DE19900709A1 - Composition for prophylaxis or therapy, particularly as an adjuvant for antitumor vaccines, comprises an active agent bound to an adsorbent - Google Patents

Abstract

A composition (A) for prophylactic or therapeutic use in humans or animals comprising an active substance (I) bound to an adsorbent (II), is new. Independent claims are also included for the following: (1) production of a vaccine by mixing (A), as an adjuvant, with an antigen preparation; and (2) preparing (A) by adsorbing a freshly prepared solution of at least one (I) on to aluminum hydroxide (IIa) or calcium phosphate (IIb), then either using the product directly or storing it cold.

Description

introduction

The development of molecular biology over the past decade has made it possible become biologically effective proteins of higher mammals in large quantities to produce in a recombinant form by genetic engineering. Especially proteins, that play a role in the interaction between cells of the immune system, such as B. the interleukins, chemokines, interferons and the colony stimulating factors (CSF), are therefore in the purest form and in pharma quality has become available. Today the application of recombi nant manufactured cytokines, such as IL-2, GM-CSF, G-CSF and the interferons, from the therapy of a number of human diseases to think. Other substances from these groups will be added to this list in the next years.

However, the use of these biologically active proteins is not a problem Come on. Most of them are intercellular messenger Molecules through which the cells of the immune system interact with each other, but also with other cells of the organism, such as endothelial cells and fibroblasts, communicate. For this reason, natural processes are usually not only one, but always several different ones from these groups of biolo gisch effective molecules in graded order and in suitable con concentrations mostly local, directly at the location of an event gene interaction, released by cells and by the same cells (autocrine) or from other nearby cells (juxtakrin) via specific receptors again recorded ("cytokine crosstalk"). This does not preclude some of these biologically active substances also hormone-like via remote effects (para act).

It is now known that lymphocytes are also receptors for messenger substances of the Nervous system, the so-called neurotransmitters, and from them if can be stimulated (Levite, 1998, Levite et al., 1998). So that will be Network of cellular interaction possibilities still expanded considerably.

The "artificial" application of such biologically active molecules, e.g. B. by intravenous, subcutaneous or intramuscular injection, which can be used with natural Do not imitate process reactions. Therefore, they are after them Applications observed effects not "natural", i.e. H. the biological Task of these molecules accordingly, but artificial and therefore can also have pathological effects. Although with some diseases also after systemic (intravenous) application of cytokines therapeuti cal effects can be achieved, such as. B. in systemic treatment treatment of carcinoma patients by intravenous or subcutaneous application  tion of IL-2 (e.g. Rosenberg et al., 1985) or of interferons is the systemi The application of most of these biologically active substances not only basically biologically nonsensical, but can also be dangerous for the patient his. The intravenous use of IL-2 can even lead to death (vasculary leak syndrome; Rosenberg et al., 1993). The real biological up gave this cytokine, the local intercellular communication between the cells len of the immune system, as well as with neighboring other cells of the Or ganism, cannot be achieved by systemic application. In the opposite part: in that the respective cytokine after systemic application in the organization ism is ubiquitous, the targeted interaction of immune cells stems with each other but also with other cells of the body (e.g. with En dothelial cells) made impossible. The result is that the immune system in certain this way becomes directionless.

Preparatory work

Based on these considerations, we have developed a method to Bring cytokine necrophee to local effect in tumor vaccines. At this Method for which under the number DE 44 11 425 (Falkenberg and Reimer, 1996) A German patent has been granted to cytokine molecules in depot bound form, mixed with inactivated tumor cells, applied locally. In preclinical animal experiments, vaccinations with Mi creations from incapable of division (irradiation with 200Gy) cells muri ner tumors and depot-linked cytokines, such as B. IL-2, IL-4, TL-12 and GM-CSF to induce permanent immunity to these tumors in mice. In some model tumors, vital tumor cells even managed to survive for a few days had been administered in a lethal dose prior to vaccination and to heal the animals from the tumor forever. Part of this Results in which liposomes were used as a depot form are published light (Falkenberg and Reimer, 1995; Krup and Falkenberg, 1997; Krup et al., 1999).

About the cellular and molecular processes taking place in this therapeutic approach Processes can only be speculated for the time being. The results suggest that it is predominantly local processes, although, as late systemic effects also play a role. We can ten clearly show that the efficacy of cytokine depot tumor vaccines not only, as expected, from the antigen (tumor cell) dose, but above all lem also depended on the dose of cytokine. That's how we examined it Cytokine depot tumor vaccines below a lower cytokine threshold dose not effective. When using IL-2 we could clearly demonstrate that it in addition to a lower threshold dose there is also an upper limit dose above whose IL-2 depot tumor vaccines are not effective. In other words: IL-2 only works in experiments with mice in IL-2 depot tumor vaccines  Apply an optimal dose within a very narrow dose range sters.

The existence of a dose window for IL-2 was confirmed by Schmidt et al. (Schmidt et al., 1995) also with the help of cytokine gene-transfected tumor cells with under different degrees of transfection and resulting different Cytokine secretion levels are shown.

With IL-2, we were able to verify the presence of a dose window when using evidence of different forms of deposit. For other cytokines appear after the available results also exist dose windows.

It is precisely because of this dose dependency that the advantage of that we develop cytokine depot tumor vaccines clearly: In contrast to the many today applied Tu consisting of cytokine gene transfected tumor cells morvakzinen, where the cytokine secretion rate is purely random depends on the transfection process, it is with the Tu we use Morvakzinen possible to fix the dose of antigen and cytokine as needed that the cytokine secretion rate for the effect of the transfected tumor cell len containing tumor vaccines, Si mons et al. (Simons et al., 1997) in a clinical study with GM-CSF gene show transfected renal carcinoma cells. Only the patient could objective improvement of the condition (reduction of lung metastases) are detected, whose tumor cells have the highest GM-CSF secretion rate had.

In the studies on the development of cytokine depot tumor vaccines we also have the effectiveness of various other forms of custody in this Vaccination approach examined.

Next

• IL-2 directly covalently bound to tumor cells,
• - IL-2 directly adsorptively bound to latex particles
• - Indirectly via cytokine antibodies to latex beads-bound IL-2 (Krup et al., 1999) and
• - Liposome-encapsulated IL-2 (Krup and Falkenberg, 1997) we have above all
• - Cytokines adsorbed on inorganic adsorbents

used as cytokine depot formulations, so-called "adsorbate cytokines".

Aluminum hydroxide has proven to be the optimal adsorbent. Calci Umphosphate showed a similar but somewhat weaker effect. Because of their good These two inorganic adsorbents are the only ones that are compatible Application in vaccines approved and for decades worldwide in milli Arden of doses of adsorbate vaccines can be used. Thereby prevails in the professional opinion that aluminum hydroxide is a particularly for the Induction of humoral (antibody) immune responses suitable adjuvant, which is suitable for the induction of cellular, also tumor-specific cytotoxic, Immune responses are not suitable. It cannot be ruled out that tumor  specific antibodies "cover" surface structures of tumor cells (mask) and that defense cells can be prevented from doing so Recognize and eliminate tumor cells. That aluminum hydroxide as ad juvans for the induction of cellular cytotoxic tumor-specific Im is not suitable, is also confirmed in the relevant literature (Souberbielle et al., 1996).

Because of the simplicity of manufacture and handling, we have above all lem on the development of cytokine depot tumor vaccines using formation of aluminum hydroxide adsorbed cytokines (adsorbate cytokines) concentrated. It has been shown that the local release of cytokines the adsorbate cytokines optimally meet the requirements for use in Cytokine-depot tumor vaccines appear to meet. As in control Investigations with mixtures of irradiated tumor cells and aluminum umhydroxid (without adsorption of IL-2) has clearly shown the aluminum minium hydroxide particles themselves make no or only a very small contribution to Induction of the observed cellular cytotoxic tumor-specific Im response.

The effects we observed are therefore not based on a possible adjuvant Effect of the aluminum hydroxide admixed to the tumor cells, but clearly on the cytokine and its delayed release from the aluminum Umhydroxid- or calcium phosphate cytokine adsorbate (depot effect) back respectively.

Description of manufacture and application of aluminum hydro xid-adsorbed biologically active substances

We also have those in the studies with adsorbate IL-2 tumor vaccines Lymphoid organs from vaccinated animals were examined and found that the local subcutaneous application of adsorbate-IL-2-containing tumor vaccines to egg extremely large increase in the size of the spleens (splenomegaly) of the behan delten animals. The cytokines applied in the form of adsorbates act al not only locally, but also have to have a long-range effect (paracrine). The The size of the spleens and the number of lymphocytes contained therein increased up to 300% too (Example 1).

example 1 Enlargement of the spleen after vaccination with tumor vaccines containing adsorbate-IL-2

To the effect of adsorbate-IL-2 on the enlargement of the spleen and the expansion of the ent to study lymphocyte populations, mice were treated with tumor vaccines, which consisted of irradiated tumor cells and adsorbate rhuIL-2.  

Test procedure

BALB / c strain mice were treated by subcutaneous injection of 10 ⁶ irradiated (200 Gy) RenCa cells mixed with 10 µg rhuIL-2 adsorbed on aluminum hydroxide. Four vaccinated animals were sacrificed 3 (animal 3 / 1-3 / 4) and 6 (animal 6 / 1-6 / 4) days after vaccination, plus four animals (animal K / 1-K / 4) one untreated Control group.

The spleens were removed, cell suspensions prepared and the number of cells determined. Diagram 1 shows the number of cells obtained per spleen.

The results shown in diagram 1 show a time-dependent course of the increase in Spleen size: after three days, the average number of spleen cells per spleen increased to 217%, increased to 262% after six days.

