the claims which follow
thereafter.
Experimental Details
Flow cYtometric analysis of an~ibodv bindincr to HIV-1
infected cells
Approximately 106 HIV-infected or control cells are stained
first with approximately 50,1 of anti-carbohydrate antibody
(~5 ~.g/mL) for approximately 20 mini at 4 °C in PBS/1%FBS/O.I%
sodium azide (assay buffer) washed with assay buffer and
then stained with a phycoerythri.n-conjugated reporter
antibody for 20 minutes at 4 °C. The cells are washed with
assay buffer, fixed overnight at: 4°C with PBS/1% FBS/1%
formaldehyde, and analyzed on a flow cytometer. The GD2-'and
GM2-positive melanoma cell line SK-MEL-31 can serve as a
positive control in assays using anti-GM2 and anti-GD2
antibodies.
The antibodies may to be monoclonal antibodies,, purified
hyperimmune immunoglobulin, or human or animal sera known to
contain high levels of carbohydrate-reactive antibodies
produced naturally or.elicited by active immunization. The
HIV-1 infected cells may be either cell lines competent for
HIV-1 infection or phytohemagglutinin-stimulated human
peripheral blood mononuclear cells (PBMC). The HIV-1
viruses may include laboratory adapted X4 viruses and
primary R5, X4 and R5X4 isolates.
Complement-mediated lysis of HIV-1.infected cells
Complement-mediated cytotoxicity assays axe performed by a
4h S~Cr release assay. 2xlC~ cells are labelled with 100~.Ci
Na25~Cr04 (New England Nuclear; Boston, MA) in 10% FCS RPMI
for lh at 37°C in a C02 incubator. The cells are viashed
twice, and 104 cells/well in 96-well round-bottom plates
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(Corning, New York, NY) are labelled and incubated with
sera, antibodies or with medium alone for lh at 37°C in a C02
incubator. The cells are washed. and incubated with human
complement (Sigma) at a dilution of 1:4 for 4h at 37°C. The
5 plates are spun at 5008 for 5 min, and a 125.1 aliquot of
supernatant of each well i.s harvested for determination of
released S~Cr. All assays axe performed in triplicate and
include control wells for maximum release in 1% NP-40
(Sigma) and for spontaneous release in the absence of
10 complement. The GD2- and GM2-po;aitive melanoma cell line
SK-MEL-31 can serve as positive control target cells for
assays using anti-GM2 and anti-GD2 antibodies.
The percentage of specific lysis is calculated as follows:
I5
Experimental release - spontaneous release
o - x 100
cytatoxicity Maximum release - spontaneous release
The antibodies may to be monoclonal antibodies, purified
hyperimmune immunoglobulin, or human or animal sera known to
contazn high levels of carbohydrate-reactive antibodies
,produced naturally or elicited by active immunization. The
25 HIV-1 infected cells may be either cell lines competent for
HIV-1 infection or PBMC. The HIV-I viruses may include
.labaratory adapted X4 viruses and primary R5, X4 arid R5X4
isolates.
30 Anti-carbohydrate antibody-dependent cell-mediated
cytotoxicitv (ADCC) assay
PBMC are suspended in RPMI media supplemented with loo heat-
inactivated FBS (assay media) at a density of 4x106 cells/ml.
These cells are then incubated overnight at 37°C prior to use
35 as effectors in the ADCC assay. The following day, target
cells are labeled with Na25~Cr04 (100~.Ci per 2x106 cells) for
approximately 3 h in RPMI 1640 assay media prior to use in
the assay. ' In each well, 104 S~Cr_-labeled target cells are
preincubated with various dilutions of sera or
40 concentrations of antibodies in 150.1 total assay volume for
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30 min at 37°C. Then 106 PBMC are added in 50,1 assay media,
and the plates are centrifuged at 3008 for 5 minutes prior
to incubation at 37°C for 4h. Plates are then centrifuged at
300g, and 1001 aliquots of cell supernatants are collected.
