EP1370289A2

EP1370289A2 - Method for producing a vaccine containing autologous antibodies

Info

Publication number: EP1370289A2
Application number: EP02706509A
Authority: EP
Inventors: Gottfried Himmler; Hans Loibner; Helmut Eckert; Otto Doblhoff-Dier; Ralf Kircheis; Manfred Schuster; Erich Wasserbauer; Günter Waxenecker
Original assignee: Igeneon Krebs-Immuntherapie Forschungs- und Entwicklungs-GmbH
Current assignee: ALTROPUS GMBH
Priority date: 2001-03-23
Filing date: 2002-03-20
Publication date: 2003-12-17
Also published as: WO2002080966A8; NO20034145L; NO20034145D0; ATA4702001A; WO2002080966A2; MXPA03008587A; CA2441499A1; SK11732003A3; BR0208641A; US20040191242A1; CN1522155A; HUP0303638A3; PL363720A1; WO2002080966A3; AT410636B; JP2004524369A; HUP0303638A2

Abstract

The invention relates to a method for producing a vaccine, which contains autologous antibodies, characterized by the following steps: preparing a liquid that contains antibodies from a body fluid containing autologous antibodies or from autologous cell preparations or tissue preparations; treating the liquid that contains antibodies with a solid support, on which ligands are immobilized that bond to a defined group of antibodies, with the stipulation that the ligands used are not antibodies or fragments thereof with the same idiotype, directed against tumor-associated antigens; obtaining the antibodies that bond to the ligands, and; processing the obtained antibodies to form an autologous vaccine, which contains an efficient amount of antibodies ranging from several micrograms to one gram.

Description

Process for the preparation of a vaccine

The present invention relates to a method for producing a vaccine.

Higher organisms are characterized by an immune system that protects them from potentially dangerous substances or microorganisms. When a substance (necessary) penetrates the body, it is recognized as "foreign" and eliminated with the help of the immune system. "Degenerate" body's own cells are also usually recognized by the immune system and withdrawn from circulation.

The ^" adaptive immune system of humans consists of two essential components, the humoral and the cellular immunity. The adaptive immune response is based on the clonal selection of B- and T-ymphocytes and allows in principle the recognition of any antigen as well as the development of an immunological memory. These characteristics of the adaptive immune system are generally addressed in a useful manner during vaccinations.

Each B cell produces an antibody with a specific binding specificity. This. Antibody is also a specific receptor in the membrane of the B cell that produces it. The humoral immune response against antigens recognized as foreign is based on the selective activation of those B cells which produce antibodies which can bind to epitopes of the respective antigen. DNA rearrangements play a decisive role in the course of B cell differentiation for the variety of antibodies.

A large number of antibodies of various specificities, isotypes and subclasses are found in human serum. The total concentration of all immunoglobulins in the serum is 15-20 mg / ml; ^' That means that approx. 100 g of immunoglobulins of various specialties circulate continuously in the blood. It is not possible to specify the exact number of all antibodies with different specificity, the repertoire of different B cell clones in a human is around 10 ⁹ . A particular antibody can generally bind different, similar antigens, albeit with different affinity and avidity. _T he _I mmunsystem must endogenous regulatory mechanisms with the help obtain a homeostasis with respect to the distribution and importance of these different specificities upright. An essential mechanism for this is the "idiotypical network" (Ann. Immuno1. 125C: 373-89 (1974)). Against every idiotype of an antibody, which determines its binding specificity, there are anti-idiotypic antibodies, which bind to the idiotype of the first antibody in the sense of antigen recognition. According to this explanatory model, the interactions between the idiotype-specific receptors on lymphocytes are responsible for the regulation of the immune system. These interactions obviously actually take place, since it has been shown that in the course of an immune response, anti-idiotypic antibodies against the antibodies primarily induced by the immune response also arise. Since there are anti-idiotypic antibodies against every antibody, lymphocytes are generally not tolerant of idiotypes of antibodies.

There are several ways to interfere with the immune system.

1. Passive antibody therapy:

For therapeutic purposes, it is possible to deliver antibodies which are necessary to fulfill a specific function within an organism to this organism. This type of application is called passive immunotherapy and can be used in various medical indications, e.g. in cancer immunotherapy (Immunol. Today (2000), 21: 403), poisoning (Toxicon (1998), 36: 823; Therapie (1994), 49:41), infections (Clin. Infect. Dis. (1995 ), 21: 150). In these cases, antibodies can be used that either come from appropriately immunized animals or can be obtained from cells by immortalizing immunoglobulin genes using various biological or molecular biological techniques (e.g. hybridoma technique, phage display technique, etc.).

Passive antibody administration has the disadvantage that it does not have a long-lasting effect, since the effect decreases with the natural degradation of the administered antibodies in the recipient organism. 2. Active immunization:

To modulate the immune system, one can immunize with antigens. Antigens are molecules, molecular complexes or entire organisms to which antibodies can bind.

Not all antigens elicit an immune response, i.e. not all antigens are immunogenic. Certain small molecules are not registered by the immune system (haptens), such smaller molecules can be presented to the immune system in a suitable form, making them immunogenic. One such method is the coupling of the hapten to an immunogenic molecule, a so-called "carrier molecule".

Other non-immunogenic antigens are so-called "self-antigens", ie structures that the immune system recognizes as the body's own substances. Immunization with such antigens usually does not result in a specific immune response. In the case of tumor-associated antigens, the fact that these antigens are actually "self-antigens" is one of the greatest difficulties in developing a potent vaccine.

Active immunization against pathogens such as viruses or bacteria using complete antigens is only possible if the pathogens are weakened or killed. In the case of weakened pathogens, there is a risk of reversion, i.e. the weakened pathogens can become virulent forms again. A solution to such a problem could be the use of anti-idiotypic antibodies as surrogate antigen for immunization (Int. Arch. Allergy Immuno1. (1994), 105: 211).

Active immunization with anti-idiotypic antibodies has also been proposed for the treatment of allergies (Int. Arch. Allergy I unol. (1999), 118: 119).

Antibodies against the body's own antigens are present in the serum of every human being and are referred to as "natural autoantibodies". _A nti-idiotypic antibodies of such 'natural autoantibodies are involved in the regulation of these autoantibodies involved (Immunol Reviews (1989) 110: 135; Eur J. Immuno1 (1993)., 23: 783rd.).

Inadequate idiotypic regulation appears to play a role in a number of autoimmune diseases:

- Systemic lupus erythematosus (Autoimmunity (1994), 17: 149);

- Autoimmune thyroiditis (Eur. J. Immunol. (1993), 23: 2945);

- Systemic vasculitis (J. Autoimmunity (1993), 6: 221);

- Guillain-Barre syndrome (Clin. Immunol. Immunopathol. (1993), 67: 192);

anti-factor VII: C autoimmune disease (Proc. Natl. Acad. Sei., USA (1987), 84: 828).