It is known that a certain enlargement of the spleen is also conventional len immunizations can be observed. So z. B. the Mil of mice after intravenous immunization with sheep erythrocytes up to 50% (Wortis et al., 1966). Even after immunization with protein Antigens and common adjuvants, such as. B. Complete Freund's Adjuvant (CFA), spleen enlargements occur on the order of a few 10% on. In no case do they reach the extent we do with those with cyto kin-depot tumor vaccine-treated mice have been observed (some 100%). Observe after intraperitoneal application of IL-2 adsorbate tumor vaccines we saw an increase in the number of accumulated in peritoneal exudate ten cells (PEC) by a factor of 5 (Example 2).

Example 2 Increase in the peritoneal exudate cell population after intraperitoneal vaccination with Ad tumor vaccines containing sorbate-IL-2

To determine the effect of adsorbate IL-2 tumor vaccines on the composition of the peritoneal To study exudate cell (PEC) population, mice of the BALB / c strain were intraperito neal tumor vaccines applied, which consisted of irradiated tumor cells and adsorbate IL-2.

Test procedure

The mice in the test group were treated by intraperitoneal injection of 10 ⁶ irradiated (200 Gy) RenCa cells mixed with 10 μg rhuIL-2 adsorbed on aluminum hydroxide. The mice in the control group were injected intraperitoneally with cell culture medium. The animals were sacrificed 7 days after the application, the PEC was obtained by lavage the peritoneum with heparin-containing medium and the number of cells in the suspensions was determined.

To determine the number of cells in the seed characterized by lymphocyte markers Aliquots of each cell suspension with lymphocyte marker-specific monoclonal samples were zen antibodies incubated. Binding of the lymphocyte marker-specific monoclonal antibodies on the cells was indirectly detected by fluorescein-labeled second antibodies. The An Parts of the individual cell types in the total population in percent was fluorescence-activated Cell sorter (FACS) determined.  

Table 1

Table 1 shows the proportion of cells carrying the lymphocyte markers characterized by lymphocyte marker-specific monoclonal antibodies in the spleens of the animals (data in%) and the yield of PEC (data in 10 ⁶ cells). Each test group consisted of three animals (medium control K / 1-K / 3, vaccination group V / 1-V / 3). The mean values are in the columns K / tot. or V / tot. listed.

In diagram 2 are the mean values of the percentages for the different lympho zytenmarker positive PEC of the animals of the medium group and the vaccination group next to each other the shown.

The results shown in Table 1 and Diagram 2 show that the yield of PEC of the animals treated with vaccines is on average more than four times as high as the yield of PEC of the animals treated with medium. It can be seen in detail that despite this increase, the percentage of most lymphocyte subpopulations has remained almost constant. It is striking that the percentage of Thy-1 ⁺ lymphocytes increased by 26% (very significant, P = 0.0020). It can be assumed that this increase in Thy-1 ⁺ cells is due to the effect of the IL-2 applied in aluminum hydroxide-adsorbed form.

We found completely unexpectedly in control examinations, in which we found the Adsorbate cytokines alone, i.e. without the addition of tumor cells, subcutaneously had applied tan, in the animals also greatly enlarged spleens. The Ver So enlargement of the spleens does not appear or not only on the actual Im immunization with the antigen (tumor cell) adsorbate-cytokine mixture to be traceable, but is obviously based on a general im munstimulierendem and the immune system expanding effect of the Ad sorbate-IL-2. Accordingly, adsorbate-IL-2 is not only "a new type of adjuvant for the Induction of cytotoxic immune responses against tumor cells ". The observer Rather, indications suggest that it is in the case of adsorbate-bound cyto kinen ("adsorbate cytokines") is a completely new type of biologically active men substances that have different properties than in free form ap duplicated cytokines. Even their local, natural mode of action of this sub punch very similar, application promotes at the same time completely new and so far not yet described the expansion of the cellular immune system and thereby improves and modulates the immune status of the treated individual to.

According to the results and observations available to us, several processes run in parallel after the application of cytokine depot tumor vaccines:

• 1. By local release of cytokines from the vaccine inoculate, directly The neighbors to the antigen, the tumor cells, become lymphocytes lured, which secrete cytokines themselves, thereby other lymphocytes attract and thus an effective cellular cytotoxic tumor-specific Im start the munesponse.
• 2. Due to the systemic release of the cyto taking place at the same time kins will, regardless of the presence of the antigen, the observed ex Treme expansion of the immune system induces splenomegaly and, possibly, leukocytosis.
• 3. It can be assumed that the second partial reaction, the expansion of the Im munsystems through the systemic effect of the cytokine, then also influence to the local reaction: that resulting from the expansion of the repertoire of the cellular immune system will become available lymphocytes participate locally in the induction of the immune response and possibly se may even be the cause of the success of the vaccination.

The Hanada group recently described an observation that in many respects coincides with and confirms the observation we have described. Hanada et al. (Hanada et al., 1996) subcutaneously injected C57BL / 6 mice with GM-CSF gene-transfected vital B16 tumor cells (B16-GM-CSF) in order to recruit the dendritic cells so important for the induction of immune reactions in the animals Stimulate cells. After the injection, the tumor cells came on, multiplied, formed large tumors, the cells of which produced GM-CSF in such quantities that in the final stage, with a tumor size of approximately 2.5 cm ³ , up to 7.8 in the peripheral blood of the animals ng / ml GM-CSF could be detected. In the course of the experiments, extremely enlarged spleens (by a factor of 5) were observed in the animals treated in this way (splenoma galie). The number of dendrific cells contained in the spleens was increased more than 10-fold to 6-10 × 10 ⁶ cells per spleen (up to 5 × 10 ⁵ dendritic cells per spleen can be obtained from the spleens of untreated animals). Through several intraperitoneal injections of free GM-CSF (three injections per day for 6 days), Metcalf et al. (Metcalf et al., 1987), however, only induce moderate splenomegaly (50% enlargement of the spleen). According to Hanada et al. The cause of the effect of the GM-CSF gene transfected tumor cells lies in the depot effect of the gene transfected cells: "...... GM-CSF-transduced B16 tumor was transplanted subcutaneously, as a conti nuous source of cytokine.".

In view of the strong increase in spleen size by a factor of 5 observed by these authors, corresponding to an increase in the spleen cell population from approximately 100 to approximately 500 million cells, the increase in the population of dendritic cells to 10 × 10 ⁶ cells observed in these experiments appears to be relatively insignificant per spleen. In order to achieve such a strong expansion of the spleen cell population, other cell populations contained in the spleen must have multiplied extremely. Hanada et al. have not examined this in more detail. However, other authors have reported that leukocytosis can be induced by G-CSF and GM-CSF-secreting tumors (Katoh et al., 1993; Suzuki et al., 1993). Tani et al. reported that the injection of G-CSF gene-transfected fibroblasts in nude mice not only increased neutrophils increased by a factor of 30 to 280 × 10 ⁶ neutrophils per ml, splenic enlargements by a factor of 10 (from 100 to 960 mg), but also a strong increase in the progenitor cells of all spleen cell types could be observed (Tani et al., 1989).

These results clearly show that the unique local (subcutaneous) application of aluminum hydroxide adsorbed cytokines, as we have used in our work, has the same effect as

• 1. the repeated systemic or local application of free cytokines,
• 2. the local application of cytokine gene-transfected fibroblasts (Tani et al.) or
• 3. the local application of replication-capable cytokine gene transfected Tumor cells (Hanada et al.).

Repeated subcutaneous applications of free cytokines result in both Experimental animals as well as humans to splenomegaly (Ratcliffe et al., 1992). In Splenomegaly by Athanassakis et al. after treatment with gamma interferon can be detected (Athanassakis et al., 1995), by Khar et al. after treatment with tumor necrosis factor-alpha (Khar et al., 1993), by Killar et al. after treatment with IL-1 (Killar et al., 1989) by Micallef et al. to Treatment with IL-18 (Metcallef et al., 1997) by Mao et al. (Mao et al., 1994), Anderson et al. (Anderson et al., 1993) and Harada et al. (Harada et al., 1993) Treatment with IL-2.

In humans, significant splenic enlargements were repeated after repeated Application of free IL-2 detected (Pozniak et al., 1995). These authors have in patients for various (non-haematological) reasons had been treated by repeated subcutaneous injections of IL-2, so Leukocytosis and splenomegaly were observed and quantified. While many other authors also reported splenomegaly after application of IL-2 and other cytokines describe this phenomenon as pathological Consider side effects of cytokine therapy, interpret Pozniak et al. the This phenomenon as a positive immunostimulating effect ("... suggest that spleenic enlargement seen in association with IL-2 therapy reflects the general immune activation seen with this treatment.

The splenomegaly described in this work is therefore often obviously a general effect of the application of IL-2 and of their cytokines. This effect is referred to in all publications as "side effect cytokine therapy ". Of course, one corresponds to the factor 5 enlarged spleen not the normal physiological state of this organ. According to the results we have received, it is the spleen size tion after cytokine application, however, is not a "side effect of the cytokine  kin application ", but with a high probability around the main effect the cytokine application. Some enlargement of the spleen after immunization tion is a physiological, by the cells running during the immunization process related process. For spleen enlargements, like ours after application of adsorbate-IL-2 tumor vaccines or of adsorbate-IL-2 al leash, by Hanada et al. after application of GM-CSF gene transfected Tu morzellen, by Tani et al. after application of G-CSF gene-transfected Fibrobla most and by the other authors cited above after repeated systemi shear or subcutaneous application of various cytokines in free form, see above has been observed in both experimental animals and humans obviously a change caused by the unphysiological application on, or by applying cytokines in unphysiological doses is caused. The results obtained can only be interpreted so that the this certainly unphysiological condition also has an important positive effect, namely the increase in the potential of lymphoid cells.

