Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-1- 1326819
PHARMACEUTICAL STRUCTURE
Backaround of the Invention
Field of the Invention
The invention concerns a pharmsceutical
structure in which haptens and/or immunogenic-
immunostimulatory substances are attached to a proteincarrier.
Backaround Information
Attempts had been made heretofore to bind
~; 20 haptens and/or immunogenic-immunostimulatory substances
(hereinafter collectively referred to as "immunoactive
substances") to proteins and thereby to increase the
immunogenicity or activity of such immunoactive
sub~tances. In the~e prlor attempts, the protein
molecules were present as monomers in solution or
dispersed as unstructured aggregates. In such
formulations, the binding sites to which immunoactive
substances can attach differ considerably with respect to
heir nature, kind, number and position within the protein
molecules, so that a chemically well-defined linkage of
immunoactive substances to the protein molecules cannot be
achieved.
~` The invention is based on the problem of
creating a pharmaceutical structure of the aforementioned
~ ~ 35 kind wherein a precisely defined number of binding qites
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are available for attachment of a precisely defined
quantity of immunoactive substances in a chemically
defined manner.
STATEMENT OF THE INVENTION
In accord with the present invention, this
problem of ill-defined coupling of immunogenic-
immunoregulatory materials is solved by using as the
protein carrier to which the immunoactive substances are
bound, crystalline or para-crystalline aggregates of
proteins or protein containing molecules. These
aggregates may also be covalently cross-linked. By virtue
of the crystalline or para-crystalline structure, the
protein molecules display a constant, precisely defined
spatial orientation with respect to each other; thus both
the nature of the linkages and the number and spatial
orientation of the immunoactive substances and the
distance between the binding sites which can carry the
immunoactive substances are always precisely defined.
Furthermore, by the practice of this invention
it is possible to choose suitable protein carriers so that
they, by virtue of their shape, size, arrangement and
surface properties, are preferentially phagocytosed.
Thus, the uptake by phagocytosing cells, e.g.,
macrophages, of the immunoactive substances is
considerably enhanced. Consequently, a more efficient
immune response is achieved.
Brief DescriPtion of the Drawinas
~he invention will be described with reference
being made to the sole figure. The figure i8 a graph
illustrating the improved analytical results possible
using the coupling methods of this invention.
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Descri~tion of the PreferreA Embodiments
Advantageously, the crystalline or para-
crystalline aggregates may consist of glycoproteins,
whereby the structure of the carriers may further
approximate the shape of a bacterium. The appropriate
aggregates may be derived from one or several microbial
cell wall layers. Thus, the glycoproteins are obtained in
an especially simple manner. Such suitable protein
a~gregates may contain other adhering cell wall
components. In certain suitable cases of microorganisms,
microbial cell wall fragments as such can be used to carry
the immunoactive substances. Particularly suitable as
sources of this type of carrier are those microorganisms
which, aside from the cry~talline surface layer proteins,
lS contain underneath additional rigid layers ~uch as those
composed of peptidoglycan or pseudo-murein.
Suitable carrier aggregates may contain the
immunoactive substances linked to a protein portion
present in the carrier. In other situation~, such as
90metimes with haptens, it may be advantageous to have
them linked to the carbohydrate portion~ of a glycoprotein
(glycoprotein glycan) carrier. The choice of these two
modes of attachment w~ll depend both upon the nature of
the immunoactive sub8tances and upon the type of
application of the pharmaceutical structure. Under
certain circumstance~, a mixed mode of attachment can be
advantageous.
Furthermore, the immunoactive substances can be
attached to the respective carrier molecule~ by way of
~30 bridging molecule~ such as homo- or heterobifunctional
cross-linking agents or peptide chains (e.g., polylysine).
The introduction of such spacers or bridging molecules
offers the ad~antage of more precise control of the
reIease of haptens, etc., and of the nature of such
fragments as would form by enzyme-catalyzed degradation
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within the endosomes (lysosomes) of macrophages or other
antigen-processing cells. Using appropriate spacer
groups, preferred sites of cleavage of the immunogenic
aggregates may be introduced.
Finally, different carriers and/or carrier
aggregates comprising different immunoactive substances
may be combined. Thereby differential functions of the
pharmaceutical structure are achieved. Thus, strongly
hydrophobic carrier molecules can be mixed with aggregates
carrying the immunoactive substances causing increased
uptake of the pharmaceutical structure by phagocytosing
cells. Furthermore, carrier aggregates comprising
different haptens, etc., can be used so that the profile
of activity of the pharmaceutical structure can be
precisely controlled.
