Note: Descriptions are shown in the official language in which they were submitted.
CA 02404111 2002-09-19
A Polysaccharide-Polypeptide Conjugate
The present invention relates to a new use of oxi-
dized polysaccharides as a carrier material for compo-
nents of vaccines, in particular to a method of
producing,a polysaccharide-polypeptide conjugate by re-
acting a polysaccharide with a polypeptide comprising
at least one free amino group, as well as to the use of
such a conjugate as a vaccine.
Vaccines are characterized in that one or more an-
tigens are administered in an immunogenic formulation
in a small amount, mostly parenteral (subcutaneously or
intramuscularly) so as to trigger a strong and protec-
tive immune response. At present, most vaccines are
produced for protecting against microbial infections.
In these instances, the antigens used are inactivated
and altered microorganisms or parts thereof, or defined
proteins from such microorganisms which are suitable to
trigger an immune response against the respective mi-
croorganism.
For years also the effectiveness of many experi-
mental vaccines against other diseases has been inves-
tigated. Among them are vaccines against cancer. In
this case, the immune system of cancer patients is to
be selectively activated so as to combat malignant
cells. This is attempted by means of the most differing
approaches. Among them are vaccinations with autologous
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or allogenic tumor cells, chemically or molecular-bio-
logically modified autologous or allogenic tumor cells,
isolated tumor-associated antigens (TAA) or tumor-asso-
ciated antigens prepared by chemical or molecular-bio-
logical methods, peptides derived therefrom, anti-
idiotypical antibodies as a surrogate of a TAA, lately
also vaccinations with DNA which codes for TAA or for
structures derived therefrom, etc. In principle, very
small amounts of a suitable vaccine will suffice to in-
duce an immunity from months up to years, since the at-
tenuation can be boosted by booster vaccinations.
Moreover, in an active immunization both a humoral and
a cellular immunity can be induced the interaction of
which can yield an effective protection against cancer.
To attain a strong immunity, antigens in vaccines
mostly are administered together with an adjuvant. As
examples of adjuvants the following may be mentioned,
without, however, being restricted thereto: aluminum
hydroxide (Alu-Gel), derivatives of lipopolysaccharide,
Bacillus Calmette Guerin (BCG), liposome preparations,
formulations with additional antigens against which the
immune system has already produced a pronounced immune
response, such as, e.g " tetanus toxoid, Pseudomonas
exotoxin or components of influenza viruses optionally
in a liposome preparation. Furthermore, it is known
that the immune response may also be enhanced by simul-
taneously administering endogenous proteins which play
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CA 02404111 2002-09-19
an important role in the build-up of an immune re-
sponse, such as, e.g., granulocyte macrophages-stimu-
lating factor (GM-CSF), interleukin 2 (IL-2),
interleukin 12 (IL-12) or gamma interferon (IFNy).
US-5,554,730-B relates to polysaccharide-protein
conjugates, wherein a particulate vaccine is to be cre-
ated. For this purpose, a polysaccharide-protein conju-
gate is created as a Schiff's base (azomethin),
primarily by reacting a protein carrier with an oxi-
dized polysaccharide antigen in the presence of a
"crowding agent" (water displacing agent), wherein the
protein carrier is immediately denatured due to the
presence of the crowding agent, and the conjugate pre-
cipitates in the form of microparticles. Although a
dissolution of the precipitated microparticles in a
strongly basic environment (0.1 N NaOH) for obtaining a
_ vaccination solution as such is possible and has also
been disclosed, it only makes sense if a polysaccharide
antigen is used, because any antigenic protein would
have lost its antigenic determinants as a consequence
of denaturing, and thus would no longer be effective.
WO 99/55715 describes polysaccharide-antigen con-
jugates in which the antigen is either bound to the
polysaccharide via a suitable bivalent linker, or via a
terminal aldehyde group. A direct binding of the anti-
gen to the polysaccharide via an azomethin bond thus is
limited to the number of the terminal aldehyde groups
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present in the polysaccharide.
Also DE-198 21 859-A1 describes polysaccharide-an-
tigen conjugates, wherein a suitable crosslinker is
bound in the polysaccharide by means of an azomethin
bond to aldehyde functions obtained by periodate oxida-
tion. In the cross-linker, a maleimido function is ad-
ditionally provided, to which an -SH group of cysteine
can add. The utilized antigens then are N- or C-termi-
nally provided with an additional Cys so as to allow
for the addition of the terminal SH function with the
cross-linker and thus the obtaining of the polysaccha-
ride-antigen conjugates described.