Adsorbate cytokines are therefore a new application form of cytokines. With a single injection of adsorbate cytokines the same effect can be achieved as by repeated applications of free cyto cytokine molecules or secreting cytokine molecules Kin gene transfected normal or malignant cells. Through the The use of adsorbate cytokines is now possible by a single injection of an ad containing one or more cytokines sorbate to activate the immune system and the cellular immune potential of the to amplify the individual concerned to an extent that was not previously was possible.

The application of adsorbate cytokines can have at least two effects:

• 1. a local effect, which is accompanied by the local presence of cellular antigens for the induction of cytotoxic cellular immune responses leads.
• 2. a systemic effect that is independent of the local presence It is the antigen that stimulates the immune system and through cytokine moles kille is induced, the circulation from the adsorbate cytokine depot range, to an unspecific and almost explosive prophecy of lymphoid cells (leukocytosis, splenomegaly) and thereby general immune activation.

The results obtained so far do not allow a precise conclusion to be drawn that whether all cytokines work in the same way. It is also not entirely clear whether all cells of the immune system are replicated to what extent they enter the affects individual cell types and whether under the action of individual adsorbate cytokines certain cells of the immune system are preferentially reproduced. According to us ren and that of Hanada et al. and by Tani et al. results reported  the application of cytokines in depot form for general multiplication all or many subpopulations of lymphoid cells, with individual sub populations such as B. in Hanada's experiments the dendritic cells, be propagated particularly strongly.

The possibilities arising from the medical application of such adsorbate Cytokines have not yet been estimated. Especially from the Use of combinations of adsorbate cytokines with other biolo gisch effective substances in aluminum hydroxide adsorbed form application options, which are discussed below. Some We have investigated these possibilities in animal experiments.

Applications of adsorbate cytokines 1. To stimulate the humoral immune response by adsorbate cytokines

Since the injection of adsorbate cytokines is obviously the general verb immune status, it should also improve humoral Lead immune responses. Therefore, we examined the sera from animals using Adsorbate-IL-2 tumor vaccines (Example 3) or with adsorbate-GM-CSF- Tumor vaccines (Example 4) had been vaccinated. We were able to demonstrate that the sera also use antibodies against antigens in the vaccines ten tumor cells contained in those with adsorbate GM-CSF tumor vaccines treated animals higher than those treated with adsorbate-IL-2 wa ren.

Example 3 Induction of a humoral and tumor immune response after vaccination with tumor cells and Ad tumor vaccines containing sorbate-IL-2

To demonstrate the humoral anti-tumor immune response, the sera from mice were treated with adsor bat-IL-2 tumor vaccines had been vaccinated, obtained and analyzed in an indirect ELISA (cell ELISA) for their content of specific antibodies reacting with the tumor cells.

Test procedure

In the example shown here, mice from the inbred strain BALB / c were mixed with be emitted (200 Gy) RenCa tumor cells and rhuIL-2 in liposome-encapsulated form or rhuIL-2 vaccinated prophylactically in the form of adsorbate-IL-2. The animals in the control groups received either only irradiated (200 Gy) tumor cells or only cell culture medium.

Blood was drawn from the animals ten days after application of the vaccines. The blood Sera were collected in a cell ELISA with vital RenCa tumor cells as antigen on their Content of tumor cell-specific antibodies examined. 14 days after vaccination Animals were given a lethal dose of vital RenCa tumor cells.  

In the diagrams, both the antibody binding curves determined in the ELISA are the serum samples (diagram 3a) as well as the Kaplan-Meier survival curves of the test animals after the Challenge presented with vital RenCa tumor cells (diagram 3b).

From the results shown in diagram 3a and diagram 3b it can clearly be seen that the Antibody titers correlate with the course of the survival curves: the higher the antibody titers the more animals survive. It is also shown that rhuIL-2 adsorbed on aluminum hydroxide for the induction of both cellular cytotoxic and humoral tumor-specific Im responses is more suitable than liposome-encapsulated rhuIL-2.

Example 4 Induction of a humoral anti-tumor immune response after vaccination with Adsorbat-GM-CSF containing tumor vaccines

In order to demonstrate the humoral anti-tumor immune response, the sera from mice that were labeled with Ad sorbate-GM-CSF tumor vaccines had been vaccinated in an indirect ELISA (cell ELISA) examined for their content of specific antibodies reacting with the tumor cells.

Test procedure

In the example shown here, C57 BL / 6 mice were exposed to irradiated (200 Gy) B16BL6.D5- Tumor cells and different doses of adsorbate-rmuGM-CSF (depot form aluminum hydroxide) vaccinated prophylactically four times a week. The animals in the control groups either kept only irradiated tumor cells or only cell culture medium. 10 days after Appli cation of the fourth vaccine, blood was taken from the animals.

The sera obtained from this blood were analyzed in a cell ELISA with vital B16BL6.D5 Tumor cells as antigen examined for their content of tumor cell-specific antibodies. 14 days After application of the fourth vaccine, the animals received a lethal dose of vital B16BL6.D5- Tumor cells.

In the diagrams, both the antibody binding curves determined in the ELISA are the serum samples (diagram 4a) as well as the Kaplan-Meier survival curves of the test animals after the Challenge shown with vital B16BL6.D5 tumor cells (diagram 4b).

The results shown in Diagram 4a and Diagram 4b show that after vaccination with Tumor vaccines containing adsorbate-rmuGM-CSF contain the humoral tumor-specific immune response depends very much on the rmuGM-CSF dose and not on the survival curves of the animals correlate with the cellular cytotoxic tumor-specific immune response.

It remains to be seen whether these antibodies are also involved in tumor defense stay. At least in the animals treated with adsorbate IL-2 tumor vaccines was able to establish a clear correlation between the measured antibody titers and the survival of the animals: the higher the proportion of survivors animals in a group, the higher the measured antibodies pertiter. This was not the case with the animals treated with GM-CSF adsorbate (Example 4). The use of adsorbate-GM-CSF in the tumor vaccines appeared  lead to a preference for the humoral immune response, while the at the same time, cytotoxic anti-tumor immune response was less effective seemed to be.

Because adsorbate cytokines obviously are not only the cellular cytotoxic, but we also promote the humoral immune response against tumor antigens is investigating whether adsorbate cytokines are also "a new type of adjuvant for induction of humoral immune responses against protein antigens ". These investigations (Example 5) were first carried out using one of the usual methods protein antigens used in the laboratory for research purposes, the bovine Serum albumin (BSA). This was the antigen, in this case BSA, adsorbed on aluminum hydroxide. Then the aluminum hydroxide adsorbed BSA mixed with aluminum hydroxide adsorbed IL-2 and more fold (primary injection: day 0; booster injections on days 28 and 56) subcu tan or intraperitoneally applied. The sera obtained were determined indirectly ELISA with BSA on the solid phase for their content of BSA-specific anti body examined. Control animals were only treated with BSA aluminum hydroxide Immunized adsorbate.

Example 5 Induction of a humoral immune response after immunization with BSA and Adsorbate IL-2

To investigate whether adsorbate-IL2 is also an adjuvant for the induction of humoral immunants words against protein antigens, test animals with aluminum hydroxide immunized adsorbed bovine serum albumin (BSA) together with adsorbate cytokines.

Test execution

Mice from the inbred strain BALB / c were treated with a mixture of 100 μg of aluminum hydroxide ad sorbed BSA and 10 µg rhuIL-2 vaccinated in the form of adsorbate rhuIL-2. Animals of another Experimental groups received immunizations with 100 µg in complete Freund's adjuvant emul certified BSA. The animals in the control groups were adsorbed with 100 μg of aluminum hydroxide Immunized with BSA or with 100 µg free BSA. Ten days after secondary immunization, the Sera from the animals were obtained and the antibody titers were determined in an ELISA using BSA as the antigen on the solid Phase determined.

Diagram 5 shows the antibody binding curves of the animal serum samples determined in the ELISA shown.

The bond curves shown in Diagram 5 allow the following conclusions:

• - By vaccination with BSA adsorbed on aluminum hydroxide (adsorbate-BSA) higher antibody titers are achieved than by vaccination with BSA in free form.
• - By adding Adsorbat-rhuIL-2 to Adsorbat-BSA, these titers can be increased even further become.
• - The induced by vaccination with a mixture of Adsorbat-rhuIL-2 and Adsorbat-BSA The antibody titers are comparable to the antibody titers that are obtained by vaccination with in CFA-emulsified BSA can be achieved.

The results (Example 5) clearly showed that adsorbate IL-2 was excellent also as an adjuvant for the induction of humoral immune responses to soluble Protein antigens are suitable. Although using the for comparison Complete Freund's Adjuvant (CFA) used faster higher anti achieve body titers than through adsorbate IL-2. However, this result is without great importance since CFA in humans because of the strong local adjunct under no circumstances can be applied. In Germany the An use of CFA because of the strong side effects in recent times No more animals allowed.

After the third vaccination, the adsorbate-IL-2 was used as an adjuvant however, antibody titers were about as high as those achieved by CFA.

In adsorbate vaccines for use in humans or animals are true, the antigen is adsorbed in its aluminum hydroxide form, without an additional adjuvant. One assumes that from that aluminum hydroxide itself has an adjuvant effect, some due to its adsorbate property, partly also due to its foreign body Property is conditional. The comparison of the anti achieved with the adsorbate IL-2 body titers with the titers obtained in this control group (BSA adsorbate) showed very clearly the advantage of the new adjuvant Adsorbat-IL-2: the one with Antibody titers obtained from aluminum hydroxide adsorbed BSA were found in al len cases significantly less than those with the mixture of aluminum Antibody titers obtained from BSA and adsorbate IL-2. The admixture of adsor bat-GM-CSF resulted in higher titers than the addition of adsorbate-IL-2.

