WO2012110639A1 - Procédé de production d'un vaccin dc contre le cancer - Google Patents

Procédé de production d'un vaccin dc contre le cancer Download PDF

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Publication number
WO2012110639A1
WO2012110639A1 PCT/EP2012/052777 EP2012052777W WO2012110639A1 WO 2012110639 A1 WO2012110639 A1 WO 2012110639A1 EP 2012052777 W EP2012052777 W EP 2012052777W WO 2012110639 A1 WO2012110639 A1 WO 2012110639A1
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Prior art keywords
interleukin
cells
treatment
tissue
vaccine
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PCT/EP2012/052777
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German (de)
English (en)
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Thomas GRAMMEL
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Grammel Thomas
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Publication of WO2012110639A1 publication Critical patent/WO2012110639A1/fr

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Definitions

  • the present invention relates to a method for producing a vaccine.
  • Vaccine d. H. Vaccines are known in various designs in the art.
  • the term vaccine refers to an antigen, usually consisting of proteins, genetic fragments, killed or attenuated pathogens that provoke an immune response of the immune system.
  • vaccines find application in the prevention of infectious diseases. But they are also used in the treatment of tumor diseases and autoimmune diseases.
  • Dendritic cells referred to as DC, are frequently used here.
  • DC therapeutic applications are based on the ability of DC to capture and present antigens and to induce or inhibit primary immune responses.
  • Myeloids DC serve as controlling mediators between innate and acquired immunity. They phagocytose antigens and migrate as accessory cells to the secondary lymphoid organs, such as lymph nodes, where they present T-lymphocyte processed antigens and initiate the proliferation and differentiation of specific T lymphocytes by costimulation.
  • DC are not only predestined for the treatment of tumors. Their field of application relates to all areas of the immune system, including the use of DC to induce an immune response in bacterial or viral infections and to develop tolerance to self-antigens in autoimmune diseases and degenerative organ diseases and in the case of foreign antigens in organ transplants.
  • WO 2005/003295 A3 relates to methods for the maturation of dendritic cells
  • WO 2003/086041 A3 a method for reducing human cells that form antigens.
  • EP 1 570 837 A1 relates to a method for processing of Dendrites
  • DE 37 51 553 T2 relates to immunomodulatory agents and their use.
  • DE 603 20 009 T2, DE 603 02 231 T2, DE 603 00 681 T2 and DE 102 14 095 Cl are to mention the details of autoimmune treatment the subject.
  • the invention relates to vaccines of DC of monocytic origin, as well as their preparation and their use for the generation of a successful tumor immunity.
  • the need to develop alternative therapies to the currently available classical tumor therapy options such as chemotherapy and radiation is to reduce the side effects and thus reduce the patient's burden of therapy. Since the therapy can be started and carried out by the veterinarian, immunosuppressive transports from the native environment of the animal are also eliminated.
  • the DC cell populations are used per se for the preparation of a pharmaceutical composition for the in v / 'vo-suppression of tumors.
  • monocytes can differentiate in vitro by the addition of GM-CSF and IL-4 in DC, the conditions leading to DC formation in vivo are still largely unknown.
  • IL-4 plays a prominent role at least in vitro and is currently essential for the generation of DC in vitro (Sallusto, Lanzavecchia, 1994).
  • CD14 monocytes or CD34 precursor cells open up the possibility of obtaining larger amounts of these antigen-presenting cells.
  • MoDC cultivating with GM-CSF and IL-4 correspond in their functions immature DC.
  • the use of IL-4 as a growth factor for DC allows their use for experimental as well as clinical purposes.
  • dendritic cells are loaded ex vivo with tumor-relevant antigens and thus activated and returned to the body.
  • the aim is to induce an immune reaction against the tumor.
  • tumor lysates apoptotic tumor cells, tumor peptides, tumor RNA, tumor DNA, liposomes with nucleic acids or tumor antigens in viral vectors are used.