S~Cr release is detected using a Wallac 1470 gamma counter.
A11 assays are performed in triplicate and include control
wells for maximum release in 1% NP-40 (Sigma) and for
spontaneous release in the absence of complement.
The percenta~ ;e of specific lysis :is calculated as follows
Experimental release - :spontaneous release
- x 100
cytotoxicity Maximum release - spontaneous release
The antibodies rnay to be monoclonal antibodies, or purified
hyperimmune immunoglobulin, or human or animal sera known to
contain high levels of carbohydrate-reactive antibodies
produced naturally or elicited by active immunization. The
HTV-1 infected cells may be either cell lines competent for
HIV-1 infection or PBMC. The HIV-1 viruses may include
laboratory adapted X4 viruses and primary R5, X4 and R5X4
isolates. The GD2- and GM2-positive melanoma cell line SK-
MEL-31 can serve as positive control target cells for assays
using anti-GM2 and anti-GD2 antibodies.
HIV-1 neutralization assays
Virus neutralization is assessed using phytohemagglutinin-
stimuiated PBMC as indicator cells, with determination of
p24 antigen production as the endpoint. PBMC are stimulated
with PHA for 48 h before removal of the mitogen by washing.
Antibodies are combined in 2-food serial dilutions with
virus and/or cells for 1 h at 3'7 °C. The virus is then
added to the PBMC at a density of 4 x 106 /ml and the
cultures incubated for 7 days. The culture supernatants are
harvested, treated with 1~ Empigen detergent before
determination of the p24 concentration by ELISA. The Leu
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3a HIV-1 inhibitory monoclonal antibody (Becton Dickinson)
may to be used as a positive control.
The antibodies may to be monoclonal antibodies, purified
hyperimmune immunoglobuli.n, or human or animal sera known to
contain high levels of carbohydrate-reactive antibodies
produced naturally or elicited by active immunization. The
HIV-1 viruses may include laboratory adapted X4 viruses and
primary R5, X4 and R5X4 isolates.
10
Preparation of Clinical Grape Ganqlioside Con~uqate Vaccine
CHEMISTRY AND MANUFACTURING
DRUG SUBSTANCE
NAME AND SOURCE
Proper name:
GM2-KLH
Chemical name:
113NeuAc-GgOse3Cer-keyhole limpet hemocyanin (KLH)
20 Manufacturer: Progenies Pharmaceuticals, Inc., 777 Old Saw
Mill River Road, Tarrytown, New York 10591, United States of
America.
MATERIALS USED FOR THE PREPARATION OF GM2
MATERIAL SUPPLIER GRADE
Acetone BDH ACS
Ammonia Solution BDH ACS
Chloroform BDH ACS
Ethanol Commercial
Alcohol Ltd.
Ethyl Ether BDH ACS
Methanol BDH ACS
2-Propanol Fisher UJ.V1219 ACS
Water Travanol sterile water
for irrigation.
Calcium Chloride Fisher Certified
(anhydrous - 20 mesh
granular)
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Dimethyl Sulfide Aldrich 99% +
GM2 Fidia ---
Oxygen Linde UN1072 ' USP
Silica Gel E Merck Kieselgel
50H Art 7736
Sodium Aldrich 95% Pure
Cyanoborohydride
TLC Plates E Merck K i a s a 1 g a 1
60H F254
MATERIALS USED IN THE CONJUGATION PROCEDURE
MATERIAL SUPPLIER GRADE
Keyhole limpet hemocyanin Perimmune, Inc. USP
(KLH) Rockville, MD
Deoxycholic acid, sodium salt Aldrich Analytical
(DOC) (monohydrate) 98%
Ethylenediamine tetraacetic Aldrich ACS
acid
di-sodium hydrogen orthophos- BDH Analytical
. 20 phate (anhydrous) (NaZHP04)
Sodium chloride BDH Analytical
(NaCl)
Potassium dihydrogen BDH Analytical
orthophosphate (KHZP04)
25Sodium hydroxide BDH Analytical
(NaOH)
Tris (hydroxymethyl) Sigman
aminomethane hydrochloride
Sodium cyanoborohydride Aldrich
3 0 ( NaBH3CN )
Sepharose CL-4B Pharmacia
Nitrogen gas (filtered) Medigas
GM2 aldehyde Progenics
35 DEVELOPMENT CHEMISTRY
Data for the GM2 and GM2 Aldehyde:
The structures of GM2 and GM2 aldehyde were characterized by
;H NMR spectroscopy, thin layer chromatography (TLC), FAB-MS
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and FT-IR.
