AU627154B2 - Pharmaceutical structure - Google Patents

Abstract

In the pharmaceutical structure haptens and/or immunogenic or immunostimulant substances are bound to a protein carrier. In order to obtain accurately definable binding sites of the haptens and/or immunogenic or immunostimulant substances on the protein carrier, the latter is formed by protein molecules or protein-containing molecules which are, where appropriate, covalently crosslinked with one another and arranged in a crystalline or paracrystalline manner. <IMAGE>

Description

S, 627154 CO14ONWEALTH OF AUSTRALIA FORM PATENTS ACT 1952 COMPLETE SPECIFICATION FOR OFFICE USE: Class Int.Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: o..Priority: .,,,.Related Art: 9 9 Name of Applicant: UWE B. SLEYTR ."*Address of Applicant: Parhamer Platz 10, A-1170 Vienna, Austria S*,Actual Inventor: Uwe B. Sleytr Address for Service: SHELSTON WATERS, 55 Clarence Street, Sydney .Complete Specification for the Invention entitled: b "PHARMACEUTICAL STRUCTURE" S The following statement is a full description of this invention, including the best method of performing it known to me:- 1 la SPharmaceutical Structure The invention concerns a pharmaceutical structure, in which haptens and/or immunostimulatory substances are attached to a protein carrier.

Attempts had been made previously to bind haptens and/or immunostimulatory substances to proteins and thereby to increase the immnunogenicity or activity of such haptens and/or immunostimulatory substances. In the cases of such known formulations, the protein molecules are present as monomers in solution or dispersed as unstructured aggregates. The binding sites of haptens and/or immunostimulatory substances can, in these known formulations differ considerably with respect to their nature, kind, number, and position within the protein molecules, so that a chemically well-defined linkage of t o haptens and/or immuno- stimulatory substances to the V protein molecules cannot be achieved.

The invention is based on the problem of creating a wherein a precisely defined number of binding s 4 tes is 4 *available for attachment of a precisely defined amount of 9.9 haptens and/or immunostimulatory substances in a chemically defined manner.

In the context of the present invention, this problem containing molecules. These aggregates may also be covalently cross-linked. By virtue of the crystalline or

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44 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 bound haptens and/or immunostimulatory substances and the distances between the binding sites of haptens and/or immunostimulatory substances are always precisely defined.

Furthermore, 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, macrophages, of the haptens and/or immunostimulatory substances is considerably enhanced. Consequently, a more efficient immune response is achieved.

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 20 derived from one or several microbial cell wall layers.

Thus the glycoproteins are obtained in an especially simple manner. Such suitable protein aggregates 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 haptens and/or immunostimulatory substances. Particularly suitable as sources of this type of immunogenic carrier are those a microorganisms which, aside from the crystalline surface

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-3layer proteins, contain underneath additional rigid layers such as those composed of peptidoglycan or pseudo-murein.

Suitable carrier aggregates may contain the haptens and/or immunostimulatory substances linked to the protein portion of the carrier(s). For other haptens, it may be advantageous to have them linked to the carbohydrate portions of the glycoproteins (glycoprotein glycans). The choice of these two modes of attachment will depend both upon the nature of the haptens and/or immunostimulatory substances and upon the type of application of the pharmaceutical structure. Under certain circumstances, a mixed mode of attachment can be advantageous.

Furthermore, the haptens and/,r immunostimulatory substances can be attached to the respective carrier 15 molecules by way of bridging molecules such as homo- or oooo heterobifunctional cross-linking agents or peptide chains o polylysine). The introduction of such spacers or!; bridging molecules offers the advantage of more precise 2" control of the release of haptens etc, and of the nature 20 of such fragments as would form by enzyme-catalyzed degradation within the endosomes (lysosomes) of j 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 haptens, etc, may be combined.

Thereby differential functions of the pharmaceutical Sstructure are achieved. Thus, strongly hydrophobic i -Al.-r 4 99 9I 9 9*99 0' 9 9s 9 99 9 99 9 *9 -9 carrier molecules can be mixed with those aggregates carrying the haptens and/or immunostimulatory substances causing increased uptake of the pharmaceutical structure by phagocytosing cells. Furthermore, carrier aggregates comprising different haptens etc, can be used whereby the profile of activity of the pharmaceutical structure can be precisely controlled.