  • tumors consist of heterogeneous cell populations and often express a wide range of tumor antigens. Often it is not known which of these antigens can cause a potent T cell response.
  • the loading of the dendritic cells with only one tumor antigen can be unsuccessful in the therapy of a tumor and may induce tumor growth through existing escape mechanisms. It has also been shown that the use of a tumor antigen-based vaccine can also induce immune responses against other tumor antigens. It is believed that the destruction of tumor cells by antigen-specific T cells can lead to the induction of other antigen-specific cytotoxic T cells. It is critical that there is a risk of induction of autoimmune reactions when using whole tissue portions. While vaccines are more commonly used to fight tumors in humans, their use in animals has not previously been described. Thus, a systematic use in veterinary medicine is not possible. On this basis, it is an object of the present invention to provide a method for obtaining a vaccine for the treatment of tumors in veterinary medicine available.
  • the present invention provides a method of obtaining a vaccine for treating an animal, comprising the steps of: providing body tissue which is frozen and thawed at least once; and providing a suitable body fluid selected from blood, bone marrow, liver or spleen obtainable by a method comprising the steps of preventing the coagulation of the body fluid, differential gravitational settling of the components of the body fluid, separation of at least a portion of the resulting supernatant, resuspension of the recovered heavy components , Determining the number of cells within the heavy components, mixing the heavy components with medium and serum, adding cytokines, incubating the mixture; and mixing the body fluid with the body tissue, comprising the steps of: incubating the mixture of prepared body fluid and treated body tissue, harvesting the recovered cells, differential gravitational settling of the cells, resuspending the recovered heavy components, determining the cell count.
  • the preferred treated animal is the mammal.
  • the present invention makes it possible now, domestic animals and farm animals, especially horses, dogs and cats targeted at tumor diseases and to treat with little side effects.
  • the body tissue to be provided is preferably tumor tissue.
  • tumor-degenerated tissue is removed with the aid of a sterile scalpel and transferred directly into a sterile vessel with sterile saline solution or PBS.
  • a wound care until healing takes place.
  • This process step has the advantage that the surgical treatment of the diseased site with the recovery of the antigenic material takes place in one step. As a result, a superfluous multiple burden of the animal to be treated is avoided while ensuring economic recovery of the material.
  • a required amount of blood, bone marrow, liver or spleen cells is provided.
  • the blood can be taken from the jugular vein (horse), or from the veins of the front or hind legs (cat, dog).
  • the use of bone marrow, liver or spleen preferably occurs in allogeneic or xenogenic use.
  • a horse requires 450 to 500 ml of blood. In a dog or a cat, correspondingly less.
  • a prevention of coagulation of the body fluid obtained is a differential gravitational displacement of the constituents of the body fluid. Due to a density gradient, the mononuclear cells (monocytes and lymphocytes) are separated from the erythrocytes and polymorphonuclear leukocytes. Erythrocytes and granulocytes accumulate as pellets due to their greater density in the lower part of the tube. After gravity settling, the supernatants containing the monocytes and lymphocytes are collected and recentrifuged.
  • monocytes and lymphocytes are resuspended with PBS, then made up to 50 ml with PBS and centrifuged. This process is repeated several times (washing step) until the supernatant appears clear and is thus free of platelets and cell debris.
  • the pellet is resuspended in medium and the cells are incubated (about 1 hour adherence time). Not adherent cells are then carefully rinsed from the plate, the adherent cells with fresh medium, serum and cytokines for 6-7 days (in dog and horse 7 days, in the cat 6 days) at 37 ° C incubated. During this time, cell cultures receive fresh medium plus cytokines on the second or third and fifth day.
  • the tissue Before the cultured cells are mixed with the tumor tissue, this is processed first.
  • the tissue is frozen at a temperature of minus 80 ° C and then thawed again. It is recommended to repeat this step and freeze and thaw several times. As a result of these repeated cycles of freezing, the greatest possible destruction of the cell structure is achieved so that the cell components are later soluble.
