WO2002077208A1 - Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation - Google Patents

Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation Download PDF

Info

Publication number
WO2002077208A1
WO2002077208A1 PCT/EP2002/003292 EP0203292W WO02077208A1 WO 2002077208 A1 WO2002077208 A1 WO 2002077208A1 EP 0203292 W EP0203292 W EP 0203292W WO 02077208 A1 WO02077208 A1 WO 02077208A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
presenting cell
monoantigenic
receptor
monoantigenic antigen
Prior art date
Application number
PCT/EP2002/003292
Other languages
German (de)
French (fr)
Inventor
Ahmed Sheriff
Birgit Vogt
Original Assignee
Genethor Gmbh
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Genethor Gmbh filed Critical Genethor Gmbh
Publication of WO2002077208A1 publication Critical patent/WO2002077208A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C12N5/064—Immunosuppressive dendritic 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4637—Other peptides or polypeptides
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464839—Allergens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to antigen-presenting cells (APC), processes for producing antigen-presenting cells, medicaments containing antigen-presenting cells and use of the antigen-presenting cells.
  • APC antigen-presenting cells
  • an allergen that gets into the blood in some way can trigger anaphylaxis: an allergic reaction in regions of the body that are far from its point of entry. Severe anaphylactic shocks can upset all normal bodily functions and can be fatal.
  • the allergic reactions manifest themselves differently, they are always started by the same mechanism: the sensitization.
  • an allergen typically a protein
  • the allergenic substance meets so-called phagocytes or macrophages. These gobble up the foreign substance, dismember it and present the fragments on the cell surface with MHC II molecules.
  • T-helper lymphocytes recognize the fragments presented and bind to them.
  • the T helper lymphocytes are activated by the macrophages and then in turn activate some B lymphocytes, which also recognize the allergen.
  • the B cells then mature into plasma cells that produce antibodies. First of all, these are antibodies of the so-called IgM type; from a certain point in time, however, the plasma cells switch to IgE antibodies.
  • IgE molecules With their Fc region, they attach themselves to IgE receptors of two different classes of immune system cells.
  • mast cells which are usually found in the body tissues near blood vessels and epithelial cells. There is contact with the outside world via the epithelium (this also includes the epithelium of the respiratory tract and gastrointestinal tract).
  • IgE antibodies also bind to basophilic granulocytes (basophils). However, these cells circulate in the bloodstream.
  • IgEs Once production of the IgEs begins, it apparently lasts for months, sometimes even years. As a result, they constantly occupy IgE receptors on mast cells and basophils - ready to take immediate action the next time they come into contact with allergens.
  • the second exposure initiates a stage of the hypersensitivity reaction, which also appears externally.
  • the allergy-causing substance binds to the IgEs of the mast cells within seconds of contact with human tissue. If he attaches himself to two or more IgE molecules at the same time, he bridges between them. Such cross-links bring the affected IgE receptors closer together, and this activates the cell so that it releases highly effective substances that directly produce allergic symptoms. (The release can also be caused in other ways; allergic reactions are only spoken if IgEs are involved). The most important of these substances is histamine.
  • the second group of mediators consists mainly of prostaglandins and leukotrienes. They are only released after the allergen molecules have attached to the IgEs on the cells. Like histamine, they narrow the bronchi and dilate the blood vessels. However, their effects last longer.
  • stimulated mast cells emit a variety of potentially toxic enzymes. They also appear to release cytokines that regulate the activities of other immune cells.
  • Antihistamines are usually effective and still serve as standard therapy. The latest variants can no longer easily cross the blood-brain barrier and no longer make the patient tired. If antihistamines are ineffective in severe inflammation, inhalable corticosteroids, which are usually prescribed to relieve chronic inflammation in asthma, often help. In severe cases, immunotherapy or hyposensitization (also known as allergy shots or desensitization), which was introduced in 1911, can provide relief in the long term. With this treatment, doctors inject patients with increasing doses of the allergen to which they are sensitive. The dose is decisive in all cases: too little allergen does not confer tolerance. In addition, protection is rarely complete.
  • Bronchodilators are the most widely used drugs for asthma. They relieve the symptoms caused by histamine and other bronchoconstrictors very quickly, but are unlikely to affect the underlying inflammation. In addition, their excessive use can cause a back reaction of the body, so that after their effects have subsided, the respiratory flow is more restricted than before. In addition, the methods listed under 1. are used.
  • Another new strategy is the use of humanized monoclonal anti-IgE antibodies against the Fc ⁇ RI binding region for IgE. This prevents the binding of IgE to the IgE receptor, so that no mediators of the allergic reaction from mast cells or basophils can be released. This strategy has shown in clinical studies in patients with allergic rhinitis and allergic asthma that these antibodies are well tolerated and reduce the allergic reactions.
  • Tissue transplantation to replace diseased organs is an important medical therapy today.
  • a response from the adaptive immune system to the graft poses the greatest threat to successful treatment.
  • Responses from the adaptable immune system are induced by antigen presenting cells by activating T helper lymphocytes.
  • the ABO and Rh blood group antigens When transfusing blood, which is the first and most commonly used graft, the ABO and Rh blood group antigens must be matched to avoid the rapid destruction of inappropriate erythrocytes.
  • the very polymorphic main histocompatibility complexes (MHC) have to be matched to one another, since these almost always trigger the immune response. Unfortunately, the perfect matching of the MHCs is almost impossible, except for relatives.
  • the product of the mouse PD-1 gene (ACCESSION NM_021893), a member of the IgG superfamily, is a receptor that is common in many tissues. Analyzes carried out by flow cytometry and immunoprecipitation with the monoclonal antibody J43mAk showed that the PD-1 gene product is a 50 to 55 kDa membrane protein. The PD-1 protein appears to be highly glycosylated since the calculated molecular weight of the amino acid sequence is 29310 Da. Normal mouse lymphoid tissue such as thymus, spleen, lymph nodes and bone marrow contain only a small number of PD-1 positive cells.
  • PD-1 positive population appears in thymocytes as well as on T cells in spleen and lymph nodes due to the in vivo treatment with anti-CD3 monoclonal antibodies.
  • PD-1 antigen expression was strongly induced by in vitro stimulation in various subgroups of thymocytes and spleen T cells, either with anti-CD3 mAb or Concavalin A, which can cause T cells to activate as well as to die , PD-1 expression on spleen B cells was similarly stimulated with anti-IgM Ab.
  • PD-1 was not significantly expressed after treatments such as growth factor deprivation, dexamethasone or lipopolysaccharide.
  • PD-1 is structurally similar to CTL-associated antigen 4 (CTLA-4), which binds B7-1 and B7-2 and plays a crucial role in the maintenance of T cell homeostasis (for reviews, see (Sperling & Bluestone 1996; Thompson & Allison 1997)).
  • CTLA-4 CTL-associated antigen 4
  • PD-1 does not contain the MYPPPY motif, which is critical for B7-1 and B7-2 binding.
  • the extracellular region of PD-1 and CTLA-4 each consist of an IgV domain with 23% identity to each other.
  • a ligand (PD-Ll) exists for PD-1. The binding of PD-1 by PD-Ll leads to the inhibition of TCR-mediated T cell proliferation and cytokine secretion (Freeman et al 2000).
  • the human and mouse PD-Ll molecule (EMBL / GenBank / DDBS under accession nos. AF233516 and AF233517, (Freeman et al 2000)) are members of the B7 gene family and have a similar structural organization, which consists of an IgV and an IgC domain in the extracellular region (Boussiotis et al 1996), a hydrophobic transmembrane domain, followed by a short, charged intracellular region.
  • Human PD-Ll is identical to B7-H1 at 290 amino acids, which is reported to activate T cell stimulation (Dong et al 1999).
  • the mouse PD-Ll cDNA encodes a polypeptide with 70% amino acid identity to the human PD-Ll.
  • PD-Ll has amino acid identities of 21, 20, and 23% to B7-1, B7-2, and ICOS (ligand of inducible co-stimulator) (Boussiotis et al 1996; Ling et al 2000; Swallow et al 1999; Yoshinaga et al 1999).
  • the expression patterns of B7-1, B7-2, and PD-Ll are different.
  • B7-2, one of the ligands of CD28 and CTLA-4, is constitutively expressed on monocytes. However, the constitutive expression of B7-1 and B7-2 cannot be observed in any organ.
  • B7-1 and B7-2 expression can be induced in dendritic cells, macrophages and B cells (Boussiotis et al 1996), as well as some types of fibroblasts and epithelial cells.
  • PD-Ll is constitutively expressed by non-lymphoid, parenchymal organs such as the heart, placenta, skeletal muscles and lungs, but not the small intestine (Dong et al 1999).
  • PD-Ll is also expressed in some cancers. These tumors may be using PD-Ll to inhibit anti-tumor immune responses.
  • PD-1 Ligand 2 (PD-L2) is a second ligand for PD-1. Binding of PD-1 to PD-L2 dramatically inhibits T cell receptor (TCR) mediated proliferation and cytokine production by CD4 T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals.
  • PD-L-PD-1 interactions lead to cell cycle residue in G 0 / Gx (cell cycle phases) but do not increase the number of dead cells.
  • PD-L2 expression on APCs is increased by treatment with interferon ⁇ and could also be detected in some other tissues and tumor cell lines.
  • PD-Ll and PD-L2 thus have overlapping functions (Latchman et al 2001).
  • mPD ⁇ L2 (previous name: Protein AF142780) codes for a polypeptide with 38% amino acid identity to mPD-Ll.
  • Murines and human PD-L2 have 70% amino acid identity.
  • the five members of the B7 family - B7-1, B7-2, ICOS-L, PD-Ll and PD-L2 - have 21-27% amino acid identity and a structural organization consisting of a signal sequence, an IgV-like one IgC-like and a transmembrane domain and a short cytoplasmic tail.
  • the cytoplasmic tail of PD-Ll is conserved between mice and humans, which is in contrast to the poor conservation of the cytoplasmic tail of PD-L2 of humans and mice (Latchman et al 2001).
  • the PD-L2 tissue distribution is similar to that of PD-Ll. There is expression in the placenta, heart, pancreas, lungs and liver, low expression in the spleen, lymph nodes and thymus, no expression in unstimulated monocytes, but this could be induced by interferon ⁇ . However, the kinetics of this induction is slower than that of PD-Ll.
  • Autoimmune diseases are chronic diseases. These include rheumatism in various clinical forms, diabetes, multiple sclerosis, certain forms of cardiac muscle inflammation and Thyroid disease. The series of autoimmune diseases could be continued as desired, although approximately 90% epidemiologically only make up a small percentage.
  • autoimmune diseases are antibody-mediated.
  • immunology this means that the organism produces antibodies for mostly unknown reasons, which are directed against the body's own cellular structures (autoantigens). Once these antibodies have been formed, their interaction and binding to the respective organ or cell-specific structures trigger a cell and tissue-destroying reaction of the immune system. Ultimately, this leads to the clinical picture of the disease (Steinman 1994).
  • PCT / EP 00/03984 discloses a method for reducing specific immune reactions by suppressing the B7 expression of cells presenting antigen while simultaneously expressing predetermined (in particular pathological) antigens.
  • WO-A-01/14557 relates to PD-1 as a receptor for B7-4.
  • B7-4 can inhibit immune cell activation due to binding to an inhibitory receptor or to an immune cell.
  • Active substances are described for the modulation of PD-1, B7-4 and their interaction in order to modulate co-stimulating or inhibiting signals in an immune cell, which results in a modulation of the immune response.
  • a problem on which the invention is based is, inter alia, to provide genetic engineering, therapeutically usable products for the reduction of specific immune reactions in which targets (antigens, autoantigens) and antibodies, autoantibodies are known.
  • the antigen-presenting cell which predominantly presents certain antigens beforehand (monoantigenic antigen-presenting cell) and is characterized in that molecules which bind PD-1 are preferably produced in the monoantigenic antigen-presenting cell and if appropriate, CTLA4-binding molecules are produced and, if appropriate, one of the functions of stimulatory receptors, such as a B7 and / or CD40 receptor, is suppressed.
  • CTLA4 (CD152) is an analogue of CD28, also binds B7, but induces apoptosis of the T cell.
  • CTLA4 occurs mainly in intracellular vesicles and is only released after activation of the cells.
  • PD-1 is a CD28 and CTLA4 analogue and develops CTLA4-like effects.
  • Fig. 3 shows schematically the function of gene manipulation at the molecular level: PD-1 is bound by a ligand (PD-Ll), which is located on the plasma membrane of the monocyte (antigen-presenting cell), and thus inactivates the T cell.
  • PD-Ll ligand
  • the effect can be intensified if B7 is additionally suppressed in the antigen-presenting cell and, if appropriate, a CTLA4-binding molecule is also introduced.
  • Fig. 4 shows the function of gene manipulation at the molecular level: the autoantigen is synthesized by the PD-Ll producing antigen presenting cells.
  • FIG. 5 and 6 show a comparison of the antigen presentation of "normal" and genetically modified monocytes.
  • Figure 5 shows autoantigen levels presented on the MHC II. Normal monocytes rarely present the autoantigen and if so, only a few MHC II present it. 6 shows that the genetically manipulated monocytes almost exclusively present the autoantigen.
  • FIG. 7a and 7b demonstrate the function of the genetically modified monocytes in the body.
  • natural autoantigen-presenting monocytes induce the production of (pathological) autoantibodies if they are B7-positive.
  • genetically manipulated monocytes compete with the natural autoantigen-presenting monocytes and reduce autoantibody production.
  • the monoantigenic antigen-presenting cell according to the invention is preferably a monocyte, a dendritic cell and / or a macrophage.
  • the APCs are the switching points of the adaptable immune system. Only they can trigger a T cell-mediated immune response. This is shown in the following figures using the example of monocytes and the antibody production triggered by T helper cells: Within the immune system, the antibodies normally play an important role as specific defense molecules ("humoral immune response"). They are produced by mature B lymphocytes. However, the induction and production of soluble antibodies is not independent of the remaining cells in the immune system; rather, this humoral immune response is controlled by other cells in the immune system.
  • the antigen is e.g. a bacterial protein
  • the immune response is useful for the organism.
  • the antigen is an endogenous structure, one speaks of a (pathological) autoimmune reaction.
  • the monoantigenic antigen-presenting cell according to the invention has an increased expression of an antigen, preferably by transfection of an antigen-presenting cell with nucleic acid-containing material, the antigen-presenting cell essentially presenting only predefined antigens.
  • the gene therapy method according to the invention is based on parallel genetic engineering interventions on the patient's own blood monocytes.
  • the interventions are carried out using suitable probes and cause the production of PD 1 binding molecules, preferably PD-L1 and possibly CTLA4 binding molecules, preferably antibodies (not shown), and possibly the reduction of B7 molecules by hindering or preventing the formation of the molecule on the surface of the monocytes (not shown) (Fig. 3) and at the same time a strong presentation of autoantigen (Fig. 4 - 6).
  • pathological autoantibodies is specifically ended by the manipulation of monocytes (or DCs) and the resulting shutdown of antigen-specific T cells. This takes advantage of the fact that T cells produce the receptor PD-1. When bound to PD-Ll, this ensures that T cells no longer proliferate. Binding of PD-1 can thus shut down T cell responses. Binding molecules other than PD-Ll can also be used for this.
  • T cells produce two receptors that recognize B7 (1 or 2).
  • One receptor is CD28
  • the other is CTLA4.
  • CD28 has a stimulating effect, while CTLA4 does not.