STRUCTURAL MOLECULAR MOLECULAR.
FORMULA FORMULA WEIGHT
GM2 - ganglioside
{compound #1)
GaINAcBI-4GaLB1-4 GlcB1-lCer C6~'~721026N3 M-1--1382 Solid
Neu5Aca2
I3NeuAc-GgOse3Cer C69~~125026N3 M-1=1410
(acid)
IO TLC: Rf=0.21 (65:35:8 CHCL3-CH30H-~H20)
Rf=0.60 (5:4:1 CHC13-CH30H-0.2% aqueous
CaCl2)
Rf=0.2 {7:1:1 {CH~)ZCHOH-NH~OH-H20)
STRUCTURAL MOLECULAR MOL. PHYSICO-CHEMICAL
FORMULA FORMULA WT. CHARACTERISTICS
GM2-aldehyde C53H9302~3 1204.29 Cream White,
(compound #2) Odorless,
Amphorous Solid
STRUCTURAL DATA
~H (DMSO-d6:D2) b : 9 . 48 (d,1H, J=2, OHz) , 4 . 79 (d, 1H, J=8 . 5Hz, III-
1), 4.26(d,lH,J=8.OHz,II-1), 4.19 (d,IH,J=8,OHZ,I-
1),2.54(dd,lH, A-3e)', 1.88{s,3H,Ac),
1.78(s,3H,Ac),0.85(t,3H,J=6.6Hz,CH3).
25 FT-IR (KBr Cast, CM-~}: 3439, 3420, 2952, 2923, 2851, 1634,
1070 (possibly the gem diol).
TLC Rf=0.5 {5:4:1 CHC13-CH30H-0.2% aquecous CaCl2)
Data for KLH and GM2-KLH:
30 Keyhole limpet hemocyanin (KLH) is a large, complex protein
composed of a number of smaller molecular weight subunits.
KLH is extracted and purified from the keyhole limpet
mollusk (Megathura crenulata).
3 S COMPOUND SEPHAROSE Ch-4B ISOET.ECTRIC RESORCIPIOL-GEL
CHROMATOGRAPHY FOCUSIHIG HCl
Molecular Weight (Isoelec. pts.) moles of GM2/
. (daltons) moles of protein
4 0 KZH Whole mol.(2):>2x106 Mult. bands
Subunits: 2-7 x 105 between pH 4.65
iii
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and pH 6
GM2-ItI~H Whole mol. t2)>2x106 Multiple bands
Subunits: 2-7 x105 between pH 4.65 200-
and pH 6 1900
GM2-KLH MANUFACTURING FLOW CHART
STEP 1 - PURIFICATION OF GM2:
10 GM2 (FIDIA)
y
Silica Gel Column Chromatography
1. 65:35 Chloroform - methanol
2. 65:35:4 chloroform - methanol - water
y
In-process QC:
1. TLC
y
STEP 2 - SYNTHESIS OF GM2 ALDEH'YDE (COMPOUND #2):
2 0 GM2 ( Compound #1 )
y
( 1 ) 03, MeoH
y
( 2 ) CH3SCH:~
y.