Finally, US-5,846,951 relates to polysaccharides
comprising at least 5 sialic acid residues which poly-
saccharides can be provided with terminal aldehyde
groups at the non-reducing ends of the polysialic acids
by means of oxidation with sodium periodate. Terminal
aldehyde groups created in this manner may then bind
amino-group-containing medicaments, e.g proteins, via
azomethine bonds.
Most antigens used for vaccines comprise struc-
tures with primary amino groups. In particular, all
protein antigens normally comprise at least one, but
mostly several, Iysines in their amino acid sequence.
The amino groups of these lysines are present in free
form.
It has long been known that primary amines can re-
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CA 02404111 2002-09-19
act with aldehydes. The product of this reaction is
called Schiff~s base. Schiff~s bases are not completely
stable compounds, they can be hydrolyzed under suitable
conditions and thus~be returned into their starting
substances.
Furthermore, it has been known that compounds com-
prising vicinal hydroxyl groups can be oxidized with
the help of suitable oxidants, in particular with peri-
odic acid or salts of periodic acid, such as sodium
metaperiodate, such that two aldehyde functions are
formed by breaking the C-C bond on which the neighbor-
ing hydroxyl groups are located.
A large number of high-molecular polysaccharides
consist of monomeric sugar units which carry vicinal
hydroxyl groups. Dextrane and mannan should be men-
tioned as two non-limiting examples. Such polysaccha-
rides thus can be oxidized with periodate in the above-
described manner without the bands between the monomers
being split. If, based on the number of monomeric
units, a stoichiometric smaller amount of periodate is
used, the oxidation will occur only partially, which
means that only so many monomers will be oxidized ac-
cording to the principle of random as corresponds to
the amount of periodate.
The present invention is based on the object of
providing further means and methods which will lead to
immunogenic formulations of vaccines.
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In a method of the initially defined type, this
object is achieved in that a polysaccharide carrier
comprising vicinal hydroxyl groups is oxidized under
ring opening to create vicinal aldehyde groups, and is
reacted with one or several base-instable antigenic
polypeptide(s) containing at least one free amino
group, wherein the polypeptide(s) is (are) bound di-
rectly to the polysaccharide carrier via at least one
azomethine bond. Partially oxidized polysaccharides
thus are a suitable carrier material for the formula-
tion of vaccines if the utilized base-instable anti-
genic polypeptides comprise one or more free primary
amino groups and thus, via an azomethine bond, can be
connected with the vicinal aldehyde groups created in
the carrier material by ring opening. Preferably, the
base-instable antigenic polypeptides used according to
the invention are stable up to a pH of approximately
11, preferably up to a pH of approximately 10, still
more preferred up to a pH of approximately 9, most pre-
ferred up to a pH of approximately 8. If polypeptides
are mentioned in the context of the present invention,
proteins having at least 6 amino acids in the chain are
to be understood. In the same way, polysaccharides are
understood to be poly-sugars comprising at least 3
monomer units in the chain. Preferably used polysaccha-
rides are mannan, e.g. having a molecular weight of at
least 70 kDa, and dextrane, e.g. having a molecular
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weight of at least 70 kDa, particularly preferred hav-
ing a molecular weight of approximately 2000 kDa.
According to a preferred embodiment of the present
invention, the vicinal hydroxyl groups originally pres-
ent in the polysaccharide carrier are at least par-
tially oxidized, preferably by at least 20~. By
controlling the rate of oxidation, e.g. by a stoichio-
metric smaller amount of oxidating agent, the amount of
aldehyde groups available for an azornethine bond be-
tween carrier and polypeptide can easily be adjusted.
Preferably, the base-instable antigenic polypep-
tide is a vaccine antigen, particularly preferred an
antibody, e.g. a monoclonal antibody, such as the
murine monoclonal antibody HE2. A new method of cancer
vaccination has been described in application
PCT/EP00/00174 (priority date: Jan. 13, 1999), "Ver-
wendung von Antikorpern zur Vakzinierung gegen Krebs"
("The Use of Antibodies for Vaccinating against Can-
cer"), the disclosure of which is included herein by
reference thereto. The monoclonal antibody HE2 de-
scribed there which is used as the vaccine antigen in a
cancer vaccination serves as a non-limiting example for
the formulation of a vaccine according to the method of
conjugation to a partially oxidized high-molecular
polysaccharide described here.