By an advantageous process for the production of
the pharmaceutical structure of this invention, such
groups on the protein or protein-containing carrier
molecules as would bind the immunoactive substances may be
activated prior to attaching the immunoactive substances.
Thereby a reliably precise and reproducibly stable
attachment of haptens, etc., to the respective groups is
safeguarded. .,
For attaching immunoactive substances to
carbohydrate portions, binding sites within the
glycoprotein glycans may be generated by oxidation, e.g.,
using periodate. Binding sites on the protein molecules
can also be generated by reacting with glutaraldehyde, the
reagent used for cross-linking and activation. Pormation
of binding sites can also occur by the introduction of
active groups, whereby a precise control of the number and
kind of binding sites can be achieved. For an especially
stable linkage, the haptens etc. can be attached by amide
linkages to the carboxyl groups of the protein carrier.
For certaln active bindlng slte~, the attachment of the
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haptens, etc., can occur in the form of Schiff bases. The
Schiff bases can also be reduced to secondary amines.
Furthermore, special linkages or linkages
amenable to a special mode of cleavage can be constructed
by the use of such bridging molecules as would contain
activated binding sites on both ends, e.g., homo- or
heterobifunctional cross-linking agents or peptide chains
(e.g.~ polylysine)~ as follows. The intermediate molecule
is attached to the carrier by one activated group whereas
the other activated group is used to attach the
immunoactive ~ubstances. In this manner, ~o-called
~pacers~' are introduced into the pharmaceutical
structure.
Finally, different carrier aggregates, or
carrier aggregates comprising different immunoactive
substances can be attached to an auxiliary matrix which,
following cross-linking of the carriers, can be removed as
the case may be. Thereby, a pharmaceutical preparation
can be generated that would combine different types of
carrier molecule~ suCh as those differing in their
crystalline structures. Also, carriers comprising
different haptens, etc., could thus be combined into new
;~ pharmaceutical structures.
With similar advantages, binding ~ites on the
immunoactive substances can be activated and the
immunoactive substances attached by means of these
activated binding sites, to the protein or glycoprotein
carriers. This, too, re~ult~ in e~pecially stable
linkages.
The invention will be further explained making
reference to the following examples.
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1 32681 q
Example 1
A. Preparation of the Carrier
Cells of Clostridium thermohydrosulfuricum L111-
69 (2.5 g) are suspended in 50 mM Tris HCl buffer, pH =7.2. and sonicated briefly (about 1 minute). Following
the addition of a 2~ solution of Triton X-100 (12.5 mL),
the suspension is incubated at 50C for 15 minutes. ay
this treatment, the cytoplasm and plasma membrane of the
organisms is disintegrated whereas the crystalline or
para-crystalline protein-containing cell wall layer
(henceforth termed "S-layer~') and the underlying
peptidoglycan layer are conserved as fragments.
Subsequently, the mixture is centrifuged at 20,000 x g and
the pellets washed three times to remove detergent. The
pellets are then suspended in 5 mM magne~ium chloride
solution (25 mL). For removal of cytopla~mic residues and
nuclelc acids, DNAse (125 ug) and RNAse (500 ug) are added
and the whole stirred for 15 minutes at 37C. The
su8pen9ion is then centrifuged at 20,000 x g and washed
three times with water. The pellet is then su~pended in
0.1 M cacodylate buffer (pH = 7.2; 20 mL) and a 50%
aqueous solution of glutaraldehyde iq added at 4C to a
final concentration of 0.5%. The suspension is well
stirred at 4C for a few minutes, centrifuged, and wa~hed
with water. The pellet is then suspended in water (25 mL)
and tri~-hydroxymethylaminomethane ("Tri~") is added.
Following 10 minutes standing at room temperature, the
suspension is again centrifuged (20,000 x g) and washed.
Ultrasonic treatment is omitted when the
cellular shape of the microorganisms is to be conserved
and only the cytoplasmic constituents are to be removed.
When the above procedure is used, the underlying
peptidoglycan layer remains associated with the protein-
containing cell wall layer. With numerous organisms, this
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may result in the formation of an additional S-layer.
Thus, the fragments or 'ghosts", consisting only of S-
layer and peptidoglycan, now display S-layers on the inner
face of the peptidoglycan layers; these additional
S-layers can also be coupled to immunoactive substances.
