CA1326819C - Pharmaceutical structure - Google Patents

Abstract

Abstract The invention concerns a pharmaceutical structure wherein immunoactive substances e.g., haptens or immunogenic (immunostimulatory) substances are bound to a protein carrier. To achieve exactly definable binding sites for the immunoactive substances, the protein carrier is formed from protein or protein-containing molecules assembled in crystalline or para-crystalline assays which may be cross-linked if so desired.

Description

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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_3_ 1326819 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 , . . . . . .. .

-4_ 1326819 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 .. . . . , -.. ~ . . .. .

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 *Trade Mark .. ~ .

_7_ 1 3268 1 9 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 .

~ ~ -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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-- -10- 132681q 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

1- 1 3268 1 q 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:

--12- 1 3268 1 q 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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, -14- 1 3268 1 q 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
~Lo O~CO~

20C_~ON ~ NscN2cN2NN2 ~/

30 H ~ C~
~ H ~N-(CH2)2S(CH2)3 Na(CN)BH3 H ~ N-CH2CH2SCH2CH2CH' , ~: , ~ .. ..

. . . -- ~ - -1 32681 q 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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-17- l 3268 1 9 O~f, N + 0 2N ~ NH-NH 2 s H~C~N-NH ~ N02 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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Claims (21)

1. In a pharmaceutical structure wherein an immunoactive substance is bound to a protein carrier, the improvement comprising employing as the protein carrier protein or protein-containing molecules assembled in arrays selected from crystalline arrays, para-crystalline arrays, covalently cross-linked crystalline arrays, and covalently cross-linked para-crystalline arrays.

2. The pharmaceutical structure according to claim 1, wherein the protein carrier comprises glycoprotein molecules assembled into crystalline or para-crystalline arrays.

3. The pharmaceutical structure according to claim 2 wherein the immunoactive substance is bound to the carbohydrate portion of the glycoprotein.

4. The pharmaceutical structure according to claim 1, wherein the protein carrier comprises crystalline arrays of proteins or protein-containing molecules derived from microbial cell wall layers.

5. The pharmaceutical structure according to claim 4, wherein the proteins or protein-containing molecules derived from microbial cell wall layers function as binding sites for the immunoactive substance.

6. The pharmaceutical structure according to claim 1, wherein the protein carrier additionally comprises cell wall constituents attached to a crystalline or para-crystalline array protein carrier.

7. The pharmaceutical structure according to claim 6, wherein the cell wall constituents function as binding sites for the immunoactive substance.

8. The pharmaceutical structure according to claim 1, wherein the protein carrier comprises protein-containing molecules and wherein the immunoactive substance is bound to the protein portion of the protein-containing.

9. The pharmaceutical structure according to claim 1 wherein the immunoactive substance is bound to the protein carrier through a bridging molecule.

10. The pharmaceutical structure according to claim 9 wherein the bridging molecule is selected from the group consisting of homo- and heterobifunctional cross-linking agents and peptide chains.

11. A pharmaceutical composition comprising an admixture of two different pharmaceutical structures according to claim 1.

12. A method for preparing a pharmaceutical structure having an immunoactive substance bound to a protein carrier comprising protein or protein-containing molecules assembled in arrays selected from crystalline arrays, para-crystalline arrays, covalently cross-linked crystalline arrays, and covalently cross-linked para-crystalline arrays, said method comprising the step of activating the binding sites on the protein or protein-containing molecules prior to binding the immunoactive substance to said binding sites.

13. The method according to claim 12 wherein the protein carrier comprises a glycoprotein and wherein the binding sites are located within the carbohydrate portion of the glycoprotein and said step of activating includes oxidation at said binding sites.

14. The method according to claim 12 wherein the protein or protein-containing molecules are assembled in arrays selected from covalently cross-linked crystalline arrays and covalently cross-linked para-crystalline arrays and the binding of the immunoactive substances to the protein or protein-containing molecules is effected by means of the same agent that effects the cross-linking of the crystalline arrays or para-crystalline arrays.

15. The method according to claim 12 additionally comprising the step of forming binding sites by introducing active groups into the protein carrier.

16. The method according to claim 12 wherein the immunoactive substance is bound through an amide bond formed by an amine on the immunoactive substance and a carboxyl group of the protein or protein-containing molecule.

17. The method of claim 12 wherein the binding of the immunoactive substance includes a step in which a Schiff base is formed.

18. A method for preparing a pharmaceutical structure having an immunoactive substance bound to a protein carrier comprising protein or protein-containing molecules assembled in arrays selected from crystalline arrays, para-crystalline arrays, covalently cross-linked crystalline arrays, and covalently cross-linked para-crystalline arrays, said method comprising the step of activating binding sites on the immunoactive substance prior to binding the immunoactive substance to said protein carrier.

19. The method according to claim 18 wherein said binding sites are aldehyde functions and wherein said aldehyde functions are bound to amine groups on said protein carrier.

20. A method for preparing a pharmaceutical structure having an immunoactive substance bound via a bridging molecule selected from the group consisting of homo- and heterobifunctional cross-linking agents and peptide chains to a protein carrier comprising protein or protein-containing molecules assembled in arrays selected from crystalline arrays, para-crystalline arrays, covalently cross-linked crystalline arrays, and covalently cross-linked para-crystalline arrays, said method comprising the step of activating binding sites on the bridging molecule prior to binding the immunoactive substance to said protein carrier.

21. A method for preparing an admixture of two or more pharmaceutical structures each having an -immunoactive substance bound to a protein carrier comprising protein or protein-containing molecules assembled in arrays selected from crystalline arrays, para-crystalline arrays, covalently cross-linked crystalline arrays, and covalently cross-linked para-crystalline arrays, said method comprising the step of assembling the protein or protein containing molecules on an auxiliary layer which is removed following binding of the immunoactive substances.