By an advantageous process for the production of the invented pharmaceutical structure, such groups on the protein molecules or protein-containing molecules as would bind the haptens and/or immunostimulatory substances may be activated prior to attaching the haptens and/or immunostimulatory substances. Thereby a reliably precise and reproducibly stable attachment of haptens etc. to the 15 respective groups is safeguarded.

For attaching haptens and/or immunostimulatory substances to carbohydrate portions, binding sites within the glycoprotein glycans may be generated by oxidation, using periodate. Binding sites on the protein 20 molecules can also be generated by reacting with glutaraldehyde, the preferred reagent used for cross-linking of the protein molecules, thus combining the steps of cross linking and activation. Formation 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 S linkages to the carboxyl groups of the protein carrier.

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5 .1 o 6 00* o 0 0 o o d e 0 0ooe 0 00 *9 a r o 90 0 9 0 oa 09* 0 r it *000 o 00 0 0 00 0 o 9 0 9 For certain active binding sites, the attachment of the haptens etc. can occur in the form of Schiff's bases. The Schiff's 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 haptens and/or immunostimulatory substances. In this manner, so-called "spacers" are introduced into the pharmaceutical structure.

Finally, different carrier aggregates, or carrier 15 aggregates comprising different haptens and/or immunostimulatory 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 20 combine different types of carrier molecules 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 sites on the haptens and/or immunostimulatory substances can be activated and the haptens and/or immunostimulatory substances attached by means of these activated binding sites, to the protein or glycoprotein carriers. This, too, results in especially stable linkages.

i ii -6- In the following, the invention is explained using examples.

Example 1 A. Preparation of the Carrier Cells of Clostridium thermohydrosulfuricum Llll-69 are suspended in 50mM Tris, HCI buffer, pH 7.2 and sonicated briefly (ca 1 min). Following the addition of a 2% solution of Triton X-100 (12.5mL) the suspension is incubated at 50°C for 15 minutes. By this treatment the cytoplasm and plasma membrane of the organisms is disintegrated whereas the crystalline or paracrystalline protein-containing cell wall layer (henceforth termed "S-layer") and the underlying peptidoglycan layer are S. conserved as fragments. Subsequently, the mixture is 15 centrifugated at 20.000xg and the pellets washed three times to remove detergent. The pellets are then suspended S° in 5mM magnesium chloride solution (25mL). For removal of 9* 4« Scytoplasmic residues and nucleic acids, DNAse (125ug) and S.o: RNAse (500ug) are added and the whole stirred for 4 20 minutes at 37 0 C. The suspension is then centrifuged at |L .20.000xg and washed three times with water. The pellet is then suspended in 0.1M cacodylate buffer, (pH 7.2; r* and a 50% aqueous solution of glutaraldehyde is added at 4 9* 4 0 C to a final concentration of The suspension is well stirred at 4°C for a few minutes, centrifuged, and washed with water. The pellet is then suspended in water and tris-hydroxymethylaminomethane ("Tris") added.

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7 4 4 f4 4 I 4t 4' I i 4( 4 t (rl 4 44 4* ta 4444 44€ t I r rr t cc t i 4 i f 4 t I I t t Following 10 minutes standing at room temperature, the suspension is again centrifuged (20.000 xg) 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 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 loaded with haptens and/or immunostimulatory substances. Should the presence 15 of the peptidoglycan be undesirable, the latter can be degraded with a peptidoglycan-degreding enzyme, e.g.

lysozyme, and removed.

To this end the material produced as under A is treated for 1 h at 36 0 C with a solution of lysozyme 20 lysozyme per mL of a 50mM solution of Tris. HC1 buffer, pH In this case, 1OmL of lysozyme solution are added per 0.5g 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 viz, the crystalline or paracrystalline S-layer is preserved intact.