Immunization with idiotypic antibodies that mimic autoantigens has already been carried out in autoimmune diseases (J. Rheumatol. (1999), 26: 2602). Peptides for the induction of anti-idiotypic antibodies (Proc. Natl. Acad. Sei., USA (1993), 90: 8747; Immunology (1999), 96: 333) have already been described.

Immunization in autoimmune diseases based on T cell receptors (J. Immunol. (1990), 144: 2167; US Pat. No. 6,090,387; US Pat. No. 6,007,815) has also already been proposed.

Active immunization is also used to protect against toxic substances (e.g. bacterial toxins). If the toxins are to be used as a vaccine, they must first be weakened or inactivated. Such inactivation can affect the effectiveness of the immune response. Anti-idiotypic antibodies as vaccines that mimic toxins have been proposed (Int J Clin Lab Res (1992) 22:28; Clin Exp Immunol. (1992) 89: 378; Immunopharmacology (1993) 26: 225).

It has also been proposed to use anti-idiotypic antibodies to immunize for the first time and then to achieve a higher specific immunization effect with a vaccine that would be too weak alone (Virology (1984) 136: 247). 3. Withdrawal of antibodies and immune complexes from the blood:

One way to remove antibodies from the blood is to use perfusion systems (US Pat. No. 5,122,112), which has been used primarily for autoimmune diseases (The Lancet (10/20/1979), 824). However, such extracorporeal immunoadsorptions represent a great burden and a high treatment risk for patients, so that this method is not suitable for broad clinical therapy.

Active immunization for modulation can currently be performed with certain antigens, which may either be too toxic or potentially infectious, or may be non-immunogenic. A partial solution to this problem is the use of anti-idiotypic antibodies for immunization. However, it is necessary to produce such antibodies either in cell culture or in vitro or to induce them in certain organisms and then to administer them to another organism.

It is therefore an object of the present invention to overcome disadvantages of the prior art and to provide treatment materials and methods for a multiplicity of diseases using the patient's immune system. In particular, efficient treatment strategies for autoimmune diseases, allergies and similar complaints are to be created.

The present invention therefore relates to a method for producing an autologous, antibody-containing vaccine, which is characterized by the following steps:

Providing an antibody-containing liquid from an autologous antibody-containing body fluid or from autologous cell or tissue preparations,

Treating the antibody-containing liquid with a solid support on which ligands are immobilized, which bind to a specific group of antibodies, with the proviso that no antibodies or fragments of the same idiotype are used as ligands, which are against tumor-associated antigens are directed

Obtain the antibodies that bind to the ligands come in, and

Processing the antibodies obtained into an autologous vaccine that contains an immunogenic amount of more than one microgram of antibody.

The vaccine thus obtained can now be administered to the patient in a suitable manner. The method according to the invention solves the problems described at the outset by using precisely these "target" antibodies from the same organism to induce antibodies against a "target" antibody in an organism. Therefore, in vitro cultivation of cells for the production of the antibodies is not necessary, nor is production in a foreign donor organism. The “target” antibodies are, for example, abl antibodies with a specificity for an antigen, if appropriate for the induction of antiidiotypic antibodies, or ab2 antibodies, that is antiidiotypic antibodies, for generating an immune response, if appropriate directed against the antiidiotypes, that is to say equally against the antigen.

Nevertheless, it is no longer necessary to obtain the antibodies obtained according to the invention from a pool or plasma pool (cf. Zouali et al., J. Immunol. 135 (2) (1985), pages 1091-1096). In contrast to such antibody preparations from pools, in which antibodies are collected from different individuals, 100% autologous antibody-containing vaccines can be produced with the antibody preparations according to the present invention, their effect in the idiotypic network of the. individual is completely specific and can therefore be more effective.

This enables an individually specific modulation of the immune system in a targeted manner for the desired purpose. By shifting the immunological equilibrium by administering an autologous vaccination with a vaccine produced according to the invention, selective stimulation of those B cells that produce antibodies with a specific specificity is ensured. The specificity can be determined by the type of isolation of the antibodies (by the choice of ligands) from an individual. _B evorzugterweise the antibody-containing body fluids, of course, blood, serum, lymphatic fluid, Cere- are brospinalflüssigkeit, colostrum, mucosal body fluids such as vaginal or nasal discharge, malignant effusions, feces or urine, but it can just as well autologous cells or tissue preparations obtained by biopsy can be processed with a variety of methods known per se for liquids containing antibodies to be used according to the invention. Above all those body fluids with a particularly high antibody content are used according to the invention as starting materials, with human serum or plasma being of course particularly preferred.

Decisive for the specificity of the vaccines obtained according to the invention in influencing the idiotypic network is of course in each case the choice of the ligand in the present immunoaffinity purification. Specific monoclonal or polyclonal antibodies or antigens can be used to obtain specific antibody fractions. Monoclonal antibodies or derivatives thereof can be obtained by methods known per se, e.g. Hybridoma technology, phage display technology or recombination can be produced. (Immunology Today, 2000, 21: entire issue 8). However, idiotype-independent ligands that can bind certain specific subclasses or subtypes of immunoglobulin, e.g. one or more of IgGl, IgG2, IgG3 or IgG4 or IgA, IgG, IgM, IgE or IgD. Furthermore, ligands can be selected which recognize a certain group of antibody fragments or chains, for example at least parts of the lambda or kappa chains, Fc or Fab fragments. Likewise suitable ligands selectively bind not only antibodies but also corresponding isotypes or paraglobulins.

The method according to the invention can also be combined with an identical method for producing an autologous vaccine, antibodies or fragments thereof with the same idiotype that are directed against tumor-associated antigens and / or antibodies being used as ligands. For a simple process, it is preferred that the corresponding ligands are used as a mixture. More complex procedures Ren include the consecutive or parallel treatment of the body fluid with the different ligands. In this way, for example, a specific antibody fraction can be obtained from serum, with a specificity for cellular adhesion proteins and / or Lewis Y carbohydrate structures, or with a specificity for B-cell lymphoma, which is then immediately made available as an autologous vaccine formulation.

Autologous vaccines produced according to the invention which contain antibodies which occur in connection with B-cell lymphoma contain in particular only certain subclasses or fractions of the serum fraction containing IgG in order to ensure the targeted immune response.

For the isolation according to the invention, antibodies or antibody fragments such as e.g. Fc or Fab fragments can be used. However, ligands can also be other substances to which immunoglobulins can bind, for example ligands for the chromatographic purification of immunoglobulins, affinity peptides, affinity polypeptides, proteins such as protein A or protein G, or ionic structures which are also used, for example, for ion exchange chromatography.

When selecting the suitable immobilized ligands, care is usually taken to ensure that undesired bleeding of the ligands or of ligand-antibody complexes into the isolated antibody fraction obtained is avoided. In some cases, serial cleaning of the antibodies may also be appropriate in order to separate any contaminants from the immobilized ligands. Bleeding out of the material can also be advantageous if certain ligand-antibody complexes are desired in the preparation.

The production of a particularly high-quality vaccine can include further purification of the antibodies obtained, known methods such as chromatography, gel permeation, precipitation, separation on a liquid or solid phase, in particular on ferromagnetic particles, or ultra / diafiltration.