In order to investigate whether adsorbate-IL-2 is also suitable as an adjuvant for viral vaccines used in humans, mice of the inbred strain C3H / HeN were immunized with a commercial hepatitis B vaccine (gene HB-Vax) intended for human application (Example 6). Such an HBV vaccine contains 10 μg HBs antigen (hepatitis B surface anti gene) adsorbed on aluminum hydroxide per dose. To immunize mice (C3H / HeN), 1/8 of the human dose was used per mouse. Before the injection, each aliquot of aluminum hydroxide adsorbed HBs antigen either

• - 10 µg of IL-2 adsorbed on aluminum hydroxide or
• - 5 µg of GM-CSF adsorbed on aluminum hydroxide or
• - 10 µg of aluminum hydroxide adsorbed IL-2 plus 5 µg of aluminum hy hydroxide-adsorbed GM-CSF

added.

Example 6 Induction of a humoral immune response after vaccination with hepatitis B antigen (HBs-Ag) and adsorbate cytokines

To investigate whether adsorbate cytokines are also suitable for vaccination with a viral antigen mice with a vaccine made of aluminum hydroxide adsorbed hepatitis B- Antigen and aluminum hydroxide adsorbed cytokines (Adsorbat-IL-2 and Adsorbat-GM-CSF) vaccinated.

Test execution

A commercially available HBV vaccine (Gen-HB-Vax) was used for the vaccination. The vaccine intended for use in humans contained 10 µg of aluminum hydroxide-adsorbed HBs antigen per dose. For use in mice, 1/8 of this vaccine dose was used for each vaccination. Each mouse was vaccinated twice (primary and 30 days later secondary) and thus received 1.25 µg HBs antigen mixed with the respective adsorbate cytokine with each vaccination:

• - 1.25 µg adsorbate HBs antigen plus 10 µg rhuIL-2 in the form of adsorbate IL-2 or
• - 1.25 µg adsorbate HBs antigen plus 5 µg rhuGM-CSF in the form of adsorbate GM-CSF or
• - 1.25 µg adsorbate HBs antigen plus 10 µg rhuIL-2 in the form of adsorbate IL-2 and 5 µg rhuGM-CSF in the form of adsorbate GM-CSF.
• - 1.25 µg adsorbate HBs antigen alone was set up as a control.

Blood was drawn 11 days after the secondary vaccination and the HBs content specific antibodies tested in an indirect ELISA with HBs antigen on the solid phase. The results are shown in the form of binding curves in diagram 6.

The results shown in diagram 6 clearly show that the antibody binding titers in the sera of the animals have been greatly increased by admixing adsorbate cytokines with the HBs antigen adsorbed to the aluminum hydroxide:

• - by a factor of about 10 by admixing Adsorbat-GM-CSF
• - by a factor of about 50 by admixing adsorbate IL-2
• By a factor of about 250 by simultaneous addition of adsorbate IL-2 and adsorbate GM-CSF

When both adsorbate cytokines are added to the aluminum hydroxide, the HBs antigen is adsorbed the effects seem to add up.

We also found corresponding results when using tetanus toxoid and of diphtheria toxoid as antigens and adsorbate IL-2. With these subs It was shown that the increase in the inducible by adsorbate-IL-2 humoral anti-toxoid immune response from both the dose of and the adjuvant adsorbate used IL-2 as well as the dose of the aluminum hydroxide adsorbed toxoid hangs.

The vaccine effect increased continuously with the dose of the IL-2 contained in the adsorbate IL-2:

• - Without the addition of adsorbate IL-2 in the vaccine, the anti were obtained body titer extremely low
• - When free IL-2 was added to the vaccine, the antibody titers obtained were also extremely low  
• - at a dose of 1 µg IL-2 per vaccine dose as adsorbate IL-2 in the vaccine the vaccination effect was better, but still low
• - at a dose of 3 µg IL-2 per vaccine dose as adsorbate IL-2 in the vaccine the vaccination effect was much better
• - at a dose of 10 µg IL-2 per vaccine dose in the form of adsorbate-IL-2 er the vaccination effect reached its maximum
• - at doses above 10 µg IL-2 per vaccine dose in the form of adsorbate-IL-2 it was no longer possible to increase the antibody titers.

A corresponding dependence of the vaccination success on the dose of the antigen used adsorbed aluminum hydroxide was also observed:

• - With a low antigen dose of e.g. B. 1 µg toxoid per animal and injection was the increase in antibody titers by adding 10 µg of IL-2 in the form strongest from adsorbate-IL-2 to vaccine (up to 10,000 times).
• - The increase was at an antigen dose of 100 µg per animal and injection the antibody titer by adding 10 µg IL-2 in the form of adsorbate IL-2 to vaccine lower, but the antibody titers were still up to 10 times higher than after vaccination with the adsorber on aluminum hydroxide based on antigens obtained from antibody titers.

This already shows an important application option for adsorbate cytokines:
as adjuvants for the induction of humoral immune responses to protein antigens.

We have examined the adjuvant properties of adsorbate-IL-2 in detail and have been able to demonstrate

• - that recombinant human IL-2 (rhuIL-2) in adsorbate form (adsorbate-IL-2) in mice as experimental animals, it is best at a dose of 10 µg per injection acts as an adjuvant to induce humoral immune responses. Low re doses lead to lower titers, higher doses intensify the effect hardly anymore, but also do not lead to the suppression of humoral Immune response.
• - That for a good adjuvant effect, the cytokine must under no circumstances be bound to an excess of aluminum hydroxide. A weight ratio of 1: 1 has proven to be optimal, at least for IL-2:
  10 µg rhuIL-2, adsorbed on 10 µg aluminum hydroxide (example 7), leads to optimal effects.
  Because of the lower protein binding capacity, calcium phosphate must be used in a large excess: 10 µg IL-2 are adsorbed on 1,000 µg calcium phosphate.
  It is possible that the same local IL-2 dose can be successful in animals with larger body masses and in humans. However, it is also possible that higher doses have to be used in accordance with the larger body and fluid volume. For safety, the IL-2 dose to be used must be determined for each species.

Example 7 Influence of the loading density of the alumirum hydroxide gel with the biologically active substance on the effectiveness of the adsorbate-bound biologically active substances

Aluminum hydroxide gel has a very high protein binding capacity, which is of the order of 1 µg protein per lug of aluminum hydroxide in the gel. Studies with IL-2 have shown that the release of IL-2 from aluminum hydroxide-adsorbed IL-2 (adsorbate-IL-2) depends on the loading density of the aluminum hydroxide with IL-2. It could be shown that

• - With a loading ratio of 10 µg IL-2 to 10 µg aluminum hydroxide 90-96% of the "ange botenen "IL-2 were bound to the aluminum hydroxide
• - With a loading ratio of 10 µg IL-2 to 30 µg aluminum hydroxide 98% of the "offers "IL-2 were bound to the aluminum hydroxide
• - with a loading ratio of 10 µg IL-2 to 100 µg aluminum hydroxide 99% of the "offers "IL-2 were bound to the aluminum hydroxide.

Correspondingly, the IL-2 adsorbed in the release of IL-2 from aluminum hydroxide also depended on the loading density. In in vitro release experiments, we were able to show that after two hours of rolling a suspension of rhuIL-2-loaded aluminum hydroxide gel at room temperature

• - Free about 11.4% of the adsorbate IL-2 loaded with 10 µg IL-2 per 10 µg aluminum hydroxide were set
• - About the adsorbate-IL-2 loaded with 10 µg IL-2 per 30 µg aluminum hydroxide 3.2% were released
• - About 2.1% free of the adsorbate-IL-2 loaded with 10 µg 1L-2 per 100 µg aluminum hydroxide were set.

Corresponding results were obtained from animal experiments in which IL-2-loaded aluminum hy droxid in various loading densities as an adjuvant for the induction of humoral immunant words was used. The highest antibody titers were obtained when 10 µg IL-2 was added to 10 µg Aluminum hydroxide adsorbed when adsorbate-IL-2 was used.

Also when using adsorbate-IL-2 together with irradiated tumor cells in tumor vaccines the best protection was obtained when the IL-2 was 1: 1 to the aluminum hydroxide was bound (10 µg IL-2 to 10 µg aluminum hydroxide).

The "mixed adsorption" of IL-2 and antigen for the preparation of immunization mixtures or vaccines did not turn out to be cheap. Better results were obtained when antigen and IL-2 separately, each in their optimal adsorption ratio to aluminum hydroxide and then mixed and applied together.

As Example 7 shows, the percentage of aluminum hydroxide ad sorbed IL-2 with a constant amount of "offered" IL-2 for adsorption with increasing amount of aluminum hydroxide and reached at one Ratio of 10 µg IL-2 to 100 µg aluminum hydroxide 99%. That is ver of course, because the larger the adsorption capacity offered, the more  IL-2 can be bound. On the other hand, the existing one then complicates Excess adsorption capacity releases the IL-2 from the adsorber asked, as was also demonstrated in Example 7.

The adjuvant effect of the adsorbate IL-2 becomes corresponding by binding of the cytokine reduced to an excess of adsorbent. This became so probably in attempts to induce humoral as well as in attempts to duction of cellular immune responses observed.