GM2 Aldehyde (Compound #2, may be the gem diol)
y
In-process tests done in-house:
1. TLC
w y
STEP 3 - CONJUGATION OF THE GM2 ADEHYDE TO KLH:
Sterile pyrogen free KLH
y
KLH added to GM2 aldehyde in 4:1 ratio (w/w).
y
Incubated at room temperature with shaking for 3 minutes
y
NaBH3CN is added to GM2 Adehyde/KLH mixture in 1:1 ratio
(w/w)
40 y
Reaction mixture is gently stirred at room temperature
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overnight then at 40°C for 4 days
y
STEP 4 - DIAFILTRATION OF THE CON~TUGATE:
Conj ugate is diaf i7.tered vs .
-PBS pH 7.5
-TRIS/EDTA pH 7.75
-TRIS/EDTA/0.05~ DOC pH 7.75
-TRIS/EDTA pH 7.5
-PBS pH 7.5
Conjugate aseptically removed from the Amicon filtration
unit
y
Centrifuged
y
Conjugate sterile filtered
In-process QC tests:
1. BioRad Protein Assay
2. Sepharose gel filtration
3. Isoelectric focusing (IEF)
y
Concentration of conjugate aseptically adjusted to 1 mg/mL
Conjugate dispensed into 1 mL sterile, pyrogen free vials
and stored at 2-8°C
y
Final QC testing
1. Enzyme immunoassay (EIA) 5. Rabbit pyrogen test
2. LAL pyrogen test 6. - General safety test
3. BioRad protein assay 7. Sterility test
4. Resorcinol-HCl assay 8. Impurity test for
cyanide
METHOD OF MANUFACTURE OF GM2-KLH (~ONJUGATE
The manufacturing of the GM2-KLH conjugate is carried out in
4 steps:
1. Purification of incoming GM2 (bovine source) (compound
## 1 ) .
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2. Synthesis of GM2 aldehyde (compound #2).
3. Conjugation of the GM2 aldehyde to KLH.
4. Diafiltration of the conjugate.
Step 1: Purification to GM2 (Compound #1}:
Name: GM2 ganglioside
Abbreviated Name: II3NeuAc-GgOse3Cer
GM2 ganglioside (bovine source) starting material is
supplied by FIDIA. All glass wares is washed with distilled
acetone followed by distiller ethanol arid then'dried (130°C)
for 18 hours prior to use. A column (Michel-Miller S 795-
10) of silica gel (30.58, Kieselgel 60H, Art 7736, E. Merck}
is packed at 75 psi (SSI Model 300 Lo pump) using 65:35
chloroform:methanol as solvent. GM2 (200 mg) is applied as
a concentrated 65:35 chlorofoz-m-methanol solution and
elution is performed with this solvent, followed by 65:35:4
chloroform-methanol-water. The fractions are analyzed by
TLC (Rf 0.6, 5x4:1 chloroform-methanol-0.2~ aqueous CaCl2).
The GM2 containing fractions are pooled and evaporated to
give a creamy white amorphous solid.
In-process testing for this material (compound #1).includes
~H NMR and thin layer chromatography (TLC) to confirm the
identity and purity of this ganc3lioside. The in-process
test results must meet the specifications listed under
developmental chemistry. If this material is found to be
impure, the above purification is repeated.
Step 2: Synthesis of GM2 Aldehyde (Compound #2):
All glassware is rinsed with distilled methanol and dried
(130°C) for 18 hours prior to use. A solution of the
purified GM2 ganglioside (compound #1) (40 mg) in distilled
methanol (10 mL) is stirred at ~7.5°C (dry ice-ethanol) and
ozone gas .(Orec 03V10-0 ozonator) is passed through the
solution for 7 minutes. A stream of argon is then passed
through the solution while the reaction is checked by TLC
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(5:4:1 chloroform-methanol- 0.2% aqueous CaCl2). The
solvents are then removed under reduced pressure and the
resulting material is dissolved i.n distilled methanol. To
this solution is added methylsulfide (200 m~) and the
5 reaction mixture is stirred at room temperature for one
hour. The solvents are then removed and the residue is
washed with ethyl ether(4 x 25 nnL). The resulting white
solid (compound #2) is dried in vacuo for 15 minutes to
remove~any remaining solvent and is then used directly in
the subsequent canjugation step.