  • the tumor tissue should be stored for extended periods in liquid nitrogen, eg for a further planned treatment. Additional mechanical homogenization achieves further destruction of the cell tissue.
  • the recovered tissue slurry is resuspended in a suitable solution, such as PBS, and subjected to differential gravitational settling. Subsequently, the protein concentration in the cell lysate can be determined.
  • the cell lysate treated in this way is added to the cell culture described above on the 5th or 6th day, depending on the animal species to be treated.
  • the cells incubate again for 24 hours at 37 ° C. Thereupon, a visual inspection of the cells which are of irregular shape and have cytoplasmic deposits is performed.
  • the plate is placed on ice for about 1 hour to allow both non-adherent and adherent cells to more easily be harvested with cold PBS by gently rinsing the bottom of the cell culture dish with cold PBS.
  • the liquid is pipetted off, transferred to a tube, centrifuged, the pellet resuspended with PBS and mounted in a syringe.
  • the cell suspension is thus injectable.
  • the reprocessing of blood and tissue took place during the entire period of cultivation to avoid infections under the safety sterile bench (class II). Subsequently, the vaccine thus obtained is supplied to the animal concerned. This is done intravenously, intracutaneously or intraperitoneally depending on the type of tumor to be treated. For superficial tumors, an intracutaneous injection of the vaccine takes place directly the previously cleaned areas of the wound edges of all surgically removed tissue parts. The treatment usually takes place in the second week, ie one week postoperatively, and in the sixth week and tenth week after the removal of the tissue.
  • viral vaccines or interferons can be used to enhance the immune response (as Danger signals). This is done either by injection into the affected organs, introduction into the culture or simultaneous use in the vaccination.
  • the body tissue used is a tumor tissue.
  • a tumor tissue There is a great need in the circles involved for an effective and low-side-effect tumor treatment.
  • treatments include invasive therapy, ligating, cryosurgery, excision, electrotomy and laser surgery.
  • Non-invasive forms of therapy include photodynamic therapy, radiation and chemotherapy.
  • the ability of the dendritic cells to accept and present antigens and to induce primary immune responses can cause activation of the immune system. This is of great value in the treatment of tumors.
  • the vaccine is suitable for autologous and / or allogenic and / or xenogeneic treatment.
  • autologous treatment tissue parts of the animal to be subsequently treated with antigens are removed and prepared for antigen treatment.
  • This treatment has the advantage that it is specially adapted to the immune system of the animal to be treated.
  • An allogeneic treatment in which, for example, a vaccine is obtained from a dog and used on another dog is much more economical.
  • Xenogenic treatment is rarely considered. For example, in xenogenic treatment, a vaccine obtained from a cat is used in a dog.
  • the selection of the method depends on the species to be treated and the severity of the disease to be treated. Furthermore, it is advantageous if the cells obtained from the body fluid are natural killer cells and / or stromal cells and / or dendritic cells and / or osteoblast cells and / or endothelial cells. Depending on the species to be treated and the severity of the disease, mixtures of the cells can be prepared in a targeted manner. Preferred here is the use of dendritic cells.
  • the cytokine used or combinations thereof are selected from a group comprising interleukin 1 (la and / or ls), interleukin 2, interleukin 3 (multipotential CSF), interleukin 4, interleukin 5, interleukin 6, interleukin 7 , Interleukin 8, Interleukin 9, Interleukin 10, Interleukin 11, Interleukin 12, Interleukin 13, Interleukin 15, Interleukin 16, Interleukin 17, Interleukin 18, Interleukin 22, Interleukin 23, Interleukin 27, Interleukin 31, Interleukin 32, Interleukin 33, Interleukin 34, Interleukin 35, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), Macrophage Colony Stimulating Factor (M-CSF), Interferon (IFN) and Tumor Necrosis Factor, and / or Vitamin D3, Rapamycin or a corticosteroid is used.