In order to achieve optimal effects, it is therefore essential to use mixtures of several biologically active substances in aluminum hydroxide-adsorbed form, as well as mixtures of aluminum hydroxide-adsorbed antigens with biologically active substances in aluminum hydroxide-adsorbed form,

• - That both the antigen and each one of the biologically active Substances separately in the for each optimal adsorption ratio Aluminum hydroxide is adsorbed and
• - That the individual adsorbates are then mixed together.
• - That the adjuvant effect of adsorbate-IL-2 is particularly good at low An doses come into play. With increasing antigen dose the effect decreases. But even with high doses of antigen, e.g. B. of 100 µg Tetanus toxoid or 100 µg diphtheria toxoid, the antibody titer is characterized by Addition of adsorbate-IL-2 to the aluminum hydroxide adsorbed anti conditions significantly increased.

In experiments with various antigens, we were able to show that adsorbate IL-2 is an excellent adjuvant for the induction of immune responses against protein antigens in mice, e.g. B. also for the extraction of spleen lines for the generation of hybridomas for the production of monoclonal antibodies, is suitable (Example 8). The antibody titers were those with adsorbate-IL-2 as Adju vans immunized mice equal to or higher than the antibody titers of the mice immunized with CFA as adjuvant. The spleens with adsorbate-IL-2 immunized animals, taken four days after the last immunization had shown splenomegaly. Also the number of after merger of the spleen cells of the immunized animals obtained hybridomas was in animals, which had been immunized with adsorbate-IL-2, larger than according to conventional Immunization using CFA. On the animals in the Immunization course with adsorbate-IL-2-containing vaccines no gravie side effects are observed.

Example 8 Generation of hybridomas after immunization with the F protein of the RS virus and adsorbate IL-2

Since adsorbate-IL-2, as could be shown in the previous examples, as an adjuvant to the induc tion of humoral immune responses, it was examined whether the splenic lymphocytes of the so im Munized animals are also suitable for cell fusion to generate hybridomas.  

Test execution

Mice of the inbred strain BALB / c were either treated with antigen emulsified in CFA or with Alu minium hydroxide-adsorbed antigen together with 10 µg adsorbate rhuIL-2 primary, after 4 weeks Chen secondary and immunized again after another four weeks. Ten days after the last one The sera of the animals were obtained by immunization and analyzed in an ELISA for their specific Ge halt on antibodies against the RSV F protein examined. 4 days after another immunization the animals were sacrificed and the spleens removed. The spleen cells obtained from it became Cell fusion to generate hybridomas for the production of monoclonal F-protein-specific An antibody used.

The following results were obtained

• 1. The binding titers determined in the sera of the animals were those with adsorbate rhuIL-2 as Animals immunized with adjuvant were higher than in animals immunized with CFA as adjuvant
• 2. From the very large spleens of the mice immunized with antigen together with 10 µg adsorbate rhuIL-2, more than twice as many (100 × 10 ⁶ ) spleen cells could be obtained and used for fusion as from the spleens with emulsified antigen immunized mice (40 × 10 ⁶ ).
• 3. The number of hybridoma clones isolated after cell fusion was with after immunization Adsorbate-rhuIL-2 as an adjuvant is twice as high as after immunization with CFA as an adjuvant.
• 4. After immunization with adsorbate-IL-2 as adjuvant, the animals showed no symptoms, which suggested serious side effects.

By immunization with vaccines that contain adsorbate-IL-2 in addition to the aluminum hydroxide-adsorbed antigen, it is therefore possible to obtain stronger humoral immune responses than with the usual adsorbate vaccines containing only the aluminum hydroxide-adsorbed antigen. This gives it a number of possible uses:

• - In people whose immune systems are unable to use vaccination the usual HBV vaccine with a high antibody response ren, it is not possible to vaccinate against protective immunity To produce hepatitis B (Zuckerman and Zuckerman, 1998). To this group of people belong to both patients with certain diseases (e.g. kidney failure) as well as normal healthy people (McDermott et al., 1997) who are genetically determined low or non-responders. By vaccination with HBV vaccines that contain adsorbate IL-2 as an adjuvant can also be used in low- or non-responders protective immunity against hepatitis B viruses indu be decorated. The same applies to vaccinations against other pathogens, in People like animals.
• - The use of adsorbate cytokines as Adju is particularly interesting vantien in vaccines also because it becomes possible, even with extreme low antigen doses to induce protective immunity.
• - Adsorbate-IL-2 can be used as an adjuvant to immunize laboratory animals instead of Complete Freund's Adjuvant (CFA) it doesn't cause the strong side effects like CFA. This is discussed in Zu  important in basic biological and medical research be because of the use of CFA because of the severe side effects Laboratory animals are already banned in Germany and in other European countries countries will be banned in the foreseeable future.
• - It is no longer permitted by law to use mice to induce humoral Immune responses, e.g. B. for the purpose of obtaining hybridomas with Immunize antigen using CFA.
• Because of its good adjuvant activity, adsorbate IL-2 is likely and other adsorbate cytokines are also useful for the development of vaccines against parasite antigens and against HIV antigens.

2. For direct (intratumoral) therapeutic treatment of existing Tumors

From the literature it is known that repeated intratumoral injection a certain, if mostly not permanent, regression of free cytokines can be achieved by tumors. Such examinations are different those cytokines, both in humans (Robinson et al., 1998; Vaquero et al., 1992) as well as in experimental animals (Kurane et al., 1997). In order to check whether the Ad used in our tumor vaccines as an adjuvant sorbate cytokines are also suitable for direct intratumoral use, ha we give animals with existing palpable tumors by repeated intratumo oral injections of adsorbate-IL-2 treated. Control animals were treated accordingly treated with intratumoral injections of medium. That get Results show that adsorbate cytokines are excellent for intratumo rale injection into existing tumors are suitable. While the murine Melanoma B16BL6.D5 only significantly extended the lifespan of the animals could be reached, 60% of the mice, the tumors of the murine RenCa renal cell carcinoma finally contributed to the tumor disease to be cured (Example 9) were against a later challenge with a lethal one Dose of vital RenCa cells were immune and were still more than 1 year after Treatment tumor-free and immune to another challenge with le tal dose of vital tumor cells.

Example 9 Tumor eradication by intratumoral injection of adsorbate IL-2

To investigate whether the local application of adsorbate IL-2 by injection into existing Tu moren (intratumoral injection) has had an effect on tumor growth in animals subcutaneous injection of a lethal dose of vital tumor cells initially induced tumors and these Tumors then treated by intratumoral injection.

Test execution

Tumors were induced in mice of the inbred strain BALB / c by subcutaneous injection of the lethal dose of 10 ⁵ vital RenCa cells. As soon as palpable tumors developed, the animals were treated as follows.

• - The animals in the test group were given 3 µg of aluminum hydroxide every 3 days adsorbed rhuIL-2 existing adsorbate-IL-2 injected intratumorally.
• - Physiological saline was injected intratumorally into the animals in the control group.

Diagram 7 shows the survival curves of the animals in the experimental and control groups poses.

While the animals in the control group "Saline" had died by the 50th day at the latest, about 60% of the animals in the group that had been treated with adsorbate rhulb-2 ("IL-2 ALU") lived. The difference between the courses of the survival curves was extremely significant (P = 0.0002).

In a further experiment (not shown) tumors were induced in mice of the inbred strain C57BL / 6 by injection of a lethal dose of 10 ⁵ vital cells of the mouse melanoma cell line B16BL6.D5. After the tumors became palpable, the animals were treated as described above by intratumoral injection of adsorbate-IL-2, in the control group by intratumoral injection of medium. Since the murine melanoma cell line is a much more aggressive tumor than the RenCa renal cell carcinoma used above, no cure could be achieved in this case, but only a significant extension of the life of the animals.

One of the most promising methods for tumor therapy is the use of factors that inhibit vascularization, angiogenesis (Boehm et al., 1997). The effect of these anti-angiogenesis factors, such as. B.

• Angiostatin protein (Kim Lee Sim et al., 1997), amino acids 93 to 470 comprehensive peptide from the plasminogen, and
• Endostatin protein (O'Reilly et al., 1997), a 20 kD C-terminal Frag ment from collagen XVIII,
• - As well as substances of small molecular size, such as
• - squalamine,
• - 2-methoxyestradiol (Klauber et al., 1997) and
• Thalidomide (D'Amato et al., 1994) or analogues thereof

is currently used in preclinical animal experiments and in the first clinical Studies examined. These factors are mainly applied systemically. With systemic application, relatively high doses of angiogenesis are required inhibiting factors are applied to locally, in / on the tumor, a sufficient effective dose. These high doses have too Side effects, because the application in general in the organism Inhibited angiogenesis. Nevertheless, the actual ones come for the effect intended place, in / on the tumor, only very small doses for effect. The lo kale intratumoral use of adsorbate anti-angiogenesis factors would here not only to weaken the systemic side effects, son at the same time lead to a significant improvement in the local impact ren.  

In particular, it is expected that the simultaneous or sequential application of several biologically active substances adsorbed on an adsorbent with different mechanisms of action not only to improve the Elimination of the tumor cells, but also for the induction of Tu Morse-specific immune responses can lead. For example, Nakayama et al. show that simultaneous intratumoral injection of alcohol and IL-2 Tumor-specific cytotoxic immune responses could be induced (Nakayama et al., 1997).

It is expected that by intratumoral injection of mixtures of adsorbent adsorbed biologically active substances with different principles of action, e.g. B.

• - of adsorbate anti-angiogenesis factors and adsorbate IL-2, or
• - of adsorbate-histamine and adsorbate-IL-2

additive effects occur and on the one hand the tumor is destroyed and on the other at the same time a tumor-specific immune response is induced (Hellstrand et al., 1998). The immunity that arises would also eli metastases be mined.

