EP1370289A2 - Procede de production d'un vaccin contenant des anticorps autologues - Google Patents

Procede de production d'un vaccin contenant des anticorps autologues

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Publication number
EP1370289A2
EP1370289A2 EP02706509A EP02706509A EP1370289A2 EP 1370289 A2 EP1370289 A2 EP 1370289A2 EP 02706509 A EP02706509 A EP 02706509A EP 02706509 A EP02706509 A EP 02706509A EP 1370289 A2 EP1370289 A2 EP 1370289A2
Authority
EP
European Patent Office
Prior art keywords
antibodies
ligands
antibody
vaccine
autologous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02706509A
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German (de)
English (en)
Inventor
Gottfried Himmler
Hans Loibner
Helmut Eckert
Otto Doblhoff-Dier
Ralf Kircheis
Manfred Schuster
Erich Wasserbauer
Günter Waxenecker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ALTROPUS GMBH
Original Assignee
Igeneon Krebs-Immuntherapie Forschungs- und Entwicklungs-GmbH
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Filing date
Publication date
Application filed by Igeneon Krebs-Immuntherapie Forschungs- und Entwicklungs-GmbH filed Critical Igeneon Krebs-Immuntherapie Forschungs- und Entwicklungs-GmbH
Publication of EP1370289A2 publication Critical patent/EP1370289A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a method for producing a vaccine.
  • 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.
  • 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.
  • 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).
  • 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.).
  • Antigens are molecules, molecular complexes or entire organisms to which antibodies can bind.
  • haptens 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”.
  • 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.
  • 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.).
  • anti-factor VII C autoimmune disease (Proc. Natl. Acad. Sei., USA (1987), 84: 828).
  • 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).
  • 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.
  • 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:
  • 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.
  • B evorzugt the antibody-containing body fluids, of course, blood, serum, lymphatic fluid, Cere- are brospinalfactkeit, 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.
  • 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.
  • 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.
  • 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.
  • antibodies or antibody fragments such as e.g. Fc or Fab fragments can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits 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).
  • the first container comprises the ligands required for the adsorption of the antibodies, bound to ferromagnetic particles (“beads”).
  • 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.
  • 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.
  • the set is made available as a container which contains the ligands and the agents b) and c).
  • this agent may be advantageous to use this agent together with the ligands in a formulation.
  • a carrier of ligands can be selected that is also used for the production of antibodies and has an adjuvant effect.
  • a carrier is a poorly soluble aluminum compound such as AluGel or aluminum hydroxide.
  • 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.
  • 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".
  • the present invention also relates to a method for producing an autologous antibody-containing vaccine, characterized by the following steps:
  • 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.
  • 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.
  • 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.
  • 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 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 ⁇ -gal epitope / as found in serum in humans are partly responsible for acute A bommeungsre force against xenografts or allografts.
  • These natural anti- ⁇ -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- ⁇ -Gal antibodies by immunization with autologous anti- ⁇ -Gal antibodies should allow a significant increase in the survival time of xenografts.
  • 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.
  • 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 with a Treated vaccine produced according to the invention.
  • the committed ⁇ set vaccines containing antibodies against the inhibiting antibodies or rheumatoid factor are associated with inhibiting antibodies against coagulation factors, insulin or rheumatoid factors.
  • anti-IgE antibodies or parts of such antibodies with the same specificity are conceivable.
  • allergens or parts of allergens are also conceivable as ligands.
  • toxin-specific antibodies can be used as ligands.
  • such antibodies are available as monoclonal.
  • 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).
  • the serial connection of several immunoadsorption steps with different specificities is preferred in the production of multi-specific vaccines according to the present invention.
  • 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).
  • SLE systemic lupus erythematosus
  • 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).
  • gangliosides can be used as a ligand (in patients with Guillain-Barre syndrome (Intern. Med. (1997) 36: 599) and other neuropathies.
  • 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.
  • antibody 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the antibodies obtained by immunoaffinity purification are formulated with a suitable vaccine adjuvant.
  • the autologous antibody fractions or their fragments and derivatives can be formulated together with vaccine adjuvants.
  • adjuvants increase the immune response.
  • 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.
  • aluminum hydroxide for example aluminum gel
  • BCG Bacillus Calmette Guerin
  • 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 lipo
  • 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
  • This cytokine stimulates an efficient immune response by activating antigen-processing cells (eg dendritic cells).
  • 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.
  • 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.
  • 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 (19
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention relates to a method for therapeutic or prophylactic vaccination against autoimmune diseases, infectious diseases, various intoxications and allergies.
  • 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.
  • 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.
  • the present invention also relates to the use of an autologous antibody preparation for producing an agent for immunomodulation.
  • Fig. 1 Scheme of obtaining the vaccine.
  • Fig. 4 Vascular system for the production of the autologous vaccine using magnetic particles
  • 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.
  • BSA Bovines Serum albumin
  • 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.
  • the vaccine was purified from the pool of sera on days -21, -14 and -7
  • 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.
  • Tris-HCl (tris (hydroxymethyl) aminomethane) buffer, pH 8.0
  • the affinity matrix for the second 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:
  • 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.
  • 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 ⁇ l of buffer and alhydrogel (27 ⁇ l (for 400 ⁇ l), 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 ⁇ l.
  • BSA solution BSA (SIGMA Cat.No A-7638); 10 ⁇ g / 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 ⁇ l / well). Incubation: 30 min at 37 ° C.
  • Serum samples were serially diluted (in 2% dry milk / PBS). The sample dilutions (100 ⁇ l / 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 ⁇ l / well). After an incubation of 30 min.
  • 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.
  • Cleaning buffer B 0.1 M glycine / HCl
  • Binding buffer 15 mM Na 2 CO 3 35 mM NaHCO 3 3 mM NaN 3 pH: 9.6
  • Blocking buffer A 5% fetal calf serum (heat inactivated) in PBS
  • Blocking buffer B 1% bovine serum albumin
  • Dilution buffer A 2% fetal calf serum (heat inactivated) in PBS
  • Dilution buffer B PBS
  • 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 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.
  • 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 ⁇ l aliquots of the mouse IgG2a antibody used for affinity purification (antibody HE2; solution with 10 ⁇ g / 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 ⁇ l of blocking buffer A were added in each case and incubated at 37 ° C. for 30 minutes.
  • the antibody binding was detected by adding 100 ⁇ l of the specific substrate and the color reaction was stopped after about 3 minutes by adding 50 ⁇ l 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).
  • OD optical density
  • 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:
  • mouse IgG2a 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.