In the course of the processing, it may also be necessary to formulation as a durable solution, for example by adding preservatives or complexing substances or adjuvants. A storage-stable embodiment of the autologous vaccine produced according to the invention is particularly desirable if the patient is to be immunized several times with the same preparation at intervals.

On the other hand, the administration of freshly produced vaccines can have the advantage that changes in the immune system are taken into account and the occurrence of escape mutants, for example of antibody-producing cells or infectious agents, can be largely prevented. The simple processing of the antibodies obtained into a vaccine is suitable for this, which in the best case can be done on site. For example, the vaccine produced according to the invention can be used immediately after taking blood within one working day, or even during patient treatment.

Another embodiment of the method according to the invention relates to the depletion of components of the body fluid that are undesirable in the vaccine. For example, ligands can be chosen which do not selectively bind certain antibodies, but which do, however, have accompanying substances in order to then obtain the specific antibodies from the unbound fraction.

According to the present invention, individuals are understood to mean individual human or animal organisms which have body fluids or tissues which contain antibodies. The production according to the invention is of course preferably used in vertebrates, particularly preferably mammals, in particular humans.

The antibodies can be isolated from animal body fluids which contain antibodies (for example human serum) by means of immunoaffinity purification by methods known to the person skilled in the art (Clin. Chem. (1999), 45: 593; J. Che. Technol. Biotechnol 48: 105 (1990). Solid phase immunoaffinity purification is particularly preferred. A specific ligand or a mixture of different ligands is immobilized on a solid phase. The solid phase can be a membrane, a gel, be a chromatography material or similar material to which ligands can be coupled without any substantial loss of the specific binding properties of these ligands (Mol. Biotechnol. (1994) 1:59).

A finished set can also be made available for the immediate production of the vaccine according to the invention. This set contains the following components: a) a device with ligands for binding a certain group of antibodies from an antibody-containing liquid of an individual, b) a means for obtaining the antibodies which bind to the ligands, and c) an agent for processing the antibodies obtained into an autologous vaccine.

The device a) is in particular a container, a device or an automat for manual or automatic actuation. Device a) contains the ligands which are optionally immobilized on a solid support. A buffer can also be included which enables the antibodies to be adsorbed after the body fluid has been absorbed into the device under controlled conditions.

The agent b) comprises substances or solutions of substances for washing or cleaning or desorbing the antibodies. These include wash buffers and / or elution buffers. In addition, a formulation agent including a possible adjuvant is also provided in the set according to the invention, provided that this is not already included as b) in the agent for obtaining the antibodies.

It has been shown, for example, that antibodies can be adsorbed on a sparingly soluble aluminum compound, whereupon they can be washed in a simple manner and in a single device, re-buffered and packaged into a finished vaccine which already contains the aluminum compound as an adjuvant. Then namely only a single means for obtaining the antibodies and processing the vaccine instead of separate components b) and c) in the set according to the invention needed. Otherwise, additional aids, such as washing and buffering substances, can be provided in the set.

Suitable ligands can also be attached to magnetic beads, which can be easily located or oriented or positioned using a magnet. A specific ligand is bound to ferromagnetic particles, for example, and placed in a sterile container to hold the body fluid. If a magnetic transport part or rod is also stored in the container, the particles on which the antibodies are bound from the body fluid can be collected on the transport part by switching the magnet on and off, for example by means of electrical pulses for actuating an electromagnet. The particles can then be separated, preferably while maintaining the magnetic field. The magnetic field can be removed again during a washing process or antibody desorption. Afterwards, immobilization of the particles on the magnets is again expedient in order to separate or obtain the washing solution or the desorbed antibodies.

A particular embodiment of a kit for producing an autologous vaccine comprises sterile, endotoxin-free containers, preferably "single-use" containers, which are intended for single use, such as syringes, which are optionally connected to one another and are equipped with a septum (see see Fig. 4).

For example, the first container comprises the ligands required for the adsorption of the antibodies, bound to ferromagnetic particles (“beads”). Commercially available "beads" include activated porous glass beads such as Prosep (Millipore, Durham, UK), Dynabeads (Deutsche Dynal GmbH, Hamburg, Germany) and the same material from Miltenyi (Bergisch Gladbach, Germany). An adsorption buffer is also placed in this container or separately. Human serum is introduced via a septum. There is also a suitable magnet in or on this vessel for immobilizing the "beads" after adsorption, possibly washing and desorption. A defined amount of washing buffer is introduced through a septum. Through a frit then the washing solution is applied again. The antibodies can be eluted in a separate elution vessel with elution buffer, into which the loaded beads are placed. After elution of the antibodies, the "beads" are separated by immobilization on the transport part and removal of the same from the solution. Then a formulation agent is added through a septum. The formulated solution is introduced into a syringe and is ready for use on the patient. The individual containers are each equipped with one or more septa for the transfer of solutions or suspensions, as well as frits for phase separation.

According to a special embodiment, the set is made available as a container which contains the ligands and the agents b) and c). In the case of a single agent instead of two different agents b) and c), it may be advantageous to use this agent together with the ligands in a formulation. For example, a carrier of ligands can be selected that is also used for the production of antibodies and has an adjuvant effect. Such a carrier is a poorly soluble aluminum compound such as AluGel or aluminum hydroxide. Although these ligands are then contained in the pharmaceutical preparation together with the antibodies obtained, this is quite desirable in the case of an antibody ligand which itself acts as a vaccine antigen.

Antibodies from the liquids of individuals to the ligands can be bound in a mixed bed ("batchwise") or in a flow-through method. Immunoaffinity cleaning can be carried out automatically on a chromatography apparatus or by means of a manual process; however, it is also conceivable that the method is carried out manually, automatically or semi-automatically using a simple device which contains the immobilized ligands.

Ultimately, it is only necessary for the isolation of antibodies from an individual according to the invention that the desired antibodies can essentially be separated from the body from undesired other substances. It is conceivable that this task can be accomplished by separation processes other than immunoaffinity purification, such as described above, can be solved, such as by reacting ligands with the antibodies and then separating the specific immune complexes from the substances not complexed with the ligands. The antibodies can also be bound to or obtained from the ligands in the liquid phase, colloidal solution, emulsion or by so-called "immune affinity partitioning".

Accordingly, the present invention also relates to a method for producing an autologous antibody-containing vaccine, characterized by the following steps:

Providing antibody-containing liquid from an individual,

Treating the liquid with ligands that bind to a specific group of antibodies, with the proviso that no antibodies or fragments of the same idiotype are used as ligands which are directed against tumor-associated antigens,

Separation of all substances that do not bind to the ligands,

Treating the antibodies bound to the ligands with an eluent so that the antibodies bound to the ligands are eluted,

Recovering the antibodies that bind to the ligands in an eluate, and

Processing of the eluate obtained into an autologous vaccine which contains an immunogenic amount of more than one microgram of antibody.