5hould the presence of the peptidoglycan be
undesirable, the latter can be degraded with a
peptidoglycan-degrading enzyme, e.g., lysozyme, and
removed. To this end, the material produced as under this
section A is treated for 1 hour at 36C with a solution of
lysozyme (O.S mg lysozyme per mL of a 50 mM ~olution of
Tris HCl buffer, pH = 7.2). In this ca~e, 10 mL of
lysozYme solution is added per 0.5 g wet pellet.
Depending on the microorganism used, the S-layer fragments
obtained consist of a simple or double S-layer. Following
ultrasonic treatment of cells, open fragments are formed
¦ whereas in the absence of ultrasonic treatment, the
cellular shape, i.e., the crystalline or paracrystalline
S-layer is preserved intact.
B. Formation of Bindinq Sites
¦ The pellet prepared according to A is suspended
in water (5 mL) and a 0.1 M solution of sodium periodate
I (5 mL) sdded. The suspension is allowed to stand for 24
1 25 hours with exclusion of light to allow oxid~tion and give
rise to binding sites. Subsequently, the suspension is
centrifuged and the pellet washed with 10 mM sodium
j chloride solution, to remove the iodine-containing salts.
~- 30 C. Bindinq of Proteins to the Modified S-LaYers
(Obtained)
The pellet of binding-site containing material
I obtained according to B (about 0.2 g) is suspended in
¦ water (1 mL) and the suspension is mixed with a solution
(1 mL\ of bovine serum albumin (50 mg) in ~ater (10 mL).
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This solution is allowed to stand at room temperature (60
minutes) and is then centrifuged.
To determine the amount of albumin bound to the
carrier, the extinction at 750 nm is measured relative to
that of a preparation wherein the periodate solution has
been replaced by water (unoxidized control). The result
of this measurement is seen in Fig. 1. Clearly, the
attachment to the carrier is significantly higher in the
case involving prior oxidation with periodate.
Example 2
A. Preparation of the Carrier
Cells of Bacillus stearothermophilus PV7.2
(2.5/g) are suspended in 50 mM Tris HCl buffer pH = 72 and
sonicated for about 1 minute. Following addition of 2%
Triton X-100 (12.5 mL), the suspension is incubated for 15
min. at 50C. By means of this treatment, the cytoplasm
of the cells i8 disintegrated while the S-layer and the
peptidoglycan layer are preserved. Thus, fragments are
formed which correqpond in shape more or less to the
original shape of the bacterial cell (so called "ghosts").
Subsequently, the suspension i5 centrifuged at
20,000 x g and the pellet washed three times with water to
remove the detergent. The pellet is then suspended in 5
mM magnesium chloride solution (25 mL), DNAse
(deoxyribonuclease,, 125 ug) and RNAse (ribonuclease, 500
uG) are added, and the mixture is stirred at 37C for 15
min. Subsequently, the pellet is washed three times with
water, centrifugation in between being at 20,000 x g. The
pellet is then suspended in 0.1 M cacodylate buffer (pH =
7.2) and the suspension mixed with a 50% solution of
glutaraldehyde in water at 4C to a final concentration of
0.5%. The suspension is then vigorously stirred at 4C
for a few minutes, centrifuged, and the pellet washed with
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water. The glutaraldehyde residues are linked through
only one of their two aldehyde functions so the remaining
aldehyde can serve as binding sites as has been achieved
by oxidation under Example l.B.
B. Bindinq of Protein(s) to the Modified S-LaYers
The modified S-layers prepared in section 2.A.
are mixed with a solution of bovine serum albumin as in
Example l.C., and the amount of protein bound is
determined as described there.
Example 3
A. Preparation of the Carrier
Cell walls of Clostridium thermohydrosulfuricum
j L111-69 are treated with glutaraldehyde (.5~ in 0.1 M
sodium cacodylate buffer, pH = 7.2) for 20 minutes at
20C, so as to stabilize the outermost cell wall layer (S-
layer). The reaction iB terminated by the addition of
excess ethanolamine. During cro~s-linking the cell wall
fragments may be either in suspension or attached to a
porous surface (S-layer ultrafiltration membrane). The
cell wall fragments a~e then washed with distilled water
to remove the reagent mixture.