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e 1- II jl:" :i 'i Y i 1 r i r r:::i 8- B. Formation of bindin sites The pellet prepared according to A is suspended in water (5mL) and a 0.1M solution of sodium periodate added. The suspension is allowed to stand for 24 h with exclusion of light. Subsequently, the suspension is centrifuged and the pellet washed with 10mM sodium chloride solution, to remove the iodine-containing salts.

C. Binding of Proteins to the Modified S-lavers 0 00 0t 000f 0 0o0 o e 15 00000 o 0 #0W 0 o 0 0 0 0 2 0«o a00 ee« 4 0 o* 0 2 2 (obtained) The pellet obtained according to B (ca. 0.2g) is suspended in water (ImL) and the suspension is mixed with a solution (ImL) of bovine serum albumin (50mg) in water (10mL). 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 750nm 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 PV 72 (2.5g) are suspended in 50mM Tris.HCl buffer pH 7.2 and sonicated for ca. 1 minute. Following addition of 2% Triton X-100 (12.5mL) the suspension is incubated for 15 min at 50 0

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By means of this treatment, the cytoplasm, of the cells is

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54 5 disintegrated while the S-layer and the peptidoglycan layer are preserved. Thus fragments are formed which correspond in shape more or less to the original shape of the bacterial cell (so called "ghosts").

Subsequently, the suspension is centrifuged at 20.000xg and the pellet washed three times with water to remove the detergent. The pellet is then suspended in magnesium chloride solution (25mL), DNAse (deoxyribonuclease, 125ug) and RNAse (ribonuclease, 500ug) are added, and the mixture is stirred at 37 0 C for 15 minutes.

Subsequently, the pellet is washed three times with water, centrifugation in between being at 20.000xg. The pellet is then suspended in 0.1M cacodylate buffer (pH 7.2) and the suspension mixed with a 50% solution of glutaraldehyde in water at 4°C to a final concentration of The suspension is then vigorously stirred at 4°C for a few minutes, centrifuged, and the pellet washed with water.

As those glutaraldehyde residues linked through only one aldehyde function will serve as free binding sites, it is 20 not necessary in this case to create special binding sites as has been achieved by oxidation under example l.B.

B Binding of Protein(s) to the Modified S-layers The modified S-layers prepared as above are mixed with a solution of bovine serum albumin as in example 1C, and the amount of protein bound is determined as described there.

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(1 ij if :I t i i ii 10 Example 3 A Preparation of the Carrier Cell walls of Clostridium thermohydrosulfuricum Llll-69 are treated with glutaraldehyde in O.1M sodium cacodylate buffer, pH 7.2) for 20 min at 20 0 C, so as to stabilize the outermost cell wall layer (S-layer).

The reaction is terminated by the addition of excess ethanolamine. During cross-linking the cell wall fragments may be either in suspension or attached to a porous surface (S-layer ultrafiltration membrane). The cell wall fragments are then washed with distilled water to remove the reagent mixture.

B Creating Binding Sites for Ligands Containing Thiol e" (SH) Groups 15 The pellet of a cross-linked preparation as under A above, is suspended in distilled water (30mL) and to the

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suspension is added l-Ethyl-3,3(dimethylaminopropyl) t carbodiimide (EDC; 60mg) maintaining a pH of 4.75. This step activates the exposed carboxyl groups of the *9*I 20 S-layer. Subsequently, an excess of hexamethylenediamine is added to the pH kept at 8.0 for 60 minutes.

Subsequently, the reaction is terminated by addition of acetic acid. The suspension is centrifuged at 20.000 xg and the pellet washed three times with distilled water.

The wet pellet (100mg) is suspended in 50mM phosphate buffer, pH 7 (9mL) and a solution of meta maleimido- Ii"; Aj benzoyl-N-hydroxysuccininide ester (50mg per rnL of tetrahydrofuran; lmL) is added, The mixture is then incubated for 30 minutes at 200C.

C. Binding of SH-containing aroteins to the S-lavers derivatized as under B Following centrifugation at 20.000 xg, the pellet is suspended in 50mM phosphate buffer (pH f-galacto- sidase (20mg) is added and the whole incubated for 2 h at 20 0 C. After centrifugation at 20.000 xg and repeated washing with phosphate buffer, the activity of the 1-galactosidase covalently linked to the protein matrix is determined.