  • the contact of CTLA4 with B7 switches off the respective T cell. This then either goes into apoptosis via programmed cell death, is switched off, or is converted into a tolerance-generating T cell. In this case, the tolerance is generated against the specific antigen recognized by this T cell.
  • CTLA4 For binding to CTLA4 instead of CD28, molecules such as antibodies are used which bind to CTLA4 but not to CD28. These can then induce the T cell response promoted by CTLA4. These CTLA4 binding molecules can preferably be antibodies.
  • the co-receptor B7 without which the antigen presentation or the induction cascade for antibody production does not start, can also be suppressed in order to suppress preferential binding of CD28.
  • the co-receptor is two different co-receptors called CD80 (B7-1) and CD86 (B7-2). Their structures are known.
  • the cells After the in vitro manipulation of the monocytes, the cells are returned to the patient's bloodstream.
  • the genetically modified monocytes now switch off the pathological T helper lymphocytes in the organism.
  • the genetically modified cells compete directly with the autoantigen-presenting monocytes already present in the organism (preferably the bloodstream and lymphatic system), which however carry B7 on the cell surface and normally activate the T helper lymphocytes and thus the antibody production (Fig 7a and 7b).
  • the cDNA of a protein that causes the production of pathological autoantibodies as an autoantigen is integrated into the monocyte genome. This genetic information then serves to overproduce the autoantigen. Peptides of this autoantigen are then preferably presented on MHC II and / or MHC I [see also FIGS. 5 and 6]. MHC II presents the peptides to the T helper cells and tries to find those that specifically recognize these presented peptides. If a PD-1 binding molecule activates PD-1 instead of CD28 and a CTLA4-binding molecule also activates CTLA4 instead of CD28 and the co-receptor B7 does not appear on the cell surface at the same time, the T helper cells are shut down and may experience premature cell death.
  • Monocytes present the autoantigen and then only at a few MHC II complexes.
  • the aim of the treatment is to displace the "normal" monocytes which present the autoantigen and activate the T helper cells in vivo by the genetically modified monocytes which have been programmed to switch off the antibody production or T cell response.
  • the monoantigenic antigen-presenting cell according to the invention can in particular show an increased number of homing receptors, such as CD44. Overexpression of homing receptors will show the monoantigenic antigen-presenting cell the way into the lymph nodes, as a result of which the genetically modified APCs increase and accumulate faster in lymph nodes.
  • the lymph nodes are where most of the responses of the adaptive immune system are triggered. There, the genetically modified APCs have a much greater effect than outside the lymph nodes.
  • transfection causes an increase in the number of homing receptors and or an increase in the number of PD-1 binding molecules and / or a suppression of functions of the B7, CD40 receptors and / or an increase in the number of CTLA4 binding molecules.
  • the PD-1 binding molecules can preferably be PD-Ll, PD-L2, antibodies, monoclonal antibodies. These can be such that they remain in the plasma membrane of the cell.
  • the CTLA4 binding molecules can preferably be antibodies, monoclonal antibodies. These can be such that they remain in the plasma membrane of the cell.
  • the B7 and / or CD40 receptor expression in the monoantigenic antigen-presenting cell according to the invention can be prevented or reduced.
  • the expression of the B7 and / or CD40 receptors can be suppressed by nucleic acids by co-suppression in the monoantigenic antigen-presenting cell according to the invention.
  • the monoantigenic antigen-presenting cell according to the invention can contain or be transfected with nucleic acids which bring about an expression of proteins or peptides which have structures affine with B7 and / or CD40 receptors. In this way, proteins are formed which, by forming complexes with B7 and / or CD40 receptors, practically neutralize these receptors.
  • CTLA4, CD28, antibodies, F (ab) 2 , scFv and / or F ab fragments can be considered as proteins.
  • the monoantigenic antigen-presenting cell according to the invention contains nucleic acids which code for a signal sequence or expression products of a signal sequence which determine whether the expression products remain in the endoplasmic reticulum, the Golgi apparatus, the Trans-Golgi network or intracellular vesicles causes.
  • the monoantigenic antigen-presenting cell according to the invention is transfected for the expression of antigens with nucleic acids which enable the transport of the expressed antigens in MHC II compartments of the cells.
  • All genetically modified monocytes present the autoantigen on most of their MHC complexes, while very few "normal” monocytes present the autoantigen at all and then only on a few MHC complexes.
  • the aim of the treatment is to displace the "normal" monocytes that present the autoantigen and activate the T helper cells by means of the genetically manipulated monocytes programmed to switch off the antibody products. It is very important to also administer the antigen (s) in a form that allows them to be transported into the MHC II endosomes. This is the only way to reliably achieve a presentation at MHC II. In the case of a genetic engineering intervention, this is usually done by manipulating the open reading frame, so that the reading frame is preceded by a signal sequence for this compartment. Genetic manipulation has the additional advantage that it has a much longer half-life than, for example, oligo- nucleotides or peptides. A stable integration of genes even leads to a permanent change in the properties of the target cells.
  • the corresponding nucleic acids can be DNA, RNA, oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • the DNA preferably has regulatory elements such as enhancers, promoters, polyA-coding 3 "ends for transcribing the DNA into RNA, the RNA regulatory elements for translating the RNA into protein.
  • the regulatory elements ensure efficient expression of the genes.
  • the monoantigenic antigen-presenting cell according to the invention is produced, for example, by ex vivo or in vivo methods.
  • An antigen-presenting cell is preferably ex vivo or in vivo by treatment with viruses, viral vectors, bacterial vectors, plasmids by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a monoantigenic antigen-presenting cell transfected.
  • an antigen-presenting cell or a monoantigenic antigen-presenting cell can be obtained by treatment with viruses, viral vectors, bacterial vectors, plasmids, by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with an increased amount of PD-1 binding molecules and optionally with an increased amount of CTLA4 binding molecules and possibly suppressed function of co-stimulatory receptors or the expression of co-stimulatory receptors by preventing their expression or the co-stimulatory receptors are reacted with affine structures prevented from stimulating T cells bound to the monoantigenic antigen presenting cell.
  • nucleic acids In this case, antisense nucleic acids must come into contact with the mRNA of the co-stimulatory molecule. You will then likely bind the molecule and prevent translation.
  • RNAs A wide range of molecules, such as RNAs, come as nucleic acids
  • DNAs, PNAs, ribozymes in question.
  • a genetic engineering intervention would ensure the longest half-life of the effect.
  • Re 3. The binding of the co-stimulatory molecule e.g. by specific antibodies prevents this molecule from contacting the notified
  • Receptor takes up on a T cell and thus prevents activation of the T cell.
  • the external addition of such binding molecules has the disadvantage that they act on all antigen-presenting cells and thus prevent any immune reaction.
  • An extension of this model is intended to ensure that the binding molecule is retained in the intracellular compartment so that the co-stimulatory receptor does not reach the plasma membrane in the first place.
  • Signal sequences are known for this, which must be added to the open reading frame of the binding molecule.
  • This intracellular approach can be carried out well on isolated cells.
  • genetic engineering intervention is preferable.
  • the desired effects can also be achieved if only one of the two goals is carried out with a genetic engineering intervention.
  • z. B. the following other molecules are used:
  • Nucleic acids (mostly complementary to the target sequence), which are e.g. B. can be oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNAs),
  • PNAs peptide nucleic acids
  • Antibodies or other molecules that bind the co-stimulatory molecules are included in the immunoglobulin analogs.
  • Proteins, peptides, peptidomimetics Proteins, peptides, peptidomimetics.
  • molecules such as antibodies, proteins, peptides, peptidomimetics, PD-L1, PD-L2, CTLA4, CD28, CD40L and / or constituents and / or combinations of these molecules, which e.g. B7-1, B7-2, CD40 bind, which prevents co-stimulation of the T cell taking place in the presence of an antigen presentation, brought into contact with the monoantigenic antigen presenting cell or the antigen presenting cell.
  • molecules such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, in particular biodegradable nanocapsules or particles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or other techniques to transfer molecules into the monoantigenic antigen-presenting cell or the antigen-presenting cell.
  • Nucleic acids can in particular by viruses, viral vectors, bacterial vectors, plasmids by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for the introduction of molecules are transferred into the monoantigenic antigen-presenting cell or the antigen-presenting cell.
  • a medicament containing at least one monoantigenic antigen-presenting cell according to the invention is further claimed.
  • the medicament according to the invention is preferably formulated as an infusion solution for intravenous or intraperitoneal administration.
  • the formulation is chosen such that when the medicament is administered there is no significant impairment of the effectiveness of the monoantigenic antigen-presenting cell according to the invention.
  • physiological saline is preferred as the infusion solution.
  • other solutions with a pH of 5.5 to 8.5 are also suitable.
  • Serum for example human serum, autologous serum or serum of other species, solutions with plasma substitutes, such as polyvinylpyrrolidone, are also suitable.
  • plasma substitutes such as polyvinylpyrrolidone
  • 0.5 ml to 500 ml should be applied.
  • the monoantigenic antigen-presenting cell according to the invention can be used in particular for the production of a medicament for the treatment of unwanted immune reactions such as autoimmune diseases and allergies or deliberately induced immune reactions such as in immunizations.
  • the monoantigenic antigen-presenting cell according to the invention can be used for the production of a medicament for the treatment of immune reactions against allologic and / or xenologic tissue characteristics.
  • the immune reactions to be treated are related to antigens or their gene sequences and / or parts thereof and are selected from the group consisting of Enzymes, their gene sequences and / or partial sequences, in particular glutamic acid decarboxylase (GAD), receptor type protein tyrosine phosphatase IA-2Beta, antigen: H + K + ATPase, U1RNP, transglutaminase, argininosuccinate lyase (ASL), tyrosinase related protein-2, thyroid peroxidase, factor VIII, factor IX;
  • GAD glutamic acid decarboxylase
  • IA-2Beta receptor type protein tyrosine phosphatase IA-2Beta
  • antigen H + K + ATPase
  • U1RNP transglutaminase
  • ASL argininosuccinate lyase
  • tyrosinase related protein-2 thyroid peroxidase, factor
  • Receptors their gene sequences and / or partial sequences, in particular acetylcholine receptor of the nicotine type, ⁇ 1-adrenergic receptor, ⁇ l-adrenergic receptor, angiotensin-2-ATl receptor, glutamate receptor, thyrotropin-stimulating hormone (TSH) receptor, LFA receptor 1,
  • HLA-B27 Epididymal Protein DE, Zona Pellucida (ZP) -3 Glycoprotein, Zona Pellucida (ZP) -4 Glycoprotein, Follicle-Stimulating Hormone (FSH) receptor, Sperm Immunogen SP-10 or Sperm Protein SP-10;
  • Hormones or messenger substances their gene sequences and / or partial sequences, in particular insulin, thyroglobulin, follicle-stimulating hormones (FSH), prostaglandin F2 alpha, gonadotropin-releasing hormones (GnRH), oestradiol-17beta, estrogen, luteinizing hormone (LH) receptor, inhibin , Testosterone, androgen, chorionic gonodrophin (CG), interleukins, interferons, cytokines, chemokines, bone morphogenetic factors, ß-interferon, estradiol;
  • Structural proteins their gene sequences and / or partial sequences, in particular myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), ⁇ -fodrin, non-erythroid ⁇ -
  • MBP myelin basic protein
  • PGP proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • ⁇ -fodrin non-erythroid ⁇ -
  • beta-amyloid precursor protein beta-APP
  • type 2 collagen type 2 collagen
  • sperm plasma membrane protein PH-20 type 2 collagen
  • Antigens their gene sequences and / or partial sequences, in particular CENP-A autoantigens, Beta2GP-I, ribosomal P protein, Ro / SSA,
  • the invention claims a use in which the immune reactions to be treated are associated with allologic and / or xenological tissue characteristics, their gene sequences and / or partial sequences, in particular MHC I, MHC II, rhesus factor.
  • the method presented here for reducing immune responses is based on the genetic manipulation of certain blood cells, preferably outside the body, which are then subsequently returned to the organism.
  • the aim of this treatment is, among other things, the specific switching off of a chronic, immune system-driven production of autoantibodies that trigger the disease.
  • DCM Dilated cardiomyopathy
  • the antibodies play an important role as defense molecules ("humoral immune response"). They are produced by mature B lymphocytes. However, the induction and production of the antibodies is not detached from the remaining cells of the immune system; rather, this humoral immune response is controlled by other cells in the immune system. The immune response cannot be maintained without the help and mediation of antigen presenting cells and T helper lymphocytes.
  • the molecular mechanisms of the interaction - of cell-cell contact - of antigen-presenting cells with the T-helper lymphocytes is known in detail at the receptor level.
  • auxiliary receptors are involved in cell-cell contact and cell activation on the cell surface.
  • An essential molecule of the intercellular interaction in antigen presentation contact of antigen-presenting cells with the T-helper lymphocytes
  • costimulatory receptors with the names PD-Ll, PD-L2, B7h (LICOS), B7-1 and B7-2 , CD40 also plays a role in this signal transduction.
  • the designed procedure is based on at least two parallel interventions on the patient's own antigen-presenting cells.
  • the interventions are carried out using suitable probes and have an effect
  • a PD-1 binding molecule e.g. PD-Ll
  • PD-1 a PD-1 binding molecule which via the action of PD-1, which occurs in T cells, prevents the T cells from activation and proliferation
  • CTLA4 binding molecule e.g. Antibodies that prevent the T cells from activating through the action of CTLA4, which occurs in T cells
  • the cells are returned to the donor's bloodstream.
  • the genetically manipulated monocytes now switch off the corresponding T helper lymphocytes in the organism.
  • the genetically manipulated cells compete directly with the autoantigen-presenting monocytes already present in the organism (preferably the bloodstream and lymphatic system), which however carry B7 and no additional PD-1 binding molecule, CTLA4 binding molecule on the cell surface and usually the T-helper - Activate lymphocytes and, among other things, antibody production.
  • autoimmune diseases and allergies do not exist. With a few exceptions (extracorporeal elimination of antibodies from the patient's blood or plasmapheresis), autoimmune diseases and allergies are treated with medication by inhibiting immune system reactions.
  • Immunosuppressive preparations such as cortisone and its derivatives, as well as cyclosporin, beta interferon or cytostatics (methotrexate)
  • cytostatics metalhotrexate
  • antibodies, antagonists and oligonucleotides against various signal components of the immune system are tested with the purpose of preventing the activation of T cells.
  • Such drugs are at best selective, but never specifically directed against the wrongly programmed autoimmune reactions.
  • Immunosuppressants therefore have a negative effect on important, necessary and useful parts of the immune system; for this reason and their side effects, their use is limited.
  • Boussiotis VA, Freeman, GJ, Gribben, JG, Nadler, LM 1996.
  • ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 397: 263-6.
  • CD28 co-stimulation stabilizes the expression of the CD40 ligand on T cells.
  • PD-L2 is a second ligand for PD-1 and inhibits T cell activation. 2: 261-268.
  • CTLA4 Cytotoxic T lymphocyte antigen 4
  • CTLA-4 ligation suppresses CD28-induced NF-kappaB and AP-1 activity in mouse T cell blasts [published erratum appears in J Biol Chem 1999 Jul 23; 274 (30): 21490]. J Biol Chem. 274: 14400-14405.