  • interleukin 1 la and
  • the invention provides a medicament for treating an animal obtainable by a method for obtaining a medicament or vaccine for treating an animal, comprising the steps of: providing body tissue which is frozen and thawed at least once; and providing blood, bone marrow, liver and spleen cells, obtainable by means of: inhibition of coagulation, differential gravitational settling of constituents of body fluid, separation of at least a portion of the resulting supernatant, resuspension of the recovered heavy components, determination of the cell number within the heavy constituents, Mixing of the heavy components with medium and serum, addition of cytokines and other mediators, incubation of the mixture, as well as the mixing of the cell culture with body tissue.
  • the method may include the steps of incubating the mixture of cell culture and treated body tissue, harvesting the harvested cells, differential gravitational settling of the cells, resuspending the recovered heavy components, and determining cell number. It will be immaterial if the pharmaceutical preparation contains cells which do not meet the criteria of the invention as long as sufficiently functioning cells with the desired criteria are present.
  • Such a medicament comprises appropriate accompanying and auxiliary substances.
  • the use of the vaccine for the treatment of squamous cell carcinoma, hemangiosarcoma, melanoma, mast cell tumor, breast carcinoma, spindle cell tumor and sarcoid and other tumor types has been shown to provide great therapeutic benefits in the form of high quality life extension and healing.
  • the vaccine or the medicament is used for the treatment of mammal carcinoma and / or ovarian carcinoma and / or chronic leukemia and / or lymphoma and / or testicular carcinoma and / or other chemotherapy-sensitive carcinomas, such as, for example, sarcomas or other types of tumors of different animal species and organ systems. It is particularly advantageous if the vaccine, which can be prepared as described above, or the medicament for the treatment of autoimmune diseases and / or for the treatment of a tissue defect by regeneration is suitable.
  • the medicament can be used for autologous (autogenic) and / or allogeneic (homogeneous) and / or xenogenic treatment.
  • therapeutic efficacy and cost-effectiveness have to be weighed against each other.
  • the medicament is used for the treatment of dogs and / or a cat and / or a horse.
  • the cells selected from the body fluid comprise natural killer cells and / or stromal cells and / or dendritic cells and / or osteoblast cells and / or endothelial cells.
  • the cytokine used or combinations thereof are selected from a group comprising interleukin 1 (la and / or ls), interleukin 2, interleukin 3 (multipotential CSF), interleukin 4, interleukin 5, interleukin 6, interleukin 7 , Interleukin 8, Interleukin 9, Interleukin 10, Interleukin 11, Interleukin 12, Interleukin 13, Interleukin 15, Interleukin 16, Interleukin 17, Interleukin 18, Interleukin 22, Interleukin 23, Interleukin 27, Interleukin 31, Interleukin 32, Interleukin 33, Interleukin 34, Interleukin 35, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), Macrophage Colony Stimulating Factor (M-CSF), Interferon (IFN) and Tumor Necrosis Factor, or Vitamin D3 , Rapamycin or a cortico steroid, or used in combination
  • GM-CSF Gran
  • the invention offers the advantage of being able to provide a functioning alternative therapy and / or therapy supplement to chemotherapy and radiation rapidly and at a reasonable cost without causing any ethical or legal problems.
  • the invention is playing a pioneering role.
  • the use with dog and / or cat and / or horse serves the intention to be able to provide the knowledge gain in the not too distant future of the human medicine, in order to take into account the demographic change, since with age the tumor risk also increases.
  • a tumor vaccine for the treatment of a horse
  • the removal of the tumor takes place on a standing horse after sedation with xylazine 2% solution for injection (0.8 mg / kg iV) and local anesthesia with mepivacaine 4% (scandicaine solution for injection) ,
  • the coat at the surgical site is shaved and then wet shaved. Subsequently, the skin is disinfected accordingly.