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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 Generatinc the Binding Sites The cell wall fragments (100mg) are suspended in anhydrous tetrahydrofuran (THF), incubated at 200 for min, centrifuged at 20.000 xg and suspended again in a solution of cyanogen bromide in anhydrous tetrahydrofuran (10mL). Following incubation for 2 h, the cell wall fragments are separated by centrifugation at 20.000 xg and washed with THF for removal of residual reagent.

C. Binding of proteins to the Derivatized S-layer The pellet is suspended in 50mM phosphate buffer pH 8.0 (10mL) containing invertase (20mg) and incubated for 18 h at 4°C. Following centrifugation at 20.000 xg the 20 pellet is washed twice with phosphate buffer and the enzyme activity of the invertase bound to the protein matrix determined.

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B Generating the binding sites *0 t 151 o#4 Cell wall fragments (0.1g) are suspended in anhydrous dimethyformamide (DMF, 2OmL) and EDC (60mg) and N-hydroxysuccinimide (0,5g) are added to the suspension. Following incubation for 1 h, the suxspension is centrifuged at ZO.O0O xg and washed twice with DMF.

C Bindincr of iproteins to the S-layer thus modified The pellet obtained as under 5B is suspended in O.1M sodium hydrogencarbonate (pH 8.8) containing dissolved dextranase (20mg) and the reaction mixture is incubated at 4 0 C for 18 h. The cell wall fragments containiing 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.

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0 14 Example 6.

Coupling of a synthetic carbohydrate antigen to oxidized S-layers.

A Prep~aration of the Carrier B Generating the binding sites The preparation of the oxidized glycoprotein S-layers was performed as described in Example 1, sections A and B.

C Bindina ot the Carbohydrate Antigen to the Carrier The oxidized (polyaldehyde) derivative of the S-layer is incubated with the 3-(2-aminoethyl) thiopropyl glycoside of a disaccharide whereby Schiff's base formation occurs. This step can also be performed with any other saccharide attached to 'an aglycone that contain's amlno groups.

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*4 t4 4 General recipe for the preparation of 3-(2-aminoethylthio)propyl glycosides from allyl glycosides.

A solution of the allyl glycoside (5mM) in a solution of cysteamine hydrochloride (15 milliequivalents of SHgroups in 10mL) is allowed to stand for 1.5 h at room temperature. The duration of this reaction may vary. The reaction mixture is subsequently separated over a column of cation exchange resin Rexyn 101, ammonium form, 200-400 mesh). The column is eluted with water, ammonia, and 1.OM ammonia. Unreacted allyl glycoside appears in the aqueous eluate, and the 3-(2-aminoethylthio)propyl glycoside is eluted in the fraction corresponding to 1.OM ammonia. Those fractions containing 15 products are subsequently evaporated to dryness.

The Schiff's 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.

These can be assayed directly if they are labelled with ferritin horseradish peroxidase, 125I or in any other appropriate manner. The bound antibodies can also be assayed via a so-called "sandwich" method by binding of labelled antibodies directed against the first, hapten-bound antibodies.

The Schiff's base derivative of the S-layers as obtained by binding of the 3-(2-aminoethylthio)propyl glycoside may be converted into a secondary amine A4 T c i_.

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The determination of the content of free aldehyde groups in the polysaccharide portion, following oxidation with periodate, is performed using phenylhydrazine or 2,4-dinitrophenylhydrazine, or other suitable reagents.

20 Suitable, carbohydrate-containing S-layers 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 solution of the corresponding hydrazine reagent in 10% acetic acid is added and the mixture is allowed to react for 1 h.

Subsequently, 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 immunostimulatory substances.

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N02N2 2 antibody titres and protective isotypes. When antibodies are used as immunogenic substances, anti-idiotypic antibodies may be prepared by this method. Furthermore, the pharmaceutical structures can be used to advantage for embodiment of the present invention are particularly primary immunization and boosting when one and the same haptens and/or immunostimulatory substance is bound to S-layer proteins or glycoproteins derived from two different strains. The structures are applicable also as immune sorbents or affinity matrices, e.g, for diagnostic kits or extracorporeal depletion of undesirable antibodies from human blood.