Abstract

The invention relates to a reduction in specific immune reactions. According to the method, antigen-producing cells are stimulated to present defined antigens with simultaneous production of a PD-1 binding molecule.

Description

Antigen-abhängige Reduktion von spezifischen Immunreaktionen durch Beeinflussung der Co-StimulationAntigen-dependent reduction of specific immune reactions by influencing the co-stimulation
Gegenstand der vorliegenden Erfindung sind antigen-präsentierende Zellen (APC), Verfahren zur Herstellung Antigen präsentierender Zellen, Arzneimittel enthaltend Antigen präsentierende Zellen sowie Verwendung der Antigen präsentierenden Zellen.The present invention relates to antigen-presenting cells (APC), processes for producing antigen-presenting cells, medicaments containing antigen-presenting cells and use of the antigen-presenting cells.
Beschreibungdescription
Seit knapp 10 Jahren werden für verschiedene Krankheiten und in Tiermodellen gentherapeutische Verfahren entwickelt und angewendet. Bisher sind sie noch nicht in die Routine überführt. Meist handelt es sich dabei um die Behandlung von genetisch bedingten, schweren Erkrankungen, für die andere Therapien nicht zur Verfügung stehen. Weiterhin werden Gentherapien zur Behandlung von schweren und ebenfalls nicht therapierbaren Krebserkrankungen eingesetzt.For almost 10 years, gene therapy methods have been developed and used for various diseases and in animal models. So far, they have not become routine. Most of the time, this is the treatment of genetically determined, serious diseases for which other therapies are not available. Furthermore, gene therapies are used to treat severe and also non-treatable cancer.
Wenig Aufmerksamkeit hat die experimentelle Medizin bisher der Behandlung von Allergien und Autoimmunerkrankungen mittels gentechnischer Methoden geschenkt.Experimental medicine has so far paid little attention to the treatment of allergies and autoimmune diseases using genetic engineering methods.
Aligemeines zu AllergienGeneral information about allergies
Allergien verursachen beträchtliche Kosten im Gesundheitswesen der Industrieländer. Immerhin sind schätzungsweise mindestens 20% der Bevölkerung gegen irgendeine Substanz allergisch. Die meisten Betroffenen leiden an aller- gischer Rhinitis, zu der insbesondere der Heuschnupfen zählt, oder an Bronchialasthma : Sie niesen oder ringen nach Luft, nachdem sie bestimmte Pollen oder andere im allgemeinen harmlose Substanzen eingeatmet haben. Viele Kinder und einige Erwachsene reagieren auch allergisch auf Nahrungsmittel. Andere erleiden Hautausschläge oder sogar einen allergischen Schock, nach- dem sie Medikamente wie Penizillin erhalten haben. Bei wieder anderen rufen Bienenstiche starke lokale Schwellungen oder schwere systemische - den gesamten Organismus erfassende - Störungen hervor. Im Extremfall können allergische Anfälle sogar zum Tod führen. Allein die unmittelbare medizinische Versorgung von Asthmapatienten hat in den USA 1990 schätzungsweise 3,6 Milliarden Dollar verschlungen und damit rund ein Prozent aller Kosten im Gesundheitswesen ausgemacht.Allergies cause considerable healthcare costs in industrialized countries. After all, it is estimated that at least 20% of the population is allergic to any substance. Most suffer from allergic rhinitis, which includes hay fever in particular, or bronchial asthma: they sneeze or gasp for breath after inhaling certain pollen or other generally harmless substances. Many children and some adults are also allergic to food. Others experience rashes or even an allergic shock after taking medication such as penicillin. Call someone else Bee stings cause severe local swelling or severe systemic disorders that affect the entire organism. In extreme cases, allergic attacks can even lead to death. Immediate medical care for asthma patients in the United States consumed an estimated $ 3.6 billion in 1990, accounting for around one percent of all healthcare costs.
Mittlerweile ist bekannt, dass einige der zellulären und molekularen Wechselwirkungen bei allergischen Reaktionen oft ähnlich ablaufen, unabhängig davon, auf welche Substanzen der einzelne anspricht und welche Symptome er entwickelt. Gewisse Begleiterscheinungen der Allergien treten normalerweise ausschließlich auf, wenn das Immunsystem Parasiten bekämpft. So reagiert der Körper auf Schmarotzer ebenso wie auf Allergene mit der massiven Produktion von Molekülen, die als Immunglobulin-E-Antikörper (IgE) bezeichnet werden. Die Produktion dieser Antikörper wird durch T-Helfer-Zellen induziert, die wiederum von Antigen präsentierenden Zellen aktiviert werden.It is now known that some of the cellular and molecular interactions in allergic reactions are often similar, regardless of which substances respond to the individual and what symptoms he develops. Certain side effects of allergies usually only occur when the immune system is fighting parasites. The body responds to parasites as well as allergens with the massive production of molecules called immunoglobulin E antibodies (IgE). The production of these antibodies is induced by T helper cells, which in turn are activated by cells presenting antigen.
Sensibilisierungsensitization
Unterschiedliche Allergene rufen unter anderem deswegen verschiedenartige Symptome hervor, weil sie mit dem Immunsystem in verschiedenen Körperregionen in Berührung kommen. In den oberen Luftwegen erzeugt die fehlgeleitete Immunreaktion Niesen und eine verstopfte Nase. In den unteren Luftwegen können dagegen die Bronchien sich verengen und verschleimen, so dass typische asthmatische Symptome auftreten. Entsprechend rufen Immunaktivitäten in den Geweben des Magen-Darm-Traktes Übelkeit, Bauchkrämpfe, Durchfall oder Erbrechen hervor.Different allergens cause different symptoms, among other things, because they come into contact with the immune system in different parts of the body. In the upper airways, the misguided immune response causes sneezing and a stuffy nose. In contrast, the bronchi in the lower airways can narrow and become mucous, so that typical asthmatic symptoms appear. Accordingly, immune activities in the tissues of the gastrointestinal tract cause nausea, abdominal cramps, diarrhea or vomiting.
Schließlich vermag ein Allergen, das auf irgendeinem Weg ins Blut gelangt, eine Anaphylaxe auszulösen: eine allergische Reaktion in weit von seiner Eintrittsstelle entfernten Körperregionen. Schwere anaphylaktische Schocks können alle normalen Körperfunktionen durcheinanderbringen und tödlich enden.After all, an allergen that gets into the blood in some way can trigger anaphylaxis: an allergic reaction in regions of the body that are far from its point of entry. Severe anaphylactic shocks can upset all normal bodily functions and can be fatal.
Auch wenn sich die allergischen Reaktionen unterschiedlich äußern, werden sie doch stets durch den gleichen Mechanismus in Gang gesetzt: die Sensibilisierung. Dazu kann bereits der einmalige Kontakt mit einem Allergen, typischer- weise einem Eiweißstoff, genügen. In den Luftwegen oder anderen Geweben trifft die allergieauslösende Substanz auf sogenannte Fresszellen oder Makrophagen. Diese verschlingen den Fremdstoff, zerstückeln ihn und präsentieren die Fragmente auf der Zelloberfläche mit MHC II-Molekülen. Im weiteren Verlauf erkennen einige T-Helfer-Lymphozyten die dargebotenen Bruchstücke und binden daran. Die T-Helfer-Lymphozyten werden von den Makrophagen aktiviert und aktivieren dann ihrerseits einige B-Lymphozyten, die gleichfalls das Allergen erkennen. Die B-Zellen reifen dann zu Antikörper produzierenden Plasmazellen aus. Zunächst sind dies Antikörper vom sogenannten IgM-Typ; ab einem bestimmten Zeitpunkt schalten die Plasmazellen jedoch auf IgE- Antikörper um.Even if the allergic reactions manifest themselves differently, they are always started by the same mechanism: the sensitization. One-time contact with an allergen, typically a protein, are sufficient. In the airways or other tissues, the allergenic substance meets so-called phagocytes or macrophages. These gobble up the foreign substance, dismember it and present the fragments on the cell surface with MHC II molecules. In the further course, some T-helper lymphocytes recognize the fragments presented and bind to them. The T helper lymphocytes are activated by the macrophages and then in turn activate some B lymphocytes, which also recognize the allergen. The B cells then mature into plasma cells that produce antibodies. First of all, these are antibodies of the so-called IgM type; from a certain point in time, however, the plasma cells switch to IgE antibodies.
Bis die Antikörper hergestellt sind, können Tage oder Wochen vergehen, und das Allergen, welches ihre Produktion in Gang gesetzt hat, ist dann womöglich schon lange verschwunden. Nicht so die IgE-Moleküle. Mit ihrer Fc-Region heften sie sich an IgE-Rezeptoren zweier unterschiedlicher Klassen von Immunsystemzellen. Bei der einen handelt es sich um Mastzellen, die sich im Körpergewebe für gewöhnlich in der Nähe von Blutgefäßen und Epithelzellen ansiedeln. Über das Epithel besteht Kontakt mit der Außenwelt (darunter fällt auch das Epithel der Atemwege und des Magen-Darm-Traktes). IgE-Antikörper binden außerdem an basophile Granulozyten (Basophile). Diese Zellen zirkulieren allerdings im Blutstrom.It can take days or weeks for the antibodies to be made, and the allergen that started their production may have long since disappeared. Not the IgE molecules. With their Fc region, they attach themselves to IgE receptors of two different classes of immune system cells. One is mast cells, which are usually found in the body tissues near blood vessels and epithelial cells. There is contact with the outside world via the epithelium (this also includes the epithelium of the respiratory tract and gastrointestinal tract). IgE antibodies also bind to basophilic granulocytes (basophils). However, these cells circulate in the bloodstream.
Hat die Produktion der IgEs einmal begonnen, hält sie offenbar Monate, ja manchmal sogar Jahre an. Folglich besetzen sie unablässig IgE-Rezeptoren auf Mastzellen und Basophilen - bereit, beim nächsten Allergenkontakt augenblicklich in Aktion zu treten.Once production of the IgEs begins, it apparently lasts for months, sometimes even years. As a result, they constantly occupy IgE receptors on mast cells and basophils - ready to take immediate action the next time they come into contact with allergens.
Akute SymptomeAcute symptoms
Während also die erste Begegnung mit einem Allergen selbst bei Personen, die sich später als Allergiker entpuppen, keine Symptome hervorruft, leitet die Zweitexposition ein Stadium der Überempfindlichkeitsreaktion ein, welches auch äußerlich in Erscheinung tritt. Innerhalb von Sekunden nach dem Kontakt mit menschlichem Gewebe bindet der allergieauslösende Stoff an die IgEs der Mastzellen. Heftet er sich dabei an zwei oder mehr IgE-Moleküle zugleich, bil- det er eine Brücke zwischen ihnen. Solche Quervernetzungen lassen die betroffenen IgE-Rezeptoren dichter zusammenrücken, und dies aktiviert die Zelle, so dass sie hochwirksame Substanzen ausschüttet, die auf direktem Wege allergische Symptome erzeugen. (Die Freisetzung kann auch auf andere Arten hervorgerufen werden; von allergischen Reaktionen spricht man nur, wenn IgEs beteiligt sind). Die wichtigste dieser Substanzen ist Histamin. Es kann sowohl die Schleimbildung in den Epithelien anregen und so zur Verstopfung der Luftwege beitragen als auch die glatte Muskulatur, die wie ein elastisches Band Bronchien und Därme umschlingt, kontrahieren lassen. Ferner vermag es die feinen Blutgefäße zu weiten und durchlässiger zu machen, so dass Flüssigkeit ins Gewebe sickern kann. Rötungen und Schwellungen sind die Folge. Betreffen diese Gefäßveränderungen große Teile des Körpers, können sie ein tödliches Kreislaufversagen auslösen: Bei einem solchen Schock fällt der Blutdruck jäh so stark ab, dass die Sauerstoffversorgung von Herz und Gehirn nicht mehr gewährleistet ist.So while the first encounter with an allergen does not cause symptoms even in people who later turn out to be allergy sufferers, the second exposure initiates a stage of the hypersensitivity reaction, which also appears externally. The allergy-causing substance binds to the IgEs of the mast cells within seconds of contact with human tissue. If he attaches himself to two or more IgE molecules at the same time, he bridges between them. Such cross-links bring the affected IgE receptors closer together, and this activates the cell so that it releases highly effective substances that directly produce allergic symptoms. (The release can also be caused in other ways; allergic reactions are only spoken if IgEs are involved). The most important of these substances is histamine. It can stimulate the formation of mucus in the epithelia and thus contribute to the blockage of the air passages, as well as the smooth muscles that wrap around the bronchial tubes and intestines like an elastic band. It is also able to dilate the fine blood vessels and make them more permeable so that fluid can seep into the tissue. The result is redness and swelling. If these vascular changes affect large parts of the body, they can cause fatal circulatory failure: With such a shock, the blood pressure suddenly drops so much that the oxygen supply to the heart and brain is no longer guaranteed.
Die zweite Gruppe von Mediatoren besteht hauptsächlich aus Prostaglandinen und Leukotrienen. Sie werden erst ausgeschüttet, nachdem die Allergenmole- küle sich an die IgEs auf den Zellen angelagert haben. Wie Histamin verengen sie die Bronchien und erweitern die Blutgefäße. Ihre Wirkung hält allerdings länger an.The second group of mediators consists mainly of prostaglandins and leukotrienes. They are only released after the allergen molecules have attached to the IgEs on the cells. Like histamine, they narrow the bronchi and dilate the blood vessels. However, their effects last longer.
Zusätzlich stoßen stimulierte Mastzellen eine Vielzahl potentiell toxischer Enzyme aus. Offenbar setzen sie ferner Cytokine frei, die die Aktivitäten anderer Immunzellen regulieren.In addition, stimulated mast cells emit a variety of potentially toxic enzymes. They also appear to release cytokines that regulate the activities of other immune cells.
Behandlung: 1. Die allergische RhinitisTreatment: 1. Allergic rhinitis
Antihistaminika erweisen sich in der Regel als wirksam und dienen immer noch als Standardtherapie. Die neuesten Varianten können die Blut-Hirn-Schranke nicht mehr ohne weiteres passieren und machen die Patienten nicht mehr müde. Wenn bei einer schweren Entzündung Antihistaminika wirkungslos bleiben, helfen oft inhalierbare Corticosteroide, die gewöhnlich zur Linderung der chronischen Entzündung bei Asthma verschrieben werden. In schweren Fällen kann die schon im Jahre 1911 eingeführte Immuntherapie oder Hyposensibilisierung (auch als Allergiespritzen oder Desensibilisierung bekannt) auf lange Sicht Erleichterung verschaffen. Bei dieser Behandlung injizieren Ärzte den Patienten steigende Dosen des Allergens, auf das diese emp- findlich reagieren. In allen Fällen ist die Dosis ausschlaggebend: Zu wenig Allergen verleiht keine Toleranz. Außerdem ist der Schutz selten vollständig.Antihistamines are usually effective and still serve as standard therapy. The latest variants can no longer easily cross the blood-brain barrier and no longer make the patient tired. If antihistamines are ineffective in severe inflammation, inhalable corticosteroids, which are usually prescribed to relieve chronic inflammation in asthma, often help. In severe cases, immunotherapy or hyposensitization (also known as allergy shots or desensitization), which was introduced in 1911, can provide relief in the long term. With this treatment, doctors inject patients with increasing doses of the allergen to which they are sensitive. The dose is decisive in all cases: too little allergen does not confer tolerance. In addition, protection is rarely complete.
2. Asthma2. Asthma
Bronchodilatatoren sind die meistverwendeten Arzneimittel bei Asthma. Sie lindern die durch Histamin und andere Bronchokonstriktoren hervorgerufenen Symptome sehr schnell, beeinflussen die zugrundeliegende Entzündung jedoch wahrscheinlich nicht. Außerdem kann ihr übermäßiger Gebrauch eine Gegenreaktion des Körpers hervorrufen, so dass nach Abklingen ihrer Wirkung der Atemstrom stärker behindert ist als zuvor. Zusätzlich kommen die unter 1. aufgezählten Methoden zur Anwendung.Bronchodilators are the most widely used drugs for asthma. They relieve the symptoms caused by histamine and other bronchoconstrictors very quickly, but are unlikely to affect the underlying inflammation. In addition, their excessive use can cause a back reaction of the body, so that after their effects have subsided, the respiratory flow is more restricted than before. In addition, the methods listed under 1. are used.
3. Anaphylaktische Reaktionen3. Anaphylactic reactions
Insektenstiche rufen bei manchen Menschen Anaphylaxien aus. In schweren Fällen führen diese zum Tod durch z.B. Kreislaufversagen oder Ersticken. Jede schwere Anaphylaxie - gleich ob sie zum ersten oder fünfzehnten Mal auftritt - ist ein Notfall, bei dem zunächst versucht werden muss, die bedrohlichsten Symptome zu beherrschen. Meist geschieht das durch Injektion von Adrenalin, welches die Freisetzung der Mediatoren hemmt, die Luftwege öffnet und der Erweiterung der Blutgefäße entgegenwirkt. Man kann vorbeugend durch eine Immunisierung z. B. mit dem Gift eines gesundheitsbedrohenden Insekts a- gieren.Insect bites cause anaphylaxis in some people. In severe cases these lead to death e.g. Circulatory failure or suffocation. Any severe anaphylaxis, whether it occurs for the first or fifteenth time, is an emergency, and one must first try to control the most threatening symptoms. This is usually done by injecting adrenaline, which inhibits the release of the mediators, opens the airways and counteracts the dilation of the blood vessels. One can preventively by immunization z. B. act with the poison of a health-threatening insect.
Neue AnsätzeNew approaches
In der Erprobung im Tiermodell befindet sich die auf DNA basierende Immunisierung mit einem Allergen (Der p 5) der Milbe Dermatophagoides pteronyssl- nus. Diese Immunisierung resultiert in einer Produktion von IgG, aber nicht IgE, und resultiert in einer 90%igen Reduktion der Mengen an spezifischem IgE, welche durch klassische Sensibilisierung mit Der p 5 und Alaun als Adju- vant oder allergen-induzierte Rhinitis hervorgerufen wurden.Animal-based immunization with an allergen (Der p 5) of the mite Dermatophagoides pteronyssl- nus is currently being tested in an animal model. This immunization results in IgG production, but not IgE, and results in a 90% reduction in the amounts of specific IgE, which were caused by classic sensitization with Der p 5 and alum as adjuvant or allergen-induced rhinitis.
Eine weitere neue Strategie ist die Verwendung von humanisierten monoklo- nalen Anti-IgE-Antikörpern gegen die FcεRI-Binderegion für IgE. Dadurch wird die Bindung von IgE an den IgE-Rezeptor verhindert, so dass keine Mediatoren der allergischen Reaktion von Mastzellen oder Basophilen ausgeschüttet werden können. Diese Strategie hat in klinischen Studien bei Patienten mit allergi- scher Rhinitis und allergischem Asthma gezeigt, dass diese Antikörper gut toleriert werden und die allergischen Reaktionen reduzieren.Another new strategy is the use of humanized monoclonal anti-IgE antibodies against the FcεRI binding region for IgE. This prevents the binding of IgE to the IgE receptor, so that no mediators of the allergic reaction from mast cells or basophils can be released. This strategy has shown in clinical studies in patients with allergic rhinitis and allergic asthma that these antibodies are well tolerated and reduce the allergic reactions.
(siehe auch L.M. Lichtenstein: "Allergie und Immunsystem"; in Spektrum der Wissenschaft Spezial: Das Immunsystem; 1994; 74-83; S-K Huang, K-Y Chua und K-H Hsieh: Allergen gene transfer. Current Opinion in Immunology 1997; 800-804; C. Heusser und P. Jardieu: Therapeutic potential of anti-IgE antibo- dies. Current Opinion in Immunology 1997; 805-814).(see also LM Lichtenstein: "Allergy and immune system"; in the spectrum of science special: The immune system; 1994; 74-83; SK Huang, KY Chua and KH Hsieh: allergen gene transfer. Current Opinion in Immunology 1997; 800-804; C. Heusser and P. Jardieu: Therapeutic potential of anti-IgE antibodies. Current Opinion in Immunology 1997; 805-814).
Allgemeines zu TransplantationenGeneral information about transplants
Die Transplantation von Geweben, um kranke Organe zu ersetzen, ist heute eine wichtige medizinische Therapie. In den meisten Fällen stellt eine Reaktion des anpassungsfähigen Immunsystems gegen das Transplantat die größte Bedrohung für eine erfolgreiche Behandlung dar. Reaktionen des anpassungsfä- higen Immunsystems werden von Antigen präsentierenden Zellen durch Aktivierung von T-Helfer-Lymphozyten induziert. Bei der Transfusionen von Blut, welches das erste und am häufigsten verwendete Transplantat ist, müssen die ABO und Rh Blutgruppenantigene abgeglichen werden, damit die schnelle Zerstörung nicht passender Erythrozyten vermieden wird. Bei anderen Geweben müssen die sehr polymorphen Haupthistokompatibilätskomplexe (MHC) aufeinander abgeglichen werden, da diese fast immer die Immunreaktion auslösen. Leider ist der perfekte Abgleich der MHCs, außer bei Verwandten, fast unmöglich.Tissue transplantation to replace diseased organs is an important medical therapy today. In most cases, a response from the adaptive immune system to the graft poses the greatest threat to successful treatment. Responses from the adaptable immune system are induced by antigen presenting cells by activating T helper lymphocytes. When transfusing blood, which is the first and most commonly used graft, the ABO and Rh blood group antigens must be matched to avoid the rapid destruction of inappropriate erythrocytes. In the case of other tissues, the very polymorphic main histocompatibility complexes (MHC) have to be matched to one another, since these almost always trigger the immune response. Unfortunately, the perfect matching of the MHCs is almost impossible, except for relatives.