  • the tumorous degenerated skin is removed by means of a sterile scalpel and transferred directly into a sterile glass vessel containing PBS with a highly concentrated antibiotic mix comprising penicillin / streptomycin (200 ⁇ g / ml G-penicillin and 200 ⁇ g / ml streptomycin). This step prevents the growth of remaining bacterial microorganisms. Following surgery, wound care is performed with direct monitoring until healing.
  • an antibiotic mix comprising Amphotericin B powder may also be 0.0013 g, ampicillin
  • the tumor material is freed of blood and other contaminants with PBS under sterile conditions and transferred to a sterile Petri dish.
  • a small portion of the tumor material is used for histopathological examination to histologically confirm the diagnosis made.
  • the remaining parts of the skin are removed and the clean tumor tissue is placed in a fresh sterile Petri dish.
  • the samples are frozen (at minus 196 ° C in liquid nitrogen). This causes a destruction of the cell structure.
  • the suspension is given and achieved by friction further destruction of the cell tissue. This is continued until a uniform fine tissue consistency is evident.
  • This tissue slurry is dissolved in PBS and transferred to a sterile 50 ml tube. There is now an addition of 1 ml of hydrogen peroxide.
  • the tumor powder thus obtained is placed on a sieve and rinsed with plenty of PBS.
  • the purified liquid is provided with the antibiotic mix described above (4 ml) and stored overnight in the refrigerator.
  • a new centrifugation and a new decantation of the antibiotic solution through a sieve, wherein the remaining tumor powder is rinsed again with PBS. It is re-centrifuged for 10 minutes and then pouring the supernatant.
  • the sterile tumor particles are placed in a cryotube and frozen two to three times at minus 196 ° C in liquid nitrogen and thawed again.
  • Blood is obtained by puncture of the jugular vein under sterile conditions after stasis of the vein to obtain the body fluid.
  • a mixture of 1: 10 sodium citrate is filled up with fresh blood, 10 ml of blood being taken per 10 kg of body weight.
  • 10 ml of blood is taken per 10 kg of body weight.
  • 50 ml of blood with 5 ml of sodium citrate are to be placed in a tube.
  • the blood is taken in B axter blood bags with a volume of 500 ml, in which the appropriate amount of sodium citrate is already included.
  • one to six serum tubes are filled with blood and the autologous serum is obtained therefrom.
  • anticoagulated blood EDTA, heparin or citrate as anticoagulants
  • Leukosep tubes are overlaid with 15 ml Biocoll solution and centrifuged briefly at 1000 rpm and room temperature. In this way, 18 leukosispt tubes corresponding to about 450 ml of blood are processed in a horse of medium size. It is then centrifuged for a further 20 minutes at 1,800 rpm and room temperature. In the density separation described here, the individual cell populations separate due to their specific density. The uppermost layer forms the cell-free plasma. Between plasma and lymphocyte separation medium is the so-called interphase with mononuclear cells (MNC, lymphocytes and monocytes) as well as fractions of the platelet fraction.
  • MNC mononuclear cells
  • the interphase is aspirated with a wide-lumen pipette and transferred to a pre-cooled 50 ml tube. If possible, two interphase are combined in one tube. With cold PBS will open 50 ml filled up. It is centrifuged again at 1800 rpm and 4 degrees Celsius for 10 minutes. The supernatant is discarded and the pellets are resuspended in cold PBS. It is then made up to 50 ml with cold PBS. The washing steps are repeated until the supernatant is free of platelets. After each step, it should be re-evaluated whether the pellets can be pooled in a tube. The goal is to unite all the pellets. When this is done, the cells are resuspended, of which 10 microliters are mixed with 5 microliters of tryptan blue and the number of cells in a counting chamber determined.
  • the cells After resuspending the pellets, the cells are placed in the incubator together with RPMI and autologous serum for about 1 hr (adherence time). Thereafter, the non-adherent cells (lymphocytes) are discarded, culture medium and cytokines are added and the cells are incubated at 37 ° C and 5% C0 2 for 6 to 7 days. One day before the harvest of the cells, the cell culture is liberated with the tumor lysate. In horses, cytokines of the type Human GmCSF Equine IL4 are preferably added, in dogs Canine GmCSF of the type Canine IL4, and in cats Feline GmCSF of the type Feline ILA For each sample, a daily microscopic control is performed.