B. Creatina Bindina Site~ for Liaands Containina Thiol
(SH) GrouPs
The pellet of a cross-linked preparation as
under A above, is suspended in distilled water (30 mL) and
to the suspension is added 1-Ethyl-3.3
(dimethylaminopropyl) carbodiimide (EDC; 60 mg)
maintaining a pH of 4.75. This step activates the exposed
carboxyl groups of the S-layer. Subsequently, an excess
of hexamethylenediamine (0.5 g) is added and the pH kept
at 8.0 for 60 minutes. Subsequently, the reaction is
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terminated by addition of acetic acid. The suspension is
centrifuged at 20,000 x g and the pellet washed three
times with distilled water. The wet pellet (100 mg) is
suspended in 50 mM phosphate buffer, pH = 7 (9 mL) and a
solution of meta maleimidobenzoyl-N-hydroxysuccinimide
ester (50 mg per mL of tetrahydrofuran; 1 mL) is added.
The mixture is then incubated for 30 minutes at 20C.
C. Bindina of SH-Containina Proteins to the S-LaYers
10Derivatized as Under B
Following centrifugation at 20,000 x g, the
pellet is suspended in 50 mM phosphate buffer (pH = 7.0),
B-galactosidase (20 mg) is added and the mixture is
incubated for 2 hours at 20C. After centrifugation at
20,000 x g and repeated washing with phosphate buffer, the
activity of the B-galactosidase covalently linked to the
protein matrix is determined.
The reactions of the Example are summarized as
follows:
1. -COOH + EDC+H2N-(CH2)6-NH2 ~ ~CI NH (CH2j6 NH2
252. -6-NN-(CN2_6~NH2 + ~ N-C-C ~ NO~
3- -6~NN~(CH2)6~NN-c ~ ~ +N-GalactosidaGe
30O O (SH-Ligand)
o
4. -C-NH-(CH2)6-NH-~CI ~ N ~
O O S-~-Galactosidase
O
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Example 4
A. For the coupling of invertase, the vicinal diol
groupings of the carbohydrate portion (glycan) of S-layer
glycoprotein are utilized. Cell wall fragments are
treated with glutaraldehyde, as described in Section A of
Example 3, to stabilize the outermost cell surface.
B. Generatinq the Bindinq Sites
The cell wall fragments from Section A (100 mg)
are suspended in anhydrous tetrahydrofuran ~THF),
incubated at 20C for 10 minutes, centrifuged at 20,000 x
g and suspended again in a 2.5% solution of cyanogen
bromide in anhydrous tetrahydrofuran (10 mL). Following
incubation for 2 hours, the cell wall frag~ents are
separated by centrifugation at 20,000 x g and wai~hed with
THF for removal of residual reagent.
C. Bindina of ~roteins to the Derivatized S-La~er
The pellet is suspended in 50 mM phosphate
buffer pH - 8.0 (10 m~) containing invertase (20 mg) and
incubated for 18 hr at 4C. Following centrifugation at
20,000 x g, the pellet is washed twice with phosphate
buffer and the enzyme activity of the invertase bound to
the protein matrix determined.
The reactions of this Example are summarized as
follows:
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S ~OH
/ + srcN--- C=NH
~OH ~ O/ (cyclic imidocarbonate)
~ / ~
OH
+ R-NH2~ ~
;~ O-C-N-R
O H .
i (N-substituted
carbamate)
I Example 5
¦ A. Cell wall fragments of Clostridium
thermohydrosulfuricum L111-69 are cross-linked with
glutaraldehyde as described in Example 3, section A.
B. Generatinq the Bindina Sites
I
Cell wall fragments (0.1 g) are suspended in
anhydrous dimethylformamide (DMF, 20 mL) and EDC (60 mg)
and N-hydroxysuccinimide (0.5 g) are added to the
suspension. Following incubation for 1 hour, the
suspension is centrifuged at 20,000 x g and washed twice
with DMF.
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C. Bindinq of Proteins to the S-Layer thus Modified
The pellet obtained as under SB is suspended in
0.1 M sodium hydrogencarbonate,(pH 8.8) containing
dissolved dextranase (20 mg) and the reaction mixture is
incubated at 4C for 18 hr. The cell wall fragments
containing the bound dextranase are obtained by
centrifugation at 20.000 xg and washed twice with
distilled water. The dextranase activity contained in the
pellet is then determined.
The reaction~ of this Example are summarized as
follows:
~1 0
~_COOH + EDC HO-N ~ O ~ + Ligand-NH2
O O
~,C-NH-Ligand
alkali
Example 6
CouPlina of a SYnthetic CarbohYdrate Antiaen
to Oxidized S-LaYers
A. Preparation of the Carrier
B. Generatina the Bindina Sites
The preparation of the o~idized glycoprotein S-
layers was performed as described in Example 1, sections A
and B.