3. To enlarge the T cell population

However, there are many other possible applications. So in In recent years, the immune deficiency disease caused by HIV has been tried can also be combated by applying free IL-2. Because of the strong side The effects of systemic (intravenous) administration have been in the last few years started to use this cytokine subcutaneously. As Kirchner et al. (Kirchner et al., 1998) could show the IL-2 concentration decreases in the peripheral Blood after subcutaneous application of 1.0 mg IL-2 within 4 hours a maximum of about 4.0 ng IL-2 / ml and then falls within 24 Hours back to normal. A similar one, albeit because of the smaller height of the animals slightly different, course of the IL-2 concentration after application of IL-2 we have also in the peripheral blood of mice can point (Example 10). Just 30 minutes after subcutaneous application of 100 µg free IL-2 was a much higher maximum value of 17 µg / ml er enough. With an assumed blood volume of the mouse of 3.0 ml about 50% of the total subcutaneously administered IL-2. Already had after 6 hours the IL-2 concentration in peripheral blood falls below the detection limit of the test taken. In contrast, when applying IL-2 in the form of aluminum sodium hydroxide-adsorbed IL-2 a maximum value of only 3.0 µg IL-2 per ml blood (Corresponding to about 10% of the total applied IL-2) reached after one hour. For this, the IL-2 content in the blood only reached the detection level after 48 hours of the test.  

Example 10 Detection of IL-2 in the peripheral blood of mice after subcutaneous application of adsorbate IL-2

To study the in vivo release of IL-2 from adsorbate-IL-2, animals were given adsorbate-IL-2 injected subcutaneously and the IL-2 concentration determined in peripheral blood samples. As a control free IL-2 was injected subcutaneously.

Test execution

Adult males of the C3H / HeN mouse strain were given 100 µg rhuIL-2 either in free Form or adsorbed on aluminum hydroxide injected subcutaneously. After the injection, the tie blood was drawn at 50p1 at different points in time, which in a sandwich ELISA on his RhuIL-2 content was examined.

Table 2 shows those in the animals of the different test groups at the time indicated measured rhuIL-2 concentrations.

Diagram 8a and diagram 8b show the course of the cytokine concentration in peripheral blood of mice as a function of time after injection for the "Adsorbate-IL-2" test batches and "free IL-2".

The diagrams differ in the scaling of the ordinates: the scaling of the Y axis in Diagram 8b ("free IL-2") covers a much larger area than the scaling of the Y- Axis in diagram Sa ("Adsorbate-IL-2").

Table 2

From the results shown in Table 2 and in Diagrams 8a and 8b, it can be clearly seen that that after subcutaneous application of 100 µg of free rhuIL-2 after 30 minutes a very high one IL-2 serum maximum value of 17.5 µg IL-2 / ml blood is reached, which, however, also very quickly decreases again and has already dropped below the detection limit of the test after 9 hours. The compared with a subcutaneous application of adsorbate IL-2, a significantly lower serum Maximum value of 3.0 µg IL-2 / ml blood already reached after 60 minutes. However, the serum IL 2 content in peripheral blood after application of 100 µg adsorbate IL-2 only after 48 hours the detection limit.  

Subcutaneous application of IL-2 in free form creates a kind of short-term depot in a “tissue pocket” that empties completely within 24 hours. Kirchner et al. could also show that repeated (every 12 hours) subcutaneous application of half the dose of IL-2 (0.5 mg / m ² ) achieves a significantly better bioavailability: the IL-2 content in the peripheral blood took on a wavy shape Curve ("sawtooth curve") and never decreased to normal. The effect of the subcutaneously applied IL-2 depot is prolonged by repeated application in a 12-hour rhythm. If this application is continued over a longer period, IL-2 is available in the organism practically over the entire period of treatment.

This type of IL-2 therapy by repeated subcutaneous injection is e.g. B. in HIV patients, in combination with other medications, used with good success (Carr et al., 1998; Witzke et al., 1998) and leads to an increase in the proportion of CD4 ⁺ cells. For example, in an article published in the November 1998 issue of Science ("News of the Week", Science, Vol. 282, Nov. 20, 1998, pages 1394-1395: "Can IL-2 smoke out HIV reservoirs? ") reported the results of studies in which the HIV patients were treated with repeated subcutaneous injections of IL-2 in medium doses (0.5 to 1.0 mg per m ² ) in addition to the multiple therapy that is common today. Some of the patients completely eradicated the virus-infected lymphocytes.

There are a number of reports on the treatment of patients with "multip le drug resistant tuberculosis "(MDR-TB). In these patients, the repeat application of low-dose IL-2 also improves the cli symptoms. Johnson et al. have reported that in MDR-TB patients who For 30 days in addition to the usual therapy ("multidrug therapy") daily received a low dose application of IL-2, not just an improvement clinical symptoms, but also a reduction in sputum detectable bacteria could be achieved (Johnson BJ et al., 1995, 1997 and 1997). The clinical improvement was also proven by X-ray diagnosis become.

Villahermosa et al. have very low dose (10 µg) more leper sick IL-2 times injected into the leprosy lesions and observed a strong T cell Infiltration and reduction of bacterial infestation (Villahermosa et al., 1997).

Replacement of repeated subcutaneous application of IL-2 in free form by applying depot-bound IL-2, e.g. B. as adsorbate-IL-2, would Treatment in HIV patients as well as in tuberculosis patients simplify considerably, reduce treatment costs and improve therapy take place lead.

Based on the experience in the literature with low-dose Repeatedly used IL-2 can be assumed to be adsorbate IL-2 not only for the two chronic infectious diseases listed,  but generally in the case of infectious diseases through expansion of the lymphocyte Population and the associated general activation of the immune system stems improve the treatment options for infectious diseases that Simplify application and reduce the cost of treatment.

4. For the treatment of immunodeficient patients, e.g. B. in tumor patients after chemotherapy or in leukemia patients

Another application is in patients with immune Deficiency diseases, e.g. B. in tumor or leukemia patients after chemo therapy. These patients receive leu to stimulate recruitment regularly subcutaneous injections of G-CSF. The effectiveness of this The factor could also be improved by application in the form of adsorbate G-CSF be tested.

Possible uses of other biologically active substances in Adsorbent adsorbed form 1. In multiple sclerosis (MS)

For the treatment of patients with multiple sclerosis, "Co polymer-1 "(COP-1) applied, a copolymer of different amino acids ren (Teitelbaum et al., 1991), whose mode of action has not yet been fully clarified, However, this leads to an improvement in the situation of these patients (Johnson et al., 1995; Johnson et al., 1998). Patients inject this preparation themselves occasionally subcutaneous. After application, the formation of antibodies against COP-1 observed (Johnson et al., 1995). Whether these antibodies are essential for the effects are not yet clear. COP-1 could also be used in Adsorbate-bound form ("Adsorbat-Cop-1") to improve the us simplification of treatment and a reduction in costs ren.

Another preparation used to treat multiple sclerosis is the beta interferon belonging to the group of cytokines / interferons, that likewise leads to improvements in the course of the disease (IFNB Multiple Sclerosis Study Group, 1993, 1993 and 1995). Here too it is to be expected that the subcutaneous appli cation of adsorbate interferon beta has a much better effect than that subcutaneous application of free IFN-beta.

2. The use of aluminum hydroxide adsorbed histamine (adsorbate Histamine)

The versatile use of adsorbate-bound biologically active substances were shown in experiments that we performed with histamine (4- Imidazolethylamine), a biologically extremely effective substance of low Mo.  reading size. Histamine is a mediator used for vasodilation leads and is able to chemotactically and activating on different cells of the immune system. It is found in secretory vesicles of Mast cells stored in large quantities (approx. 0.6 pg per mast cell) and in the frame allergic reactions after contact with a sensitized individual with the allergen locally, at the point of entry of the allergen into the organ mus, released. Release occurs when the allergen arrives with the the Fcε receptors of the mast cell bound IgE antibodies reacted. The at The processes involved in the release of histamine can be very violent. With massive systemic release of histamine, it can lead to shock and even die of allergy sufferers. What role mast cells and Hista min play in "normal" immunological reactions is still largely unsettled. However, it is known that there are different types of mast cells, which ver different tissues localized and at different locally running physio logical and pathological reactions are involved.

The reaction initiated by histamine release is a the most violent reactions caused by immune phenomena. With massive Systemic release of histamine can occur within a few minutes Death. Therefore, there are research approaches that try to do this Immune phenomenon for the treatment of tumors, e.g. B. with the help of tumor specific monoclonal IgE antibodies, directly in / on the tumor for action bring to. So it is Luiten et al. (Luiten et al. 1997) succeeded in recombinant to produce monoclonal antibodies on the constant part of human lichen epsilon chain the variable part of a tumor-specific monoclonal Wear antibody. The injection of such antibodies into tumor patients should according to the concept of tumor targeting to bind the antibodies to the Tumor cells and for local triggering of allergic immune phenomena to lead. It is hoped that the IgE bound to the tumor cells Activate antibodies directly on the tumor mast cells, which lead to histamine Release and lead to the complex allergic reaction. It is expected that the effector cells flowing into the tumor area initiate a response that will ultimately destroy the tumor leads.

This approach is being worked on in some laboratories, although so far it has been completely It is unknown whether there is any hope for the immune response and above all can lead to a tumor-specific cytotoxic immune response. Man knows that the release of histamine to the inflow of the different leads cells of the immune system. However, it has never been proven the fact that it actually triggers immune reactions.

Based on the knowledge that the local release of biologically active seed molecules is the natural biological route through which these molecules we mimicked the local release of histamine by using Hi  stamin adsorbed on aluminum hydroxide and together with aluminum hy injected subcutaneously with hydroxide-adsorbed antigen (Example 11).

It has been shown completely unexpectedly that adsorbate histamine is a very good one Adjuvant action for induction of humoral immune responses against has a protein antigen. In its immunity-inducing effect, Ad sorbate-histamine has practically the same effect as adsorbate-IL-2. Although it is assume that the mechanism of action taking place is a completely different one is the one that occurs during the injection of adsorbate-IL-2. The effect, namely the very effective induction of a humoral immune response, however, is the same. Such use of histamine has not yet been described in the literature been written.