  • RPMI and cytokines are added.
  • the Petri dish On the day of the cell harvest, the Petri dish is placed on ice for one to two hours to dissolve adherent cells.
  • the cell culture dish is rinsed gently with 25 ml of cold PBS and the cell suspension transferred to a 50 ml tube, counted and filled up with PBS (1 ⁇ 10 6) 1 ⁇ 10 8 cells / ml, preferably 1 ⁇ 10 6 cells / ml). From this tube, the cell suspension is taken injectable with a syringe. Until injection, the syringe should be stored with the cell suspension on ice.
  • the vaccination is carried out 3 times at intervals of 4-5 weeks.
  • a V2 annual vaccination is carried out.
  • the horse is sometimes sedated by intravenous injection of xylazine (1, 1 mg / kg body weight) or because of the difficult to reach Lokali organization of the tumor under general anesthesia.
  • xylazine 1, 1 mg / kg body weight
  • between 0.2 ml and 0.4 ml of vaccine are injected per site of application.
  • the invention described above makes it possible to significantly improve the treatment situation in domestic animals, in particular horses, cats and dogs.
  • Monocytes of the subject can be separated by a hypertonic, 50% Percoll gradient in good purity and yield and able to differentiate into identical cells in vitro. Characterization of the dendritic cells is possible both morphologically and phenotypically by means of surface markers. In a mixed leukocyte reaction, the antigen presenting function of the in vitro generated dendritic cells was confirmed. It could be shown that autologous dendritic cells are effective stimulators, assuming the absorption of suitable antigens.
  • the invention relates to tolerogenic DC monocytic origin, as well as to their production and their use for producing a successful immune tolerance.
  • DC cell populations of the invention for the preparation of a vaccine and / or medicament for in v / Vivo Suppression of autoimmune reactions are used. Blood collection and workup are as described above. The culture, however, no additional tissues or cells are added, but the monocytes are cultured only with medium and cytokines and / or other mediators. It is irrelevant if the pharmaceutical preparation contains cells that do not meet the criteria of the invention as long as the desired criteria corresponding cells are present.
  • the vaccine produced contains vital tolerogenic DC cell populations and is processed to injectability as described above (1 ⁇ 10 6 to 10 ⁇ 10 8 cells / ml in PBS). The vaccine has the ambition to be a pioneer. The use in dog and / or cat and / or horse serves the purpose of being able to provide the knowledge gain in the not too distant future of human medicine, since autoimmune diseases and allergies, two sides of a coin, are still mainly treated only symptomatically can be.
  • the invention offers the possibility of being able to provide a functioning therapy alternative and / or a therapy supplement for immunosuppression rapidly and at a reasonable cost without causing ethical or legal problems.
  • Another embodiment relates to adult dedifferentiated programmable stem cells derived from human monocytes, as well as their production and use for the production of specific body cells and tissues.
  • Preferred is the form of autologous use, i. Cell donors and recipients represent one and the same individual.
  • the cells may be derived from animals and / or humans.
  • Stem cells are cells that have a high potential for self-renewal and produce at least one cell type in numerous forms through asymmetric division behavior. Since the use of human embryonic stem cells, which can differentiate into any cell type, is ethically and legally problematic in Germany, the use of pluripotent stem cells of embryonic origin has narrow limits. Furthermore, by using allogeneic or xenogenic Use of embryonic stem cells in the recipient of unwanted rejection reactions. Such legal limits do not exist with cells of animal origin. Stem cells can be obtained from different differentiated tissues. These are multipotent adult stem cells, which occur as a cell reservoir in each organ and form specific organ cells. The main difference to embryonic stem cells, however, lies in the limited range of cells that can be differentiated from these organ-specific stem cells.