According to a further preferred embodiment of the
present invention, the base-instable antigenic polypep-
CA 02404111 2002-09-19
tide has the same binding fine specificity as the anti-
body HE2.
It is also suitable if in addition to the respec-
tive base-instable antigenic polypeptide substances are
conjugated which cause an enhancement of the immune re-
sponse, e.g. GM-CSF, IL-2, IL-12 or Gamma-Interferon,
or a mixture of these substances.
Moreover, it is preferred if the polysaccharide-
polypeptide conjugate according to the invention is ad-
ditionally adsorbed on aluminum hydroxide and/or mixed
with pharmaceutically acceptable carriers.
Finally, it is preferred if the polysaccharide-
polypeptide conjugate obtained according to the inven-
tion is formulated as a vaccine formulation to be ad-
ministered by subcutaneous, intradermal or
intramuscular injection, e.g. by dissolving or suspend-
ing the optionally, e.g., aluminum-hydroxide-adsorbed
conjugate in a suitable physiological buffer and the
like.
In general, the following advantages and specific
properties of the conjugate according to the invention
should be mentioned:
~ The components coupled to the oxidized polysaccha-
rides via primary amines (conjugate and adjuvants and
additives, respectively) are slowly released in the
presence of an excess of molecules with free primary
amines, e.g. serum proteins. The slow release effect
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CA 02404111 2002-09-19
thus forming is desired for vaccines, since by this
antigen-presenting cells are able to locally receive
the vaccination antigens at the site of vaccination
for a longer period of time.
~ By the choice of the polysaccharide, the properties
of the conjugate can be influenced. This applies both
to the molecular size of the polysaccharide and to
its chemical composition. If, e.g., mannan is chosen
as the polysaccharide, the corresponding conjugate
preferably will be taken up by cells of the immune
system which carry the mannose receptor. Among them
are, in particular, macrophages and dendritic cells
as professional antigen-presenting cells. In this
manner, an increased immune response is attained.
~ Several components can simultaneously be bound to
partially oxidized polysaccharides. These may be sev-
eral differing vaccine antigens, or vaccine antigens
together with components enhancing the immune re-
sponse, such as, e.g., the proteins GM-CSF, IL-2, IL-
12 or gamma interferon .
The enclosed Fig. 1 shows the comparison of two
formulations as regards the induction of antibodies
against HE2 (ELISA) in rhesus monkeys.
E x a m p 1 a .
At first, dextrane having a molecular weight of
2000 kDa (SIGMA D-5376) is oxidized by 20~ with sodium
metaperiodate. For this purpose, 324 mg of dextrane are
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CA 02404111 2002-09-19
dissolved with stirring in 4 mI of distilled water. To
this solution, 86 mg of sodium metaperiodate previously
dissolved in 0.6 ml of distilled water are admixed, and
incubated in the dark at 37°C for 30 minutes. 25 mg of
the antibody HE2 (PCT/EP00/00174) are brought to pH 7.4
with 1 M NaaHP04, and 45 u1 of a thimerosal solution
(20 mg/ml) are added.
To this solution, 1.675 1 of the above-obtained
oxidized dextrane solution are added and incubated in
the dark at 37°C for 2 days. The completeness of the
reaction is analytically checked by chromatography on a
molecular weight column (Zorbax 450). The signal corre-
sponding to a molecular weight of 150 kDa (monomeric
HE2) has disappeared, and in its place a signal occurs
in the exclusion volume of the column which corresponds
to a molecular weight of >2000 kDa.
The solution obtained is chromatographed by means
of a preparative molecular weight column which is
equilibrated with the final buffer (1 mM phosphate
buffer in physiological saline, pH=5.5). The material
obtained in the exclusion volume consists of the high-
molecular conjugate of the antibody HE2 on partially
oxidized dextrane. The content of conjugated HE2 can be
determined by integration of the signal after analyti-
cal chromatography on a molecular weight column as com-
pared to monomeric HE2. The solution obtained is mixed
with an aqueous aluminum hydroxide such that the final
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concentration is 0.5 mg of HE2 on 1.67 mg of aluminum
hydroxide in 0.5 ml of buffer.
Four rhesus monkeys are subcutaneously immunized
with 0.5 ml of the above formulation on days 1, 15, 29
and 57. The sera of various points of time were assayed
by means of ELISA for an induction of antibodies
against monomeric HE2. As a comparison, four rhesus
monkeys were vaccinated in the same manner with a stan-
dard formulation of 0.5 mg of monomeric HE2 adsorbed on
1.67 mg of aluminum hydroxide.