Obwohl die Immunreaktion Organtransplantationen schwierig macht, gibt es wenige Alternativen bei Organausfällen. Die Verwendung von potenten im- munsuppressiven Wirkstoffen, besonders Cyclosporin A und FK-506, die die Aktivierung von T-Zellen verhindern, macht Organtransplantationen erfolgreich. Trotzdem entstehen hier einige Probleme, da das Leiden, welches das eigene Organ zerstört hat, auch oft das fremde Organ zerstört. Außerdem steigt durch die Unterdrückung des Immunsystems, das Risiko an Krebs oder Infektionen zu erkranken. Zudem ist die Prozedur sehr kostspielig (Janeway and Travers 1997).Although the immune response makes organ transplantation difficult, there are few alternatives to organ failure. The use of potent im- Organ-suppressive agents, especially cyclosporin A and FK-506, which prevent the activation of T cells, make organ transplants successful. Nevertheless, some problems arise here, since the suffering that has destroyed one's own organ often also destroys the foreign organ. The suppression of the immune system also increases the risk of developing cancer or infections. The procedure is also very expensive (Janeway and Travers 1997).
Co-stimulatorische MoleküleCo-stimulatory molecules
Das Produkt des Maus PD-1 Gens (ACCESSION NM_021893), einem Mitglied der IgG-Superfamilie, ist ein Rezeptor mit Verbreitung in vielen Geweben. Analysen, die durch Durchflusszytometrie und Immunopräzipitation mit dem monoklonalen Antikörper J43mAk durchgeführt wurden, zeigten, dass das PD- 1 Genprodukt ein 50 bis 55 kDa großes Membranprotein ist. Das PD-1 Protein scheint stark glycosyliert zu sein, da das berechnete Molekulargewicht der A- minosäuresequenz 29310 Da ist. Normales lymphoides Gewebe der Maus wie Thymus, Milz, Lymphknoten und Knochenmark enthalten nur eine geringe Anzahl an PD-1-positiven Zellen. Trotzdem erscheint eine signifikante PD-1- positive Population in Thymozyten ebenso wie auf T-Zellen in Milz- und Lymphknoten durch die in vivo -Behandlung mit Anti-CD3 monoklonalen Antikörpern. Weiterhin wurde die PD-1-Antigenexpression durch in vitro - Stimulation in verschiedenen Untergruppen von Thymozyten und Milz-T-Zellen stark induziert, entweder mit Anti-CD3 mAk oder Concavalin A, welches T-Zellen sowohl zur Aktivierung als auch zum Zelltod bringen kann. Die PD-1- Expression auf Milz-B-Zellen wurde in ähnlicher Weise mit Anti-IgM Ak stimuliert. Im Gegensatz dazu wurde PD-1 nach Behandlungen wie Wachstumsfaktorentzug, Dexamethason oder Lipopolysaccharid nicht signifikant exprimiert. Diese Ergebnisse legen die Vermutung nahe, dass die Expression von PD-1 strikt reguliert ist und durch Signaltransduktion über den Antigenrezeptor induziert wird und schließt nicht die Möglichkeit aus, dass das PD-1 Antigen in der klonalen Selektion von Lymphozyten eine Rolle spielt, obwohl für den normalen Ablauf der Apoptose PD-1 Expression nicht erforderlich ist (Agata et al 1996; Ishida et al 1992; Nishimura et al 1996). Der PD-1 Rezeptor induziert die Verminderung von Immunantworten und sein Verlust führt zum Zusammenbruch der Toleranz in peripheren Geweben. PD-1 defiziente Mäuse entwickeln Lupus-artige proliferative Arthritis und Glomerulo- nephritis mit prädominater IgG3 Ablagerung während des Alterns. PD-1 ist von der Struktur ähnlich zum CTL-assozierten Antigen 4 (CTLA-4), welches B7-1 und B7-2 bindet und eine entscheidende Rolle bei der Instandhaltung der T- Zell-Homeostase spielt (für Reviews, siehe (Sperling & Bluestone 1996; Thompson & Allison 1997)). PD-1 enthält das MYPPPY Motiv nicht, welches kritisch für die B7-1 und B7-2 Bindung ist. Die extrazelluläre Region von PD-1 und CTLA-4 bestehen jeweils aus einer IgV-Domäne mit 23% Identität zueinander. Zu PD-1 existiert ein Ligand (PD-Ll). Die Bindung von PD-1 durch PD- Ll führt zur Inhibition der TCR-vermittelten T-Zell-Proliferation und Cytokin- sekretion (Freeman et al 2000).The product of the mouse PD-1 gene (ACCESSION NM_021893), a member of the IgG superfamily, is a receptor that is common in many tissues. Analyzes carried out by flow cytometry and immunoprecipitation with the monoclonal antibody J43mAk showed that the PD-1 gene product is a 50 to 55 kDa membrane protein. The PD-1 protein appears to be highly glycosylated since the calculated molecular weight of the amino acid sequence is 29310 Da. Normal mouse lymphoid tissue such as thymus, spleen, lymph nodes and bone marrow contain only a small number of PD-1 positive cells. Nevertheless, a significant PD-1 positive population appears in thymocytes as well as on T cells in spleen and lymph nodes due to the in vivo treatment with anti-CD3 monoclonal antibodies. Furthermore, PD-1 antigen expression was strongly induced by in vitro stimulation in various subgroups of thymocytes and spleen T cells, either with anti-CD3 mAb or Concavalin A, which can cause T cells to activate as well as to die , PD-1 expression on spleen B cells was similarly stimulated with anti-IgM Ab. In contrast, PD-1 was not significantly expressed after treatments such as growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of PD-1 is strictly regulated and induced by signal transduction via the antigen receptor and does not exclude the possibility that the PD-1 antigen plays a role in the clonal selection of lymphocytes, although for the normal course of apoptosis PD-1 expression is not required (Agata et al 1996; Ishida et al 1992; Nishimura et al 1996). The PD-1 receptor induces the decrease in immune responses and its loss leads to the breakdown of tolerance in peripheral tissues. PD-1 deficient mice develop lupus-like proliferative arthritis and glomerulonephritis with predominant IgG3 deposition during aging. PD-1 is structurally similar to CTL-associated antigen 4 (CTLA-4), which binds B7-1 and B7-2 and plays a crucial role in the maintenance of T cell homeostasis (for reviews, see (Sperling & Bluestone 1996; Thompson & Allison 1997)). PD-1 does not contain the MYPPPY motif, which is critical for B7-1 and B7-2 binding. The extracellular region of PD-1 and CTLA-4 each consist of an IgV domain with 23% identity to each other. A ligand (PD-Ll) exists for PD-1. The binding of PD-1 by PD-Ll leads to the inhibition of TCR-mediated T cell proliferation and cytokine secretion (Freeman et al 2000).
Das humane und Maus PD-Ll Molekül (EMBL/GenBank/DDBS unter accession nos. AF233516 und AF233517, (Freeman et al 2000)) sind Mitglieder der B7 Genfamilie und haben eine ähnliche strukturelle Organisation, die aus einer IgV und einer IgC Domäne in der extrazellulären Region (Boussiotis et al 1996), einer hydrophoben Transmembrandomäne, gefolgt von einer kurzen, geladenen intrazellulären Region besteht. Das humane PD-Ll ist mit 290 Aminosäuren identisch zu B7-H1, von welchem berichtet wird, daß es die T-Zell- Stimulation aktiviert (Dong et al 1999). Die Maus PD-Ll cDNA kodiert ein Po- lypeptid mit 70% Aminosäureidentität zum humanen PD-Ll. PD-Ll hat Aminosäureidentitäten von 21, 20, und 23% zu B7-1, B7-2, und ICOS (ligand of in- ducible co-stimulator) (Boussiotis et al 1996; Ling et al 2000; Swallow et al 1999; Yoshinaga et al 1999). Die Expressionsmuster von B7-1, B7-2, und PD-Ll sind verschieden. B7-2, einer der Liganden von CD28 und CTLA-4, wird konstitutiv auf Monozyten exprimiert. Die konstitutive Expression von B7-1 und B7-2 kann allerdings in keinem Organ beobachtet werden. Die B7-1 und B7-2 Expression kann in Dendritischen Zellen, Macrophagen und B-Zellen (Boussiotis et al 1996), sowie einigen Typen von Fibroblasten und Epithelzellen induziert werden. Im Kon- trast dazu wird PD-Ll konstitutive von nicht-lymphoiden, parenchymalen Organen wie dem Herzen, der Placenta, Skeletmuskeln und Lunge, nicht aber dem Dünndarm exprimiert (Dong et al 1999). PD-Ll wird auch in einigen Krebsarten exprimiert. Möglicherweise benutzen diese Tumoren PD-Ll, um Antitumorimmunantworten zu inhibieren. Da PD-1 auf aktivierten T- als auch B-Zellen exprimiert wird (2), könnte die Expression von PD-Ll in nicht- lymphoiden Geweben, sowie auf dendritischen Zellen die Regulation von po- tentiell autoreaktiven Lymphozyten ermöglichen. Das könnte ein wichtiger Mechanismus sein, um Aktivitäten von T- und B-Zellen im Herzen, in der Lunge, in den Nieren und in der Placenta zu limitieren, wo PD-Ll hoch exprimiert ist (Freeman et al 2000). PD-1 Ligand 2 (PD-L2) ist ein zweiter Ligand für PD-1. Die Bindung von PD-1 mit PD-L2 inhibiert dramatisch die T-Zellrezeptor (TCR) -vermittelte Proliferation und Cytokinproduktion durch CD4 T-Zellen. Bei niedriger Antigenkonzent- ration inhibieren PD-L2-PD-l-Interaktionen starke B7-CD28-Signale. Bei hohen Antigenkonzentration reduzieren sie die Cytokinproduktion aber inhibieren nicht die T-Zellproliferation. PD-L-PD-1-Interaktionen führen zum Zellzyclu- sarrest in G0 /Gx (Zellzyklusphasen) erhöhen aber nicht die Anzahl der toten Zellen. Die PD-L2-Expression auf APCs wird durch Behandlung mit Interferon γ erhöht und konnte auch in einigen anderen Geweben und Tumorzellinien de- tektiert werden. PD-Ll und PD-L2 haben also überlappende Funktionen (Latchman et al 2001). mPD~L2 (bisheriger Name: Protein AF142780) kodiert für ein Polypeptide mit 38% Aminosäureidentität zu mPD-Ll. Murines und humanes PD-L2 haben 70% Aminosäureidentität. Die fünf Angehörigen der B7-Familie — B7-1, B7-2, ICOS-L, PD-Ll und PD-L2— haben 21-27% Aminosäureidentität und eine strukturelle Organization, die aus einer Signalsequenz, einer IgV-artigen, einer IgC-artigen und einer transmembran Domäne und einem kurzen cytoplasmati- schen Schwanz besteht. Der cytoplasmatische Schwanz von PD-Ll ist zwischen Mäusen und Menschen konserviert, was im Kontrast zu der geringen Konservierung des cytoplasmatischen Schwanz von PD-L2 von Menschen und Mäusen steht (Latchman et al 2001).The human and mouse PD-Ll molecule (EMBL / GenBank / DDBS under accession nos. AF233516 and AF233517, (Freeman et al 2000)) are members of the B7 gene family and have a similar structural organization, which consists of an IgV and an IgC domain in the extracellular region (Boussiotis et al 1996), a hydrophobic transmembrane domain, followed by a short, charged intracellular region. Human PD-Ll is identical to B7-H1 at 290 amino acids, which is reported to activate T cell stimulation (Dong et al 1999). The mouse PD-Ll cDNA encodes a polypeptide with 70% amino acid identity to the human PD-Ll. PD-Ll has amino acid identities of 21, 20, and 23% to B7-1, B7-2, and ICOS (ligand of inducible co-stimulator) (Boussiotis et al 1996; Ling et al 2000; Swallow et al 1999; Yoshinaga et al 1999). The expression patterns of B7-1, B7-2, and PD-Ll are different. B7-2, one of the ligands of CD28 and CTLA-4, is constitutively expressed on monocytes. However, the constitutive expression of B7-1 and B7-2 cannot be observed in any organ. B7-1 and B7-2 expression can be induced in dendritic cells, macrophages and B cells (Boussiotis et al 1996), as well as some types of fibroblasts and epithelial cells. In contrast, PD-Ll is constitutively expressed by non-lymphoid, parenchymal organs such as the heart, placenta, skeletal muscles and lungs, but not the small intestine (Dong et al 1999). PD-Ll is also expressed in some cancers. These tumors may be using PD-Ll to inhibit anti-tumor immune responses. Since PD-1 is expressed on activated T and B cells (2), the expression of PD-Ll in non-lymphoid tissues and on dendritic cells could regulate po- enable potentially autoreactive lymphocytes. This could be an important mechanism to limit T and B cell activity in the heart, lungs, kidneys and placenta where PD-Ll is highly expressed (Freeman et al 2000). PD-1 Ligand 2 (PD-L2) is a second ligand for PD-1. Binding of PD-1 to PD-L2 dramatically inhibits T cell receptor (TCR) mediated proliferation and cytokine production by CD4 T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. At high antigen concentrations, they reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle residue in G 0 / Gx (cell cycle phases) but do not increase the number of dead cells. PD-L2 expression on APCs is increased by treatment with interferon γ and could also be detected in some other tissues and tumor cell lines. PD-Ll and PD-L2 thus have overlapping functions (Latchman et al 2001). mPD ~ L2 (previous name: Protein AF142780) codes for a polypeptide with 38% amino acid identity to mPD-Ll. Murines and human PD-L2 have 70% amino acid identity. The five members of the B7 family - B7-1, B7-2, ICOS-L, PD-Ll and PD-L2 - have 21-27% amino acid identity and a structural organization consisting of a signal sequence, an IgV-like one IgC-like and a transmembrane domain and a short cytoplasmic tail. The cytoplasmic tail of PD-Ll is conserved between mice and humans, which is in contrast to the poor conservation of the cytoplasmic tail of PD-L2 of humans and mice (Latchman et al 2001).
Die PD-L2 Gewebeverteilung ist ähnlich wie die von PD-Ll. Man findet eine Expression in Plazenta, Herz, Pankreas, Lunge und Leber, eine niedrige Expression in Milz, Lymphknoten und Thymus, keine Expression in unstimulierten Monozyten, die aber durch Interferon γ induziert werden konnte. Die Kinetik die- ser Induktion ist allerdings langsamer als die von PD-Ll.The PD-L2 tissue distribution is similar to that of PD-Ll. There is expression in the placenta, heart, pancreas, lungs and liver, low expression in the spleen, lymph nodes and thymus, no expression in unstimulated monocytes, but this could be induced by interferon γ. However, the kinetics of this induction is slower than that of PD-Ll.
Allgemeines zu AutoimmunerkrankungenGeneral information on autoimmune diseases
Autoimmunerkrankungen gehören zu den chronischen Erkrankungen. Zu ihnen zählen Rheuma in verschiedensten klinischen Ausprägungen, Diabetes, Multiple Sklerose, bestimmte Formen von Herzmuskelentzündungen und von Schilddrüsenerkrankungen. Die Reihe von Autoimmunerkrankungen ließe sich beliebig fortsetzen, wobei ca. 90% allerdings epidemiologisch nur einen kleinen Prozentsatz ausmachen.Autoimmune diseases are chronic diseases. These include rheumatism in various clinical forms, diabetes, multiple sclerosis, certain forms of cardiac muscle inflammation and Thyroid disease. The series of autoimmune diseases could be continued as desired, although approximately 90% epidemiologically only make up a small percentage.
Die klinischen Verläufe von Autoimmunerkrankungen selbst bei ein- und derselben Diagnose können sehr unterschiedlich sein. Z. T. werden schubartige Krankheitsverläufe beobachtet. Entsprechend werden die Behandlungen vom Arzt individuell gestaltet.The clinical course of autoimmune diseases, even with one and the same diagnosis, can be very different. Relapsing courses of the disease are sometimes observed. Accordingly, the treatments are individually designed by the doctor.
Einige der Autoimmunerkrankungen sind antikörpervermittelt. Darunter wird in der Immunologie verstanden, dass der Organismus aus meist nicht bekannten Ursachen Antikörper produziert, welche sich gegen körpereigene zelluläre Strukturen (Autoantigene) richten. Sind diese Antikörper einmal gebildet, lösen sie durch ihre Interaktion und Bindung an die jeweiligen organ- oder zell- spezifischen Strukturen eine zell- und gewebszerstörende Reaktion des Immunsystems aus. Letztendlich führt diese dann zu dem klinischen Bild der Erkrankung (Steinman 1994).Some of the autoimmune diseases are antibody-mediated. In immunology, this means that the organism produces antibodies for mostly unknown reasons, which are directed against the body's own cellular structures (autoantigens). Once these antibodies have been formed, their interaction and binding to the respective organ or cell-specific structures trigger a cell and tissue-destroying reaction of the immune system. Ultimately, this leads to the clinical picture of the disease (Steinman 1994).
Beispiel hierfür ist die Schilddrüsenerkrankung Morbus Basedow, aber auch eine Form der Herzmuskelentzündung, die zur Dilatativen Cardiomyopathie führt. In einigen Fällen sind sowohl die Targets, gegen die sich die Autoantikörper richten, als auch die Autoantikörper selbst, genau charakterisiert.An example of this is the thyroid disease Graves' disease, but also a form of cardiac muscle inflammation that leads to dilated cardiomyopathy. In some cases, both the targets against which the autoantibodies are directed and the autoantibodies themselves are precisely characterized.
PCT/EP 00/03984 offenbart ein Verfahren zur Reduzierung von spezifischen Immunreaktionen mittels Unterdrückung der B7-Expression von Antigen präsentierenden Zellen bei gleichzeitiger Expression vorbestimmter (insbesondere pathologischer) Antigene.PCT / EP 00/03984 discloses a method for reducing specific immune reactions by suppressing the B7 expression of cells presenting antigen while simultaneously expressing predetermined (in particular pathological) antigens.
Die WO-A-01/14557 betrifft PD-1 als Rezeptor für B7-4. Die Druckschrift of- fenbart, dass B7-4 Immunzellaktivierung inhibieren kann aufgrund von Bindung an einen inhibitorischen Rezeptor oder an eine Immunzelle. Es werden Wirkstoffe beschrieben für die Modulierung von PD-1, B7-4 sowie deren Wechselwirkung, um co-stimulierende oder inhibierende Signale in einer Immunzelle zu modulieren, welches in einer Modulation der Immunreaktion resultiert. Ein der Erfindung zu Grunde liegendes Problem besteht unter anderem darin, gentechnologische, therapeutisch nutzbare Produkte für die Reduktion von spezifischen Immunreaktionen zur Verfügung zu stellen, bei denen Targets (Antigene, Autoantigene) und Antikörper, Autoantikörper bekannt sind.WO-A-01/14557 relates to PD-1 as a receptor for B7-4. The document discloses that B7-4 can inhibit immune cell activation due to binding to an inhibitory receptor or to an immune cell. Active substances are described for the modulation of PD-1, B7-4 and their interaction in order to modulate co-stimulating or inhibiting signals in an immune cell, which results in a modulation of the immune response. A problem on which the invention is based is, inter alia, to provide genetic engineering, therapeutically usable products for the reduction of specific immune reactions in which targets (antigens, autoantigens) and antibodies, autoantibodies are known.
Gelöst wird das Problem durch die erfindungsgemäße antigen-präsentierende Zelle, die überwiegend vorher bestimmte Antigene präsentiert (monoantigene antigen-präsentierende Zelle) und dadurch gekennzeichnet ist, dass in der monoantigenen antigen-präsentierenden Zelle PD-1 bindende Moleküle vor- zugsweise Antikörper produziert werden und gegebenenfalls CTLA4 bindende Moleküle produziert werden und gegebenenfalls eine der Funktionen co- stimulatorischer Rezeptoren, wie ein B7- und/oder CD40-Rezeptor supprimiert ist.The problem is solved by the antigen-presenting cell according to the invention, which predominantly presents certain antigens beforehand (monoantigenic antigen-presenting cell) and is characterized in that molecules which bind PD-1 are preferably produced in the monoantigenic antigen-presenting cell and if appropriate, CTLA4-binding molecules are produced and, if appropriate, one of the functions of stimulatory receptors, such as a B7 and / or CD40 receptor, is suppressed.
Fig. 1 zeigt ein Schema zur Funktion der Genmanipulation auf zellulärer Ebene:1 shows a diagram of the function of gene manipulation at the cellular level:
a) normaler Mechanismus der T-Helfer-Zell-Aktivierung über Monozytena) Normal mechanism of T helper cell activation via monocytes
b) Mechanismus der Monozyten nach Genmanipulation. Ausbleiben der T-Helfer-Zell-Aktivierung nach Genmanipulation der Monozyten.b) Mechanism of monocytes after genetic engineering. Absence of T helper cell activation after gene manipulation of the monocytes.
Fig. 2 zeigt eine "Normale" Immunreaktion zur Antikörperbildung, antigen- präsentierende Monozyten als Induktoren der Antikörperproduktion. Monozy- ten nehmen als fremd erkannte Moleküle auf, zerlegen sie und bringen die Bruchstücke an MHC II-Molekülen auf die Zelloberfläche (Antigenpräsen- tation). Gleichzeitig wird ein Oberflächenmolekül (B7) exprimiert, das sowohl an CD28 als auch an CTLA4 binden kann. Das präsentierte Antigen wird von T- Helfer-Lymphozyten erkannt, die ihrerseits B-Zellen zur Antikörperproduktion anregen. Das Schema ist stark vereinfacht. CTLA4 (CD152) ist ein Analogon zu CD28, bindet auch B7, induziert aber die Apoptose der T-Zelle. CTLA4 kommt hauptsächlich in intrazellulären Vesikeln vor und wird erst nach Aktivierung der Zellen freigesetzt. PD-1 ist ein CD28 und CTLA4 Analogon und entfaltet CTLA4- artige Effekte. Fig. 3 zeigt schematisch die Funktion der Genmanipulation auf molekularer Ebene: PD-1 wird durch einen Liganden (PD-Ll), der sich auf der Plasmamembran des Monozyten (antigen-präsentierende Zelle) befindet, gebunden und die T-Zelle so inaktiviert.2 shows a “normal” immune reaction for antibody formation, antigen-presenting monocytes as inducers of antibody production. Monocytes take up molecules recognized as foreign, disassemble them and bring the fragments of MHC II molecules onto the cell surface (antigen presentation). At the same time, a surface molecule (B7) is expressed that can bind to both CD28 and CTLA4. The antigen presented is recognized by T helper lymphocytes, which in turn stimulate B cells to produce antibodies. The scheme is very simplified. CTLA4 (CD152) is an analogue of CD28, also binds B7, but induces apoptosis of the T cell. CTLA4 occurs mainly in intracellular vesicles and is only released after activation of the cells. PD-1 is a CD28 and CTLA4 analogue and develops CTLA4-like effects. Fig. 3 shows schematically the function of gene manipulation at the molecular level: PD-1 is bound by a ligand (PD-Ll), which is located on the plasma membrane of the monocyte (antigen-presenting cell), and thus inactivates the T cell.