By adding Adsorbat-IL-2 to a mixture of Aluminumhy hydroxide-adsorbed antigen and adsorbate histamine could not provide any additional Increase in humoral immune response can be induced.

Example 11 Evidence of the adjuvant effect of adsorbate-bound histamine ("adsorbate histamine") in the induction of a humomile immune response against a protein antigen

The adjuvant effect of adsorbate-IL-2 for the induction of the humoral immune response against Pro tein antigens have already been detected in Examples 5, 6 and 8. In the following example the adjuvant effect of another biologically active substance, histamine (4- Imidazolethylamine), in adsorbate-bound form in the induction of the humoral immune response against a protein antigen. For comparison, adsorbate IL-2 is used in one animal group Adjuvant used.

Test execution

Groups of C3H / HeN mice (6 animals per group) were either

• 1. 1 µg of diphtheria toxoid adsorbed on aluminum hydroxide alone or
• 2. 1 µg diphtheria toxoid adsorbed on aluminum hydroxide and 10 µg of rhuIL-2 (adsorbate-IL-2) adsorbed on aluminum hydroxide or
• 3. 1 µg diphtheria toxoid adsorbed on aluminum hydroxide and 100 µg of histamine adsorbed on aluminum hydroxide (adsorbate histamine) or
• 4. 1 µg diphtheria toxoid adsorbed on aluminum hydroxide, 10 µg of rhuIL-2 (adsorbate-IL-2) adsorbed on aluminum hydroxide and 100 µg histamine adsorbed on aluminum hydroxide (adsorbate histamine)

injected.

After 4 weeks, the injections were repeated (boost), 10 days after the 2nd injection, the Blood was drawn from animals and the serum samples in the ELISA with solid phase-bound diphtheria Toxoid examined for their content of diphtheria-toxoid-specific antibodies.

The results shown in diagram 9 show that the results obtained by immunization with 1 µg Alumi nium hydroxide adsorbed diphtheria toxoid along with aluminum hydroxide adsorbed Histamine ("adsorbate histamine") induced humoral immune response against the antigen (Diphthe rie toxoid) in the sera of the animals of the 3 experimental groups (groups 2, 3, 4) about the same level, but much higher than in the sera of diphtheria only adsorbed on aluminum hydroxide Toxoid immunized animals of the control group (group 1).  

The adjuvant property of histamine described here has not yet been described in the literature knows.

Because of its vasodilator and the blood flow and infiltration of the be affected tissue area extremely strong promoting properties, it is very true Apparently, adsorbate histamine is also tumor-specific when inducing cytotoxic immune responses is effective. Therefore we have tests carried out in which irradiated tumor cells mixed with adsorbate Histamine when tumor vaccines were used (Example 12). By the Be the action could obviously have cytotoxic immunity to the animal Tumor induced, because in 6 of the 14 animals treated before the Vaccination in lethal dose applied to vital tumor cells, and the animals survived.

Example 12 Evidence of the adjuvant effect of adsorbate-bound histamine ("adsorbate histamine") in the induction of a cellular cytotoxic tumor-specific immune response

To investigate whether histamine in adsorbate-bound form also has adjuvant properties for the Has induction of cellular cytotoxic anti-tumor immune responses, we have histamine in Aluminum hydroxide adsorbed form (adsorbate histamine), together with irradiated RenCa- Tumor cells used in tumor vaccines.

Test execution

Four days before the application of the vaccine consisting of 10 ⁶ irradiated RenCa tumor cells and adsorbate histamine (100 µg histamine adsorbed on 100 µg aluminum hydroxide), a lethal dose of 10 ⁵ vital RenCa tumor cells was administered to mice of the inbred strain BALB / c. The animals of a control group were given tumor vaccines that only contained irradiated tumor cells.

Treatment with vaccines from irradiated tumor cells and adsorbate histamine led to almost same results as treatment with vaccines from irradiated tumor cells and adsorbate IL-2. The result of this experiment has shown that aluminum hydroxide adsorbed Hista min also has a limited effect in tumor vaccines. Six of the 14 behan Deleted animals survived the experiment and were immune to the tumor (diagram 10).

It can be assumed that many other biologically active substances small molecular size due to the application in the form of adsorbates show biological effects. So z. B. Serotonin in its effect Histamine very similar substance, and should have similar effects. In particular special is to be expected that the application of several biologically active to substances with different activity spectra in adsorbent form dit effects.  

3. Peptides and other biological molecules adsorbed on aluminum hydroxide oils that can stimulate or inhibit the immune response

These include immunostimulating peptides such as. B. the tetrapeptide tuftsin (Spirer et al., 1989) and thymus peptides such as thymosin alpha 1 or abge thereof directed shorter peptides. For thymosin alpha, as well as for those derived from it thymosin peptides described that they respond to various immune parameters can act (Birr et al., 1992; Lopez et al., 1994). Other peptides can again inhibit the immune response. By applying such the Im munesponse stimulating or inhibiting substances in aluminum hydro xid-adsorbed form, it should be possible to control the immune response at will ern. So it succeeded one through a mixture of aluminum hydroxide adsorbed antigen and adsorbate IL-2 induced humoral immune response by adding an inhibitor, also in aluminum hydroxide adsorbed form to completely inhibit.

In experiments with biologically active molecules that inhibit the immune response We were able to show that locally, by adding aluminum hydroxide adsorbed inhibitors (adsorbate inhibitors) to tumor vaccines, which from be emitted tumor cells and adsorbate IL-2 passed, including the cellular cytotoxi tumor-specific immune response could be quantitatively suppressed.

It is known that lymphocyte receptors for chemokines, complement Have fragments and neuropeptides and that the nervous system has such Neuropeptides can also influence the immune system. So through the application of neuropeptides to regulate the inflammatory response. For example, neuropeptides can be chemotacic on eosinophilic granulocytes act table and stimulate them to secrete cytokines (Dunzendorfer et al., 1998; Levite, 1998; Levite et al., 1998). Ad induce sorbate-bound neurotransmitter substances locally.

So can

• - chemokines,
• - eicosanoids,
• - Complement fragments
• - substance P (SP),
• - substance Y,
• - somatostatin
• - dopamine
• - the calcitonin gene-related peptide (CGRP),
• - secretoneurin or
• - the vasoactive intestinal peptide (VIP) and others

act on cells of the immune system and in this way local immune actions such as the inflammatory response. It is expected to be not only be possible by using these substances in adsorbate form is to bring them to bear locally, but also to decide their effect  dend to improve. The particular advantage of this application form is in that it is possible to use several of these substances simultaneously or in desired to bring the local sequence into effect.

4. Increasing the immune response by aluminum hydroxide adsorbed bacteria Real immunostimulating substances

Muramyl dipeptide (MDP) is a pep derived from the bacterial cell wall tid (Sano et al., 1987), which has an immunostimulating effect, without itself as an to induce an immune response and that as an immune stimulant is used. However, it is in free form, mixed with that if applied in free form added antigen.

To investigate whether adsorbate MDP is also suitable for inducing humoral We have mice with immune responses to soluble protein antigens Aluminum hydroxide adsorbed antigen mixed with Adsorbat-MDP immu nized. As an antigen, aluminum hydroxide adsorbed diphtheria toxoid used in low doses (see above). The Er obtained in these experiments Results (Example 13) show that adsorbate MDP is a suitable for immunization suitable adjuvant. In this approach we were able to demonstrate that adsorbate IL-2 and adsorbate MDP act synergistically.

Example 13 Evidence of the adjuvant effect of adsorbate-bound muramyl dipeptide ("adsorbate MDP ") in the induction of a humoral immune response against a protein antigen

It is known from the literature that muramyl dipeptide has adjuvant properties. In attempts with MDP this is generally in free form, mixed with antigen, or in liposomes encapsulated form used.

To investigate whether MDP is also in aluminum hydroxide adsorbed form (adsorbate MDP) in the Induction of humoral immune responses has adjuvant effects, gulls with aluminum hydro oxide-adsorbed antigen and adsorbate-MDP (25 µg MDP per 25 µg aluminum hydroxide) several times immunized.

Experimental setup

Groups of C3H / HeN mice (4 animals per group) were either

• 1. 1 µg of diphtheria toxoid adsorbed on aluminum hydroxide alone or
• 2. 1 µg diphtheria toxoid adsorbed on aluminum hydroxide and 10 µg of rhuIL-2 (adsorbate-IL-2) adsorbed on aluminum hydroxide or
• 3. 1 µg diphtheria toxoid adsorbed on aluminum hydroxide and 25 µg of aluminum hydroxide adsorbed muramyl dipeptide (adsorbate MDP) or
• 4. 1 µg diphtheria toxoid adsorbed on aluminum hydroxide, 10 µg of rhuIL-2 (adsorbate-IL-2) adsorbed on aluminum hydroxide and 25 µg of aluminum hydroxide adsorbed muramyl dipeptide (adsorbate MDP)

injected.  

After 4 weeks, the injections were repeated (boost), 10 days after the 2nd injection, the Blood was drawn from animals and examined in an ELISA with solid-phase-bound diphtheria toxoid halt on diphtheria toxoid-specific antibodies.

The results obtained (diagram 11) show that adsorbate MDP also has adjuvant activity and leads to the induction of humoral immune responses. The by immunization with aluminum However, hydroxide-adsorbed antigen (DT) and adsorbate-MDP induced immune response less than the immune response induced by adsorbate IL-2 as an adjuvant. But shit the effects of the two adjuvants when added together add up.