In essence, the vaccine according to the invention downregulates unwanted antibody activities. Inhibiting antibodies are, for example, a "target" and, according to the invention, can be isolated and formulated into a vaccine against these undesirable inhibiting antibodies. Essentially, the vaccine produced according to the invention is used for prophylactic and / or therapeutic use in clinical pictures which are associated with tumor diseases, autoimmune diseases, allergies or infectious diseases. Unwanted immune reactions that can occur in the course of transplants are another area of indication. _S o can be about patients to be treated develop antibodies to sperm and therefore have an acquired sterility. If these autologous antibodies are removed from a body fluid, such as vaginal secretions, and are used according to the invention to produce a vaccine, this vaccine can be used immediately to treat the patient in order to suppress the unwanted antibodies. The vaccine preparation obtained contains, in particular, IgA antibodies which, above all, can be taken up again via the mucosa. The preferred administration is therefore nasal or vaginal.

The rhesus factor intolerance reaction of patients can also be treated with the help of an antibody vaccine produced according to the invention. Women who are rhesus negative and who have been in contact with the blood or tissue of rhesus positive people develop antibodies against this rhesus factor. For example, a rhesus-negative patient who was pregnant with a rhesus-positive fetus may develop antibodies to the rhesus factor. This has to be suppressed in order to avoid intolerance reactions during a second pregnancy with a rhesus factor positive child. Antibodies against the rhesus factor are therefore obtained from serum according to the invention and formulated into an immunogenic vaccine. Active immunization is preferably carried out as prophylaxis before a planned pregnancy.

The transplantation of allogeneic material, such as bone marrow or stem cells, can also be supported by a vaccine produced according to the invention. Any rejection reactions by HLA antigen structures can be suppressed by administering a vaccine that contains the autologous antibodies against the foreign HLA.

The preparation of xenografting is another area of application to prevent possible intolerance reactions due to the graft. High titers of natural antibodies against the well-known α-gal epitope / as found in serum in humans are partly responsible for acute _A bstoßungsreaktionen against xenografts or allografts. These natural anti-α-Gal antibodies are formulated with the aid of the method according to the invention into a vaccine and administered to the patient who is being prepared for the transplantation of tissue, bone marrow or stem cells. The downregulation of the anti-Gal activity should help to avoid rejection reactions. Lowering the titer of the circulating anti-α-Gal antibodies by immunization with autologous anti-α-Gal antibodies should allow a significant increase in the survival time of xenografts.

The selection of the ligands for the isolation of antibodies according to the invention depends on the corresponding application. Some applications are mentioned below as examples:

• Use in autoimmune diseases:

Auto-antigens as ligands can be used, for example, to isolate autoimmune-specific antibodies from an individual. Antibodies obtained in this way can, after use in accordance with the invention, produce an immune response in an individual, which in particular down-regulates the production of the specific autoantibodies by idiotypical interactions.

The exact specificity of the autoreactive antibodies is not known in many autoimmune diseases. However, it is not absolutely necessary for autoantigens to be used as ligands for isolating autoimmune-specific antibodies according to the present invention. In the case of an autoimmune disease, an antibody specificity can be extremely disproportionate, so that by cleaning the total immunoglobulin fraction (according to known biochemical methods) from an individual and then formulating it as a vaccine with subsequent application to the donor individual, above all anti-idiotypic antibodies against the disproportionately represented antibody specificity.

For example, patients with disease states that are associated with inhibiting antibodies against coagulation factors, insulin or rheumatoid factors, with a Treated vaccine produced according to the invention. The committed ^¬ set vaccines containing antibodies against the inhibiting antibodies or rheumatoid factor.

• Use for allergies:

To produce a patient-specific antibody vaccine against allergies, e.g. anti-IgE antibodies or parts of such antibodies with the same specificity are conceivable. However, allergens or parts of allergens are also conceivable as ligands.

• Use with toxic substances:

To purify antibodies from an individual that can trigger a toxin-specific immune response, toxin-specific antibodies can be used as ligands. In many cases, such antibodies are available as monoclonal. However, it is also conceivable that it is not antibodies but rather other molecules which can bind very specific specific toxins (for example bacterial toxins or low molecular weight poisons) are used as ligands for cleaning.

Instead of only one ligand species, two or more ligands with different specificity can of course also be immobilized on the solid support with the present method and in this way antibodies can be obtained in preparations with specificities which are enriched or depleted in terms of several binding properties (antibodies with different specificity). Similarly, the serial connection of several immunoadsorption steps with different specificities is preferred in the production of multi-specific vaccines according to the present invention.

It is also possible to provide a solid support with several different ligands, all of which are directed to the same or similar target substance (in the simplest case a polyclonal mixture of antibodies against a specific class of antibody). Nevertheless, different monoclonal antibodies against one and the same target structure can, for example, also be present on the solid support. will see.

Particularly preferred ligands according to the present invention are autoantigens such as e.g. double-stranded DNA to treat patient-specific antibodies against ds-DNA from patients with systemic lupus erythematosus (SLE). Factor VIII or parts thereof can be used as a ligand to isolate strongly factor VIII-binding antibodies from an individual and to down-regulate the pathogenic anti-factor VIII reactivity of a patient with the vaccine formulated therefrom (Semin. Thromb. Hemost. (2000) 26: 151) , Individual antibodies can be purified from patients with myasthenia gravis by immunoaffinity purification with acetylcholine receptor or parts thereof (Proc. Natl. Acad. Sci. USA (1993) 90: 8747), which according to the invention can be vaccinated back to the same patient as an autologous vaccine.

Insulin as a ligand can be used in the production of an autologous vaccine against autoimmune diabetes (type insulin-dependent diabetes mellitus) (Diabetes Metab. Res. Rev. (2000): 16: 338).

Myelin basic protein (MBP) or parts thereof can be used as a ligand in the production of an autologous vaccine for multiple sclerosis patients or patients with other immunologically related neurological disorders (J. Neuroimmunol. (2001) 113: 163).

Various gangliosides can be used as a ligand (in patients with Guillain-Barre syndrome (Intern. Med. (1997) 36: 599) and other neuropathies.

The advantage of this autologous type of immunization lies in the inherent consideration of the patient-specific idiotypes.

Another preferred ligand according to the present invention is an anti-human IgE antibody with which very specific IgE fractions can be purified and which in turn can be administered to the patient in a suitable, immunogenic form in order to inhibit specific gE-producing cells in the patient , Nature- parts of specific anti-IgE antibodies, provided that they still have the desired specificity, can also be used as a ligand.

In the field of allergy, it is conceivable to use very specific allergens or parts thereof or anti-idiotypic antibodies or other molecules that mimic allergens as ligands.