Der Effekt kann verstärkt werden, wenn zusätzlich B7 in der antigen- präsentierenden Zelle unterdrückt wird und gegebenenfalls auch noch ein CTLA4 bindendes Molekül eingeschleust wird.The effect can be intensified if B7 is additionally suppressed in the antigen-presenting cell and, if appropriate, a CTLA4-binding molecule is also introduced.
Fig. 4 zeigt die Funktion der Genmanipulation auf molekularer Ebene: das Autoantigen wird von den PD-Ll produzierenden antigen-präsentierenden Zellen synthetisiert.Fig. 4 shows the function of gene manipulation at the molecular level: the autoantigen is synthesized by the PD-Ll producing antigen presenting cells.
Die Fig. 5 und 6 zeigen einen Vergleich der Antigenpräsentation von "normalen" und gentechnisch veränderten Monozyten. Fig. 5 zeigt Autoantigenmengen, die an den MHC II präsentiert werden. Normale Monozyten präsentieren selten das Autoantigen und wenn, dann wird es nur von wenigen MHC II präsentiert. Fig. 6 zeigt, dass die genmanipulierten Monozyten fast ausschließlich das Autoantigen präsentieren.5 and 6 show a comparison of the antigen presentation of "normal" and genetically modified monocytes. Figure 5 shows autoantigen levels presented on the MHC II. Normal monocytes rarely present the autoantigen and if so, only a few MHC II present it. 6 shows that the genetically manipulated monocytes almost exclusively present the autoantigen.
Fig. 7a und 7b demonstrieren die Funktion der genetisch veränderten Monozyten im Körper. Gemäß Fig. 7a induzieren natürliche autoantigen- präsentierende Monozyten die Produktion von (pathologischen) Autoantikörpern, wenn sie B7-positiv sind. Gemäß Fig. 7b konkurrieren genmanipulierte Monozyten mit den natürlichen autoantigen-präsentierenden Monozyten und drosseln die Autoantikörperproduktion.7a and 7b demonstrate the function of the genetically modified monocytes in the body. According to FIG. 7a, natural autoantigen-presenting monocytes induce the production of (pathological) autoantibodies if they are B7-positive. According to FIG. 7b, genetically manipulated monocytes compete with the natural autoantigen-presenting monocytes and reduce autoantibody production.
Vorzugsweise ist die erfindungsgemäße monoantigene antigen-präsentierende Zelle ein Monozyt, eine dendritische Zelle und/oder ein Makrophage.The monoantigenic antigen-presenting cell according to the invention is preferably a monocyte, a dendritic cell and / or a macrophage.
Die APCs sind die Schaltstellen des anpassungsfähigen Immunsystems. Nur sie können eine T-Zell-vermittelte Immunantwort auslösen. Dies sei in folgenden Abbildungen am Beispiel von Monozyten und der durch T-Helfer-Zellen ausgelösten Antikörperproduktion gezeigt: Innerhalb des Immunsystems spielen die Antikörper normalerweise als spezifische Abwehrmoleküle ("humorale Immunreaktion") eine wichtige Rolle. Sie werden von ausgereiften B-Lymphozyten produziert. Die Induktion und Produktion der löslichen Antikörper erfolgt allerdings nicht unabhängig von den restlichen Zellen des Immunsystems; vielmehr wird diese humorale Immunreaktion von anderen Zellen des Immunsystems gesteuert.The APCs are the switching points of the adaptable immune system. Only they can trigger a T cell-mediated immune response. This is shown in the following figures using the example of monocytes and the antibody production triggered by T helper cells: Within the immune system, the antibodies normally play an important role as specific defense molecules ("humoral immune response"). They are produced by mature B lymphocytes. However, the induction and production of soluble antibodies is not independent of the remaining cells in the immune system; rather, this humoral immune response is controlled by other cells in the immune system.
Die Antikörperproduktion - und so auch die Produktion der pathologischen Autoantikörper - lässt sich nicht aufrechterhalten ohne die Hilfe und Vermitt- lung von Monozyten und T-Helfer-Lymphozyten, die ihrerseits antigenspezi- fisch die B-Lymphozyten aktivieren (Fig.l).Antibody production - and thus also the production of pathological autoantibodies - cannot be maintained without the help and mediation of monocytes and T-helper lymphocytes, which in turn activate the B-lymphocytes in an antigen-specific manner (Fig. 1).
Die molekularen Mechanismen der Interaktion - des Zell-Zell-Kontakts - von Monozyten mit den T-Helfer-Lymphozyten ist auf Rezeptorebene bis ins Detail bekannt (Fig. 2).The molecular mechanisms of the interaction - of cell-cell contact - of monocytes with the T helper lymphocytes is known in detail at the receptor level (FIG. 2).
Handelt es sich bei dem Antigen z.B. um ein Bakterienprotein, ist die Immunreaktion für den Organismus nützlich. Ist das Antigen allerdings eine körpereigene Struktur, spricht man von einer (pathologischen) Autoimmunreaktion.If the antigen is e.g. a bacterial protein, the immune response is useful for the organism. However, if the antigen is an endogenous structure, one speaks of a (pathological) autoimmune reaction.
Neben dem zu präsentierenden Antigen (gegen welches sich die zu produzierenden Antikörper richten) sind auf der Zelloberfläche verschiedene Rezeptoren und Hilfsrezeptoren am Zell-Zell-Kontakt und der Zellaktivierung beteiligt. Ein essentielles Molekül der interzellulären Wechselwirkung bei der Antigenprä- sentation (Kontakt von Monozyt mit den T-Helfer-Lymphozyten) ist ein costi- mulierender Rezeptor mit der Bezeichnung B7 (Fig. 2).In addition to the antigen to be presented (against which the antibodies to be produced are directed), various receptors and auxiliary receptors on the cell surface are involved in cell-cell contact and cell activation. An essential molecule of the intercellular interaction in the antigen presentation (contact of monocyte with the T helper lymphocytes) is a costingulatory receptor with the designation B7 (FIG. 2).
Die erfindungsgemäße monoantigene antigen-präsentierende Zelle weist, vorzugsweise durch eine Transfektion einer antigen-präsentierenden Zelle mit nukleinsäurehaltigem Material, eine erhöhte Expression eines Antigens auf, wobei die antigen-präsentierende Zelle im wesentlichen nur vordefinierte Antigene präsentiert.The monoantigenic antigen-presenting cell according to the invention has an increased expression of an antigen, preferably by transfection of an antigen-presenting cell with nucleic acid-containing material, the antigen-presenting cell essentially presenting only predefined antigens.
Das erfindungsgemäße gentherapeutische Verfahren beruht auf parallelen gentechnologischen Eingriffen an patienteneigenen Blutmonozyten. Die Eingriffe erfolgen mittels geeigneter Sonden und bewirken die Produktion von PD- 1 bindenden Molekülen vorzugsweise PD-Ll und gegebenenfalls CTLA4 bindenden Molekülen, vorzugsweise Antikörper (nicht abgebildet), und gegebenenfalls die Verminderung von B7-Molekülen durch die Behinderung bzw. Verhinderung der Ausbildung des Moleküls auf der Oberfläche der Monozyten (nicht abgebildet) (Fig. 3) und gleichzeitig eine starke Präsentation des Autoantigens (Fig. 4 - 6).The gene therapy method according to the invention is based on parallel genetic engineering interventions on the patient's own blood monocytes. The interventions are carried out using suitable probes and cause the production of PD 1 binding molecules, preferably PD-L1 and possibly CTLA4 binding molecules, preferably antibodies (not shown), and possibly the reduction of B7 molecules by hindering or preventing the formation of the molecule on the surface of the monocytes (not shown) (Fig. 3) and at the same time a strong presentation of autoantigen (Fig. 4 - 6).
Verminderung der spezifischen Antikörperproduktion durch GentherapieReduction of specific antibody production through gene therapy
Die Produktion pathologischer Autoantikörper wird durch die Manipulation von Monozyten (bzw. DCs) und die daraus resultierende Abschaltung antigen- spezifischer T-Zellen spezifisch beendet. Hierbei wird ausgenutzt, dass T- Zellen den Rezeptor PD-1 produzieren. Dieser sorgt bei Bindung an PD-Ll da- für, dass T-Zellen nicht mehr proliferieren. Eine Bindung von PD-1 kann also für das Abstellen von T-Zell-Antworten sorgen. Hierfür können auch andere Bindemoleküle als PD-Ll eingesetzt werden.The production of pathological autoantibodies is specifically ended by the manipulation of monocytes (or DCs) and the resulting shutdown of antigen-specific T cells. This takes advantage of the fact that T cells produce the receptor PD-1. When bound to PD-Ll, this ensures that T cells no longer proliferate. Binding of PD-1 can thus shut down T cell responses. Binding molecules other than PD-Ll can also be used for this.
Zusätzlich kann ausgenutzt werden, dass T-Zellen zwei Rezeptoren produzie- ren, die B7 (1 oder 2) erkennen. Der eine Rezeptor ist CD28, der andere ist CTLA4. CD28 entfaltet eine stimulierende Wirkung, während CTLA4 eine solche Wirkung nicht entfaltet. Der Kontakt von CTLA4 mit B7 sorgt für das Abschalten der jeweiligen T-Zelle. Diese geht dann entweder in die Apoptose über programmierten Zelltod, wird abgeschaltet, oder wird in eine toleranzerzeu- gende T-Zelle umgewandelt. Die Toleranz wird in diesem Fall gegen das spezielle von dieser T-Zelle erkannte Antigen erzeugt. (Gribben et al., 1994; Jud- ge et al., 1999; Lee et al., 1998; Lin et al., 1998; Metzler et al., 1997; Olsson et al., 1999; Oosterwegel et al., 1999a; Oosterwegel et al., 1999b; Peach et al., 1994; Walunas et al., 1996; Wu et al., 1997; Wulfing and Davis, 1998).In addition, it can be exploited that T cells produce two receptors that recognize B7 (1 or 2). One receptor is CD28, the other is CTLA4. CD28 has a stimulating effect, while CTLA4 does not. The contact of CTLA4 with B7 switches off the respective T cell. This then either goes into apoptosis via programmed cell death, is switched off, or is converted into a tolerance-generating T cell. In this case, the tolerance is generated against the specific antigen recognized by this T cell. (Gribben et al., 1994; Judge et al., 1999; Lee et al., 1998; Lin et al., 1998; Metzler et al., 1997; Olsson et al., 1999; Oosterwegel et al., 1999a; Oosterwegel et al., 1999b; Peach et al., 1994; Walunas et al., 1996; Wu et al., 1997; Wulfing and Davis, 1998).
Zur Bindung an CTLA4 anstatt von CD28 werden Moleküle, wie Antikörper, eingesetzt, die an CTLA4, nicht aber an CD28 binden. Diese können dann die von CTLA4 beförderte Reaktion der T-Zellen induzieren. Diese CTLA4 bindenden Moleküle können vorzugsweise Antikörper sein. Der Co-Rezeptor B7, ohne den die Antigenpräsentation bzw. die Induktionskaskade zur Antikörperproduktion nicht anläuft, kann zusätzlich unterdrückt werden, um eine präferentielle Bindung von CD28 zu unterdrücken. Der Co- Rezeptor stellt zwei unterschiedliche Co-Rezeptoren, die als CD80 (B7-1) und CD86 (B7-2) bezeichnet werden, dar. Ihre Strukturen sind bekannt.For binding to CTLA4 instead of CD28, molecules such as antibodies are used which bind to CTLA4 but not to CD28. These can then induce the T cell response promoted by CTLA4. These CTLA4 binding molecules can preferably be antibodies. The co-receptor B7, without which the antigen presentation or the induction cascade for antibody production does not start, can also be suppressed in order to suppress preferential binding of CD28. The co-receptor is two different co-receptors called CD80 (B7-1) and CD86 (B7-2). Their structures are known.
Nach dem Abschalten der Antikörperproduktion verschwinden die im Blut zirkulierenden Autoantikörper aufgrund des natürlichen Abbaus und des fehlenden Nachschubs.After the antibody production is switched off, the autoantibodies circulating in the blood disappear due to the natural degradation and the lack of replenishment.
Nach der in vitro Manipulation der Monozyten werden die Zellen in die Blutbahn des Patienten zurückgegeben. Die gentechnisch veränderten Monozyten schalten nunmehr die im Organismus befindlichen pathologischen T-Helfer- Lymphozyten ab. Die gentechnisch veränderten Zellen treten dabei unmittel- bar in Konkurrenz zu den bereits im Organismus (vorzugsweise Blutbahn und Lymphsystem) vorhandenen Autoantigen-präsentierenden Monozyten, die allerdings B7 auf der Zelloberfläche tragen und normalerweise die T-Helfer- Lymphozyten und damit die Antikörperproduktion aktivieren (Fig. 7a und 7b).After the in vitro manipulation of the monocytes, the cells are returned to the patient's bloodstream. The genetically modified monocytes now switch off the pathological T helper lymphocytes in the organism. The genetically modified cells compete directly with the autoantigen-presenting monocytes already present in the organism (preferably the bloodstream and lymphatic system), which however carry B7 on the cell surface and normally activate the T helper lymphocytes and thus the antibody production (Fig 7a and 7b).
Antigenpräsentation bei gentechnisch veränderten MonozytenAntigen presentation in genetically modified monocytes
Die cDNA eines Proteins, das als Autoantigen die Produktion pathologischer Autoantikörper hervorruft, wird in das Monozytengenom integriert. Diese Erbinformation dient dann zur Überproduktion des Autoantigens. Peptide dieses Autoantigens werden daraufhin bevorzugt an MHC II und/oder MHC I präsentiert [siehe auch Fig. 5 und 6]. MHC II präsentiert die Peptide den T- Helferzellen und versucht solche zu finden, die spezifisch diese präsentierten Peptide erkennen. Wenn gleichzeitig durch ein PD-1 bindendes Molekül PD-1 anstelle von CD28 aktiviert wird und eventuell zusätzlich ein CTLA4 bindendes Molekül CTLA4 anstelle von CD28 aktiviert und eventuell zusätzlich der Co- Rezeptor B7 nicht gleichzeitig an der Zelloberfläche erscheint, werden die T- Helferzellen stillgelegt und können eventuell einen vorzeitigen Zelltod erleiden.The cDNA of a protein that causes the production of pathological autoantibodies as an autoantigen is integrated into the monocyte genome. This genetic information then serves to overproduce the autoantigen. Peptides of this autoantigen are then preferably presented on MHC II and / or MHC I [see also FIGS. 5 and 6]. MHC II presents the peptides to the T helper cells and tries to find those that specifically recognize these presented peptides. If a PD-1 binding molecule activates PD-1 instead of CD28 and a CTLA4-binding molecule also activates CTLA4 instead of CD28 and the co-receptor B7 does not appear on the cell surface at the same time, the T helper cells are shut down and may experience premature cell death.
Alle gentechnisch veränderten Monozyten präsentieren an den meisten ihrer MHC II-Komplexe das Autoantigen, während in vivo nur sehr wenige "normale"All genetically modified monocytes present the autoantigen on most of their MHC II complexes, while very few "normal" in vivo
Monozyten das Autoantigen präsentieren und dann auch nur an wenigen MHC II-Komplexen. Es ist Ziel der Behandlung, in vivo die "normalen" Monozyten, die das Autoantigen präsentieren und die T-Helfer-Zellen aktivieren, zu verdrängen durch die auf Abschaltung der Antikörperproduktion bzw. T- Zellantwort programmierten, gentechnisch veränderten Monozyten.Monocytes present the autoantigen and then only at a few MHC II complexes. The aim of the treatment is to displace the "normal" monocytes which present the autoantigen and activate the T helper cells in vivo by the genetically modified monocytes which have been programmed to switch off the antibody production or T cell response.
Die erfindungsgemäße monoantigene antigen-präsentierende Zelle kann insbesondere eine erhöhte Anzahl von Homing-Rezeptoren, wie CD44, aufzeigen. Eine Überexpression von Homing-Rezeptoren wird der monoantigenen anti- gen-präsentierenden Zelle den Weg in die Lymphknoten weisen, wodurch die gentechnisch veränderten APCs sich vermehrt und schneller in Lymphknoten ansammeln. Die Lymphknoten sind der Ort, an dem die allermeisten Reaktionen des anpassungsfähigen Immunsystems hervorgerufen werden. Dort entfalten die gentechnisch veränderten APCs daher eine um ein vielfaches höhere Wirkung als außerhalb der Lymphknoten.The monoantigenic antigen-presenting cell according to the invention can in particular show an increased number of homing receptors, such as CD44. Overexpression of homing receptors will show the monoantigenic antigen-presenting cell the way into the lymph nodes, as a result of which the genetically modified APCs increase and accumulate faster in lymph nodes. The lymph nodes are where most of the responses of the adaptive immune system are triggered. There, the genetically modified APCs have a much greater effect than outside the lymph nodes.
In einer bevorzugten Ausführungsform der erfindungsgemäßen monoantigenen antigen-präsentierenden Zelle bewirkt eine Transfektion eine Erhöhung der Anzahl von Homing-Rezeptoren und oder eine Erhöhung der Anzahl von PD-1 bindenden Molekülen und/oder eine Suppression von Funktionen der B7-, CD40-Rezeptoren und/oder eine Erhöhung der Anzahl von CTLA4 bindenden Molekülen.In a preferred embodiment of the monoantigenic antigen-presenting cell according to the invention, transfection causes an increase in the number of homing receptors and or an increase in the number of PD-1 binding molecules and / or a suppression of functions of the B7, CD40 receptors and / or an increase in the number of CTLA4 binding molecules.
Die PD-1 bindenden Moleküle können vorzugsweise PD-Ll, PD-L2, Antikörper, monoklonale Antikörper sein. Diese können so geartet sein, dass sie in der Plasmamembran der Zelle verbleiben.The PD-1 binding molecules can preferably be PD-Ll, PD-L2, antibodies, monoclonal antibodies. These can be such that they remain in the plasma membrane of the cell.
Die CTLA4 bindenden Moleküle können vorzugsweise Antikörper, monoklonale Antikörper sein. Diese können so geartet sein, dass sie in der Plasmamembran der Zelle verbleiben.The CTLA4 binding molecules can preferably be antibodies, monoclonal antibodies. These can be such that they remain in the plasma membrane of the cell.
Insbesondere mit Antisense-Nukleinsäuren kann die B7- und/oder CD40- Rezeptorexpression in der erfindungsgemäßen monoantigenen antigen- präsentierenden Zelle verhindert oder reduziert werden. Alternativ kann mit Nukleinsäuren eine Unterdrückung der Expression der B7- und/oder CD40-Rezeptoren durch Co-Suppression in der erfindungsgemäßen monoantigenen antigen-präsentierenden Zelle bewirkt werden.In particular with antisense nucleic acids, the B7 and / or CD40 receptor expression in the monoantigenic antigen-presenting cell according to the invention can be prevented or reduced. Alternatively, the expression of the B7 and / or CD40 receptors can be suppressed by nucleic acids by co-suppression in the monoantigenic antigen-presenting cell according to the invention.
Die erfindungsgemäße monoantigene antigen-präsentierende Zelle kann Nukleinsäuren enthalten oder damit transfiziert sein, die eine Expression von mit B7- und/oder CD40-Rezeptoren affinen Strukturen aufweisenden Proteinen oder Peptiden bewirken. Damit werden Proteine gebildet, die durch Komplexbildung mit B7 - und/oder CD40-Rezeptoren diese Rezeptoren praktisch neut- ralisieren. Insbesondere kommen dazu CTLA4, CD28, Antikörper, F(ab)2, scFv und/oder Fab-Fragmente als Proteine in Betracht.The monoantigenic antigen-presenting cell according to the invention can contain or be transfected with nucleic acids which bring about an expression of proteins or peptides which have structures affine with B7 and / or CD40 receptors. In this way, proteins are formed which, by forming complexes with B7 and / or CD40 receptors, practically neutralize these receptors. In particular, CTLA4, CD28, antibodies, F (ab) 2 , scFv and / or F ab fragments can be considered as proteins.
In einer weiteren bevorzugten Ausführungsform enthält die erfindungsgemäße monoantigene antigen-präsentierende Zelle Nukleinsäuren, die für eine Signalsequenz kodieren oder Expressϊonsprodukte einer Signalsequenz, die den Verbleib der Expressionsprodukte im endoplasmatischen Retikulum, dem Gol- gi-Apparat, dem Trans-Golgi-Netzwerk oder intrazellulären Vesikeln bewirkt.In a further preferred embodiment, the monoantigenic antigen-presenting cell according to the invention contains nucleic acids which code for a signal sequence or expression products of a signal sequence which determine whether the expression products remain in the endoplasmic reticulum, the Golgi apparatus, the Trans-Golgi network or intracellular vesicles causes.
Die erfindungsgemäße monoantigene antigen-präsentierende Zelle wird zur Expression von Antigenen mit Nukleinsäuren transfiziert, die den Transport der exprimierten Antigene in MHC II-Kompartimente der Zellen ermöglichen.The monoantigenic antigen-presenting cell according to the invention is transfected for the expression of antigens with nucleic acids which enable the transport of the expressed antigens in MHC II compartments of the cells.