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Abstract

L'invention concerne un procédé permettant de produire un vaccin contenant des anticorps autologues, qui se caractérise en ce qu'il comporte les étapes suivantes : préparer un liquide contenant des anticorps, à partir d'un liquide organique contenant des anticorps autologues ou à partir de préparations cellulaires ou tissulaires autologues ; traiter le liquide contenant les anticorps avec un support solide, sur lequel sont immobilisés des ligands qui induisent des liaisons avec un groupe déterminé des anticorps, sous réserve que les ligands utilisés ne soient par des anticorps ou leurs fragments de même idiotype, dirigés contre des antigènes associés à des tumeurs ; produire les anticorps qui induisent une liaison avec les ligands ; traiter les anticorps obtenus pour former un vaccin autologue contenant une quantité efficace de l'ordre de quelque microgrammes à un gramme d'anticorps.
EP02706509A 2001-03-23 2002-03-20 Procede de production d'un vaccin contenant des anticorps autologues Withdrawn EP1370289A2 (fr)

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AT4702001 2001-03-23
AT0047001A AT410636B (de) 2001-03-23 2001-03-23 Verfahren zur herstellung eines impfstoffes
PCT/AT2002/000091 WO2002080966A2 (fr) 2001-03-23 2002-03-20 Procede de production d'un vaccin

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AT410637B (de) * 2001-06-01 2003-06-25 Igeneon Krebs Immuntherapie Verwendung von polyklonalen immunglobulinen
WO2005090403A2 (fr) * 2004-03-12 2005-09-29 Biovest International, Inc. Procede et appareil de purification de proteines
JP5415963B2 (ja) * 2007-02-21 2014-02-12 オスロ ユニベルシテートシケヒュース ホーエフ 新しい癌マーカー
EP2236520A1 (fr) 2009-03-31 2010-10-06 Leukocare Ag Composition de stabilisation pour biomolécules immobilisées
WO2022103871A1 (fr) * 2020-11-10 2022-05-19 Wyomingv Immune, Inc. Compositions thérapeutiques pour le traitement de la covid-19
CN114887047A (zh) * 2022-06-21 2022-08-12 苏州工业园区唯可达生物科技有限公司 基于ny-eso-1肿瘤抗原的疫苗及制备方法

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DE4436558A1 (de) * 1994-10-13 1996-04-18 Vitorgan Arzneimittel Gmbh Verfahren zur Herstellung von humanen Impfstoffen gegen allergieauslösende Substanzen
CA2205138A1 (fr) * 1994-11-16 1996-05-23 Baxter International Inc. Anticorps humains pour les peptides recepteurs de lymphocytes t et leur methode de preparation
DE19538641C2 (de) * 1995-10-05 2000-09-21 Privates Inst Bioserv Gmbh Patientenspezifische Immunadsorber für die extrakorporale Apherese und Verfahren für deren Herstellung
WO1998026086A1 (fr) * 1996-12-11 1998-06-18 University Of Florida Methodes et compositions aux fins du traitement de maladies auto-immunes
US7022036B2 (en) * 2003-05-21 2006-04-04 Prototoy Llc Electronic throw-and-catch game

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BERGENBRANT ET AL: "Modulation of anti-idiotype immune response by immunization with the autologous M-component protein in multiple myeloma patients", BRITISH JOURNAL OF HAEMATOLOGY, vol. 92, 1996, pages 840 - 846 *

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AT410636B (de) 2003-06-25
NO20034145D0 (no) 2003-09-17
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WO2002080966A3 (fr) 2003-09-12
WO2002080966A2 (fr) 2002-10-17
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US20040191242A1 (en) 2004-09-30
CN1522155A (zh) 2004-08-18
HUP0303638A2 (hu) 2004-01-28
PL363720A1 (en) 2004-11-29
SK11732003A3 (sk) 2004-03-02
HUP0303638A3 (en) 2004-10-28
CA2441499A1 (fr) 2002-10-17
WO2002080966A8 (fr) 2003-01-23

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