  • This type of vaccination has the ambition to be a pioneer.
  • the intended use in the dog and / or cat and / or horse is intended to provide insight in the not-too-distant future of human medicine, to accommodate demographic change, e.g. in the therapy of age-related degenerative diseases of various organs, eg. Brain, such as Alzheimer's disease.
  • the production of dedifferentiated, reprogrammable monocytes can contribute to this.
  • these cells carry the monocyte marker CD14.
  • the once differentiated monocytes are thereby brought to a stage where they can develop through the influence of various mediators (e.g., cytokines, and the like) or combinations thereof in a direction desired by the experimenter.
  • the essential steps for the production according to the invention of dedifferentiated reprogrammable stem cells of monocytic origin from dog and / or cat and / or horse include:
  • the cells thus obtained can be further processed or frozen immediately.
  • the stem cells produced by this method can be reprogrammed to any body cells by the addition of suitable mediators, eg cytokines.
  • suitable mediators eg cytokines.
  • the methods for this can, for example, be taken from the following literature: Weissmann IL, Science 287: 1442-1446 (2000) and Insight Reviews Articles Nature 414: 92-131 (2001) and Handbook "Methods of Tissue Engineering", Ed. Atala, A. , Lanza, RP, Academic Press, ISBN 0-12-436636-8.
  • the subject matter of said publication is incorporated by reference in its entirety into the disclosure of the present invention.
  • the dedifferentiated, reprogrammable stem cell populations of monocytic origin can be used to produce a vaccine for the in vivo induction of tissue regeneration following tissue injury and / or loss, and thus for in vivo production of target cells and target tissue.
  • Blood collection and workup are as described above.
  • the culture however, no additional tissues or cells are added, but the monocytes are cultured only with medium and cytokines and / or other mediators. It will be immaterial if the pharmaceutical preparation cells contains that do not meet the criteria of the invention as long as enough cells corresponding to the desired criteria are present.
  • the vaccine contains vital dedifferentiated reprogrammable stem cell populations of monocytic origin and is processed to injectability as described above (10x10 6 to 10x10 8 cells / ml in PBS).
  • Stem cells have the ability to spontaneously differentiate in vivo into a cell of this tissue type by direct contact with a cell assemblage.
  • hematopoietic stem cells from the bone marrow can differentiate into liver cells: Lagasse et al: Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nature Medicine, 6 (11): 1229-1234 (2000).
  • blood stem cells also appear to serve the body as a reservoir for non-blood cells: Nakajima, H.: Role of Transcription Factors in Differentiation and Reprogramming of Hematopoietic Cells. Keio J Med 2011; 60 (2): 47-55.
  • the preferred site of application for the stem cells of monocytic origin or hematopoietic stem cells from the bone marrow is the injection into a specific cell group so that the stem cells can settle there and differentiate into the resident cell type.
  • Examples of the use of stem cells of monocytic origin are the treatment of insufficiencies of liver, pancreas, heart, kidney.
  • Diagnosis entire body urticaria, limb edema
  • Type Dog, Mixed Breed
  • the present invention provides improved compositions for the treatment of tumors, autoimmune diseases and tissue defects with simultaneously reduced side effects.

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Abstract

La présente invention concerne un procédé pour obtenir un vaccin permettant de traiter un animal, comprenant les étapes consistant à : préparer un tissu organique qui est au moins congelé une fois et décongelé, préparer un liquide organique approprié, sélectionner parmi le sang, la moelle osseuse ou la moelle épinière, et mélanger le liquide organique avec le tissu organique.
PCT/EP2012/052777 2011-02-17 2012-02-17 Procédé de production d'un vaccin dc contre le cancer WO2012110639A1 (fr)

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DE102011004335.7 2011-02-17
DE102011004335A DE102011004335A1 (de) 2011-02-17 2011-02-17 Verfahren zur Herstellung eines Vakzins

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Citations (7)

* Cited by examiner, † Cited by third party
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