Claims (1)

1. 944.rl 44- 4I 9 9 90 C19. 4 9* 49, 4. 4, 9 94 44. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. Pharmaceutical structure wherein haptens and/or immunostimulatory substances are bound to a protein carrier wherein the protein carrier consists of protein or protein-containing molecules assembled in crystalline or para-crystalline arrays which may be covalently cross-linked. 2. Pharmaceutical structure according to Claim 1, wherein the protein-containing molecules assembled into crystalline or para-crystalline arrays are glycoproteins. 3. Pharmaceutical structure according to Claims 1 or 2, wherein the molecules forming the crystalline arrays are derived from one or more microbial cell wall layers. 4. Pharmaceutical structure according to one of Claims 1 through 3, wherein additional cell wall constituents are attached to the crystalline or para-crystalline protein carrier. Pharmaceutical structure according to Claims 3 or 4, wherein fragments of microbial cell walls function as carriers of the haptens and/or immunostimulatory substances. 6. Pharmaceutical structure according to one of the Claims 1 through 5, wherein the haptens and/or immunostimulatory substances are bound to the protein portion of the carrier. 19 7. Pharmaceutical structure according to one of the Claims 2 through 5, wherein the haptens and/or immunostimulatory substances are bound to the carbohydrate portion of the glycoproteins. 8. Pharmaceutical structure according to Claim 6 or 7, wherein the haptens and/or immunostimulatory substances are linked to the respective carrier by means of bridging molecules such as homo- or heterobifunctional cross-linking agents or peptide chains polylysine). 9. Pharmaceutical structure according to one of Claims 1 though 8, wherein different carriers and/or carriers bound to different haptens and/or immunostimulatory substances, are combined. 0 M 10. Process for preparation of a pharmaceutical structure according to one of Claims 1 through 9, wherein those 9 groups of the protein- or protein-containing molecules, that would bind the haptens and/or immunostimulatory i 5 substances, are activated prior to the addition of the Shaptens and/or immunostimulatory substances. 11. Process according to Claim 10, wherein binding sites within the carbohydrate portions of the glycoproteins are created by oxidation, e.g. using periodate. 12. Process according to Claim 10, wherein binding of haptens and/or immunostimulatory substances to the protein molecules is effected by glutaraldehyde, I i 20 13. Process according to Claim 12, wherein formation of binding sites is effected by the introduction of active groups. 14. Process according to Claim 10, wherein the haptens and/or immunostimulatory substances are bound to carboxyl groups of the protein (or protein containing carrier) in amide linkages. Process according to Claim 11, wherein the binding of the haptens and/or immunostimulatory substances occurs in the form of Schiff's bases, the Schiff's bases being reduced to secondary amines if desirable. 16. Process for preparing a pharmaceutical structure .according tc one of Claims 1 through 9, wherein binding sites (eg. aldehyde functions) are created within the haptens and/or immunostimulatory substance, the haptens and/or immunostimulatory substance, being bound by means of these activated groups to amino groups of the protein carrier. o 4 S17. Process for preparing a pharmaceutical structure o0 according to Claim 8, wherein such bridging molecules may possess activated binding sites at both ends (eg. homo- or heterobifunctional cross-linking agents) or peptide chains polylysine), are attached to the carrier by one end, whereupon the haptens and/or immunostimulatory substance is attached to the other end of the bridging molecule. i .r I f ik ;r ii ji ti~ c;i r ir: C;Ii 21 18. Process for preparing a pharmaceutical structure according to Claim 9, wherein different carriers and/or such carriers are containing different, bound haptens and/or immunostimulatory substances, are allowed to assembly together on an auxiliary layer which may be removed following cross linking to the carrier(s) if desired. DATED this 29th day of MAY, 1992 UWE B. SLEYTR Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS *C *I 0 S. 0i 0 0 0 4l *0 brO C 4 0O 4 4 to C S 9C* 4 IC I 0 0 V r 1 i--