Since the immune response induced by vaccination with autologous antibodies is due to the binding region of these antibodies, i.e. is determined by their idiotype, in principle fragments or derivatives of these antibodies can be used for immunization instead of intact antibody fractions, as long as they contain the idiotype of the respective starting antibody. The term “antibody” therefore also includes fragments or derivatives of such antibodies with the same binding specificity. The following may be mentioned as examples, but not restricted to these: F (ab) 2 'fragments, F (ab)' fragments, which e.g. can be produced by known biochemical methods (for example by enzymatic cleavage). The term "derivative" includes e.g. Antibody derivatives that can be produced according to known chemical or biochemical methods, e.g. Antibodies amidated with fatty acids on free amino functions in order to increase the lipophiles for incorporation into liposomes. In particular, the term also includes products that can be produced by chemically coupling antibodies or antibody fragments with molecules that can enhance the immune response, such as e.g. Tetanus toxoid, Pseudomonas exotoxin, derivatives of Lipid A, GM-CSF, IL-2, IL-12, C3d.

The shift in the immunological balance caused by a first vaccination can be further strengthened by repeating this process, for example, a few weeks after the first autologous vaccine has been obtained by immunoaffinity cleaning, body fluid, for example blood, can be drawn off again and an autologous vaccine can be prepared and administered again. This also ensures that the respective status of the immunological balance in the individual vaccine is always taken into account. This process can be carried out again and again at suitable intervals (for example, every 4-8 weeks Chen, subsequently every 6 months), according to a follow-up check of the immune status of the respective patient through appropriate specific testing. The new composition and method for vaccination with autologous antibodies described here is fundamentally suitable for both therapeutic and prophylactic purposes.

A general advantage of the strategy of individual autologous vaccination described here lies in the fact that the immunological status of the individual in relation to the idiotypic network is taken into account, since the corresponding vaccine is in each case derived from the individual body fluid, e.g. Serum that is produced. Furthermore, the immunized individual does not come into contact with any foreign antigens, but is treated in a suitable form only with the body's own constituents, which bring about a modulation of the immunological balance.

A special application is in the treatment of patients who have developed specific antibodies due to immunization. The so-called hyperimmune serum is then used to produce an autologous vaccine in order to provoke an immune response against the autologous antibodies at a given time.

In a preferred embodiment, the antibodies obtained by immunoaffinity purification are formulated with a suitable vaccine adjuvant.

As is usual with vaccines, the autologous antibody fractions or their fragments and derivatives can be formulated together with vaccine adjuvants. Such adjuvants increase the immune response. Examples of adjuvants, but not limited to these, are: aluminum-containing adjuvants, in particular aluminum hydroxide (for example aluminum gel), derivatives of lipopolysaccharide, Bacillus Calmette Guerin (BCG), saponin and derivatives thereof (for example QS-21 ), Liposome preparations, formulations with additional antigens against which the immune system has already given a strong immune response, such as tetanus toxoid or components of influenza viruses, possibly in a liposome preparation. To boost the immune response, the vaccine preparation can also be administered with appropriate, preferably human, cytokines that support the development of an immune response. Granulocyte macrophage stimulating factor (GM-CSF) should be mentioned here in particular, if not exclusively. This cytokine stimulates an efficient immune response by activating antigen-processing cells (eg dendritic cells).

If appropriate, the autologous antibody fractions can also be incubated with autologous, ex vivo-cultivated dendritic cells according to known and published methods. The dendritic cells pulsed in this way are then re-administered to the individual concerned. In this way, a particularly efficient immune response can be achieved.

In a preferred method according to the present invention, working up the antibody eluates accordingly includes adding a substance selected from the group of adjuvants, in particular aluminum-containing adjuvants, lipopolysaccharide derivatives, Bacillus Calmette Guerin, liposomes or QS-21 ( further preferred adjuvants are described, inter alia, in Singh et al., Nat. Biotechnol. 17 (1999), pages 1075-1081), immunostimulating cells, in particular dendritic cells or other antigen-presenting cells, active agents, preferably cytokines, in particular granulocyte-macrophage-stimulating factor, formulation auxiliaries, in particular buffer substances, stabilizers or solubilizers, or mixtures of these substances.

In a preferred embodiment of the method according to the invention, the antibodies contained in the composition are mixed with an adjuvant and then subjected to a heat treatment, preferably at a temperature of above 80 ° C., in particular between 90 ° C. and 130 ° C. The adjuvant used is preferably an aluminum-containing adjuvant. It is possible that such a heat treatment denatures the protein antigen, but the immunogenic parts of the protein can be presented to the immune system in the correct form by binding to the adjuvant. But it is not absolutely necessary that Denature proteins to get the benefits of heat treatment. It is known that the thermal denaturation of proteins depends not only on the temperature, but also on the time to which the protein is exposed to this temperature. In addition, other physico-chemical parameters such as ionic strength, ion composition, pH, type and amount of the active surface in the mixture are responsible for the denaturation of a protein. Conditions are known and can easily be optimized by the person skilled in the art for any eluate in which the antibodies are not or not completely denatured and / or other effects, such as weaker desorption from the surface of the adjuvant, can be used.

Another advantage of such a way of producing a vaccine formulation with an adjuvant and the subsequent heat treatment is that infectious pathogens could be weakened or inactivated throughout the formulation. This advantage can play a role in the manufacture as well as in the storage and distribution of the vaccine formulation. There is therefore greater certainty with regard to known and unknown pathogens of communicable diseases. With appropriate packaging, it is also possible to fill without preservatives, since the microbial preservation of the vaccine is carried out by heat.

Another advantage of such a formulation is the possible increased immunogenicity of the antibodies, since the heating can cause the antibodies to be at least partially denatured. This increased antigenicity can increase the immunogenicity in particular in the case of proteins which the immune system would recognize as their own proteins.

Another advantage is the additional stabilization of the antibody-adjuvant complex by heat inactivation, i.e. desorption of the protein antigen is no longer rapid, as in antigen adjuvant formulations that have not been heat treated. This advantage also allows a longer time interval between the individual immunizations.

Accordingly, a particular embodiment of the invention relates to According to the method, a method in which a protein denaturation step, in particular a heat treatment, is carried out, in which the three-dimensional structure of the proteins contained in the eluates are at least partially changed, their immunogenic properties preferably being enhanced.

The composition prepared according to the invention can be administered by conventional methods, e.g. as a vaccine by subcutaneous, intramuscular, or intradermal injection. Another type of administration works via the mucosal route, such as vaccination by nasal or oral administration.

The present invention also relates to pharmaceutical compositions containing antibodies obtained from animal body fluids containing antibodies by immunoaffinity purification for use as autologous vaccines.

Furthermore, the present invention relates to a method for therapeutic or prophylactic vaccination against autoimmune diseases, infectious diseases, various intoxications and allergies.

Accordingly, the present invention also relates to an autologous vaccine which can be obtained by the method according to the invention.

The present invention also relates to a method for treating individuals, in which a preparation according to the invention is administered in an efficient amount, preferably a few micrograms to ten grams, to the individual from whom the body fluid has been removed. The efficiency has to be assessed especially with regard to immunogenicity. It has proven useful to use at least one microgram of antibody in a vaccine dose, which can be administered in finished dose units from 0.01 to 1 ml, preferably in the range from 0.1 to 0.5 ml. The preferred amount is determined especially according to the supportive effect of adjuvants and ranges from 3 micrograms to 1 gram, more preferably 10 micrograms to 750 micrograms, most preferably 250 micrograms to 500 Micrograms.

This treatment method is especially useful for autoimmune diseases such as Systemic lupus erythematosus, autoimmune thyroiditis, systemic vasculitis, Guillain-Barre syndrome and anti-factor VII: C autoimmune disease, for allergies, for tumor diseases or for the prophylaxis of intolerance reactions in the context of transplants and poisoning (such as with bacterial toxins) are particularly effective.

According to a further aspect, the present invention also relates to the use of an autologous antibody preparation for producing an agent for immunomodulation.

The invention is explained in more detail with reference to the following examples and the drawing figures, to which, however, it should not be restricted.

Show it:

Fig. 1: Scheme of obtaining the vaccine.

2: Change in the specific antibody reactivities after immunization with an autologous vaccine, produced via anti-bovine serum albumin as ligand or Sepharose.

3: Change in the specific antibody reactivity after immunization with autologous vaccine, produced via mouse IgG2a as a ligand.

Fig. 4: Vascular system for the production of the autologous vaccine using magnetic particles

Example:

Example 1 :

This example is intended to illustrate that it is possible to boost the immune system against any protein (in this case Bovines Serum albumin, BSA) to be specifically modulated.

Two groups of 3 rabbits each were immunized with a vaccine formulation prepared according to the invention.

The autologous vaccine for the first group was prepared by purifying rabbit serum immunoglobulin on an affinity chromatography column (rabbit anti-BSA immobilized on Sepharose). The autologous vaccine for the second group was prepared by purifying rabbit serum immunoglobulin over another affinity chromatography column (Sepharose without specific ligands).

The immunoglobulins obtained in this way were formulated as vaccines by adsorption on aluminum hydroxide gel and administered subcutaneously to the respective rabbits.

Blood Collection and Vaccination Scheme:

Day -21: Serum Collection Day -14: Serum Collection Day -7: Serum Collection

The vaccine was purified from the pool of sera on days -21, -14 and -7

Day 0: Serum collection

Day 0: immunization, subcutaneously

Day 14: Serum Collection Day 21: Serum Collection

Preparation of the affinity matrix:

In a first step the rabbit anti-BSA serum was purified:

For this purpose, the polyclonal rabbit anti-BSA antibodies (in 0.1M glycine / HCl buffer, pH: 2.9; volume = 4 ml) against dialysis buffer (0.1 M NaHC0 ₃ + 0.5 M NaCl pH = 8.0) dialyzed (Slide-A-LyzerÖ 10K; Pierce / USA). Method:

• Dialysis was carried out in an 800 ml beaker with magnetic stirrers on a magnetic stirrer at 4 ° C, the dialysis buffer being renewed four times.

• The samples were then concentrated by centrifugation (with Centricon 10K (Amicon)), final volume: 0.4-0.6 ml.

Immobilization to activated CH Sepharose:

Materials:

• Activated CH-Sepharose 4B; Pharmacia Biotech (Code No.: 17- 0490-01)

Ligand: polyclonal anti-BSA antibody (6, 6 mg / ml, in coupling buffer)

Coupling buffer: 0.1 M NaHC0 ₃ + 0.5 M NaCl, pH = 8.0

1 mM HCl

• 1 M ethanolamine solution

■ 0.1 M Tris-HCl (tris (hydroxymethyl) aminomethane) buffer, pH = 8.0

0.1 M Tris-HCl buffer + 0.5 M NaCl, pH = 8.0

0.1 M acetate buffer + 0.5 M NaCl, pH = 4.0

Coupling method:

0.25 g freeze-dried CH-Sepharose 4B was suspended in approx. 20 ml ImM HCl, washed with 50 ml 1 mM HCl, then washed with 50 ml coupling buffer. The Sepharose is transferred to a 50 ml Falcon tube and the antibody solution is added. A ratio of gel: buffer = 1: 2 results in a suitable suspension for the coupling. The suspension was shaken for about 1 hour. Excess antibody was removed by washing with 3 x 10 ml coupling buffer. Remaining active binding parts were blocked by incubation on a shaker with 1 M ethanolamine for one hour. This was followed by an incubation of the Sepharose with 0.1 M Tris-HCl buffer (pH = 8.0) on the shaker and the following washing cycle: first washing with 0.1 M acetate buffer (pH = 4.0) + 0, 5 M NaCl, then with 0.1 M Tris-HCl buffer (pH * = 8.0) + 0.5 M NaCl. The wash cycle was carried out 3 times. Preparation of the Affinity Matrix for the Second Group of Rabbits:

The affinity matrix for the second group (control group) was prepared according to the same procedure as described above. Only buffer was used instead of the antibody solution. Activated Sepharose is only blocked with ethanolamine:

Production of autologous vaccines:

In each case, 10 ml of serum from the corresponding rabbits (pool of days -21, -14, and -7) were purified with the respective affinity matrices (0.5 ml column volume).

Materials:

Application buffer: PBS + 0.2 M NaCl, pH = 7.2 Elution buffer: 0, IM glycine / HCl buffer, pH = 2, 9

Method:

Application to the column was carried out with an incubation period of 30 minutes at + 4 ° C. Washing was carried out with application buffer (1 washing step = 5 ml; 5 washing steps). Elution was carried out with 1 ml of elution buffer.

The eluate was neutralized with carbonate solution (0.5M NaHC0 ₃ ), the proteins purified in this way were analyzed by means of size separation chromatography.

Größentrennungschromatographie:

Chromatography was performed on a ZORBAX GF-250 column on a DIONEX HPLC system. The following immunoglobulins were used as quantitative standards:

- human IgG standard (20 mg / ml; sandoglobulin, 3.590.009.0)

- human IgM standard (1.1 mg / ml; SIGMA, cat.1-8260) column: ZORBAX GF 250 (PN: 884973.901)

Running buffer: 220 mol NaP0 buffer, pH = 7.0 + 10% acetonitrile Flow rate: 1,000 ml / min Wavelength: 214 nm Bandwidth: 5 nm Injection volume: 50 μl

Formulation with aluminum hydroxide:

The formulation of the purified, neutralized immunoglobulins as a vaccine was carried out according to the following procedure:

A Centricon ultrafiltration unit (Centricon 10K from Amicon, USA) was used for each vaccine. The ultrafiltration unit was first washed (centrifuging through 1 mM Na phosphate buffer, 0.86% NaCl, pH 6 (NBK)). 400 μl of buffer and alhydrogel (27 μl (for 400 μl), Superfos, Denmark) were then introduced, the neutralized eluate was added, centrifuged and washed (with 5 ml of buffer), so that the final volume was approximately 300 μl. This was followed by resuspending the vaccine and filling up to 396 μl with buffer, adding 4 μl of thimerosal stock solution (10 mg / ml, Sigma), 350 μl of which were filled sterile. The protein concentrations of the individual vaccines were 40-50 μg each.

BSA-EL SA:

The following samples were analyzed in the ELISA: Day 0 and a pool of day 14 and day 21. ELISA plates (NUNC, Maxisorp (F = 96); Denmark) were coated with 100 μl BSA solution (per well). (BSA solution: BSA (SIGMA Cat.No A-7638); 10 μg / ml in coating buffer). It was incubated at 37 ° C for 1 hour. After washing, blocking was carried out with 5% dry milk in PBS (200 μl / well). Incubation: 30 min at 37 ° C.

Washing scheme:

- After coating, blocking and sample incubation: 6 times with washing buffer, after 4 times incubated for one minute.

- after conjugate: 4 times with washing buffer, 2 times with coloring buffer.

Serum samples were serially diluted (in 2% dry milk / PBS). The sample dilutions (100 μl / well) were incubated for 1 hour at 37 ° C. cubed. A dilution series of the polyclonal rabbit anti-BSA serum, which had been used for affinity purification, served as positive control and as standard for a quantitative evaluation of the ELISA. After washing, the enzyme conjugate (Anti rabbit Ig HRP (Nordic Immunology, # 4694)) was applied in an appropriate dilution (1: 1000 dilution buffer) (100 μl / well). After an incubation of 30 min. At 37 ° C., the mixture was washed again, substrate was added (per well: 100 μl TMB Microwelll (BioFX, Cat-No: TMBW-0100-01, # 0034302)), and the reaction was stopped after appropriate staining (with 30% H ₂ S0 ₄ 50 μl / well), then measured the color in the photometer (450 nm). The titer for the half-maximum adsorption of each dilution series was determined. The relative titer change to zero serum (day 0; = time of immunization) for the individual rabbits is shown in FIG. 2. It can be seen that the rabbits which show an autologous vaccine which was produced by means of an anti-BSA affinity chromatography show a clear titer shift in the BSA ELISA.

Example 2:

This example should show that it is possible to specifically lower an already existing immune response in an individual. A rhesus monkey, which had been immunized with a monoclonal antibody (HE2, mouse IgG2a) and had developed a strong IgG immune response against mouse IgG2a, was used for this. The serum of this monkey was immobilized on an immuno-affinity column to ^* the mouse IgG2a (HE2) as a ligand cleaned. The immunoglobulins purified in this way were formulated as a vaccine on aluminum hydroxide and subcutaneously inoculated into the donor monkey. Blood was drawn at the time of immunization (before vaccination) and 2 weeks afterwards in order to determine the specific immune response against mouse IgG2a.

Material and methods:

Microtiter plates for ELISA: Immuno Plate F96 MaxiSorp (Nunc)

Coupling buffer: 0.1 M NaHC0 ₃

0.5 M NaCl pH 8.0

Cleaning buffer A: PBS + 0.2 M NaCl

Cleaning buffer B: 0.1 M glycine / HCl

0.2 M NaCl pH 2.9

Binding buffer: 15 mM Na ₂ CO ₃ 35 mM NaHCO ₃ 3 mM NaN ₃ pH: 9.6

PBS: 138 mM NaCl 2.7 mM KC1 6.5 mM NaHP0 ₄ pH 7.2

Wash buffer A: 2% NaCl

0.2% Triton X-100 in PBS

Wash Buffer B: 0.05% Tween 20 in PBS

Blocking buffer A: 5% fetal calf serum (heat inactivated) in PBS

Blocking buffer B: 1% bovine serum albumin

0.1% NaN ₃ in PBS

Dilution buffer A: 2% fetal calf serum (heat inactivated) in PBS

Dilution buffer B: PBS

Coloring buffer: 24.3 mM citric acid

51.4 mM Na ₂ HP0 ₄ pH: 5.0 Substrate: 40 mg o-phenylenediamine dihydrochloride

100 ml staining buffer 20 μl H ₂ 0 ₂ (30%)

Stop solution: 4 N H2SO4

Formulation buffer: 10% PBS, pH = 5.5

90% physiological NaCl solution

Execution:

The method for autologous vaccination described here was tested on a rhesus monkey that had a strong immune response against mouse IgG2a (0.5 mg mouse IgG2a (HE2) absorbed on 1.67 mg aluminum hydroxide in 0.5 ml 1 mM phosphate). Buffer, pH 6.0 / 155 mM NaCl) was inoculated subcutaneously to a rhesus monkey on days 1, 15, 29 and 57 respectively. The sera from different time points were tested for mouse IgG2a-specific antibodies by ELISA (see below). The antibodies were mainly of the IgG type at the end of the vaccination scheme. 10 ml of peripheral blood were taken from this rhesus monkey and serum was obtained therefrom. For immunoaffinity purification of the antibody fraction from the serum of this rhesus monkey, an immunoaffinity matrix was first prepared in accordance with the following instructions.

The whole procedure was carried out under sterile conditions: 7.5 g of CH-Sepharose 4B (Pharmacia) were suspended in 20 ml of 1 mM HCl for 15 minutes. The gel was then washed with 1 liter of 1 mM HCl and then with 200 ml of coupling buffer on a sintered glass filter AG3. 100 mg of murine antibody HE2 (mouse IgG2a) was dialyzed against 5 liters of coupling buffer and adjusted to 5 mg / ml with coupling buffer. This solution was mixed with the gel suspension in a sealed vessel. A gel: buffer ratio of 1: 2 results in a suspension suitable for coupling. This suspension was rotated at 4 ° C for 24 hours. The excess of the ligand was then washed away with 3 × 30 ml of coupling buffer. Remaining reactive groups were blocked by incubation for 1 hour at 4 ° C. with 1 M ethanolamine. Then the gel was left at room temperature for one hour. rotated with a 0.1 M Tris-HCl buffer. Finally the gel was washed with 3 cycles of alternating pH buffers. Each cycle consists of 0.1 M sodium acetate buffer, pH 4, with 0.5 M NaCl, and then 0.1 M Tris-HCl buffer, pH 8, with 0.5 M NaCl. The gel was stored at 4 ° C. The immunoaffinity cleaning of the antibody fraction from serum of a rhesus monkey was carried out according to the following procedure under sterile conditions: The immunoaffinity cleaning was carried out on an FPLC system (Pharmacia). 1 ml of the gel obtained according to the above instructions was filled into a Pharmacia HR5 / 5 column. 5 ml of serum were diluted 1:10 with cleaning buffer A. This solution was pumped over the column at 1 ml / minute and washed with cleaning buffer A until the UV baseline of the detector was reached again (280 nm). Bound immunoglobulins were then eluted with cleaning buffer B and the fraction neutralized with 1 M Na ₂ HP0 ₄ immediately after desorption. 50 μl of the antibody fraction purified in this way were analyzed on a size separation column (SEC, Zorbax 250 GF). 220 mM phosphate buffer, pH 7 + 10% acetonitrile was used as the eluent. The total amount of the antibody fraction was approximately 40 μg (determined by SEC compared to a standard). The antibody fraction thus obtained was tested in an ELISA for binding to antibody HE2 (which was used as a ligand for affinity purification): 100 μl aliquots of the mouse IgG2a antibody used for affinity purification (antibody HE2; solution with 10 μg / l in binding buffer) incubated in the wells of a micro titer plate for 1 hour at 37 ° C. After washing the plate six times with washing buffer A, 200 μl of blocking buffer A were added in each case and incubated at 37 ° C. for 30 minutes. After washing the plate as described above, 100 μl aliquots of the affinity-purified antibody fraction to be tested as well as normal human immunoglobulin in the same concentration were incubated as a negative control in dilutions of 1: 4 to 1:65 000 in dilution buffer A for 1 hour at 37 ° C. After washing the plate as described above, 100 μl of the peroxidase-conjugated goat anti-Huan-Ig antibody (Zymed) were added at a dilution of 1: 1000 in dilution buffer A and incubated at 37 ° C. for 30 minutes. The plate was washed four times with wash buffer A and twice with stain buffer. The antibody binding was detected by adding 100 μl of the specific substrate and the color reaction was stopped after about 3 minutes by adding 50 μl of stop solution. The evaluation was carried out by measuring the optical density (OD) at 490 nm (wavelength of the reference measurement is 620 nm). The affinity-purified antibody fraction shows a clear binding to the mouse IgG2a antibody, whereas normal human immunoglobulin practically does not bind.