Alle gentechnisch veränderten Monozyten präsentieren an den meisten ihrer MHC -Komplexe das Autoantigen, während nur sehr wenige "normale" Mono- zyten überhaupt das Autoantigen präsentieren und dann auch nur an wenigen MHC -Komplexen. Es ist Ziel der Behandlung, die "normalen" Monozyten, die das Autoantigen präsentieren und die T-Helfer-Zellen aktivieren, zu verdrängen durch die auf Abschaltung der Antikörperprodukte programmierten, genmanipulierten Monozyten. Es ist sehr wichtig, das Antigen (die Antigene) auch in einer Form zu verabreichen, die den Transport in die MHC II-Endosomen ermöglicht. Nur so kann zuverlässig eine Präsentation an MHC II erreicht werden. Bei einem gentechnischen Eingriff erfolgt dies in der Regel durch Manipulation des offenen Leserasters, so dass dem Leseraster eine Signalsequenz für dieses Kompartiment vorgeschaltet wird. Die genetische Manipulation hat den zusätzlichen Vorteil, dass sie eine viel längere Halbwertzeit hat, als z.B. Oligo- nukleotide oder Peptide. Eine stabile Integration von Genen führt sogar zu einer permanenten Veränderung der Eigenschaft der Zielzellen.All genetically modified monocytes present the autoantigen on most of their MHC complexes, while very few "normal" monocytes present the autoantigen at all and then only on a few MHC complexes. The aim of the treatment is to displace the "normal" monocytes that present the autoantigen and activate the T helper cells by means of the genetically manipulated monocytes programmed to switch off the antibody products. It is very important to also administer the antigen (s) in a form that allows them to be transported into the MHC II endosomes. This is the only way to reliably achieve a presentation at MHC II. In the case of a genetic engineering intervention, this is usually done by manipulating the open reading frame, so that the reading frame is preceded by a signal sequence for this compartment. Genetic manipulation has the additional advantage that it has a much longer half-life than, for example, oligo- nucleotides or peptides. A stable integration of genes even leads to a permanent change in the properties of the target cells.
Die entsprechenden Nukleinsäuren können dabei DNA, RNA, Oligonukleotide, Polynukleotide, Ribozyme, Peptidnukleinsäuren (PNA) sein.The corresponding nucleic acids can be DNA, RNA, oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNA).
Vorzugsweise besitzt die DNA Regulationselemente wie Enhancer, Promotoren, polyA-kodierende 3 " -Enden zur Transkription der DNA in RNA, die RNA Regulationselemente zur Translation der RNA in Protein. Die Regulations-elemente sorgen für eine effiziente Expression der Gene.The DNA preferably has regulatory elements such as enhancers, promoters, polyA-coding 3 "ends for transcribing the DNA into RNA, the RNA regulatory elements for translating the RNA into protein. The regulatory elements ensure efficient expression of the genes.
Die Herstellung der erfindungsgemäßen monoantigenen antigen- präsentierenden Zelle erfolgt zum Beispiel durch ex vivo oder in vivo Verfahren. Dabei wird vorzugsweise eine antigen-präsentierende Zelle ex vivo oder in vivo durch Behandlung mit Viren, viralen Vektoren, bakteriellen Vektoren, Plasmiden, die durch Elektroporationstechniken, Iontophorese, ballistischen Methoden und/oder anderen Techniken zur Einschleusung von Molekülen in eine monoantigene antigen-präsentierende Zelle transfiziert.The monoantigenic antigen-presenting cell according to the invention is produced, for example, by ex vivo or in vivo methods. An antigen-presenting cell is preferably ex vivo or in vivo by treatment with viruses, viral vectors, bacterial vectors, plasmids by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a monoantigenic antigen-presenting cell transfected.
In einer weiteren Ausführungsform kann eine antigen-präsentierende Zelle oder eine monoantigene antigen-präsentierende Zelle durch Behandlung mit Viren, viralen Vektoren, bakteriellen Vektoren, Plasmiden, die durch Elektroporationstechniken, Iontophorese, ballistischen Methoden und/oder anderen Techniken zur Einschleusung von Molekülen in eine Zelle mit erhöhter Menge von PD-1 bindenden Molekülen und gegebenenfalls mit erhöhter Menge von CTLA4 bindenden Molekülen und gegebenenfalls supprimierter Funktion co- stimulatorischer Rezeptoren transformiert werden oder die Expression co- stimulatorischer Rezeptoren durch Verhinderung von deren Expression oder die co-stimulatorischen Rezeptoren werden durch Reaktion mit affinen Struk- turen an einer Stimulation von T-Zellen, die an die monoantigene antigen- präsentierende Zelle gebunden sind, gehindert.In another embodiment, an antigen-presenting cell or a monoantigenic antigen-presenting cell can be obtained by treatment with viruses, viral vectors, bacterial vectors, plasmids, by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with an increased amount of PD-1 binding molecules and optionally with an increased amount of CTLA4 binding molecules and possibly suppressed function of co-stimulatory receptors or the expression of co-stimulatory receptors by preventing their expression or the co-stimulatory receptors are reacted with affine structures prevented from stimulating T cells bound to the monoantigenic antigen presenting cell.
Für die Unterdrückung der Co-Stimulation, die letztendlich ein Unterdrücken der Produktion eines oder mehrerer Proteine oder die Behinderung des Prote- ins oder der Proteine bedeutet, kommen verschiedene Strategien in Frage: 1. Antisense-AnsatzDifferent strategies can be used to suppress co-stimulation, which ultimately means suppressing the production of one or more proteins or hindering the protein or proteins: 1. Antisense approach
2. Co-Suppression2. Co-suppression
3. Bindung des co-stimulatorischen Moleküls.3. Binding of the co-stimulatory molecule.
Zu 1. : In diesem Fall müssen Antisense-Nukleinsäuren in Kontakt mit der mRNA des co-stimulatorischen Moleküls kommen. Sie binden dann wahrscheinlich das Molekül und verhindern die Translation. Als Nuk- leinsäuren kommen eine große Bandbreite von Molekülen, wie RNAs,Re 1.: In this case, antisense nucleic acids must come into contact with the mRNA of the co-stimulatory molecule. You will then likely bind the molecule and prevent translation. A wide range of molecules, such as RNAs, come as nucleic acids
DNAs, PNAs, Ribozyme, in Frage. Auch hier würde ein gentechnischer Eingriff für die größte Halbwertszeit des Effekts sorgen.DNAs, PNAs, ribozymes, in question. Here, too, a genetic engineering intervention would ensure the longest half-life of the effect.
Zu 2. : Es hat sich inzwischen herausgestellt, dass man die Produktion eines Genproduktes auch durch Integration einer möglichst homologenRe 2.: It has now been found that the production of a gene product can also be achieved by integrating a homologous one
Sense-Gensequenz erreichen kann. Der Mechanismus ist noch völlig unbekannt.Sense gene sequence can achieve. The mechanism is still completely unknown.
Zu 3. : Die Bindung des co-stimulatorischen Moleküls z.B. durch spezifische Antikörper verhindert, dass dieses Molekül Kontakt mit dem avisiertenRe 3.: The binding of the co-stimulatory molecule e.g. by specific antibodies prevents this molecule from contacting the notified
Rezeptor auf einer T-Zelle aufnimmt und verhindert so die Aktivierung der T-Zelle. Die externe Zugabe solcher Bindemoleküle hat den Nachteil, dass sie auf alle antigen-präsentierenden Zellen wirken und damit jede Immunreaktion verhindern. Um Immunreaktionen spezifisch zu behindern, haben wir ein Modell entworfen, bei dem die co- stimulatorischen Moleküle bereits in der Zelle, in einem intrazellulären Kompartiment gebunden werden. In einer Erweiterung dieses Modells soll dafür gesorgt werden, dass das Bindemolekül im intrazellulären Kompartiment zurückgehalten wird, so dass der co-stimulatorische Re- zeptor erst gar nicht zur Plasmamembran gelangt. Hierfür sind Signalsequenzen bekannt, die dem offenen Leseraster des Bindemoleküls zugefügt werden müssen. Dieser intrazelluläre Ansatz lässt sich gut an i- solierten Zellen durchführen. Auch hier ist ein gentechnischer Eingriff zu bevorzugen. Die gewünschten Effekte können auch erreicht werden, wenn nur eines der beiden Ziele mit einem gentechnischen Eingriff vorgenommen wird. In diesem Fall können statt der Gene z. B. folgende andere Moleküle eingesetzt werden:Receptor takes up on a T cell and thus prevents activation of the T cell. The external addition of such binding molecules has the disadvantage that they act on all antigen-presenting cells and thus prevent any immune reaction. In order to specifically hinder immune reactions, we have designed a model in which the co-stimulatory molecules are bound in the cell, in an intracellular compartment. An extension of this model is intended to ensure that the binding molecule is retained in the intracellular compartment so that the co-stimulatory receptor does not reach the plasma membrane in the first place. Signal sequences are known for this, which must be added to the open reading frame of the binding molecule. This intracellular approach can be carried out well on isolated cells. Here, too, genetic engineering intervention is preferable. The desired effects can also be achieved if only one of the two goals is carried out with a genetic engineering intervention. In this case, z. B. the following other molecules are used:
1. Behinderung der Co-Stimulation durch1. Impairment of co-stimulation by
Nukleinsäuren (zumeist komplementär zur Zielsequenz), bei denen es sich z. B. um Oligonukleotide, Polynukleotide, Ribozyme, Peptidnukleinsäuren (PNAs) handeln kann,Nucleic acids (mostly complementary to the target sequence), which are e.g. B. can be oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNAs),
Antikörper oder andere Moleküle, die die co-stimulatorischen Moleküle binden.Antibodies or other molecules that bind the co-stimulatory molecules.
2. Antigen Kontaktierung durch2. Antigen contacting through
Proteine, Peptide, Peptidomimetica.Proteins, peptides, peptidomimetics.
Es werden nach dem erfindungsgemäßen Verfahren insbesondere Moleküle wie Antikörper, Proteine, Peptide, Peptidomimetica, PD-Ll, PD-L2, CTLA4, CD28, CD40L und/oder Bestandteile und/oder Kombinationen dieser Moleküle, die z.B. B7-1, B7-2, CD40 binden, welche eine in Gegenwart einer Antigenpräsentation stattfindende Co-Stimulation der T-Zelle behindert, mit der monoantigenen antigen-präsentierenden Zelle oder der antigen-präsentierenden Zelle in Kontakt gebracht.In particular, molecules such as antibodies, proteins, peptides, peptidomimetics, PD-L1, PD-L2, CTLA4, CD28, CD40L and / or constituents and / or combinations of these molecules, which e.g. B7-1, B7-2, CD40 bind, which prevents co-stimulation of the T cell taking place in the presence of an antigen presentation, brought into contact with the monoantigenic antigen presenting cell or the antigen presenting cell.
Es kann vorteilhaft sein, die Moleküle mittels Vehikeln, wie Liposomen, Hydro- gelen, Zyklodextrinen, Nanokapseln, Nanopartikel, insbesondere biologisch abbaubaren Nanokapseln oder -partikel, bio-adhäsiven Mikrokugeln und/oder durch Elektroporationstechniken, Iontophorese, ballistische Methoden und/oder andere Techniken zur Einschleusung von Molekülen in die monoantigene antigen-präsentierende Zelle oder die antigen-präsentierende Zelle zu transferieren.It can be advantageous to use molecules such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, in particular biodegradable nanocapsules or particles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or other techniques to transfer molecules into the monoantigenic antigen-presenting cell or the antigen-presenting cell.
Nukleinsäuren können insbesondere durch Viren, virale Vektoren, bakterielle Vektoren, Plasmide, die durch Elektroporationstechniken, Iontophorese, ballistische Methoden und/oder andere Techniken zur Einschleusung von Molekülen in die monoantigene antigen-präsentierende Zelle oder die antigen- präsentierende Zelle transferiert werden.Nucleic acids can in particular by viruses, viral vectors, bacterial vectors, plasmids by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for the introduction of molecules are transferred into the monoantigenic antigen-presenting cell or the antigen-presenting cell.
Erfindungsgemäß beansprucht wird weiterhin ein Arzneimittel, enthaltend min- destens eine erfindungsgemäße monoantigene antigen-präsentierende Zelle. Vorzugsweise ist das erfindungsgemäße Arzneimittel als Infusionslösung zur intravenösen oder intraperitonealen Applikation formuliert. Die Formulierung ist so gewählt, dass bei Verabreichung des Arzneimittels keine wesentliche Beeinträchtigung der Wirksamkeit der erfindungsgemäßen monoantigenen anti- gen-präsentierenden Zelle erfolgt.A medicament containing at least one monoantigenic antigen-presenting cell according to the invention is further claimed. The medicament according to the invention is preferably formulated as an infusion solution for intravenous or intraperitoneal administration. The formulation is chosen such that when the medicament is administered there is no significant impairment of the effectiveness of the monoantigenic antigen-presenting cell according to the invention.
So kommt als Infusionslösung vorzugsweise physiologische Kochsalzlösung in Betracht. Grundsätzlich sind auch andere Lösungen, die einen pH-Wert von 5,5 bis 8,5 aufweisen, geeignet. Auch Serum, beispielsweise humanes Serum, autologes Serum oder Serum anderer Spezies, Lösungen mit Plasmaersatzstoffen, wie Polyvinylpyrrolidon, kommen in Betracht. Typischerweise sollen 0,5 ml bis 500 ml appliziert werden. Diese Mengen und pH-Werte sind selbstverständlich nicht absolut, sondern können vom Fachmann, je nach Bedingungen und Anforderungen, variiert und an die spezifischen Bedürfnisse eines Patienten angepasst werden.For example, physiological saline is preferred as the infusion solution. In principle, other solutions with a pH of 5.5 to 8.5 are also suitable. Serum, for example human serum, autologous serum or serum of other species, solutions with plasma substitutes, such as polyvinylpyrrolidone, are also suitable. Typically 0.5 ml to 500 ml should be applied. These quantities and pH values are of course not absolute, but can be varied and adapted to the specific needs of a patient by a specialist, depending on the conditions and requirements.
Die erfindungsgemäße monoantigene antigen-präsentierende Zelle kann insbesondere zur Herstellung eines Arzneimittels zur Behandlung von ungewollten Immunreaktionen, wie Autoimmunerkrankungen und Allergien oder gewollt hervorgerufenen Immunreaktionen, wie bei Immunisierungen verwendet werden.The monoantigenic antigen-presenting cell according to the invention can be used in particular for the production of a medicament for the treatment of unwanted immune reactions such as autoimmune diseases and allergies or deliberately induced immune reactions such as in immunizations.
Weiterhin kann die erfindungsgemäße monoantigene antigen-präsentierende Zelle zur Herstellung eines Arzneimittels zur Behandlung von Immunreaktio- nen gegen allologe und/oder xenologe Gewebsmerkmale verwendet werden.Furthermore, the monoantigenic antigen-presenting cell according to the invention can be used for the production of a medicament for the treatment of immune reactions against allologic and / or xenologic tissue characteristics.
Erfindungsgemäß beansprucht werden insbesondere Verwendungen, bei denen die zu behandelnden Immunreaktionen in Verbindung mit Antigenen oder deren Gensequenzen und/oder Teilen davon stehen und ausgewählt sind aus der Gruppe bestehend aus Enzymen, deren Gensequenzen und/oder Teilsequenzen, insbesondere Glutamic acid decarboxylase (GAD), Rezeptor-Typ Protein Tyro- sin Phosphatase IA-2Beta, Antigen: H+K+ATPase, U1RNP, Transglu- taminase, Argininosuccinatlyase (ASL), Tyrosinase-related protein-2, Thyroid Peroxidase, Faktor VIII, Faktor IX;In particular, uses are claimed according to the invention in which the immune reactions to be treated are related to antigens or their gene sequences and / or parts thereof and are selected from the group consisting of Enzymes, their gene sequences and / or partial sequences, in particular glutamic acid decarboxylase (GAD), receptor type protein tyrosine phosphatase IA-2Beta, antigen: H + K + ATPase, U1RNP, transglutaminase, argininosuccinate lyase (ASL), tyrosinase related protein-2, thyroid peroxidase, factor VIII, factor IX;
Rezeptoren, deren Gensequenzen und/oder Teilsequenzen, insbesondere Acetylcholinrezeptor vom Nicotintyp, ßl-adrenerger-Rezeptor, αl-adrenerger-Rezeptor, Angiotensin-2-ATl-Rezeptor, Glutamat- Rezeptor, Thyrotropin-stimulierendes Hormon (TSH)-Rezeptor, LFA-1,Receptors, their gene sequences and / or partial sequences, in particular acetylcholine receptor of the nicotine type, β1-adrenergic receptor, αl-adrenergic receptor, angiotensin-2-ATl receptor, glutamate receptor, thyrotropin-stimulating hormone (TSH) receptor, LFA receptor 1,
HLA-B27, Epididymal Protein DE, Zona Pellucida (ZP)-3 Glycoprotein, Zona Pellucida (ZP)-4 Glycoprotein, Follicle-Stimulating Hormone (FSH) Rezeptor, Sperm Immunogen SP-10 oder Sperm Protein SP- 10;HLA-B27, Epididymal Protein DE, Zona Pellucida (ZP) -3 Glycoprotein, Zona Pellucida (ZP) -4 Glycoprotein, Follicle-Stimulating Hormone (FSH) receptor, Sperm Immunogen SP-10 or Sperm Protein SP-10;
Hormone oder Botenstoffe, deren Gensequenzen und/oder Teilsequenzen, insbesondere Insulin, Thyroglobulin, Follicle-Stimulating Hormone (FSH), Prostaglandin F2 alpha, Gonadotropin-Releasing Hormone (GnRH), Oestradiol-17beta, Oestrogen, Luteinizing Hormon (LH) Rezeptor, Inhibin, Testosteron, Androgen, Chorionic Gona- dotrophin (CG), Interleukine, Interferone, Cytokine, Chemokine, Bone Morphogenetic Factors, ß-Interferon, Estradiol;Hormones or messenger substances, their gene sequences and / or partial sequences, in particular insulin, thyroglobulin, follicle-stimulating hormones (FSH), prostaglandin F2 alpha, gonadotropin-releasing hormones (GnRH), oestradiol-17beta, estrogen, luteinizing hormone (LH) receptor, inhibin , Testosterone, androgen, chorionic gonodrophin (CG), interleukins, interferons, cytokines, chemokines, bone morphogenetic factors, ß-interferon, estradiol;
Strukturproteine, deren Gensequenzen und/oder Teilsequenzen, insbesondere Myelin Basic Protein (MBP), Proteolipid protein (PLP), Mye- lin oligodendrocyte glycoprotein (MOG), α-Fodrin, Nicht-erythroides α-Structural proteins, their gene sequences and / or partial sequences, in particular myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), α-fodrin, non-erythroid α-
Spectrin, Beta-Amyloid Precursor Protein (beta-APP), Typ 2 Kollagen, Sperm Plasma Membran Protein PH-20;Spectrin, beta-amyloid precursor protein (beta-APP), type 2 collagen, sperm plasma membrane protein PH-20;
Antigene, deren Gensequenzen und/oder Teilsequenzen, insbeson- dere CENP-A Autoantigen, Beta2GP-I, ribosomales P Protein, Ro/SSA,Antigens, their gene sequences and / or partial sequences, in particular CENP-A autoantigens, Beta2GP-I, ribosomal P protein, Ro / SSA,
La/SSB, Sm/RNP, Sm, Scl-70, Jo-1, BCOADC-E2, Albumin, Glucagon, Inselzellantigene, Retinal S Ag;La / SSB, Sm / RNP, Sm, Scl-70, Jo-1, BCOADC-E2, albumin, glucagon, islet cell antigens, Retinal S Ag;
Allergene, die eine IgE-Antwort auslösen, deren Gensequenzen und/oder Teilsequenzen, insbesondere Der f 1, Der f 2, Der f 3, Der pAllergens that trigger an IgE response, their gene sequences and / or partial sequences, in particular Der f 1, Der f 2, Der f 3, Der p
1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 8, Eur m 1, Lep d 2, Fei d 1, Can f 1, Can f 2, Mus m 1, Rat n 1, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Per a 1, Bienengift Phospholipase A2 (PLA2), Group V major aller- gen Phl p 5b von Timothy Grass Pollen, Hom s 1.1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 8, Eur m 1, Lep d 2, Fei d 1, Can f 1, Can f 2, Mus m 1, Rat n 1, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Per a 1, Bee venom Phospholipase A 2 (PLA 2 ), Group V major allergen Phl p 5b by Timothy Grass Pollen, Hom s 1.
Weiterhin wird erfindungsgemäß beansprucht eine Verwendung, bei der die zu behandelnden Immunreaktionen in Verbindung mit allologen und/oder xenolo- gen Gewebsmerkmalen, deren Gensequenzen und/oder Teilsequenzen, insbesondere MHC I, MHC II, Rhesus Faktor stehen.Furthermore, the invention claims a use in which the immune reactions to be treated are associated with allologic and / or xenological tissue characteristics, their gene sequences and / or partial sequences, in particular MHC I, MHC II, rhesus factor.
Entwicklung einer GentherapieDevelopment of a gene therapy
Das hier vorgestellte Verfahren zur Reduzierung von Immunantworten, z. B. zur Behandlung von Autoimmunerkrankungen mit klar definiertem Autoantikörperprofil, basiert auf der gentechnischen Manipulation von bestimmten Blutzellen vorzugsweise außerhalb des Körpers, die dann anschließend wieder in den Organismus zurückgeführt werden. Ziel dieser Behandlung ist unter anderem das spezifische Abschalten einer chronischen, durch das Immunsystem getriebenen Produktion von Autoantikörpern, welche die Krankheit auslösen.The method presented here for reducing immune responses, e.g. B. for the treatment of autoimmune diseases with a clearly defined autoantibody profile, is based on the genetic manipulation of certain blood cells, preferably outside the body, which are then subsequently returned to the organism. The aim of this treatment is, among other things, the specific switching off of a chronic, immune system-driven production of autoantibodies that trigger the disease.
Anwendungsgebiete der GentherapieAreas of application of gene therapy
Das Verfahren zur spezifischen Abschaltung von Immunreaktionen ist immer dort sinnvoll, wo ein oder mehrere molekular definierte Targets (Antigene, Autoantigene) bekannt sind. Diese können beispielsweise mit einer Autoim- munerkrankung oder Allergie assoziiert sein.The procedure for the specific deactivation of immune reactions always makes sense where one or more molecularly defined targets (antigens, autoantigens) are known. These can be associated with an autoimmune disease or allergy, for example.
Z. B. handelt es sich dabei um Krankheiten mit Autoantikörper-vermittelten Autoimmunreaktionen, d.h. um Krankheiten, bei denen die "Bindungsstrukturen" dieser Autoantikörper (Autoantigene, Targets) bekannt sind und pathogenetische Bedeutung haben.For example, these are diseases with autoantibody-mediated autoimmune reactions, i.e. diseases in which the "binding structures" of these autoantibodies (autoantigens, targets) are known and have pathogenetic significance.
Beispiele für Krankheiten mit bekannten, die Autoantikörper-bindenden molekularen Strukturen (Epitope) sind:Examples of diseases with known molecular structures (epitopes) that bind autoantibodies are:
• Myasthenia gravis (Autoantikörper gegen den Acetylcholinrezeptor) • Morbus Basedow (Autoantikörper gegen den TSH-Rezeptor der Schilddrüse)• myasthenia gravis (autoantibodies against the acetylcholine receptor) • Graves' disease (autoantibody against the TSH receptor of the thyroid gland)
• Dilatative Cardiomyopathie (DCM, Autoantikörper gegen den ßl-adrenergen Rezeptor der Herzmuskelzellen)Dilated cardiomyopathy (DCM, autoantibodies against the β1-adrenergic receptor of the heart muscle cells)
• bestimmte Formen des insulinabhängigen Diabetes (Autoantikörper gegen Insulin)• certain forms of insulin-dependent diabetes (autoantibodies against insulin)
• bestimmte Formen des malignen Bluthochdrucks (Autoantikörper gegen den Angiotensin- und/oder αl-adrenergen-Rezeptor).• certain forms of malignant hypertension (autoantibodies against the angiotensin and / or αl adrenergic receptor).
Behandlung mit genmanipulierten patienteneigenen Blutzellen: Prinzip der vorgeschlagenen GentherapieTreatment with genetically manipulated patient's own blood cells: principle of the proposed gene therapy
Innerhalb des Immunsystems spielen die Antikörper als Abwehrmoleküle ("humorale Immunreaktion") eine wichtige Rolle. Sie werden von ausgereiften B-Lymphozyten produziert. Die Induktion und Produktion der Antikörper erfolgt allerdings nicht losgelöst von den restlichen Zellen des Immunsystems; vielmehr wird diese humorale Immunreaktion von anderen Zellen des Immunsystems gesteuert. Die Immunreaktion lässt sich nicht aufrechterhalten ohne die Hilfe und Vermittlung von antigen-präsentierenden Zellen und T-Helfer- Lymphozyten. Die molekularen Mechanismen der Interaktion - des Zell-Zell- Kontakts - von antigen-präsentierenden Zellen mit den T-Helfer-Lymphozyten ist auf Rezeptorebene bis ins Detail bekannt.Within the immune system, the antibodies play an important role as defense molecules ("humoral immune response"). They are produced by mature B lymphocytes. However, the induction and production of the antibodies is not detached from the remaining cells of the immune system; rather, this humoral immune response is controlled by other cells in the immune system. The immune response cannot be maintained without the help and mediation of antigen presenting cells and T helper lymphocytes. The molecular mechanisms of the interaction - of cell-cell contact - of antigen-presenting cells with the T-helper lymphocytes is known in detail at the receptor level.
Neben dem zu präsentierenden Antigen sind auf der Zelloberfläche verschiedene Rezeptoren und Hilfsrezeptoren am Zell-Zell-Kontakt und der Zellaktivierung beteiligt. Ein essentielles Molekül der interzellulären Wechselwirkung bei der Antigenpräsentation (Kontakt von antigen-präsentierenden Zellen mit den T-Helfer-Lymphozyten) sind costimulierende Rezeptoren mit den Bezeichnungen PD-Ll, PD-L2, B7h (LICOS), B7-1 und B7-2. Auch CD40 spielt bei dieser Signaltransduktion eine Rolle. Die Funktionen von ICOS, PD-1, CD28, CTLA4, B7 und CD40 sind Inhalt zahlreicher Publikationen (Daikh et al., 1997; Greenfield et al., 1998; Johnson- Leger et al., 1998; McAdam et al., 1998; Vyth-Dreese et al., 1995, (Freeman et al 2000; Hutloff et al 1999; Kopf et al 2000; Tamatani et al 2000)) und werden (außer ICOS, PDl) auch umfangreich in Lehrbüchern abgehandelt [s. z.B. (Janeway and Travers, 1997)].In addition to the antigen to be presented, various receptors and auxiliary receptors are involved in cell-cell contact and cell activation on the cell surface. An essential molecule of the intercellular interaction in antigen presentation (contact of antigen-presenting cells with the T-helper lymphocytes) are costimulatory receptors with the names PD-Ll, PD-L2, B7h (LICOS), B7-1 and B7-2 , CD40 also plays a role in this signal transduction. The functions of ICOS, PD-1, CD28, CTLA4, B7 and CD40 are the contents of numerous publications (Daikh et al., 1997; Greenfield et al., 1998; Johnson-Leger et al., 1998; McAdam et al., 1998 ; Vyth-Dreese et al., 1995, (Freeman et al 2000; Hutloff et al 1999; Kopf et al 2000; Tamatani et al 2000)) and (apart from ICOS, PDl) are also extensively dealt with in textbooks [see eg (Janeway and Travers, 1997)].
Das konzipierte Verfahren beruht auf mindestens zwei parallelen Eingriffen an patienteneigenen antigen-präsentierenden Zellen. Die Eingriffe erfolgen mittels geeigneter Sonden und bewirkenThe designed procedure is based on at least two parallel interventions on the patient's own antigen-presenting cells. The interventions are carried out using suitable probes and have an effect
• die Produktion eines PD-1 bindenden Moleküls, wie z.B. PD-Ll, die über die Wirkung von PD-1, welches in T-Zellen vorkommt, die T-Zellen an der Aktivierung und Proliferation hindertThe production of a PD-1 binding molecule, e.g. PD-Ll, which via the action of PD-1, which occurs in T cells, prevents the T cells from activation and proliferation
• gleichzeitig eine starke Präsentation des Autoantigens auf den antigen- präsentierenden Zellen• At the same time, a strong presentation of autoantigen on the antigen-presenting cells
• und gegebenenfalls die Produktion eines CTLA4 bindenden Moleküls, wie z.B. Antikörpern, die über die Wirkung von CTLA4, welches in T-Zellen vorkommt, die T-Zellen an der Aktivierung hindertAnd optionally the production of a CTLA4 binding molecule, e.g. Antibodies that prevent the T cells from activating through the action of CTLA4, which occurs in T cells
• und gegebenenfalls die Abschaltung von B7 und damit die Verhinderung der Ausbildung des Moleküls auf der Oberfläche der antigen-präsentierenden Zellen und/oder die Abschaltung von CD40.• and, if necessary, the shutdown of B7 and thus the prevention of the formation of the molecule on the surface of the antigen-presenting cells and / or the shutdown of CD40.
Sollte die Manipulation der Zellen, die z. B. Monozyten sind, ex vivo erfolgen, werden die Zellen in die Blutbahn des Spenders zurückgegeben. Die genmanipulierten Monozyten schalten nunmehr die im Organismus befindlichen ent- sprechenden T-Helfer-Lymphozyten ab. Die genmanipulierten Zellen treten dabei unmittelbar in Konkurrenz zu den bereits im Organismus (vorzugsweise Blutbahn und Lymphsystem) vorhandenen Autoantigen-präsentierenden Monozyten, die allerdings B7 und kein zusätzliches PD-1 bindendes Molekül, CTLA4 bindendes Molekül auf der Zelloberfläche tragen und normalerweise die T-Helfer-Lymphozyten und damit unter anderem die Antikörperproduktion aktivieren. Behandlung von Autoimmunerkrankungen, Allergien und Transplantationen: Stand der TechnikShould the manipulation of the cells z. B. are monocytes, ex vivo, the cells are returned to the donor's bloodstream. The genetically manipulated monocytes now switch off the corresponding T helper lymphocytes in the organism. The genetically manipulated cells compete directly with the autoantigen-presenting monocytes already present in the organism (preferably the bloodstream and lymphatic system), which however carry B7 and no additional PD-1 binding molecule, CTLA4 binding molecule on the cell surface and usually the T-helper - Activate lymphocytes and, among other things, antibody production. Treatment of autoimmune diseases, allergies and transplants: state of the art
Kausaltherapien zur Behandlung von Autoimmunerkrankungen und Allergien existieren nicht. Von wenigen Ausnahmen abgesehen (extrakorporale Elimination der Antikörper aus dem Blut der Patienten bzw. Plasmapherese), erfolgt die Behandlung von Autoimmunerkrankungen und Allergien medikamentös durch eine Hemmung von Reaktionen des Immunsystems.Causal therapies for the treatment of autoimmune diseases and allergies do not exist. With a few exceptions (extracorporeal elimination of antibodies from the patient's blood or plasmapheresis), autoimmune diseases and allergies are treated with medication by inhibiting immune system reactions.
Nach wie vor am gebräuchlichsten ist die medikamentöse Behandlung von Autoimmunerkrankungen, Allergien und Transplantationen mit immunsuppres- siven Präparaten wie Cortison und dessen Abkömmlingen, sowie Cyclosporin, Beta-Interferon oder Zytostatika (Methothrexat). Des Weiteren werden Anti- körper, Antagonisten und Oligonukleotide gegen verschiedene Signalkomponenten des Immunsystems mit dem Zweck erprobt, die Aktivierung von T- Zellen zu verhindern. Solche Arzneimittel sind bestenfalls selektiv, nie aber spezifisch gegen die falsch programmierten Autoimmunreaktionen gerichtet. Immunsuppressiva haben daher auf wichtige, notwendige und nützliche Teile des Immunsystems eine negative Wirkung; aus diesem Grund und ihrer Nebenwirkungen wegen ist ihr Einsatz begrenzt.Drug treatment of autoimmune diseases, allergies and transplants with immunosuppressive preparations such as cortisone and its derivatives, as well as cyclosporin, beta interferon or cytostatics (methotrexate) is still the most common. Furthermore, antibodies, antagonists and oligonucleotides against various signal components of the immune system are tested with the purpose of preventing the activation of T cells. Such drugs are at best selective, but never specifically directed against the wrongly programmed autoimmune reactions. Immunosuppressants therefore have a negative effect on important, necessary and useful parts of the immune system; for this reason and their side effects, their use is limited.
Von den immunsuppressiven Therapien unterscheidet sich das vorgelegte Konzept einer Gentherapie zur Reduktion von spezifischen Immunreaktionen grundlegend. Es wird eine spezifische Abschaltung fehlgeleiteter immunologischer Reaktionen durch gentechnisch umprogrammierte patienteneigene Blutzellen durchgeführt.The concept of gene therapy for the reduction of specific immune reactions differs fundamentally from immunosuppressive therapies. A specific shutdown of misdirected immunological reactions by genetically reprogrammed patient's own blood cells is carried out.
Die aufgezeigte Methodik ist als allgemein gültiges Konzept zur Reduzierung von Immunantworten gedacht. Es sollte mit diesem Grundkonzept möglich sein, jede Immunreaktion des anpassungsfähigen Immunsystems wieder abzustellen. Außerdem können nach Belieben solche Immunreaktionen hervorgerufen und dann wieder abgestellt werden. LiteraturThe methodology shown is intended as a generally applicable concept for reducing immune responses. With this basic concept it should be possible to stop any immune reaction of the adaptable immune system. In addition, such immune reactions can be triggered at will and then switched off again. literature
Agata, Y., Kawasaki, A., Nishimura, H., Ishida, Y., Tsubata, T., et al. 1996. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 8:765-72.Agata, Y., Kawasaki, A., Nishimura, H., Ishida, Y., Tsubata, T., et al. 1996. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 8: 765-72.
Boussiotis, V. A., Freeman, G. J., Gribben, J. G., Nadler, L. M. 1996. The role of B7-l/B7-2:CD28/CLTA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune response. Immunol Rev 153:5-26.Boussiotis, VA, Freeman, GJ, Gribben, JG, Nadler, LM 1996. The role of B7-l / B7-2: CD28 / CLTA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune response. Immunol Rev 153: 5-26.
Daikh D., Wofsy D. and Imboden J. (1997) The CD28-B7 constimulatory pathway and its role in autoimmune disease. J Leukoc Biol 62, 165-62. Dong, H., Zhu, G., Tamada, K., Chen, L. 1999. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5: 1365-9.Daikh D., Wofsy D. and Imboden J. (1997) The CD28-B7 constimulatory pathway and its role in autoimmune disease. J Leukoc Biol 62, 165-62. Dong, H., Zhu, G., Tamada, K., Chen, L. 1999. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 5: 1365-9.
Freeman, G. J., Long, A. J., Iwai, Y., Bourque, K., Chernova, T., et al. 2000. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192: 1027-34.Freeman, G.J., Long, A.J., Iwai, Y., Bourque, K., Chernova, T., et al. 2000. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192: 1027-34.
Greenfield, E., K. Nguyen, and V. Kuchroo. 1998. CD28/B7 costimulation: a review. C t Rev Immunol. 18:389-418.Greenfield, E., K. Nguyen, and V. Kuchroo. 1998. CD28 / B7 costimulation: a review. C t Rev Immunol. 18: 389-418.
Gribben, J., G. Freeman, L. Nadler, P. Rennert, C. Jellis, E. Greenfield, and G. Gray. 1994. Ligands for induction of antigen specific apoptosis in T cells. In Patent. Repligen Corp. (US); Dana Faber Cancer Inst. Inc. (US). Janeway J.C.A. and Travers P. (1997) Immunobiology - The immune System in health and disease. Current Biology Ltd./Garland Publishing Inc., London, San Francisco and New YorkGribben, J., G. Freeman, L. Nadler, P. Rennert, C. Jellis, E. Greenfield, and G. Gray. 1994. Ligands for induction of antigen specific apoptosis in T cells. In patent. Repligen Corp. (US); Dana Faber Cancer Inst. Inc. (US). Janeway J.C.A. and Travers P. (1997) Immunobiology - The immune System in health and disease. Current Biology Ltd./Garland Publishing Inc., London, San Francisco and New York
Hutloff, A., Dittrich, A. M., Beier, K. C, Eljaschewitsch, B., Kraft, R., et al. 1999. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 397:263-6.Hutloff, A., Dittrich, A.M., Beier, K.C, Eljaschewitsch, B., Kraft, R., et al. 1999. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 397: 263-6.
Ishida, Y., Agata, Y., Shibahara, K., Honjo, T. 1992. Induced expression of PD- 1, a novel member of the immunoglobulin gene superfamily, upon program- med cell death. Embo J 11 :3887-95.Ishida, Y., Agata, Y., Shibahara, K., Honjo, T. 1992. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon program-med cell death. Embo J 11: 3887-95.
Johnson-Leger, C, J. Christensen, and G. Klaus. 1998. CD28 co-stimulation stabilizes the expression of the CD40 ligand on T cells. Int Immunol. 10: 1083- 1091.Johnson-Leger, C, J. Christensen, and G. Klaus. 1998. CD28 co-stimulation stabilizes the expression of the CD40 ligand on T cells. Int Immunol. 10: 1083-1091.
Judge, T.A., Z. Wu, X.G. Zheng, A.H. Sharpe, M.H. Sayegh, and L.A. Turka. 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival. J Immunol. 162: 1947-1951. Kopf, M., Coyle, A. J., Schmitz, N., Barner, M., Oxenius, A., et al. 2000. Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection. J Exp Med 192:53-61.Judge, TA, Z. Wu, XG Zheng, AH Sharpe, MH Sayegh, and LA Turka. 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival. J Immunol. 162: 1947-1951. Kopf, M., Coyle, AJ, Schmitz, N., Barner, M., Oxenius, A., et al. 2000. Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection. J Exp Med 192: 53-61.
Latchman, Y., Wood, C. R., Chernova, T., Chaudhary, D., Borde, M., et al. 2001. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. 2:261- 268.Latchman, Y., Wood, C.R., Chernova, T., Chaudhary, D., Borde, M., et al. 2001. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. 2: 261-268.
Lee, K.M., E. Chuang, M. Griffin, R. Khattri, D.K. Hong, W. Zhang, D. Straus, L.E. Samelson, C.B. Thompson, and J.A. Bluestone. 1998. Molecular basis of T cell inactivation by CTLA-4. Science. 282:2263-2266.Lee, K.M., E. Chuang, M. Griffin, R. Khattri, D.K. Hong, W. Zhang, D. Straus, L.E. Samelson, C.B. Thompson, and J.A. Bluestone. 1998. Molecular basis of T cell inactivation by CTLA-4. Science. 282: 2263 to 2266.
Lin, H., J.C. Rathmell, G.S. Gray, C.B. Thompson, J.M. Leiden, and M.L. Alegre. 1998. Cytotoxic T lymphocyte antigen 4 (CTLA4) blockade accelerates the a- cute rejection of cardiac allografts in CD28-deficient mice: CTLA4 can function independently of CD28. J Exp Med. 188: 199-204.Lin, H., J.C. Rathmell, G.S. Gray, C.B. Thompson, J.M. Leiden, and M.L. Alegre. 1998. Cytotoxic T lymphocyte antigen 4 (CTLA4) blockade accelerates the a- cute rejection of cardiac allografts in CD28-deficient mice: CTLA4 can function independently of CD28. J Exp Med. 188: 199-204.
Ling, V., Wu, P. W., Finnerty, H. F., Bean, K. M., Spaulding, V., et al. 2000. Cutting edge: identification of GL50, a novel B7-Iike protein that functionally binds to ICOS receptor. J Immunol 164: 1653-7.Ling, V., Wu, P.W., Finnerty, H.F., Bean, K.M., Spaulding, V., et al. 2000. Cutting edge: identification of GL50, a novel B7-Iike protein that functionally binds to ICOS receptor. J Immunol 164: 1653-7.
McAdam, A., A. Schweitzer, and A. Sharpe. 1998. The role of B7 co- stimulation in activation and differentiation of CD4+ and CD8+ T cells. Immu- nol Rev. 165:231-247.McAdam, A., A. Schweitzer, and A. Sharpe. 1998. The role of B7 co-stimulation in activation and differentiation of CD4 + and CD8 + T cells. Immunol Rev. 165: 231-247.
Metzler, W.J., J. Bajorath, W. Fenderson, S.Y. Shaw, K.L. Constantine, J. Naemura, G. Leytze, R.J. Peach, T.B. Lavoie, L. Mueller, and P.S. Linsley. 1997. Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28 [letter]. Nat Struct Biol. 4:527-531.Metzler, W.J., J. Bajorath, W. Fenderson, S.Y. Shaw, K.L. Constantine, J. Naemura, G. Leytze, R.J. Peach, T.B. Lavoie, L. Mueller, and P.S. Linsley. 1997. Solution structure of human CTLA-4 and delineation of a CD80 / CD86 binding site conserved in CD28 [letter]. Nat Struct Biol. 4: 527-531.
Nishimura, H., Agata, Y., Kawasaki, A., Sato, M., Imamura, S., et al. 1996. Developmentally regulated expression of the PD-1 protein on the surface of double-negative (CD4-CD8-) thymocytes. Int Immunol 8:773-80.Nishimura, H., Agata, Y., Kawasaki, A., Sato, M., Imamura, S., et al. 1996. Developmentally regulated expression of the PD-1 protein on the surface of double-negative (CD4-CD8-) thymocytes. Int Immunol 8: 773-80.
Olsson, C, K. Riesbeck, M. Dohlsten, E. Michaelsson, and K. Riebeck. 1999. CTLA-4 ligation suppresses CD28-induced NF-kappaB and AP-1 activity in mouse T cell blasts [published erratum appears in J Biol Chem 1999 Jul 23;274(30): 21490]. J Biol Chem. 274: 14400-14405.Olsson, C, K. Riesbeck, M. Dohlsten, E. Michaelsson, and K. Riebeck. 1999. CTLA-4 ligation suppresses CD28-induced NF-kappaB and AP-1 activity in mouse T cell blasts [published erratum appears in J Biol Chem 1999 Jul 23; 274 (30): 21490]. J Biol Chem. 274: 14400-14405.
Oosterwegel, M.A., R.J. Greenwald, D.A. Mandelbrot, R.B. Lorsbach, and A.H. Sharpe. 1999a. CTLA-4 and T cell activation. Curr Opin Immunol. 11 :294-300. Oosterwegel, M.A., D.A. Mandelbrot, S.D. Boyd, R.B. Lorsbach, D.Y. Jarrett, A.K. Abbas, and A.H. Sharpe. 1999b. The role of CTLA-4 in regulating Th2 differentiation. J Immunol. 163:2634-2639. Peach, R.J., J. Bajorath, W. Brady, G. Leytze, J. Greene, J. Naemura, and P.S. Linsley. 1994. Complementarity determining region 1 (CDR1)- and CDR3- analogous regions in CTLA-4 and CD28 determine the binding to B7-1. J Exp Med. 180:2049-2058.Oosterwegel, MA, RJ Greenwald, DA Mandelbrot, RB Lorsbach, and AH Sharpe. 1999a. CTLA-4 and T cell activation. Curr Opin Immunol. 11: 294-300. Oosterwegel, MA, DA Mandelbrot, SD Boyd, RB Lorsbach, DY Jarrett, AK Abbas, and AH Sharpe. 1999b. The role of CTLA-4 in regulating Th2 differentiation. J Immunol. 163: 2634-2639. Peach, RJ, J. Bajorath, W. Brady, G. Leytze, J. Greene, J. Naemura, and PS Linsley. 1994. Complementarity determining region 1 (CDR1) - and CDR3- analogous regions in CTLA-4 and CD28 determine the binding to B7-1. J Exp Med. 180: 2049-2058.
Sperling, A. I., Bluestone, J. A. 1996. The complexities of T-cell co-stimulation: CD28 and beyond. Immunol Rev 153: 155-82.Sperling, A.I., Bluestone, J.A. 1996. The complexities of T-cell co-stimulation: CD28 and beyond. Immunol Rev 153: 155-82.
Steinman L. (1994) Autoimmunerkrankungen. Spektrum der Wissenschaften (Spezial: Das Immunsystem) 64-73.Steinman L. (1994) Autoimmune Diseases. Spectrum of Sciences (special: the immune system) 64-73.
Swallow, M. M., Wallin, J. J., Sha, W. C. 1999. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFalpha. Immunity 11 :423-32. Tamatani, T., Tezuka, K., Hanzawa-Higuchi, N. 2000. AILIM/ICOS: a novel lymphocyte adhesion molecule. Int Immunol 12:51-5.Swallow, M.M., Wallin, J.J., Sha, W.C. 1999. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFalpha. Immunity 11: 423-32. Tamatani, T., Tezuka, K., Hanzawa-Higuchi, N. 2000. AILIM / ICOS: a novel lymphocyte adhesion molecule. Int Immunol 12: 51-5.
Thompson, C. B., Allison, J. P. 1997. The emerging role of CTLA-4 as an immune attenuator. Immunity 7:445-50. Yoshinaga, S. K., Whoriskey, J. S., Khare, S. D., Sarmiento, U., Guo, J., et al. 1999. T-cell co-stimulation through B7RP-1 and ICOS. Nature 402:827-32.Thompson, C.B., Allison, J.P. 1997. The emerging role of CTLA-4 as an immune attenuator. Immunity 7: 445-50. Yoshinaga, S.K., Whoriskey, J.S., Khare, S.D., Sarmiento, U., Guo, J., et al. 1999. T-cell co-stimulation through B7RP-1 and ICOS. Nature 402: 827-32.
Vyth-Dreese, F., T. Dellemijn, D. Majoor, and D. de Jong. 1995. Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue. Eur J Immunol. 25:3023-3029.Vyth-Dreese, F., T. Dellemijn, D. Majoor, and D. de Jong. 1995. Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue. Eur J Immunol. 25: 3023 to 3029.
Walunas, T.L., CY. Bakker, and J.A. Bluestone. 1996. CTLA-4 ligation blocks CD28-dependent T cell activation [published erratum appears in J Exp Med 1996 Jul 1;184(1) :301]. J Exp Med. 183:2541-2550.Walunas, T.L., CY. Bakker, and J.A. Bluestone. 1996. CTLA-4 ligation blocks CD28-dependent T cell activation [published erratum appears in J Exp Med 1996 Jul 1; 184 (1): 301]. J Exp Med. 183: 2541-2550.
Wu, Y., Y. Guo, A. Huang, P. Zheng, and Y. Liu. 1997. CTLA-4-B7 interaction is sufficient to costimulate T cell clonal expansion. J Exp Med. 185: 1327-1335.Wu, Y., Y. Guo, A. Huang, P. Zheng, and Y. Liu. 1997. CTLA-4-B7 interaction is sufficient to costimulate T cell clonal expansion. J Exp Med. 185: 1327-1335.
Wulfing, C, and M.M. Davis. 1998. A receptor/cytoskeletal movement trigge- red by costimulation during T cell activation. Science. 282:2266-2269. Wulfing, C, and M.M. Davis. 1998. A receptor / cytoskeletal movement triggered by costimulation during T cell activation. Science. 282: 2266-2269.