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C. Bindinq of the CarbohYdrate Antiaen to the Carrier
The oxidized (polyaldehyde) derivative of the S-
layer prepared in Sections A/B~is incubated with the 3-(2-
aminoethyl) thiopropyl glycoside of a disaccharide whereby
Schiff base formation occurs. This step can also be
performed with any other saccharide attached to an
aglycone that contains amine groups.
These reactions are shown as follows:
~KDOp2 KDOp( 2 2C 2SCH2CH2NH3)
[3-(2-ammDnioethyl)thiopropylglycoside of a
disaccharide oonsisting of two 3-deoxy-D-
mano-2-octulopyranosylono residues
H
HO-C-CH20H
HO
~Lo
O~CO~
/ 1 2
20C_~ON ~ NscN2cN2NN2 ~/
25 HO-C-H
CH2H
30 H ~ C~
~ H ~N-(CH2)2S(CH2)3 Na(CN)BH3 H ~ N-CH2CH2SCH2CH2CH'
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1 32681 q
-15-
General recipe for the preparation of 3-(2-
aminoethylthio)propyl glycosides from allyl glycosides.
A solution of the allyl glycoside (5 mM) in a
solution of cysteamine hydrochloride (15 milliequivalents
of SH-groups in 10 mL) is allowed to stand for 1.5 hours
at room temperature. The duration of this reaction may
vary. The reaction mixture is subsequently separated over
a column of cation exchange resin (e.g.,*Rexyn 101,
ammonium form, 200-400 mesh). The column i5 eluted with
water, 0.5/M ammonia, and 1.0 M ammonia. Unreacted allyl
glycoside appears in the aqueous eluate, and the 3-(2-
aminoethylthio) propyl glycoside is eluted in the fraction
corre~ponding to 1.0 M ammonia. Those fractions
containing products are subsequently evaporated to
dryness.
The Schiff base derivative of the S-layer, as
obtained by binding of the 3-(2-aminoethylthio)propyl
glycoside can be used directly for binding of antibodies.
The e can be assayed directly if they are labeled with
ferritin, horseradish peroxidaqe, 125T or in any other
appropriate manner. The bound antibodie~ can also be
assayed via a so-called ~sandwichll method by binding of
labeled antibodies directed against the first, hapten-
bound antibodies.
The Schiff base derivative of the S-layers as
obtained by binding of the 3-(2-aminoethylthio)propyl
glyco~ide may be converted into a secondary amine
derivative of the S-layers by reacting it with sodium
cyanoborohydride or other suitable reducing agent.
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H~C~,N-CH2CH2scH2cH2cH2
NaCNBH 3\~
~H2NH-CH2CH2SCH2CH2CH2-0-
The secondary amine derivative of the S-layer
would be more stable to acid than the Schiff base
derivative.
The determination of the content of free
aldehyde groups in the polysaccharide portion, following
oxidation with periodate, is performed using
phenylhydrazine or 2,4-dinltrophenylhydrazine, or other
suitable reagents.
Suitable carbohydrate-containing S-layer~ are
oxidized with sodium metaperiodate as described in Example
1, sections A and B. Iodine-containing salts are removed
by dialysis against water. Subsequently, a ~olution of
the corresponding hydrazine reagent in 10% acetic acid is
added and the mixture is allowed to react for 1 hour.
Then the excess reagent is removed by dialysis and the
amount of hydrazone groups determined by colorimetry.
This method can also be applied to determine residual free
aldehyde groups after binding of a hapten-containing amino
groups, or of the immunoactive substances.
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O~f, N + 0 2N ~ NH-NH 2
N02
s
H~C~N-NH ~ N02
02N
The pharmaceutical structures constituting the
embodiment of the present invention are particularly
suitable as immunizing antigens for achieving high
antibody titres and protective isotypes. When antibodies
are used as immunoactive substances, anti-idiotypic
antibodies may be prepared by this method. Furthermore,
j the pharmaceutical structures can be used to advantage for
primary immunization and boosting when one and the same
immunoactive substance i8 bound to S-layer proteins or
glycoproteins derived from two different strains. The
I structures are applicable also as immune sorbents or
affinity matrice~, e.g., for diagnostic kits or
extracorporeal depletion of undesirable antibodies from
human blood.
While the invention has been described with
reference to the above embodiments, it will be understood
that its scope is defined by the following claims.
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