The. Antibody fraction obtained by affinity purification was formulated with aluminum hydroxide as an adjuvant according to the following procedure:

3 ml of the antibody solution obtained after affinity chromatography (contains approx. 40 μg antibody) were mixed with 1 mg aluminum hydroxide (aqueous suspension; Alhydrogel, Superfos) and the suspension added in a "FILTRON" centrifuge tube (Microsep TM, cut-off 10KD) Centrifuge 4000 xg for 30 minutes. The mixture was then slurried twice with 1 ml of the formulation buffer and centrifuged at 4000 x g for 30 minutes. The suspension was made up to 0.5 ml with formulation buffer and the suspension thus obtained was filled sterile. The rhesus monkey, from whose serum the above autologous vaccine was obtained, was vaccinated subcutaneously in the back with this vaccine. Before this first vaccination, 5 ml of blood was taken for serum extraction (to determine the initial value for the characterization of the immune response). Two weeks later, 10 ml of blood were taken again to obtain serum.

The binding of the serum immunoglobulin of this immunized monkey to mouse IgG2a was determined as above in the ELISA. As can be seen in FIG. 3, the mouse IgG2a reactivity decreases after the immunization with the autologous vaccine.

Claims

Patent claim:

1. A method for producing an autologous antibody-containing vaccine, characterized by the following steps:

- Treating the antibody-containing liquid with a solid support on which ligands are immobilized, which form bonds with a specific group of antibodies, with the proviso that no antibodies or fragments of the same idiotype are used as ligands, which against Tumor or -associated antigens are targeted,

Obtaining the antibodies which bind to the ligands, and

- Processing of the antibodies obtained into an autologous vaccine which contains an immunogenic amount of more than one microgram of antibody.

2. A process for the preparation of an autoantibody-containing vaccine, characterized by the following steps:

Provision of antibody-containing liquid from an individual,

Treating the liquid with ligands which bind to a specific group of antibodies, with the proviso that no antibodies or fragments of the same idiotype are used as ligands which are directed against tumor-associated antigens,

- separation of all substances which do not bind to the ligands,

Obtaining the antibodies which bind to the ligands in an eluate, and

- Processing of the eluate obtained into an autologous vaccine that has an immunogenic amount of more than one microgram Contains antibodies.

3. The method according to any one of claims 1 or 2, characterized in that an autologous vaccine containing an immunogenic amount of antibodies in the range of 3 micrograms to 3 grams is produced.

4. The method according to any one of claims 1 to 3, characterized in that a means for binding antibodies are selected as ligands, which occur in autoimmune diseases.

5. The method according to any one of claims 1 to 3, characterized in that a means for binding antibodies are selected as ligands, which occur in allergic diseases.

6. The method according to any one of claims 1 to 3, characterized in that a means for binding. Antibodies which are directed against the α-gal epitope are selected as * ligands.

7. A method according to any one of ^claims' 1 to 3, characterized in that a means as ligands are selected for binding antibodies which are directed against human sperm.

8. The method according to any one of claims 1 to 3, characterized in that a means for binding antibodies are selected as ligands that are specific for B-cell lymphoma diseases.

9. The method according to any one of claims 1 to 8, characterized in that the body fluid is serum or plasma.

10. The method according to any one of claims 1 to 9, characterized in that the individual is a human.

11. The method according to any one of claims 1 to 10, characterized in that the ligands are selected from more than one type, in particular ligands that bind antibodies with different specificity.

12. The method according to any one of claims 1 to 11, characterized in that the ligands bind a certain subclass of immunoglobulins.

13. The method according to any one of claims 1 to 12, characterized in that the ligands bind certain immunoglobulin chains.

14. The method according to any one of claims 1 to 13, characterized in that antibodies, in particular monoclonal antibodies, antigens, autoantigens, haptens, allergens or mixtures thereof are used as ligands.

15. The method according to any one of claims 1 to 14, characterized in that working up the addition of a substance selected from the group of adjuvants, in particular aluminum-containing adjuvants, lipopolysaccharide derivatives, Bacillus Calmette Guerin, liposomes, saponins and derivatives thereof, immunostimulatory cells, in particular dendritic cells, active agents, preferably cytokines, in particular granulocyte macrophage stimulating factor, formulation auxiliaries, in particular buffer substances, stabilizers or solubilizers, or mixtures of these substances.

16. The method according to any one of claims 1 to 15, characterized in that a protein denaturation step, in particular a heat treatment, is carried out.

17. Autologous vaccine, characterized in that it is obtainable by a method according to any one of claims 1 to 16.

18. Use of an autologous vaccine according to claim 17 for the preparation of an agent for immunomodulation, in particular for downregulating undesirable antibody activities.

19. Set for the production of an autologous vaccine containing the components a) a device with ligands for binding a specific group of antibodies from an antibody-containing Liquid of an individual, b) a means for obtaining the antibodies which bind to the ligands, and c) a means for processing the obtained antibodies into an autologous vaccine.

20. Set according to claim 19, characterized in that component a) comprises a container for holding the ligands and the antibody-containing liquid.

21. Set according to claim 20, characterized in that the container contains the ligands bound to a solid support.

22. Set according to claim 21, characterized in that the container contains magnetic beads, and the set further comprises a magnet for locating the beads.

23. Set according to one of claims 19 to 22, characterized in that component b) comprises a means for purifying the antibodies.

24. Set according to one of claims 19 to 23, characterized in that component c) comprises an adjuvant.

25. Set according to one of claims 19 to 24, characterized in that component b) and c) contain a single agent for obtaining and working up the antibodies obtained and optional auxiliaries.

26. Set according to claim 25, characterized in that the agent contains a poorly soluble aluminum compound for obtaining and processing the antibodies obtained.

27. Set according to one of claims 19 to 26, characterized in that the device a) comprises a container containing the ligands, the agent b) and c).

28. Set according to claim 27, characterized in that the device comprises a syringe containing a vaccine and an adjuvant.