Due to the unstable nature of the resulting aldehyde (B-
elimination), compound #2 is identified on a routine basis
only by TLC. The TLC of a typical run generally indicates
15 the presence of a small amount of sphinganine or
phytosphingosine analog (same Rf a;s compound #1) and a small
amount of reducing sugar (Rf 0.32).
Step 3: Conjugation of GM2 Alde:hyde to KLH:
All manipulations are done in a Class 100 biological safety
cabinet.
The KLH protein (160 mg) is aseptically measured and added
to the flask containing the lyophilized GM2 Aldehyde and a
magnetic stir bar. The solution is gently ag~.tated at room
temperature for 3 minutes until all of the GM2 Aldehyde has
gone into solution.
30 The sodium cyanoborohyd~ide (NaBH~CN) (40 mg) is added to the
GM2 Aldehyde/KLH solution then tile flask is sealed with a
stopper equipped with a sterile filter needle. The solution
is gently shaken then incubated overnight at room
temperature. The solution is then further incubated at 40°C
3 5 f or 4 days .
Step 4: Diafiltration of the Glycoconjugates (GM2-KLH):
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The contents of the GM2/KLH reaction vial are aseptically
transferred to a sterile, pyrogen-free Amicon
ultrafiltration unit with a YM-30 :filter. Filtered nitrogen
is used to provide an operating pressure of 16 psi for the
5 Amicon unit. The conjugate is thE~n diafiltered against the
following sterile, pyrogen-free ar. low pyrogen content
buffers successively:
1. 2 complete changes of PBS pH 7.5 (sterile, pyrogen-
free)
10 2. 2 complete changes of TRIS-HCI, EDTA pH 7.75 (sterile,
low pyrogen content)
3. 2 complete changes of TRIS-HCI pH 7.75 with 0.50
Deoxycholic acid (DOC) (sterile, low pyrogen content)
4. 4 complete changes of TRIS-:EiCI pH 7.75 (sterile, low
15 pyrogen content)
5. 3 complete changes of PBS pH 7.5 (sterile, pyrogen-
free)
The glycoconjugate is then aseptically removed from the
20 filtration unit and spun at 2000 rpm for 30 minutes. The
supernatant is thenwsterile filtered with a 0.22 mm low
protein binding filter.
.A sample of the glycoconjugate is obtained and the following
25 'in-process QC tests are done:
1. Sepharose gel filtration
2. Isoelectric focusing (IEF)
3. BioRad protein assay
30 Based on the results of the protean assay, the final volume
of the glycoconjugate is adjusted with sterile, pyrogen-free
pH 7.5 PBS buffer to yield a protein concentration of
lmg/mL.
35 Inside of a Class 100 biological safety cabinet, the final
glycoconjugate is then dispensed :in 1.0 mL aliquots with an
overfill volume of 0.1 mL into 2 mL sterile, pyrogen-free,
clear, borosilicate serum vials with rubber stoppers and
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stored at 2-8°C. During the-f~.lling procedure, the air
inside the filing area is monitored by exposing two blood
agar plates to the air near the work area inside of the hood
for a minimum of thirty minutes. These plates are then
5 transferred to a 37°C incubator and incubated for 1-2 days.
The plates are then examined for any bacterial or fungal
colonies.
The product is labeled by the manufacturing personnel and
the labeling is verifiedyby the Quality Control department.
The product is then stored at 2-8°C.
Each lot of GM2 - KLH goes through the following Final
Quality Control tests:
15 1. Enzyme Immunoassay (EIA)
2. T;AT, pyrogen test
3. BioRad protein assay
4. Resorcinol-HC1 carbohydrate assay
5. Rabbit pyrogen test
20 6. General safety test
7. Sterility test
8. Impurity testing for cyanide