The ELISA was carried out as follows:
100 dal aliquots of the MAb HE2 (solution with
10 ug/ml in binding buffer) are incubated in the wells
of a microtiter plate for 1 h at 37°C. After having
washed the plate six times with washing buffer A,
200 u1 each of blocking buffer A are added and incu-
bated at 37°C for 30 minutes. After washing the plate
as described above, 100 u1 aliquots each of the monkey
sera to be tested are incubated in dilutions of 2:100
to 1:1 000 000 in dilution buffer A at 37°C for 1 h.
After having washed the plate as described above,
100 u1 each of the peroxidase-conjugated goat anti-hu-
man-Ig antibody (Zymed) is added in a dilution of
1:1000 in dilution buffer A and incubated at 37°C for
30 minutes. The plate is washed four times with washing
buffer A, and twice with staining buffer. The antibody
bond is detected by the addition of 100 u1 each of the
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specific substrate, and the staining reaction is
stopped after 10 minutes by adding 50 u1 each of stop-
ping solution. The evaluation is effected by measuring
the optical density (OD) at 490 nm (wave length of the
reference measurement is 620 nm).
The results of the ELISA are illustrated in Fig.
1. The animals vaccinated with the conjugate of HE2 on
dextrane developed comparable titers of antibodies
against HE2 as the monkeys vaccinated with the standard
formulation.
Moreover, it was investigated to which extent HE2
after application can be found again in the serum of
the monkeys. For this purpose, again an ELISA was used,
which was carried out as follows:
100 u1 aliquots of a purified polyclonal anti-idi-
otypic goat antibody against HE2 (solution with
10 ug/ml in binding buffer) are incubated in the wells
of a microtiter plate at 37°C for 1 h. After having
washed the plate six times with washing buffer A,
200 u1 each of the blocking buffer are admixed and in-
cubated at 37°C for 30 minutes. After having washed the
plate as described above, 100 u1 aliquots each of the
monkey sera to be tested are incubated in dilutions of
1:4 to 1:100 000 in dilution buffer A at 37°C for 1 h.
After having washed the plate as described above,
100 u1 each of a peroxidase-conjugated goat anti-mouse-
IgG antibody (Zymed) are added in a dilution of 1:1000
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in diluting buffer and incubated at 37°C for 30 min.
The plate is washed four times with washing buffer, and
twice with staining buffer. The antibody bond is de-
tected by the addition of 100 u1 each of the specific
substrate, and the staining reaction is stopped after
10 minutes by adding 50 u1 each of stopping solution.
The evaluation is effected by measuring the optical
density (OD) at 490 nm (wave length of the reference
measurement is 620 nm).
the results are illustrated in the following ta-
ble:
Time Conjugate vaccine Standard formulation
0 0;0;0;0 ng/ml 0;0;0;0 ng/ml
1 h 0;0;0;0 ng/ml 13;17;74;2$0 ng/ml
4 h 0;0;0;0 ng/ml 200,280,400,740 ng/ml
!,24 h 0;0;0;0 ng/ml 960,960,1000,740 ng/ml~
After 24 h, a trace of a HE2 concentration can be
recognized in the serum of those animals which had been
vaccinated with the conjugate, yet this concentration
is below the detection limit of approximately 10 ng of
HE2/ml serum. Apparently, the desorption of the vaccine
antigen has clearly been reduced by the conjugation to
partially oxidized dextrane, as compared to the stan-
Bard formulation. Thereby probably fewer vaccinations
will suffice to obtain a comparable titer than with a
standard formulation on aluminum hydroxide.
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Materials used:
Microtiter Immuno Plate F96 MaxiSorp (Nunc) for
plates: ELISA
Binding buf fer : 15 mM NazC03
35 mM NaHCOa
3 mM NaNs
pH: 9.6
PBS deficient: 138 mM NaCl
1.5 mM KHzPOa
2.7 mM KCl
6.5 mM NazHP04
pH: 7.2
tnlashing buffer: 0.05 Tween 20 in PBS deficient
Blocking buffer: 5~ fetal calf serum (heat-inactivated)
in PBS deficient
Dilution buffer: 2~ fetal calf serum (heat-inactivated)
in PBS deficient
Staining buffer: 24.3 mM citric acid
51.4 mM NazHP04
pH: 5.0
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Substrate: 40 mg o-phenylene-diamine-dihydrochlo-
ride
100 ml staining buffer
20 ~.t1 HzOz (30~)
Stopping solu- 4 N HzS04
Lion:
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