In a further test approach (Example 14) Adsorbat-MDP with be beamed mixed tumor cells and applied to mice. The results of this Experiments show that adsorbate MDP is also used as an adjuvant for induction lär cytotoxic tumor-specific immune responses is suitable. Its us kung is not as strong as that of adsorbate IL-2, but follows a completely different one ren mechanism of action and can therefore be used for additional Improvement of the induction of cytotoxic immune responses against tumo be used.

Example 14 Evidence of the adjuvant effect of adsorbate-bound muramyl dipeptide ("adsorbate MDP ") in the induction of a cellular cytotoxic tumor-specific immune response

To investigate whether MDP in adsorbate-bound form also has adjuvant properties for the In production of cellular cytotoxic anti-tumor immune responses, we have MDP in aluminum minium hydroxide-adsorbed form (adsorbate-MDP), together with irradiated RenCa- Tumor cells, used in tumor vaccines.

Test execution

Four days before the application of the vaccine consisting of 10 ⁶ irradiated RenCa tumor cells and adsorbate MDP (25 µg histamine adsorbed on 25 µg aluminum hydroxide), a lethal dose of 10 ⁵ vital RenCa tumor cells was administered to mice of the inbred BALB / c strain. The animals in a control group were given tumor vaccines which only contained irradiated tumor cells.

The results shown in diagram 12 show that treatment with vaccines from be radiated tumor cells and adsorbate MDP to a significant extension of the life of the animals leads. Thus, by using this adjuvant in tumor vaccines, similar, if not get as good results as when using adsorbate-IL-2. So the result of this experiment has demonstrated that aluminum hydroxide adsorbed MDP is also a tumor vaccine if also has limited effect. Seven of the 12 treated animals lived significantly longer than the animals the control group.

To this group of bacterial immunostimulating biologically active Tuberculin also belongs to substances.  

5. Cytostatics adsorbed on aluminum hydroxide

By using cytotoxic substances in the treatment of Tumor diseases can have the desired effect depending on the dose be amplified or suppressed. Systemic (e.g. intraperito neale) application of the cytotoxic substances cyclophosphamide or mito mycin C in a small dose the immune from suppressor cells suppressive effect canceled and thereby the effectiveness of tumor vaccines are significantly improved (Tsung et al., 1998).

On the other hand, we were able to show (Example 15) that locally, by adding Aluminum hydroxide desorbed cyclophosphamide to an irradiated one Tumor cells and adsorbate IL-2 existing tumor vaccine, the effect of Tumor vaccine can be lifted. It turned out that the first Application of the mixture of cytokine-containing tumor vaccine and Ad sorbate-cyclophosphamide is the induction of a tumor-specific immunant word even in later vaccinations without the addition of aluminum hydroxide adsorbed cyclophosphamide inhibits the induced immunosuppression is permanent.

Example 15 Evidence of the immunoprohibitory effect of adsorbate-bound cyclophosphamide ("Ad sorbate-cyclophosphamide ") on the induction of a cellular cytotoxic tumor-specific Immune response

Cyclophosphamide is a cytostatic that is used to treat tumors. In low Thursday Cyclophosphamide can neutralize the immune-inhibiting effect of suppressor cells and thereby strengthen cytotoxic immune responses. In experiments in which the experimental animals 1 Day before application of the tumor vaccine consisting of irradiated tumor cells and adsorbate IL-2 2.0 mg mitomycin C per kg body weight had been applied, it was shown that the by the tumor vaccine induced immune response was significantly stronger and more animals attempted survived than without this pretreatment.

The transfer of this test approach to the local application of the cytostatic resulted in a full lig different result. Animals of the inbred strain C57BL / 6 were given 150 mg of cyclophosphamide per kg Body weight in aluminum hydroxide adsorbed form along with that from irradiated MCA Tumor cells and adsorbate IL-2 existing tumor vaccine administered subcutaneously.

As the results shown in Diagram 13 show, the animals developed once locally treated with the tumor vaccine and cyclophosphamide adsorbed with aluminum hydroxide large tumors, whereas the animals treated only with the vaccine had none or only developed very small tumors.

The result of this experiment shows that cyclophosphamide at a dose in which it is systemically ap the immunosuppression, once applied locally, induces an immune response prevented or at least inhibited.  

It is expected that it will be by simultaneous or sequential local application of adsorbate-bound substances with immunostimulating and immune suppressive effects is possible to immune responses at will modulate and control. Such an approach is e.g. B. for the treatment of Autoimmune diseases conceivable. By vaccinating patients with egg a mixture of the auto-antigen in adsorbate form and an immune inhibitory biologically active substance, also in adsorbate form, should it may be possible for the patient to have immunity to the autoantigen at least to decrease.

Cimetidine, an antagonist of histamine, could also be immune-inhibiting Have an effect. Other substances that negatively affect the immune system, such as the corticosteroids, cyclosporin A and used as immunosuppressants others can also be used for local regulation of the immune response be. So it is conceivable that by vaccination with a mixture of Adsorbent-bound autoantigens and adsorbent-bound immune suppressants or cytostatics autoimmune diseases can be treated nen.

6. Growth factors adsorbed on aluminum hydroxide

Also the effectiveness of others on various illnesses regularly by subcutaneous injections of biologically active substances (e.g. erythropoiesis factors, etc.) could be applied in adsorbate form be improved.

The use of neuronal growth factors or neurotransmit ters in adsorbate-bound form could regenerate damaged ones Nerves are used.

In patients where aluminum hydroxide triggers severe side effects, should be calcium phosphate or another inorganic or organic adsorber bens used as an adsorbent or the substance in question in liposomes closed.  

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Claims (23)

1. Means for use for prophylactic and therapeutic purposes in humans and animals, characterized in that a biologically active substance is bound to an adsorbent. 2. Composition according to claim 1, characterized in that Aluminumhy as the adsorbent hydroxide or calcium phosphate is used. 3. Composition according to claim 1 or 2, characterized in that act as biologically same substance in vertebrates naturally occurring and in vertebrates biologically active substances are used. 4. Composition according to claim 1 or 2, characterized in that act as biologically same substance artificially (e.g. recombinantly) produced or genetically modified other variants of biologically occurring naturally in vertebrates effective substances are used. 5. Agent according to one of claims 1 to 4, characterized in that as a biolo genetically active substance proteins or peptides (e.g. from the class of cytokines, interferons, colony stimulating factors, chemokines, wax factors, anti-angiogenesis factors, hormones or neurotrans middle) can be used. 6. Composition according to claim 1 or 2, characterized in that act as biologically same substance in vertebrates not naturally occurring synthetically manufactured and used in vertebrates biologically active substances (pharmaceuticals) become. 7. Composition according to claim 1 or 2, characterized in that act as biologically same substance in vertebrates not naturally occurring from others Organisms (e.g. bacteria) that are biologically active in vertebrates Substances are used that are either from the corresponding organisms won or synthetically produced or which are syn theoretically produced analogues. 8. Composition according to claim 2, characterized in that the particular biolo used gically active substance in such a concentration to the adsorbent ad is sorbed that there is no excess of the adsorbent. 9. Composition according to claims 1 and 8, characterized in that in vertebrate Ren biologically active substance in one for the substance and for the organ mus to which the substance is to be used, optimal dose used  becomes. 10. Agent according to one or more of claims 1 to 9, characterized in that that several biologically active substances are adsorbed on the adsorbent. 11. Composition according to claim 10, characterized in that different in vertebrate Ren biologically active substances are adsorbed separately on the adsorbent, with the proviso that each substance has an optimal characteristic Quantity is included. 12. Composition according to one of claims 3, 5, 8, 9 and 11, characterized in that as a biologically active substance which is recombinant human IL-2 in vertebrates (rhuIL-2) in adsorbent-bound form (Adsorbat-IL-2) for use with Humans, mice and other species in which this cytokine is biologically active is used. 13. Composition according to claim 12, characterized in that for use in mice per mouse between 1 and 30 µg rhuIL-2 in the form of adsorbate IL-2 at 10 µg Aluminum hydroxide or adsorbed on 1,000 µg calcium phosphate is used. 14. A method for producing vaccines, characterized in that the after claims 1-13 produced biologically adsorbed on an adsorbent who added effective substances as adjuvants to antigen preparations the. 15. The method according to 14, characterized in that for the manufacture of the vaccines Antigen preparations by adsorption of antigens on aluminum hydroxide or Calcium phosphate can be produced. 16. The method according to 14 and 15, characterized in that for use in vac zinen provided antigens in such a concentration to the adsorbent be that there is no excess of the adsorbent. 17. A method for producing vaccines according to 14, characterized in that as Adjuvants Adsorbaf-IL-2, Adsorbat-GM-CSF or mixtures of Adsorbat-IL-2 and Adsorbat-GM-CSF can be used. 18. A method for producing an agent according to one or more of the claims 1 to 13, characterized in that at least one biologically active Substance in freshly prepared solution on one of the adsorbents aluminum hy droxide or calcium phosphate is adsorbed and that the agent so produced either used immediately or stored refrigerated until use. 19. The method according to claim 18, characterized in that by adsorption means produced on an adsorbent by freezing or by freezing drying is made durable.   20. Use of the adsorbate prepared according to one of claims 14 to 17 Cytokine vaccines for vaccinations with low doses of antigen. 21. Use of the adsorbate prepared according to one of claims 14 to 17 Cytokine vaccines for the vaccination of low-responder individuals. 22. Use of the adsorbate prepared according to one of claims 14 to 17 Cytokine vaccines for the vaccination of pets or as a replacement for Komplet Freund's adjuvant for vaccination of experimental animals. 23. Use of an agent according to one or more of claims 1 to 13 prophylactic or therapeutic purposes in humans or animals.