Claims

Patentansprüche claims
1. Antigen-präsentierende Zelle, die überwiegend vorher bestimmte Antigene präsentiert (monoantigene antigen-präsentierende Zelle), dadurch gekennzeichnet, dass in der monoantigenen antigen-präsentierenden Zelle ein PD-1 bindendes Molekül produziert wird und gegebenenfalls ein CTLA4 bindendes Molekül produziert wird und gegebenenfalls eine der Funktionen co-stimulatorischer Rezeptoren, wie B7- und/oder CD40- Rezeptoren supprimiert sind.1. Antigen-presenting cell which predominantly presents certain antigens beforehand (monoantigenic antigen-presenting cell), characterized in that a PD-1 binding molecule is produced in the monoantigenic antigen-presenting cell and, if appropriate, a CTLA4-binding molecule is produced and, if appropriate one of the functions of co-stimulatory receptors, such as B7 and / or CD40 receptors, are suppressed.
2. Monoantigene antigen-präsentierende Zelle nach Anspruch 1, dadurch gekennzeichnet, dass die monoantigene antigen-präsentierende Zelle ein Monozyt, eine dendritische Zelle und/oder ein Makrophage ist.2. Monoantigenic antigen-presenting cell according to claim 1, characterized in that the monoantigenic antigen-presenting cell is a monocyte, a dendritic cell and / or a macrophage.
3. Monoantigene antigen-präsentierende Zelle nach Anspruch 1 und/oder 2, dadurch gekennzeichnet, dass durch Transfektion einer antigen- präsentierenden Zelle mit nukleinsäurehaltigem Material eine erhöhte Expression der so vordefinierten Antigene erfolgt und die antigen- präsentierende Zelle im wesentlichen nur diese Antigene präsentiert.3. Monoantigen antigen-presenting cell according to claim 1 and / or 2, characterized in that by transfection of an antigen-presenting cell with nucleic acid-containing material an increased expression of the predefined antigens takes place and the antigen-presenting cell presents essentially only these antigens.
Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die monoantigene antigen-präsentierende Zelle eine erhöhte Anzahl von Homing-Rezeptoren, wie CD44, aufweist.Monoantigenic antigen-presenting cell according to at least one of claims 1 to 3, characterized in that the monoantigenic antigen-presenting cell has an increased number of homing receptors, such as CD44.
5. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass eine Transfektion eine Expression von PD-1 bindenden Molekülen und gegebenenfalls von CTLA4 bindenden Molekülen und gegebenenfalls eine Suppression von Funktionen der B7-, CD40-Rezeptoren und gegebenenfalls eine Erhöhung der Anzahl von Homing-Rezeptoren bewirkt.5. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 4, characterized in that a transfection an expression of PD-1 binding molecules and optionally of CTLA4 binding molecules and optionally a suppression of functions of the B7, CD40 receptors and possibly increasing the number of homing receptors.
6. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 5 enthaltend Nukleinsäuren, die PD-1 bindende Mole- küle kodieren und gegebenenfalls CTLA4 bindende Moleküle kodieren und gegebenenfalls Antisense-Nukleinsäuren zur Verhinderung der B7- und/oder CD40-Rezeptor Expression.6. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 5 containing nucleic acids that encode PD-1 binding molecules and optionally encode CTLA4 binding molecules and optionally antisense nucleic acids to prevent B7 and / or CD40 receptor expression.
7. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 6 enthaltend Nukleinsäuren, die für PD-1 bindende Moleküle kodieren, die in der Plasmamembran der Zelle verbleiben.7. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 6 containing nucleic acids which code for PD-1 binding molecules which remain in the plasma membrane of the cell.
8. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 6 bei denen die PD-1 bindenden Moleküle vorzugsweise PD-Ll, PD-L2, Antikörper, monoklonale Antikörper sind.8. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 6, in which the PD-1 binding molecules are preferably PD-L1, PD-L2, antibodies, monoclonal antibodies.
9. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 8 enthaltend Nukleinsäuren, die eine Unterdrückung der Expression der B7- und/oder CD40-Rezeptoren durch Co-Suppression bewirken.9. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 8 containing nucleic acids, which suppress the expression of the B7 and / or CD40 receptors by co-suppression.
10. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 1 bis 9 enthaltend Nukleinsäuren, die eine Expression von mit B7- und/oder CD40-Rezeptoren affinen Strukturen aufweisenden Proteinen oder Peptiden bewirken.10. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 9 containing nucleic acids which cause expression of proteins or peptides having structures affine with B7 and / or CD40 receptors.
11. Monoantigene antigen-präsentierende Zelle nach Anspruch 10, wobei die Proteine, die affine Strukturen zu B7-Rezeptoren aufweisen CTLA4, CD28, Antikörper, F(ab)2A scFv und/oder Fab-Fragmente sind.11. Monoantigenic antigen-presenting cell according to claim 10, wherein the proteins which have structures affine to B7 receptors are CTLA4, CD28, antibodies, F (ab) 2A scFv and / or F ab fragments.
12. Monoantigene antigen-präsentierende Zelle nach Anspruch 10 und/oder 11, wobei die Nukleinsäuren für eine Signalsequenz kodieren oder die Expressionsprodukte eine Signalsequenz besitzen, die den Verbleib der Expressionsprodukte im endoplasmatischen Retikulum, dem Golgi- Apparat, dem Trans-Golgi-Netzwerk oder intrazellulären Vesikeln bewirkt.12. Monoantigenic antigen-presenting cell according to claim 10 and / or 11, wherein the nucleic acids code for a signal sequence or the expression products have a signal sequence which indicates the whereabouts of the expression products in the endoplasmic reticulum, the Golgi apparatus, the Trans-Golgi network or intracellular vesicles.
13. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 3 bis 12, dadurch gekennzeichnet, dass die monoantigene antigen-präsentierende Zelle zur Expression von Antigenen mit Nuklein- säuren transfiziert ist, die den Transport der exprimierten Antigene in13. Monoantigenic antigen-presenting cell according to at least one of claims 3 to 12, characterized in that the monoantigenic antigen-presenting cell for the expression of antigens is transfected with nucleic acids, which in the transport of the expressed antigens
MHC II-Kompartimente der Zellen ermöglicht. MHC II compartments of the cells enabled.
14. Monoantigene antigen-präsentierende Zelle nach mindestens einem der Ansprüche 3 bis 13, dadurch gekennzeichnet, dass die Nukleinsäuren DNA, RNA, Oligonukleotide, Polynukleotide, Ribozyme, Peptidnuklein- säuren (PNA) sind.14. Monoantigenic antigen-presenting cell according to at least one of claims 3 to 13, characterized in that the nucleic acids are DNA, RNA, oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNA).
15. Monoantigene antigen-präsentierende Zelle nach Anspruch 14, dadurch gekennzeichnet, dass die DNA Regulationselemente wie Enhancer, Promotoren, polyA-kodierende 3 '-Enden zur Transkription der DNA in RNA enthält, die RNA Regulationselemente zur Translation der RNA in Protein enthält.15. Monoantigenic antigen-presenting cell according to claim 14, characterized in that the DNA regulatory elements such as enhancers, promoters, polyA-coding 3 'ends for the transcription of the DNA into RNA contains the RNA regulatory elements for translating the RNA into protein.
16. Verfahren zur Herstellung der monoantigenen antigen-präsentierenden Zelle nach mindestens einem der Ansprüche 1 bis 15 durch ex vivo oder in vivo Ve rf a h re n .16. A method for producing the monoantigenic antigen-presenting cell according to at least one of claims 1 to 15 by ex vivo or in vivo procedures.
17. Verfahren nach Anspruch 16, wobei eine antigen-präsentierende Zelle ex vivo oder in vivo durch Behandlung mit Viren, viralen Vektoren, bakteriellen Vektoren, Plasmiden, die durch viralen Gentransfer, E- lektroporationstechniken, Iontophorese, ballistischen Methoden und/oder anderen Techniken zur Einschleusung von Molekülen in eine monoantigene antigen-präsentierende Zelle transfiziert wird.17. The method according to claim 16, wherein an antigen-presenting cell ex vivo or in vivo by treatment with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for Molecular introduction into a monoantigenic antigen-presenting cell is transfected.
18. Verfahren nach Anspruch 16 und/oder 17, wobei eine antigen- präsentierende Zelle oder eine monoantigene antigen-präsentierende18. The method according to claim 16 and / or 17, wherein an antigen-presenting cell or a monoantigenic antigen-presenting
Zelle durch Behandlung mit Viren, viralen Vektoren, bakteriellen Vektoren, Plasmiden die durch viralen Gentransfer, Elektroporationstechniken, Iontophorese, ballistischen Methoden und/oder anderen Techniken zur Einschleusung von Molekülen in eine Zelle mit erhöhter Produktion von PD-1 bindenden Molekülen und gegebenenfalls CTLA4 bindenden Molekülen und gegebenenfalls mit supprimierter Funktion co-stimulatorischer Rezeptoren transfiziert wird, die Expression co-stimulatorischer Rezeptoren durch Verhinderung deren Expression oder die co-stimulatorischen Rezeptoren durch Reaktion mit affinen Strukturen an einer Stimulation von T-Zellen, die an die monoantigene antigen-präsentierende Zelle gebunden sind, verhindert wird. Cell by treatment with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with increased production of PD-1 binding molecules and possibly CTLA4 binding molecules and optionally transfected with suppressed function of co-stimulatory receptors, the expression of co-stimulatory receptors by preventing their expression or the co-stimulatory receptors by reacting with affine structures to stimulate T cells bound to the monoantigenic antigen presenting cell are prevented.
19. Verfahren nach mindestens einem der Ansprüche 16 bis 18, wobei Moleküle wie Antikörper, Proteine, Peptide, Peptidomimetica, PD-Ll, PD-L2, PD-1 bindende Moleküle, CTLA4 bindende Moleküle, CTLA4, CD28, CD40L und/oder Bestandteile und/oder Kombinationen dieser Moleküle, die z.B. PD-1, CTLA4, B7-1, B7-2, CD40 binden, welche eine in Gegenwart einer Antigenpräsentation stattfindende Co-Stimulation der T-Zelle behindert, mit der monoantigenen antigen-präsentierenden Zelle oder der antigen-präsentierenden Zelle in Kontakt gebracht werden.19. The method according to at least one of claims 16 to 18, wherein molecules such as antibodies, proteins, peptides, peptidomimetics, PD-Ll, PD-L2, PD-1 binding molecules, CTLA4 binding molecules, CTLA4, CD28, CD40L and / or constituents and / or combinations of these molecules, for example PD-1, CTLA4, B7-1, B7-2, CD40, which prevents co-stimulation of the T cell taking place in the presence of an antigen presentation, are brought into contact with the monoantigenic antigen-presenting cell or the antigen-presenting cell ,
20. Verfahren nach mindestens einem der Ansprüche 16 bis 19, wobei die Moleküle gemäß Anspruch 17 durch Vehikel, wie Liposomen, Hydrogele, Zyklodextrine, Nanokapseln, Nanopartikel, bio-adhäsive Mikrokugeln und/oder durch Elektroporationstechniken, Iontophorese, ballistische Methoden und/oder andere Techniken zur Einschleusung von Molekülen in die monoantigene antigen-präsentierende Zelle oder die antigen- präsentierende Zelle transferiert werden.20. The method according to at least one of claims 16 to 19, wherein the molecules according to claim 17 by vehicles such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or others Techniques for introducing molecules into the monoantigenic antigen-presenting cell or the antigen-presenting cell are transferred.
21. Verfahren nach mindestens einem der Ansprüche 16 bis 20, wobei Nuk- leinsäuren durch Viren, virale Vektoren, bakterielle Vektoren, Plasmide die durch viralen Gentransfer, Elektroporationstechniken, Iontophorese, ballistische Methoden und/oder andere Techniken zur Einschleusung von Molekülen in die monoantigene antigen-präsentierende Zelle oder die antigen-präsentierende Zelle transferiert werden.21. The method according to at least one of claims 16 to 20, wherein nucleic acids by viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for the introduction of molecules into the monoantigenic antigen presenting cell or the antigen presenting cell.
22. Arzneimittel enthaltend mindestens eine monoantigene antigen- präsentierende Zelle nach mindestens einem der Ansprüche 1 bisl5.22. Medicament containing at least one monoantigenic antigen-presenting cell according to at least one of claims 1 to 15.
23. Arzneimittel nach Anspruch 22, wobei mindestens eine monoantigene antigen-präsentierende Zelle als Infusionslösung zur intravenösen oder intraperitonealen Applikation formuliert ist.23. Medicament according to claim 22, wherein at least one monoantigenic antigen-presenting cell is formulated as an infusion solution for intravenous or intraperitoneal application.
24. Verwendung mindestens einer monoantigenen antigen-präsentierenden Zelle nach mindestens einem der Ansprüche 1 bis 15 zur Herstellung ei- nes Arzneimittels zur Behandlung von ungewollten Immunreaktionen, wie Autoimmunerkrankungen und Allergien oder gewollt hervorgerufenen Immunreaktionen, wie bei Immunisierungen.24. Use of at least one monoantigenic antigen-presenting cell according to at least one of claims 1 to 15 for the manufacture of a medicament for the treatment of unwanted immune reactions, such as autoimmune diseases and allergies or deliberately induced immune reactions, such as immunizations.
25. Verwendung mindestens einer monoantigenen antigen-präsentierenden Zelle nach mindestens einem der Ansprüche 1 bis 15 zur Herstellung eines Arzneimittels zur Behandlung von Immunreaktionen gegen allologe und/oder xenologe Gewebsmerkmale.25. Use of at least one monoantigenic antigen-presenting cell according to at least one of claims 1 to 15 for the manufacture of a medicament for the treatment of immune reactions against allologic and / or xenologic tissue characteristics.
26. Verwendung nach Anspruch 24, wobei die zu behandelnden Immunre- aktionen in Verbindung mit Antigenen oder deren Gensequenzen und/oder Teilen davon stehen ausgewählt aus der Gruppe bestehend aus26. Use according to claim 24, wherein the immune reactions to be treated are associated with antigens or their gene sequences and / or parts thereof are selected from the group consisting of
Enzymen, deren Gensequenzen und/oder Teilsequenzen, insbesondere Glutamic acid decarboxylase (GAD), Rezeptor-Typ Protein Tyrosin Phosphatase IA-2Beta, H+K+ATPase, U1RNP, Transglu- taminase, Argininosuccinatelyase (ASL), Tyrosinase-related pro- tein-2, Thyroid Peroxidase, Faktor VIII, Faktor IX;Enzymes, their gene sequences and / or partial sequences, in particular glutamic acid decarboxylase (GAD), receptor type protein tyrosine phosphatase IA-2Beta, H + K + ATPase, U1RNP, transglutaminase, argininosuccinatease (ASL), tyrosinase-related protein -2, thyroid peroxidase, factor VIII, factor IX;
Rezeptoren, deren Gensequenzen und/oder Teilsequenzen, insbe- sondere Acetylcholinrezeptor vom Nicotintyp, Myasthenia gravis, ßl-adrenerger Rezeptor, αl-adreneger-Rezeptor, Angiotensin-2- ATl-Rezeptor, G I Uta mat- Rezeptor, Thyrotropin-stimulierendes Hormon (TSH)- Rezeptor, LFA-1, HLA-B27, Epididymal Protein DE, Zona Pellucida (ZP)-3 Glycoprotein, Zona Pellucida (ZP)-4 Gly- coprotein, Follicle-Stimulating Hormone (FSH) Rezeptor, SpermReceptors, their gene sequences and / or partial sequences, in particular acetylcholine receptor of the nicotine type, myasthenia gravis, β1-adrenergic receptor, αl-adrenergic receptor, angiotensin-2-ATl receptor, GI Uta mat receptor, thyrotropin-stimulating hormone (TS ) - Receptor, LFA-1, HLA-B27, Epididymal Protein DE, Zona Pellucida (ZP) -3 Glycoprotein, Zona Pellucida (ZP) -4 Glycoprotein, Follicle-Stimulating Hormone (FSH) Receptor, Sperm
Immunogen SP-10 oder Sperm Protein SP-10;Immunogen SP-10 or Sperm Protein SP-10;
Hormone oder Botenstoffe, deren Gensequenzen und/oder Teilsequenzen, insbesondere Insulin, Thyroglobulin, Follicle-Stimulating Hormone (FSH), Prostaglandin F2 alpha, Gonadotropin-ReleasingHormones or messenger substances, their gene sequences and / or partial sequences, especially insulin, thyroglobulin, follicle-stimulating hormones (FSH), prostaglandin F2 alpha, gonadotropin-releasing
Hormone (GnRH), Oestradiol-17beta, Oestrogen, Luteinizing Hormon (LH) Rezeptor, Inhibin, Testosteron, Androgen, Chorionic Gonadotrophin (CG), Interleukine, Interferone, Cytokine, Chemokine, Bone Morphogenetic Factors, ß-Interferon, Estradiol; Strukturproteine, deren Gensequenzen und/oder Teilsequenzen, insbesondere Myelin Basic Protein (MBP), Proteolipid protein (PLP), Myelin oligodendrocyte glycoprotein (MOG), α-Fodrin, Nicht-erythroides α-Spectrin, Beta-Amyloid Precursor Protein (beta-APP), Typ 2 Kollagen, Sperm Plasma Membran Protein PH-Hormones (GnRH), oestradiol-17beta, estrogen, luteinizing hormone (LH) receptor, inhibin, testosterone, androgen, chorionic gonadotrophin (CG), interleukins, interferons, cytokines, chemokines, bone morphogenetic factors, ß-interferon, estradiol; Structural proteins, their gene sequences and / or partial sequences, in particular myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), α-fodrin, non-erythroid α-spectrin, beta-amyloid precursor protein (beta-APP ), Type 2 collagen, sperm plasma membrane protein PH-
20;20;
Antigene, deren Gensequenzen und/oder Teilsequenzen, insbesondere CENP- A Autoantigen, Beta2GP-I, ribosomales P Protein, Ro/SSA, La/SSB, Sm/RNP, Sm, Scl-70, Jo-1, BCOADC-E2, Albumin, Glucagon, Inselzellantigene, Retinal S Ag;Antigens, their gene sequences and / or partial sequences, in particular CENP-A autoantigen, Beta2GP-I, ribosomal P protein, Ro / SSA, La / SSB, Sm / RNP, Sm, Scl-70, Jo-1, BCOADC-E2, albumin , Glucagon, islet cell antigens, Retinal S Ag;
Allergene, die eine IgE-Antwort auslösen, deren Gensequenzen und/oder Teilsequenzen, insbesondere Der f 1, Der f 2, Der f 3, Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 8, Eur m 1, Lep d 2, Fei d 1, Can f 1, Can f 2, Mus m 1, Rat n 1, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Per a 1, Bienengift Phospholipase A2 (PLA2),Allergens that trigger an IgE response, their gene sequences and / or partial sequences, in particular Der f 1, Der f 2, Der f 3, Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 8, Eur m 1, Lep d 2, Fei d 1, Can f 1, Can f 2, Mus m 1, Rat n 1, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Per a 1 , Bee venom phospholipase A2 (PLA 2 ),
Group V major allergen Phl p 5b von Timothy Grass Pollen, Hom s 1.Group V major allergen Phl p 5b by Timothy Grass Pollen, Hom s 1.
27. Verwendung nach Anspruch 25, wobei die zu behandelnden Immunreaktionen in Verbindung mit allologen und/oder xenologen Gewebs- merkmalen, deren Gensequenzen und/oder Teilsequenzen, insbesondere MHC I, MHC II, Rhesus Faktor steht. 27. Use according to claim 25, wherein the immune reactions to be treated are associated with allologic and / or xenologic tissue features, their gene sequences and / or partial sequences, in particular MHC I, MHC II, rhesus factor.
PCT/EP2002/003292 2001-03-27 2002-03-23 Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation WO2002077208A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP01107578.5 2001-03-27
EP01107578 2001-03-27

Publications (1)

Publication Number Publication Date
WO2002077208A1 true WO2002077208A1 (en) 2002-10-03

Family

ID=8176950

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/003292 WO2002077208A1 (en) 2001-03-27 2002-03-23 Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation

Country Status (1)

Country Link
WO (1) WO2002077208A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006636A1 (en) * 2001-07-12 2003-01-23 Genethor Gmbh Reduction of the stimulatory capacity of antigen-presenting cells
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US8652465B2 (en) 2005-06-08 2014-02-18 Emory University Methods and compositions for the treatment of persistent infections
US9598491B2 (en) 2008-11-28 2017-03-21 Emory University Methods for the treatment of infections and tumors
CN114805591A (en) * 2022-03-31 2022-07-29 浙江大学 Bispecific antibody targeting CTLA-4 and ligand CD80 or CD86, screening method, composition and application

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066715A1 (en) * 1999-05-04 2000-11-09 Genethor Gmbh Method for diminishing specific immune reactions
WO2001014557A1 (en) * 1999-08-23 2001-03-01 Dana-Farber Cancer Institute, Inc. Pd-1, a receptor for b7-4, and uses therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066715A1 (en) * 1999-05-04 2000-11-09 Genethor Gmbh Method for diminishing specific immune reactions
WO2001014557A1 (en) * 1999-08-23 2001-03-01 Dana-Farber Cancer Institute, Inc. Pd-1, a receptor for b7-4, and uses therefor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AGATA Y ET AL: "EXPRESSION OF THE PD-1 ANTIGEN ON THE SURFACE OF STIMULATED MOUSE T AND B LYMPHOCYTES", INTERNATIONAL IMMUNOLOGY, OXFORD UNIVERSITY PRESS, GB, vol. 8, no. 5, May 1996 (1996-05-01), pages 765 - 772, XP000971773, ISSN: 0953-8178 *
FREEMAN G ET AL: "ENGAGEMENT OF THE PD-1 IMMUNOINHIBITORY RECEPTOR BY A NOVEL B7 FAMILY MEMBER LEADS TO NEGATIVE REGULATION OF LYMPHOCYTE ACTIVATION", JOURNAL OF EXPERIMENTAL MEDICINE, TOKYO, JP, vol. 192, no. 7, 2 October 2000 (2000-10-02), pages 1027 - 1034, XP000971650, ISSN: 0022-1007 *
NISHIMURA H ET AL: "PD-1: an inhibitory immunoreceptor involved in peripheral tolerance", TRENDS IN IMMUNOLOGY, ELSEVIER, CAMBRIDGE, GB, vol. 22, no. 5, 1 May 2001 (2001-05-01), pages 265 - 268, XP004235272, ISSN: 1471-4906 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006636A1 (en) * 2001-07-12 2003-01-23 Genethor Gmbh Reduction of the stimulatory capacity of antigen-presenting cells
US8652465B2 (en) 2005-06-08 2014-02-18 Emory University Methods and compositions for the treatment of persistent infections
US9457080B2 (en) 2005-06-08 2016-10-04 Emory University Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (PD-1) pathway
US10370446B2 (en) 2005-06-08 2019-08-06 Emory University Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (PD-1) pathway
US11359013B2 (en) 2005-06-08 2022-06-14 Emory University Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (PD-1) pathway
US9598491B2 (en) 2008-11-28 2017-03-21 Emory University Methods for the treatment of infections and tumors
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN114805591A (en) * 2022-03-31 2022-07-29 浙江大学 Bispecific antibody targeting CTLA-4 and ligand CD80 or CD86, screening method, composition and application

Similar Documents

Publication Publication Date Title
DE69910362T2 (en) APPLICATION OF THE MESENCHYMAL STEM CELLS AS IMMUNE SUPPRESSIVA
Greenfield et al. CD28/B7 costimulation: a review
Ding et al. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression.
Lenschow et al. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse.
McCoy et al. The role of CTLA‐4 in the regulation of T cell immune responses
Cobbold et al. Infectious tolerance
Streilein Ocular immune privilege: therapeutic opportunities from an experiment of nature
KR102033276B1 (en) Method for in vivo expansion of t regulatory cells
DE69935318T2 (en) IMMUNE RESTRICTION THROUGH BLOCKING OF T-CELL COSTIMULATION SIGNAL 2 (B7 / CD28 INTERACTION)
EP1115427B1 (en) Time-staggered utilization of tumor cells in combination with intact antibodies for immunization
DE69332921T2 (en) Inhibition of tumor cell growth by administration of B7-transfected cells
Ferguson et al. The role of Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) in the ocular immune response
Gery et al. Autoimmunity in the eye and its regulation
Goleva et al. Our current understanding of checkpoint inhibitor therapy in cancer immunotherapy
Filippi et al. Transforming growth factor-β suppresses the activation of CD8+ T-cells when naive but promotes their survival and function once antigen experienced: a two-faced impact on autoimmunity
EP0885614A2 (en) Method of ex vivo immunizing using heterologous intact bispecific and/or trispecific antibodies
JPH09510211A (en) Method of regulating T cell unresponsiveness
DE102004063494A1 (en) antibody
DE69535713T2 (en) METHOD FOR MODULATING T-CELL REACTIONS BY MANIPULATING A COMMON CYTOKIN RECEPTOR GAMMA CHAIN
WO2003006636A1 (en) Reduction of the stimulatory capacity of antigen-presenting cells
WO2002077208A1 (en) Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation
EP1173550B1 (en) Method for diminishing specific immune reactions
DE19951970A1 (en) Medicines for tolerance induction
DE10038722C2 (en) Procedure for the reduction of specific immune reactions
Wang et al. Exploration and Evaluation of Therapeutic Efficacy of Drug‐Free Macromolecular Therapeutics in Collagen‐Induced Rheumatoid Arthritis Mouse Model

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP