WO2004056873A1 - Increase of the immune response by substances influencing the function of natural killer cells - Google Patents

Increase of the immune response by substances influencing the function of natural killer cells Download PDF

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Publication number
WO2004056873A1
WO2004056873A1 PCT/DE2003/004268 DE0304268W WO2004056873A1 WO 2004056873 A1 WO2004056873 A1 WO 2004056873A1 DE 0304268 W DE0304268 W DE 0304268W WO 2004056873 A1 WO2004056873 A1 WO 2004056873A1
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active ingredient
cells
hla
cell
antibody
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PCT/DE2003/004268
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German (de)
French (fr)
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Markus Jensen
Hans-Harald Sedlacek
Frank Berthold
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Medinnova Ges Med Innovationen
Markus Jensen
Hans-Harald Sedlacek
Frank Berthold
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Priority to AU2003296553A priority Critical patent/AU2003296553A1/en
Priority to EP03813541A priority patent/EP1590370A1/en
Publication of WO2004056873A1 publication Critical patent/WO2004056873A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to active substances and pharmaceutical compositions which stimulate the immune system.
  • the immune response against infectious agents and tumors can be divided into an innate defense, in which the natural killer cells play a special role, and an acquired antigen-specific defense, in which cytotoxic T-lymphocytes and specific antibodies play a central role.
  • NK cells Natural killer cells
  • TCR T cell receptor
  • the NK1.1 T cells are said to have a regulatory, in some cases inhibitory, effect on cytotoxic T cells, in particular in autoimmune reactions (Zhang et al., J Exp Med 186: 1677-1687, (1997); Ben - lagha et al., Science 296: 481-482, (2002); Shlomai et al., J Path 195: 498-507, (2001)).
  • the human correlate of the NKl .1 T cell is not yet known.
  • NK cells are enriched in the placenta and may appear to play a role in controlling placentation here. However, it is completely unknown how the NK cells could play this role (Mofett-King Nature Reviews Immunology
  • NK cells 15 2/9; 656-663, 2002.
  • those receptors on NK cells have already been analyzed with the aid of which NK cells can interact with ligands on cells of the allogeneic placenta, in particular the extravillous trophoblast cells.
  • ligands include
  • NK cells are able to kill tumor cells.
  • Experiments on mouse tumor models have shown that removal of NK cells leads to an increase in tumor growth (Karre et al., Nature 319: 675-678 (1986); Smyth et al., Nature Immunol. 2: 293 -299 (2001)).
  • the number of CD57 positive lymphocytes (which include NK cells) in the tumor was directly correlated with survival time (Villegas et al., Lung Cancer 35: 23-28, (2002)). The same could be demonstrated for gastric carcinoma (Ishigami et al. F Cancer 20 88: 577-583, (2000)).
  • NK cells play a decisive role in the induction of cytotoxic T cells and in the control of viral infections. So it was
  • NK cells 30 which no NK cells were detectable (Biron et al., N. Engl. J. Med. 320, 1731-1735 (1989).
  • CMV infections are known to cause the expression of MHC class I Down-regulate molecules as well Express substances that are believed to inhibit NK cells. These substances include UL18, UL40 and UL16 (overview from: Cerwenka et al., Nature Reviews Immunology 1: 41-49, (2001)).
  • NK cells are therefore particularly cytotoxically active for those cells which do not expose MHC class I molecules on their cell membrane.
  • the cytotoxicity-inhibiting receptors on the NK cells could be identified in the mouse Ly49 and CD94 / NKG2A and in humans KIR2DL, KIR3DL, CD 159a, CD853 and CD85d (review by: Cerwenka et al., Nature Reviews Immunology 1: 41- 49 (2001)).
  • NK cells are able to kill tumor cells, although they express MHC class I molecules.
  • the cause was identified on the membrane of NK cells, the cytotoxicity-activating receptors, the activation of which clearly dominates the inhibition by the cytotoxicity-inhibiting receptors of NK cells.
  • These cytotoxicity-activating receptors include NKR-P1C, Ly49D, Ly49H in the mouse and CD16, NKp30, NKp46, KIR2DS, CD94 / NKG2C, NKp44, NKG2D, and CD244 in humans (review by: Cerwenka et al., Nature Reviews Immunology 1: 41-49 (2001)).
  • NK cells The activation of NK cells is thought to be caused by the concerted action of these cytotoxicity-activating receptors with cytokm receptors, adhesion molecules and chemokm receptors (Cerwenka et al., Nature Reviews Immunology 1: 41-49 (2001))
  • Human NK cells are known to express the adhesion molecule N-CAM (CD56, Leu-19, NKHl) and additionally CD7, CD38, CDIla, CDllb and CD45RA.
  • CDllc and CD57 is heterogeneous and variable. After activation, the expression of CDIIa, CD38 and HLADR is increased and CD11b and CD45RA are decreased.
  • Chronically activated NK cells additionally express particularly CD2 and CD57 (Lima et al., Blood Cells Mol Dis 28: 181-190, 2002)).
  • N-CAM is not only found on NK cells, but also on T cells, on nerve cells, and especially on tumor cells of the neuroectoderm (CNS tumors, melanomas, small cell bronchial carcinoma).
  • Antibodies against N-CAM can inhibit the growth of neuroblastoma cells and cells
  • Inhibit glioblasto cells (Krushel et al., PN ⁇ S USA, 95: 2592-2596, (1998); Dehal et al., Biochem. Soc. Trans 30/4: 518-520, (2002)) and thus for diagnostics and therapy of these tumors can be used.
  • the invention is based on the technical problem of specifying pharmaceutical compositions for stimulating the immune system.
  • the invention teaches in particular active ingredients and pharmaceutical compositions according to the claims.
  • the invention is based based on the surprising finding that the inhibition of the function of human NK cells by an inhibiting active ingredient leads to an increase in the antigen-specific immune defense.
  • the invention thus also relates to a method for strengthening the immune defense, the function of NK cells being inhibited by at least one active ingredient.
  • Active substance in the sense of the invention is a binding substance which binds to an NK cell and inhibits it, or a substance which, after administration in an organism, causes the formation of a binding substance in this organism which binds to an NK cell and inhibits it.
  • the invention is based on the further surprising finding that the antigen-specific immune defense is particularly enhanced when the binding substance according to this invention is coupled to a molecule which binds to a T lymphocyte.
  • a molecule which binds to a T lymphocyte can be antigens, cytokines, chemokines, growth factors or antibodies, or fragments of antibodies which bind to the T cell receptor, to an adhesion molecule, to a cytokine receptor or to a chemokine receptor Bind T cells.
  • CD2 Molecules which are preferably used on CD2, CD3, CD4, CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD26, CD27, CD28, CD30, CD31, CD37, CD38, CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDw60, CD621, CD70, CD73, CD75, CD76, CD83, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD101, CD107a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152, CD153, CD154, CD155, CD160, CD161, CD166, CD226, CD245, CD246, CD247, or to a component of the T cell receptor complex tie.
  • the invention is further based on the surprising finding that the strengthening of the antigen-specific immune defense by
  • active substances according to the invention which are binding substances, are ligands which, by binding to inhibiting receptors of NK cells, inhibit their function.
  • the inhibitory receptors on NK cells and the associated ligands include:
  • binding substances are: partial sequences of these ligands and peptidomimetics of these ligands which bind to inhibiting receptors of the NK cells and inhibit their function, antibodies, antibody fragments and fusion proteins containing at least one antibody or an antibody fragment which bind to the inhibiting receptors of NK cells bind and inhibit their function, substances that inhibit their function by binding to activating receptors of the NK cells.
  • Activating receptors on NK cells and the associated ligands include:
  • UL16 Binding Proteins (ULBPl, -2, -3)
  • the binding substances within the meaning of the invention include mutations or cleavage products or peptidomimetics of the ligands for activating receptors on NK cells which bind to activating receptors on NK cells without activating them, antibodies, antibody fragments or fusion proteins containing at least one antibody or an antibody fragment which bind to activating receptors on NK cells without activating them or which ligands bind to activating receptors and inhibit their binding to these receptors, the ULI ⁇ protein of the CMV, which binds to ULBP-1, ULBP-2 and to MICB, homologs of the UL16 protein and peptidomimetics of the ULl ⁇ protein, antibodies, antibody fragments or fusion proteins containing at least one antibody or at least one antibody fragment which bind to an adhesion molecule on NK cells and thereby inhibits the function of the NK cell.
  • Adhesion molecules of this type are, for example, N-CAM (CD56) and CD57.
  • Examples of antibody fragments specific for an adhesion molecule are peptide sequences which bind to N-CAM and are described by Whittington et al., Medical and Pediatric Oncology 36: 243-246 (2001).
  • Examples of active substances in the sense of the invention which can trigger the formation of a binding substance in the sense of this invention in an organism are membrane components of NK cells which, after injection into an organism, lead to a specific immune reaction, in particular to the appearance of antibodies, which bind to inhibiting receptors on NK cells, or to adhesion molecules on NK cells and thereby inhibit the function of NK cells, ligands for activating receptors on NK cells and complexes of at least one ligand and at least one activating receptor, which after injection in an organism lead to a specific immune reaction, in particular to the appearance of antibodies which inhibit the activation of the activating receptors on NK cells.
  • Active substances in the sense of the invention are added to line preparations from the blood, from organs or from body cavities.
  • Such cell preparations are, for example Leukocyte preparations or lymphocyte preparations.
  • the preparation of such cell preparations from the blood, from organs or from body cavities is familiar to the person skilled in the art.
  • the mixture of this cell preparation and the active ingredient is then administered to an organism in order to strengthen its antigen-specific immune response.
  • Active substances in the sense of the invention are also used as medicaments.
  • the active ingredient according to the invention is mixed with a suitable pharmaceutical auxiliary known to the person skilled in the art.
  • Such pharmaceutical auxiliaries can be, for example, physiological aqueous injection solutions.
  • Active ingredients which are injected in order to produce an immune reaction, in particular an antibody reaction are preferably treated with an ad uvans.
  • adjuvants are aluminum hydroxide or CpG.
  • Medicaments containing an active ingredient according to the invention are administered locally or injected into the bloodstream, into an organ, into a body cavity, into the connective tissue or into the muscles for the prophylaxis or therapy of a disease. Examples of such diseases or therapies are explained below.
  • Active substances according to this invention can be used for T-cell-mediated immunotherapy of malignant tumors.
  • all T cells of the body can be activated; the specificity of the T cell receptor is irrelevant for T cell activation.
  • T cells activated in this way are able to inhibit tumor growth.
  • the inhibition of Tumor growth can be achieved directly by cytotoxically active T cells or indirectly, by activating other components of the immunological network, such as B cells or macrophages.
  • an immunological memory can be built up.
  • T cells In the case of direct antitumor activity of cytotoxic T cells, the tumor cells are attacked by T cells which either use their tumor-specific T Zeil receptor or, regardless of their TcR specificity, use tumor-specific antibodies or tumor and T cell (bi-) specific antibodies or fusion proteins recognize the tumor.
  • Active substances according to the invention can furthermore be used for the therapy of conditions of the T cell deficiency or the T cell underfunction.
  • a decrease in T cell function occurs in various diseases, e.g. as a result of chemotherapy, radio therapy or immunotherapy, under medicinal immunosuppressive therapy, as an accompanying condition of diseases that affect the spleen, lymph nodes or bone marrow, in the case of congenital immune defects or as a concomitant symptom of hematological neoplasia, chronic inflammation reactions or infections.
  • NK cell activity against NK cells in diseases with benign or malignant NK cell proliferation Benign or malignant proliferative diseases of the NK cell system, such as chronic NK cell lymphocytosis, NK cell leukemia or that NK cell lymphoma can preferably be treated with active substances according to the invention which simultaneously have a molecule Bind NK cells as well as a molecule on T cells. These active substances suppress the NK cells, thereby strengthen the T cell activity and target it to NK cells.
  • autoimmune diseases the cause of which is an NK cell hyperfunction
  • Another preferred area of application of an active substance according to the invention is those autoimmune diseases in which overactivity of the NK cells and a consequent malregulation of the T cells occurs.
  • These autoimmune diseases include so-called antibody mediated autoimmune diseases, such as the myasthenia gravis or an allergy.
  • Example 1 Correlation of NK cell depletion to T cell proliferation.
  • PBMCs mononuclear leukocytes
  • Anti-NCAM antibody (clone ERIC-1) were used in a concentration of 1000 ng / ml, as well as anti-CD3 (clone 0KT3) and anti-CD28 antibody (clone 15E8) to specify a T-cell-specific stimulus.
  • the leukocyte subpopulations were determined in the flow cytometer.
  • CD4-FITC, CD8-FITC and CD16-PE conjugated antibodies (Becton Dickmson, Heidelberg, Germany) were used for staining. The measurement was carried out in a FACSCalibur TM flow cytometer (Becton Dickmson) using TruCount TM measuring tubes (Becton Dickmson) to determine the absolute cell numbers.
  • T cells showed a marked increase in their proliferation.
  • the proliferation indices were between 1.14 and 1.73.
  • the correlation value was -0.727.
  • Example 2 Increase in T-cell proliferation by anti-NCAM antibodies which additionally bind to T-lymphocytes.
  • T cell proliferation could be achieved by antibodies that bind to NCAM and CD3 on T cells simultaneously.
  • a bispecific antibody was additionally tested with a specificity against NCAM and against CD3 (clone OE-1). This bispecific antibody was again used in a concentration of 1000 ng / ml together with an anti-CD28 antibody (clone 15E8).
  • the experiments were carried out as described in Example 1, the calculated T cell number being the technical one
  • CD8 + (bright) and CD8 + (dim) population plus the CD4 + cells are based.
  • the mean of the calculated proliferation indices was 2.16.
  • Control examinations with monospecific anti-NCAM antibodies not binding to T cells achieved an average proliferation index of 1.73.
  • AIMV LifeTechnologies. Egganh ⁇ im, Germany, 200 icrollter per nap
  • medium - an antibody against the CD3 ⁇ chain of T-lymphocytes (clone OKT3, 1 ⁇ g / l), the bispecific ⁇ antibody OE-1 (1 ⁇ g / ml ) or a high dose of tetanus toxoid (10 Lf / ml).
  • An anti-CD56 antibody (clone ERIC-1, 1 ⁇ g / ml) or a ⁇ dium control without the addition of antigens or antibodies was used for the negative control INF-y (number of spots) was determined using an IFN- ⁇ ELISPOT kit (Diaclone Research).
  • PBMC from persons vaccinated with tetanus toxoid were exposed for three days in RPMM640 medium (PAA, Linz, Austria) ⁇ 10% fetal calf serum (FCS, Biochrom AG) using the previously determined optimal stimulation conditions (TT 1 Lf / ml + OE- 2 ng / ml; or to control antl-CD5 ⁇ antibody 2 ng ml + TT 1 Lf / ml; or no antibody + TT 1 Lf / ml).
  • RPMM640 medium PAA, Linz, Austria
  • FCS fetal calf serum
  • the cells were washed twice with medium and incubated for 24 hours in an incubator in medium (RPMI1 ⁇ 40 + 10% FCS) without the addition of tetanus toxoid or antibody.
  • medium RPMI1 ⁇ 40 + 10% FCS
  • tetanus toxoid or antibody was followed by an ELISPOT test (3 days incubation period) on human IFN ⁇ y (diaclone re-search) using serum-free AIMV medium and addition (a high dosage) of TT (10 Lf / ml), or omitting TT (medium control) ,
  • the IFN-7 ELISPOT test showed that only the PBMC stimulated with TT + OE-1 were refractory 24 hours after the pre-stimulation against renewed stimulation with TT (10 Lf / ml), ie only very small amounts of IFN- ⁇ were secreted. during controls (PBMC stimulated with TT alone or in Combination with anti-CD56 antibodies) showed a normal TT response. Interestingly, the TT + OE-1 premilled cells were not refractory to non-specific stimulation with, for example, OKT3 antibodies 100 ng / ml.
  • PBMC from persons vaccinated with tetanus toxoid were exposed for three days in RPMI1640 medium + 10% fetal calf serum using the optimal stimulation conditions (TT 1 Lf / ml + OE-1 2 ng / ml; or as a control as a control) -CDS6 antibody 2 ng / ml + TT 1 Lf / m); or no antibody + TT 1 Lf / ml), then letfoch not (as in the previous experiment) incubated for 24 h in medium without TT or antibody, but now for another three days with (an optimal dosage of) 10 Lf / ml TT (in RPM11640 + 10% FCS) incubated to check whether the anergy related to the TT response can be broken.
  • the optimal stimulation conditions TT 1 Lf / ml + OE-1 2 ng / ml; or as a control as a control
  • an active ingredient according to the invention is able to increase the immune response to an antigen and, at the same time, is also able, depending on the use, to trigger an antigen-specific anergy.
  • the triggering of anergy is of particular therapeutic importance in the treatment of autoimmune diseases, allergies and organ rejection.

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Abstract

The invention relates to an active ingredient comprising a first molecular constituent which binds to a first molecule selected from the group consisting of 'NCAM, KIR2DL, KIR2DS, KIR3DL, CD94/CD159, CD57, CD85, CD16, NKp30, NKp44, NKp46, NKG2D and CD244', and a second molecular constituent which binds to a second molecule selected from the group consisting of 'CD2, CD4, CD44, CD69 and T-cell-receptor', the first and the second molecular constituents being covalently bonded. The invention also relates to the use of said active ingredient for producing a pharmaceutical composition for stimulating the immune system.

Description

Steigerung der Immunantwort durch Substanzen, welche d e Funktion von Natürlichen Killerzellen beeinflussen Increasing the immune response through substances that affect the function of natural killer cells
Gebiet der Erfindung.Field of the Invention.
Die Erfindung betrifft Wirkstoffe und pharmazeutische Zusammensetzungen, welche das Immunsystem stimulieren.The invention relates to active substances and pharmaceutical compositions which stimulate the immune system.
Hintergrund der Erfindung und Stand der Technik.Background of the Invention and Prior Art.
Trotz aller Forschungsanstrengungen in den vergangenen Jahren konnte bislang kein Durchbruch erzielt werden in der Immuntherapie von Tumoren. Gleichermaßen stellen Vi- rusinfektionen, wie beispielsweise CMV-Infektionen bei immunsupprimierten Patienten, immer noch eine lebens- bedrohliche Erkrankung dar.Despite all research efforts in recent years, no breakthrough has so far been achieved in the immunotherapy of tumors. Similarly, virus infections, such as CMV infections in immunosuppressed patients, are still a life-threatening disease.
Die Immunreaktion gegen Infektionserreger und Tumore kann unterteilt werden in eine angeborene Abwehr, bei welcher die Natürlichen Killerzellen eine besondere Rolle spielen, und eine erworbene Antigen-spezifische Abwehr, bei der zytotoxische T-Lymphozyten und spezifische Antikörper eine zentrale Rolle spielen.The immune response against infectious agents and tumors can be divided into an innate defense, in which the natural killer cells play a special role, and an acquired antigen-specific defense, in which cytotoxic T-lymphocytes and specific antibodies play a central role.
Natürliche Killer Zellen (NK-Zellen) stellen eine Lym- phozytenart dar, d e etwa 15% der penpheren Blutlym- phozyten ausmachen. NK-Zellen sind in allen Geweben und Korperhohlen einschließlich der Leber, der Peritonealhohle und der Plazenta anzutreffen. Von den NK-Zellen sind zumindest bei der Maus die NKl .1-T-Zellen zu unterscheiden. Diese NKl .1-T-Zellen exprimieren einen T-Zellrezeptor (TCR) , sind CD1 restringiert und CD4-positiv/CD8-negatιv oder CD4/CD8 doppelt negativ. Den NKl .1-T-Zellen wird ein regulativer, zum Teil hemmender Einfluss auf cytotoxische T-Zellen, im Besonderen bei Autoimmunreaktionen zugespro- 5 chen (Zhang et al . , J Exp Med 186:1677-1687,(1997); Ben- lagha et al., Science 296: 481-482,(2002); Shlomai et al., J Path 195:498-507,(2001)). Das menschliche Korrelat der NKl .1-T-Zelle ist bislang noch nicht bekannt.Natural killer cells (NK cells) represent a type of lymphocyte that makes up about 15% of penpheric blood lymphocytes. NK cells can be found in all tissues and body cavities including the liver, peritoneal cavity and placenta. At least in the mouse, the NK1.1 T cells can be distinguished from the NK cells. These NK1.1 T cells express a T cell receptor (TCR), CD1 are restricted and CD4-positive / CD8-negative or CD4 / CD8 are double-negative. The NK1.1 T cells are said to have a regulatory, in some cases inhibitory, effect on cytotoxic T cells, in particular in autoimmune reactions (Zhang et al., J Exp Med 186: 1677-1687, (1997); Ben - lagha et al., Science 296: 481-482, (2002); Shlomai et al., J Path 195: 498-507, (2001)). The human correlate of the NKl .1 T cell is not yet known.
10 Beim Menschen sind maternale NK-Zellen in der Plazenta angereichert und scheinen hier möglicherweise eine Rolle bei der Kontrolle der Plazentation zu spielen. Jedoch ist vollkommen unbekannt, wie die NK-Zellen diese Rolle ausüben konnten (Mofett-King Nature Reviews Immunology10 In humans, maternal NK cells are enriched in the placenta and may appear to play a role in controlling placentation here. However, it is completely unknown how the NK cells could play this role (Mofett-King Nature Reviews Immunology
15 2/9; 656- 663, 2002) . Jedoch sind diejenigen Rezeptoren auf NK-Zellen bereits analysiert worden, mit Hilfe derer NK-Zellen mit Liganden auf Zellen der allogenen Plazenta, im Besonderen den extravillόsen Trophoblast-Zellen, in Wechselwirkung treten können. Zu diesen Liganden gehören15 2/9; 656-663, 2002). However, those receptors on NK cells have already been analyzed with the aid of which NK cells can interact with ligands on cells of the allogeneic placenta, in particular the extravillous trophoblast cells. These ligands include
20 besonders die MHC-Klasse-I Moleküle HLA-C, HLA-E und HLA- G. Die Wechselwirkung dieser Liganden mit den zugehörigen Rezeptoren auf den NK-Zellen hat entweder eine aktivierende oder eine inhibierende Wirkung auf die NK-Zellen (Mofett-King Nature Reviews Immunology 2/9; 656- 663,20 especially the MHC class I molecules HLA-C, HLA-E and HLA-G. The interaction of these ligands with the associated receptors on the NK cells has either an activating or an inhibiting effect on the NK cells (Mofett King Nature Reviews Immunology 2/9; 656-663,
25 2002) . Zu diesen Rezeptoren gehören25 2002). These receptors include
Inhibierende Rezeptoren aufNK-zellen Liganden aufTrophoblast-ZellenInhibitory receptors on NC cell ligands on trophoblast cells
CD94/NKG2A HLA-E (+ HLA-G/C)CD94 / NKG2A HLA-E (+ HLA-G / C)
30 KIR2DL2/3 HLA-C30 KIR2DL2 / 3 HLA-C
KIR2DL1 HLA-CKIR2DL1 HLA-C
ILT2 HLA-G Aktivierende Rezeptoren auf NK-ZellenILT2 HLA-G Activating receptors on NK cells
CD94/NKG2C HLA-E (+HLA-G/C)CD94 / NKG2C HLA-E (+ HLA-G / C)
KIR2DL4 HLA-GKIR2DL4 HLA-G
KIR2DS HLA-GKIR2DS HLA-G
NKG2D MICA/B, ULBPNKG2D MICA / B, ULBP
NKp30 unbekanntNKp30 unknown
NKp46 unbekanntNKp46 unknown
Zellkulturexperimente belegen, dass NK-Zellen in der Lage 3_0 sind, Tumorzellen abzutöten. Aus Experimenten an Tumormodellen in der Maus ist bekannt, dass eine Entfernung von NK-Zellen zu einer Verstärkung des Tumorwachstums fuhrt (Karre et al., Nature 319: 675-678 (1986); Smyth et al . , Nature Immunol . 2:293-299(2001)). Bei Patienten mit Lun- _5 genkarzmomen war die Anzahl von CD57 positiven Lym- phozyten (welche NK-Zellen einschließen) im Tumor direkt korreliert mit der Uberlebenszeit (Villegas et al., Lung Cancer 35: 23-28, (2002)). Ähnliches konnte für das Magenkarzinom nachgewiesen werden (Ishigami et al.f Cancer 20 88:577-583, (2000) ) .Cell culture experiments prove that NK cells are able to kill tumor cells. Experiments on mouse tumor models have shown that removal of NK cells leads to an increase in tumor growth (Karre et al., Nature 319: 675-678 (1986); Smyth et al., Nature Immunol. 2: 293 -299 (2001)). In patients with lung cancer, the number of CD57 positive lymphocytes (which include NK cells) in the tumor was directly correlated with survival time (Villegas et al., Lung Cancer 35: 23-28, (2002)). The same could be demonstrated for gastric carcinoma (Ishigami et al. F Cancer 20 88: 577-583, (2000)).
Des Weiteren wird angenommen, dass NK-Zellen entscheidend an der Induktion von cytotox schen T-Zellen und an der Kontrolle von Virusinfektionen teilhaben. So wurdeFurthermore, it is assumed that NK cells play a decisive role in the induction of cytotoxic T cells and in the control of viral infections. So it was
25 berichtet, dass die Hemmung von NK-Zellen durch Anti-NCAM Antikörper zu einer Hemmung der Induktion Alloantigen- spezifischer T-Zellen fuhrt (Kos und Engleman, J Immunology 155:578-584,(1995)). Weiterhin wurde von einem Patienten mit tödlicher Herpesvirus-Infektion berichtet, bei25 reports that the inhibition of NK cells by anti-NCAM antibodies leads to an inhibition of the induction of alloantigen-specific T cells (Kos and Engleman, J Immunology 155: 578-584, (1995)). A patient with fatal herpes virus infection has also been reported in
30 welchem keine NK-Zellen nachweisbar waren (Biron et al., N. Engl. J. Med. 320,1731-1735 (1989). Anderseits ist von CMV-Infektionen bekannt, dass diese Viren die Expression von MHC-Klasse-I Molekülen herunterregulieren wie auch Substanzen exprimieren, von welchen angenommen wird, dass sie NK-Zellen hemmen können. Zu diesen Substanzen gehören UL18, UL40 und UL16 (Übersicht bei: Cerwenka et al., Nature Reviews Immunology 1:41- 49, (2001)).30 which no NK cells were detectable (Biron et al., N. Engl. J. Med. 320, 1731-1735 (1989). On the other hand, CMV infections are known to cause the expression of MHC class I Down-regulate molecules as well Express substances that are believed to inhibit NK cells. These substances include UL18, UL40 and UL16 (overview from: Cerwenka et al., Nature Reviews Immunology 1: 41-49, (2001)).
Es ist bekannt, dass MHC-Klasse-I Moleküle die Zytotoxizitat von NK-Zellen inhibieren können, NK-Zellen somit besonders zytotoxisch aktiv sind für solche Zellen, welche keine MHC-Klasse-I Moleküle auf ihrer Zellmembran expnm- leren. Als die Zytotoxizitat inhibierende Rezeptoren auf den NK-Zellen konnten bei der Maus Ly49 und CD94/NKG2A und beim Menschen KIR2DL, KIR3DL, CD 159a, CD853 und CD85d identifiziert werden (Übersicht bei: Cerwenka et al . , Nature Reviews Immunology 1:41- 49 (2001)).It is known that MHC class I molecules can inhibit the cytotoxicity of NK cells, NK cells are therefore particularly cytotoxically active for those cells which do not expose MHC class I molecules on their cell membrane. The cytotoxicity-inhibiting receptors on the NK cells could be identified in the mouse Ly49 and CD94 / NKG2A and in humans KIR2DL, KIR3DL, CD 159a, CD853 and CD85d (review by: Cerwenka et al., Nature Reviews Immunology 1: 41- 49 (2001)).
Andererseits sind NK-Zellen in der Lage, Tumorzellen abzutöten, obwohl diese MHC-Klasse I Moleküle exprimieren. Als Ursache wurden auf der Membran von NK-Zellen die Zytotoxizitat aktivierende Rezeptoren identifiziert, deren Aktivierung offensichtlich dominiert über die Inhibition durch die Zytotoxizitat inhibierenden Rezeptoren von NK- Zellen. Zu diesen Zytotoxizitat-aktivierenden Rezeptoren zahlen bei der Maus NKR-P1C, Ly49D, Ly49H und beim Menschen CD16, NKp30, NKp46, KIR2DS, CD94/NKG2C, NKp44, NKG2D, und CD244 (Übersicht bei: Cerwenka et al., Nature Reviews Immunology 1:41-49 (2001)).On the other hand, NK cells are able to kill tumor cells, although they express MHC class I molecules. The cause was identified on the membrane of NK cells, the cytotoxicity-activating receptors, the activation of which clearly dominates the inhibition by the cytotoxicity-inhibiting receptors of NK cells. These cytotoxicity-activating receptors include NKR-P1C, Ly49D, Ly49H in the mouse and CD16, NKp30, NKp46, KIR2DS, CD94 / NKG2C, NKp44, NKG2D, and CD244 in humans (review by: Cerwenka et al., Nature Reviews Immunology 1: 41-49 (2001)).
Es wird vermutet, dass die Aktivierung von NK-Zellen verursacht wird durch die konzertierte Aktion dieser Zytotoxizitat-aktivierenden Rezeptoren mit Zytokm- Rezeptoren, Adhesionsmolekulen und Chemokm-Rezeptoren (Cerwenka et al., Nature Reviews Immunology 1:41-49 (2001) ) . Von humanen NK-Zellen ist bekannt, dass sie in besonderem Maße das Adhäsionsmolekul N-CAM (CD56, Leu-19, NKHl) und zusatzlich CD7, CD38, CDlla, CDllb und CD45RA exprimieren. Die Expression von CDllc und von CD57 ist dagegen hetero- gen und variable. Nach Aktivierung wird die Expression von CDlla, CD38 und HLADR gesteigert und von CDllb und CD45RA vermindert. Chronisch aktivierte NK-Zellen exprimieren zusatzlich besonders CD2 und CD57 (Lima et al . , Blood Cells Mol Dis 28:181-190, 2002)).The activation of NK cells is thought to be caused by the concerted action of these cytotoxicity-activating receptors with cytokm receptors, adhesion molecules and chemokm receptors (Cerwenka et al., Nature Reviews Immunology 1: 41-49 (2001)) , Human NK cells are known to express the adhesion molecule N-CAM (CD56, Leu-19, NKHl) and additionally CD7, CD38, CDIla, CDllb and CD45RA. The expression of CDllc and CD57, on the other hand, is heterogeneous and variable. After activation, the expression of CDIIa, CD38 and HLADR is increased and CD11b and CD45RA are decreased. Chronically activated NK cells additionally express particularly CD2 and CD57 (Lima et al., Blood Cells Mol Dis 28: 181-190, 2002)).
N-CAM ist nicht nur auf NK-Zellen, sondern auch auf T- Zellen, auf Nervenzellen, und besonders auf Tumorzellen des Neuroektoderms (CNS-Tumore, Melanome, kleinzelliges Bronchiakarzinom) anzutreffen. Antikörper gegen N-CAM kon- nen das Wachstum von Neuroblastom-Zellen und vonN-CAM is not only found on NK cells, but also on T cells, on nerve cells, and especially on tumor cells of the neuroectoderm (CNS tumors, melanomas, small cell bronchial carcinoma). Antibodies against N-CAM can inhibit the growth of neuroblastoma cells and cells
Glioblasto -Zellen hemmen (Krushel et al., PNÄS USA, 95: 2592-2596, (1998); Dehal et al., Biochem.Soc. Trans 30/4: 518-520, (2002)) und damit für die Diagnostik und Therapie dieser Tumoren verwendet werden.Inhibit glioblasto cells (Krushel et al., PNÄS USA, 95: 2592-2596, (1998); Dehal et al., Biochem. Soc. Trans 30/4: 518-520, (2002)) and thus for diagnostics and therapy of these tumors can be used.
Technisches Problem der Erfindung.Technical problem of the invention.
Der Erfindung liegt das technische Problem zu Grunde, pharmazeutische Zusammensetzungen zur Stimulation des Immunsystems anzugeben.The invention is based on the technical problem of specifying pharmaceutical compositions for stimulating the immune system.
Grundzuge der Erfindung und Ausfuhrungsformen.Main features of the invention and embodiments.
Zur Losung des technischen Problems lehrt die Erfindung insbesondere Wirkstoffe und pharmazeutische Zusammensetzungen gemäß der Patentansprüche. Die Erfindung beruht auf der überraschenden Erkenntnis, dass die Inhibition der Funktion von humanen NK-Zellen durch einen inhibierenden Wirkstoff zu einer Verstärkung der antigenspezifischen Immunabwehr fuhrt . Gegenstand der Erfindung ist somit auch ein Verfahren zur Verstärkung der Immunabwehr, wobei die Funktion von NK- Zellen durch mindestens einen Wirkstoff inhibiert wird. Wirkstoff im Sinne der Erfindung ist eine Bindesubstanz, welche an eine NK-Zelle bindet und diese inhibiert oder eine Substanz, welche nach Gabe in einem Organismus in diesem Organismus die Bildung einer Bindesubstanz bewirkt, welche an eine NK-Zelle bindet und diese inhibiert Neben.To solve the technical problem, the invention teaches in particular active ingredients and pharmaceutical compositions according to the claims. The invention is based based on the surprising finding that the inhibition of the function of human NK cells by an inhibiting active ingredient leads to an increase in the antigen-specific immune defense. The invention thus also relates to a method for strengthening the immune defense, the function of NK cells being inhibited by at least one active ingredient. Active substance in the sense of the invention is a binding substance which binds to an NK cell and inhibits it, or a substance which, after administration in an organism, causes the formation of a binding substance in this organism which binds to an NK cell and inhibits it.
Die Erfindung beruht auf der weiteren überraschenden Erk- enntnis, dass die antigenspezifische Immunabwehr im besonderen Maße dann verstärkt wird, wenn die Bindesubstanz gemäß dieser Erfindung gekoppelt ist mit einem Molekül, welches an einen T-Lymphozyten bindet. Derartige, an T- Lymphozyten bindende Moleküle können sein Antigene, Cy- tokme, Chemokine, Wachstumsfaktoren oder Antikörper, oder Fragmente von Antikörpern, welche an den T-Zellrezeptor, an ein Adhasionsmolekul, an einen Cytokin-Rezeptor oder an einen Chemokin-Rezeptor auf T-Zellen binden. Bevorzugt werden solche Moleküle verwendet, welche an CD2, CD3, CD4, CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD26, CD27, CD28, CD30, CD31, CD37, CD38, CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDw60, CD621, CD70, CD73, CD75, CD76, CD83, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD101, CD107a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152, CD153, CD154, CD155, CD160, CD161, CD166, CD226, CD245, CD246, CD247, oder an einen Bestandteil des T-Zell- Rezeptor-Komplexes binden. Die Erfindung beruht des weiteren auf der überraschenden Erkenntnis, dass die erstärkung der antigenspezifischen Immunabwehr durch einen erfindungsgemäßen Wirkstoff zu einer befristeten antigenspezifischen Anergie führen kann.The invention is based on the further surprising finding that the antigen-specific immune defense is particularly enhanced when the binding substance according to this invention is coupled to a molecule which binds to a T lymphocyte. Such molecules which bind to T lymphocytes can be antigens, cytokines, chemokines, growth factors or antibodies, or fragments of antibodies which bind to the T cell receptor, to an adhesion molecule, to a cytokine receptor or to a chemokine receptor Bind T cells. Molecules which are preferably used on CD2, CD3, CD4, CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD26, CD27, CD28, CD30, CD31, CD37, CD38, CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDw60, CD621, CD70, CD73, CD75, CD76, CD83, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD101, CD107a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152, CD153, CD154, CD155, CD160, CD161, CD166, CD226, CD245, CD246, CD247, or to a component of the T cell receptor complex tie. The invention is further based on the surprising finding that the strengthening of the antigen-specific immune defense by an active substance according to the invention can lead to a limited antigen-specific anergy.
Beispiele für Wirkstoffe gemäß der Erfindung, welche Bindesubstanzen darstellen, sind Liganden, welche durch ihre Bindung an inhibierende Rezeptoren von NK-Zellen deren Funktion inhibieren. Zu den inhibierenden Rezeptoren auf NK-Zellen und den zugehörigen Liganden gehören:Examples of active substances according to the invention, which are binding substances, are ligands which, by binding to inhibiting receptors of NK cells, inhibit their function. The inhibitory receptors on NK cells and the associated ligands include:
Spezies Rezeptor LigandenSpecies receptor ligands
Maus Ly49 H-2K, H-2DMouse Ly49 H-2K, H-2D
Maus CD94/NKG2A Qa-1Mouse CD94 / NKG2A Qa-1
Mensch KIR2DL- l; - 2; und - 3; HLA-CHuman KIR2DL- l; - 2; and - 3; HLA-C
Mensch KIR3DL HLA-Bw4, HLA-4Human KIR3DL HLA-Bw4, HLA-4
Mensch CD94/NKG2A (CD159a) HLA-EHuman CD94 / NKG2A (CD159a) HLA-E
UL40 von CMVUL40 from CMV
Mensch CD85J; CD85d HLA-Klasse I;Human CD85J; CD85d HLA class I;
ULlδ von CMVULlδ from CMV
Mensch ILT2 HLA-GHuman ILT2 HLA-G
Weitere Beispiele für Bindesubstanzen sind: Teilsequenzen dieser Liganden und Peptidomimetika dieser Liganden, welche an inhibierende Rezeptoren der NK-Zellen binden und deren Funktion inhibieren, Antikörper, Antikörperfragmente und Fusionsproteine enthaltend mindestens einen Antikörper oder ein Antikörperfragment, welche an die inhibierende Rezeptoren von NK-Zellen binden und deren Funktion inhibieren, Substanzen, welche durch ihre Bindung an aktivierende Rezeptoren der NK-Zellen deren Funktion inhibieren. Zu den aktivierenden Rezeptoren auf NK-Zellen und den zugehörigen Liganden gehören:Further examples of binding substances are: partial sequences of these ligands and peptidomimetics of these ligands which bind to inhibiting receptors of the NK cells and inhibit their function, antibodies, antibody fragments and fusion proteins containing at least one antibody or an antibody fragment which bind to the inhibiting receptors of NK cells bind and inhibit their function, substances that inhibit their function by binding to activating receptors of the NK cells. Activating receptors on NK cells and the associated ligands include:
Spezies Rezeptor LigandSpecies receptor ligand
Maus NKR-P1C UnbekanntMouse NKR-P1C Unknown
Maus Ly49D H-2DMouse Ly49D H-2D
Maus Ly49H M-CytomegalovirusMouse Ly49H M cytomegalovirus
Mensch, Maus CD16 IgGHuman, mouse CD16 IgG
Mensch, Maus NKp46 Influenza- Virus Haemagglutinin ; Haemagglutinin-Neuraminidase von Parainfluenza VirusHuman, mouse NKp46 influenza virus hemagglutinin; Haemagglutinin neuraminidase from Parainfluenza virus
Mensch, CD94/NKG2C HLA-E ; HLA-G; HLA-C (Maus) (Qa-1)Human, CD94 / NKG2C HLA-E; HLA-G; HLA-C (mouse) (Qa-1)
Mensch, NKG2D MHC-classI chain related antigens (Maus) (MIC-A, MIC-B);Human, NKG2D MHC-classI chain related antigens (mouse) (MIC-A, MIC-B);
UL16 Binding Proteins (ULBPl,-2,-3)UL16 Binding Proteins (ULBPl, -2, -3)
(RAE-1, H60)(RAE-1, H60)
Mensch, CD244 CD48 (Maus)Human, CD244 CD48 (mouse)
Mensch NKp30; NKp30; Unbekannt NKp46Human NKp30; NKp30; Unknown NKp46
Mensch KIR2DS HLA-CHuman KIR2DS HLA-C
Mensch KIR2DL4 HLA-GHuman KIR2DL4 HLA-G
Mensch NKp44 Influenza-Virus HaemagglutininHuman NKp44 influenza virus hemagglutinin
Zu den Bindesubstanzen im Sinne der Erfindung gehören demnach Mutationen oder Spaltprodukte oder Peptidomimetika der Liganden für aktivierende Rezeptoren auf NK-Zellen, welche an aktivierende Rezeptoren auf NK-Zellen binden ohne diese zu aktivieren, Antikörper, Antikörperfragmente oder Fusionsproteine enthaltend mindestens einen Antikörper oder ein Antikörperfragment, welche an aktivierende Rezeptoren auf NK-Zellen binden ohne diese zu aktivieren oder welche Liganden für aktivierende Rezeptoren binden und deren Bindung an diese Rezeptoren inhibieren, das ULlβ Protein des CMV, welches an ULBP-1, ULBP-2 und an MICB bindet, Homologe des UL16 Proteins und Peptidomimetika des ULlβ Proteins, Antikörper, Antikorperfragmente oder Fu- sionsproteme enthaltend mindestens einen Antikörper oder mindestens ein Antikorperfragment, welche an ein Ad- hasionsmolekul auf NK-Zellen binden und hierdurch die Funktion der NK-Zelle inhibiert. Derartige Adhasionsmo- lekule sind beispielsweise N-CAM (CD56) und CD57. Beispiele für Antikorperfragmente spezifisch für ein Ad- hasionsmolekul sind an N-CAM bindende Peptidsequenzen beschrieben von Whittington et al., Medical and Pediatric Oncology 36: 243-246 (2001).Accordingly, the binding substances within the meaning of the invention include mutations or cleavage products or peptidomimetics of the ligands for activating receptors on NK cells which bind to activating receptors on NK cells without activating them, antibodies, antibody fragments or fusion proteins containing at least one antibody or an antibody fragment which bind to activating receptors on NK cells without activating them or which ligands bind to activating receptors and inhibit their binding to these receptors, the ULIβ protein of the CMV, which binds to ULBP-1, ULBP-2 and to MICB, homologs of the UL16 protein and peptidomimetics of the ULlβ protein, antibodies, antibody fragments or fusion proteins containing at least one antibody or at least one antibody fragment which bind to an adhesion molecule on NK cells and thereby inhibits the function of the NK cell. Adhesion molecules of this type are, for example, N-CAM (CD56) and CD57. Examples of antibody fragments specific for an adhesion molecule are peptide sequences which bind to N-CAM and are described by Whittington et al., Medical and Pediatric Oncology 36: 243-246 (2001).
Beispiele für Wirkstoffe im Sinne der Erfindung, welche in einem Organismus die Bildung einer Bindesubstanz m Sinne dieser Erfindung auslosen können, sind Membranbestandteile von NK-Zellen, welche nach Injektion in einen Organismus zu einer spezifischen Immunreaktion, im Besonderen zu dem Auftreten von Antikörpern fuhren, welche an inhibierende Rezeptoren auf NK-Zellen, oder an Adhasionsmolekule auf NK-Zellen binden und hierdurch die Funktion von NK-Zellen inhibieren, Liganden für aktivierende Rezeptoren auf NK- Zellen und Komplexe aus mindestens einem Liganden und mindestens einem aktivierenden Rezeptor, welche nach Injektion in einen Organismus zu einer spezifischen Immunreaktion, im Besonderen zu dem Auftreten von Antikörpern fuhren, welche die Aktivierung der aktivierenden Rezeptoren auf NK-zellen inhibieren.Examples of active substances in the sense of the invention which can trigger the formation of a binding substance in the sense of this invention in an organism are membrane components of NK cells which, after injection into an organism, lead to a specific immune reaction, in particular to the appearance of antibodies, which bind to inhibiting receptors on NK cells, or to adhesion molecules on NK cells and thereby inhibit the function of NK cells, ligands for activating receptors on NK cells and complexes of at least one ligand and at least one activating receptor, which after injection in an organism lead to a specific immune reaction, in particular to the appearance of antibodies which inhibit the activation of the activating receptors on NK cells.
Wirkstoffe im Sinne der Erfindung werden Zeilzubereitungen aus dem Blut, aus Organen oder aus Korperhohlen hinzugegeben. Solche Zellzubereitungen sind beispielsweise Leukozytenpraparationen oder Lymphozytenpraparationen . Die Herstellung solcher Zellzubereitungen aus dem Blut, aus Organen oder aus Korperhohlen ist dem Fachmann gelaufig. Nachfolgend wird das Gemisch aus dieser Zellzubereitung und dem Wirkstoff einem Organismus verabreicht, um seine antigenspezifische Immunreaktion zu starken.Active substances in the sense of the invention are added to line preparations from the blood, from organs or from body cavities. Such cell preparations are, for example Leukocyte preparations or lymphocyte preparations. The preparation of such cell preparations from the blood, from organs or from body cavities is familiar to the person skilled in the art. The mixture of this cell preparation and the active ingredient is then administered to an organism in order to strengthen its antigen-specific immune response.
Wirkstoffe im Sinne der Erfindung werden des Weiteren als Arzneimittel verwendet. Hierzu wird beispielsweise der erfmdungsgemaße Wirkstoff mit einem geeigneten, dem Fachmann bekannten galenischen Hilfsmittel versetzt. Derartige galenische Hilfsmittel können beispielsweise sein physiologische wassrige Injektionslosungen. Wirkstoffe, welche injiziert werden, um eine Immunreak- tion, im besonderen eine Antikorperreaktion zu erzeugen, werden vorzugsweise mit einem Ad uvans versetzt. Beispiele für Adjuvantien sind Aluminiumhydroxyd oder CpG.Active substances in the sense of the invention are also used as medicaments. For this purpose, for example, the active ingredient according to the invention is mixed with a suitable pharmaceutical auxiliary known to the person skilled in the art. Such pharmaceutical auxiliaries can be, for example, physiological aqueous injection solutions. Active ingredients which are injected in order to produce an immune reaction, in particular an antibody reaction, are preferably treated with an ad uvans. Examples of adjuvants are aluminum hydroxide or CpG.
Arzneimittel, enthaltend einen erfindungsgemaßen Wirkstoff werden lokal verabreicht oder in den Blutkreislauf, in ein Organ, in eine Korperhohle, in das Bindegewebe oder in die Muskulatur injiziert zur Prophylaxe oder Therapie einer Erkrankung. Beispiele für derartige Erkrankungen bzw. Therapien sind folgend erläutert.Medicaments containing an active ingredient according to the invention are administered locally or injected into the bloodstream, into an organ, into a body cavity, into the connective tissue or into the muscles for the prophylaxis or therapy of a disease. Examples of such diseases or therapies are explained below.
a) Antigen-spezifische Immuntherapie maligner Tumoren: Wirkstoffe gemäß dieser Erfindung können für die T-Zell vermittelte Immuntherapie maligner Tumoren verwendet werden. Nach Verabreichung des Wirkstoffes können prinzipiell alle T-Zellen des Korpers aktiviert werden; die Spezifitat des T-Zell Rezeptors spielt für die T-Zell Aktivierung keine Rolle. Derartig aktivierte T-Zellen sind in der Lage, das Tumorwachstum zu hemmen. Die Hemmung des Tumorwachstums kann dabei direkt durch zytotoxisch aktive T-Zellen erreicht werden oder indirekt, durch Mitaktivierung anderer Komponenten des immunologischen Netzwerkes, wie z.B. von B-Zellen oder Makrophagen. Zugleich kann ein immunologisches Gedächtnis aufgebaut werden. Bei der direkten antitumoralen Aktivität zytotox- ischer T Zellen werden die Tumorzellen durch solche T- Zellen angegriffen, die entweder mit ihrem tumorspezifischen T Zeil Rezeptor oder unabhängig von ihrer TcR Spezifitat über tumorspezifische Antikörper oder über Tumor- und T-Zell- (bi-) spezi ische Antikörper oder Fusionsproteine den Tumor erkennen.a) Antigen-specific immunotherapy of malignant tumors: Active substances according to this invention can be used for T-cell-mediated immunotherapy of malignant tumors. In principle, after administration of the active ingredient, all T cells of the body can be activated; the specificity of the T cell receptor is irrelevant for T cell activation. T cells activated in this way are able to inhibit tumor growth. The inhibition of Tumor growth can be achieved directly by cytotoxically active T cells or indirectly, by activating other components of the immunological network, such as B cells or macrophages. At the same time, an immunological memory can be built up. In the case of direct antitumor activity of cytotoxic T cells, the tumor cells are attacked by T cells which either use their tumor-specific T Zeil receptor or, regardless of their TcR specificity, use tumor-specific antibodies or tumor and T cell (bi-) specific antibodies or fusion proteins recognize the tumor.
b) Therapie einer T-Zell Unterfunktion oder eines T-Zell Mangels: Wirkstoffe gemäß der Erfindung können desweiteren eingesetzt werden für die Therapie von Zustanden des T- Zell Mangels oder der T-Zell Unterfunktion. Eine Verminderung der T-Zell Funktion kommt bei verschiedenen Erkrankungen vor, so z.B. als Folge einer Chemo-, Radio-, oder Immuntherapie, unter medikamentöser immunsuppressiver Therapie, als Begleitzustand von Erkrankungen, die Milz, Lymphknoten oder Knochenmark beeinträchtigen, bei angeborenen Immundefekten oder als Begleiterscheinung hama- tologischer Neoplasien, chronischer Entzundungsreaktionen oder Infekte.b) Therapy of a T cell underfunction or a T cell deficiency: Active substances according to the invention can furthermore be used for the therapy of conditions of the T cell deficiency or the T cell underfunction. A decrease in T cell function occurs in various diseases, e.g. as a result of chemotherapy, radio therapy or immunotherapy, under medicinal immunosuppressive therapy, as an accompanying condition of diseases that affect the spleen, lymph nodes or bone marrow, in the case of congenital immune defects or as a concomitant symptom of hematological neoplasia, chronic inflammation reactions or infections.
c) Steigerung der T-Zell Aktivität gegen NK-Zellen bei Erkrankungen mit benigner oder maligner NK-Zell Vermehrung: Benigne oder maligne proliferative Erkrankungen des NK-Zell Systems, wie beispielsweise die chronische NK-Zell Lymphozytose, die NK-Zell Leukämie oder das NK-Zell Lym- phom können bevorzugt mit Wirkstoffen gemäß der Erfindung therapiert werden, welche gleichzeitig ein Molekül auf NK-Zellen wie auch ein Molekül auf T-Zellen binden. Diese Wirkstoffe supprimieren die NK-Zellen, verstarken hierdurch die T-Zell Aktivität und richten diese aus auf NK-Zellen.c) Increase in T cell activity against NK cells in diseases with benign or malignant NK cell proliferation: Benign or malignant proliferative diseases of the NK cell system, such as chronic NK cell lymphocytosis, NK cell leukemia or that NK cell lymphoma can preferably be treated with active substances according to the invention which simultaneously have a molecule Bind NK cells as well as a molecule on T cells. These active substances suppress the NK cells, thereby strengthen the T cell activity and target it to NK cells.
d) Modulation der T-Zell Aktivität bei Autoimmunerkrankun- gen, deren Ursache in einer NK-Zell Überfunktion liegt: Ein weiteres bevorzugtes Anwendungsgebiet eines erfmd- ungsgemaßen Wirkstoffes sind solche Autoimmunerkrankungen, bei welchen eine Uberaktivitat der NK-Zellen und eine hieraus folgende Fehlregulation der T-Zellen vorkommt. Zu diesen Autoimmunerkrankungen gehören besonders sogenannte Antikörper mednerte Autoimmunerkrankungen, wie z.B. die Myasthenia gravis oder einer Allergie. Durch Verabreichung des erfindungsgemaßen Wirkstoffes kann eine Verstärkung der T-Zell Aktivität und nachfolgend ggfs. eine antigen- spezifische Anergie erreicht werden und hierdurch die Produktion der krankmachender Antikörper vermindert werden.d) Modulation of the T cell activity in autoimmune diseases, the cause of which is an NK cell hyperfunction: Another preferred area of application of an active substance according to the invention is those autoimmune diseases in which overactivity of the NK cells and a consequent malregulation of the T cells occurs. These autoimmune diseases include so-called antibody mediated autoimmune diseases, such as the myasthenia gravis or an allergy. By administering the active ingredient according to the invention, an increase in T-cell activity and subsequently, if necessary, an antigen-specific anergy can be achieved and the production of the disease-causing antibodies can thereby be reduced.
Beispiel 1: Korrelation von NK-Zell Depletion zu T-Zell Proliferation .Example 1: Correlation of NK cell depletion to T cell proliferation.
In einer Serie von Experimenten mit frisch isolierten mononuklearen Leukozyten (PBMCs) aus dem peripheren Blut gesunder Spender konnte eine Korrelation zwischen NK-Zell Depletion und T-Zell Proliferation gezeigt werden. Es wurden PBMCs von acht gesunden Spendern gewonnen und in einer Dichte von 1 x 106/ml in AIM-V Medium (LifeTechnology, Eg- genheim, Deutschland) + 10 % humanes AB Serum (PAA, Linz, Osterreich) + 10 mg/1 Ciprofloxacin (Bayer, Leverkusen, Deutschland) in Rundbodenmikrotiterplatten für 6 Tage im C02-Brutschrank inkubiert. Anti-NCAM Antikörper (Klon ERIC-1) wurden in einer Konzentration von 1000 ng/ml eingesetzt, ebenso antι-CD3 (Klon 0KT3) und antι-CD28 Antikörper (Klon 15E8) um einen T-Zell spezifischen Stimulus vorzugeben. Vor Beginn des Experimentes sowie an Tag 6 wurden die Leukozytensubpopulationen im Durchflusszytome- ter bestimmt. Zu Färbung dienten CD4-FITC, CD8-FITC und CD16-PE konjugierte Antikörper (Becton Dickmson, Heidelberg, Deutschland) . Die Messung erfolgte in einem FACSCalibur™ Durchflusszytometer (Becton Dickmson) unter Verwendung von TruCount™ Messrohrchen (Becton Dickmson) zur Ermittlung der absoluten Zellzahlen. Alle Tests wurden als Dreifachbestimmung durchgeführt und jeweils gemittelt. Die Absolutzahlen der T-Zellen ergaben sich aus der Summe der gemessenen CD4+ und CD8+(bπght) T-Zellen. Alle Ex- perimente erfolgten in Dreifachbestimmung. Der Prolifera- tionsmdex wurde aus den Mittelwerten nach der Formel Proliferationsmdex = Zellzahl an Tag 6 / Zellzahl an Tag 0 berechnet. Statistische Kalkulationen wurden mit Hilfe des Programms EXEL™ (Microsoft Corp., USA) durchgeführt. In allen auswertbaren Experimenten (Anzahl =6) konnte eine NK-Zell-Depletion von unterschiedlichem Ausmaß dokumentiert werden. Die Depletion von NK-Zellen zeigt sich in den unter 1,0 sinkende Proliferationsindizes (0,17 - 0,52) nach Zugabe des Anti-NCAM Antikörpers. Im Gegensatz hierzu zeigten die T-Zellen eine deutliche Verstärkung ihrer Proliferation. Die Proliferationsindizes lagen zwischen 1,14 und 1,73. Je geringer der verbliebene NK-Zell Anteil war, desto ausgeprägter war die T-Zell Proliferation. Statistisch ergab sich ein Korrelationswert von -0,727. Beispiel 2: Steigerung der T-Zell Proliferation durch anti-NCAM Antikörper die zusatzlich an T- Lymphozyten binden.A series of experiments with freshly isolated mononuclear leukocytes (PBMCs) from the peripheral blood of healthy donors showed a correlation between NK cell depletion and T cell proliferation. PBMCs were obtained from eight healthy donors and at a density of 1 x 10 6 / ml in AIM-V medium (LifeTechnology, Eggenheim, Germany) + 10% human AB serum (PAA, Linz, Austria) + 10 mg / 1 ciprofloxacin (Bayer, Leverkusen, Germany) incubated in round-bottom microtiter plates for 6 days in the C0 2 incubator. Anti-NCAM antibody (clone ERIC-1) were used in a concentration of 1000 ng / ml, as well as anti-CD3 (clone 0KT3) and anti-CD28 antibody (clone 15E8) to specify a T-cell-specific stimulus. Before the start of the experiment and on day 6, the leukocyte subpopulations were determined in the flow cytometer. CD4-FITC, CD8-FITC and CD16-PE conjugated antibodies (Becton Dickmson, Heidelberg, Germany) were used for staining. The measurement was carried out in a FACSCalibur ™ flow cytometer (Becton Dickmson) using TruCount ™ measuring tubes (Becton Dickmson) to determine the absolute cell numbers. All tests were carried out in triplicate and averaged in each case. The absolute numbers of the T cells resulted from the sum of the measured CD4 + and CD8 + (bπght) T cells. All experiments were carried out in triplicate. The proliferation mdex was calculated from the mean values using the formula proliferation mdex = cell number on day 6 / cell number on day 0. Statistical calculations were carried out using the EXEL ™ program (Microsoft Corp., USA). In all evaluable experiments (number = 6), NK cell depletion of various degrees was documented. The depletion of NK cells can be seen in the proliferation indices (0.17 - 0.52) falling below 1.0 after the addition of the anti-NCAM antibody. In contrast, the T cells showed a marked increase in their proliferation. The proliferation indices were between 1.14 and 1.73. The lower the remaining NK cell content, the more pronounced the T cell proliferation. Statistically, the correlation value was -0.727. Example 2: Increase in T-cell proliferation by anti-NCAM antibodies which additionally bind to T-lymphocytes.
Eine weitere Steigerung der T-Zell Proliferation konnte durch Antikörper erreicht werden, die gleichzeitig an NCAM und an CD3 auf T-Zellen binden. In der in Beispiel 1 beschriebenen Serie von Messungen wurde zusatzlich ein bispezifischer Antikörper getestet mit einer Spezifitat gegen NCAM und gegen CD3 (Klon OE-1) . Dieser bispezifische Antikörper wurde in einer Konzentration von 1000 ng/ml wiederum zusammen mit einem anti-CD28 Antikörper (Klon 15E8) eingesetzt wurde. Die Durchführung der Experimente erfolgte wie im Beispiel 1 beschrieben, wobei der berechneten T-Zellzahl aus technischen Gründen dieA further increase in T cell proliferation could be achieved by antibodies that bind to NCAM and CD3 on T cells simultaneously. In the series of measurements described in Example 1, a bispecific antibody was additionally tested with a specificity against NCAM and against CD3 (clone OE-1). This bispecific antibody was again used in a concentration of 1000 ng / ml together with an anti-CD28 antibody (clone 15E8). The experiments were carried out as described in Example 1, the calculated T cell number being the technical one
CD8+(bright) und CD8+(dim) Population plus die CD4+ Zellen zugrunde liegen. In 7 auswertbaren Experimenten betrug das Mittel der berechneten Proliferationsindizes 2,16. Kontrolluntersuchungen mit monospezifischen, nicht an T- Zellen bindenden anti-NCAM Antikörpern erreichten im Mittel einen Proliferationsindex von 1,73. In Negativkontrollen, bei denen weder monospezifische noch bispezifische Antikörper gegen N-CAM zugefügt wurden (sog. Mediumkontrolle) konnte keine T-Zell Proliferation festgestellt werden (Proliferationsindex = 0,85). Beispiel 3CD8 + (bright) and CD8 + (dim) population plus the CD4 + cells are based. In 7 experiments that could be evaluated, the mean of the calculated proliferation indices was 2.16. Control examinations with monospecific anti-NCAM antibodies not binding to T cells achieved an average proliferation index of 1.73. In negative controls, in which neither monospecific nor bispecific antibodies against N-CAM were added (so-called medium control), no T cell proliferation could be determined (proliferation index = 0.85). Example 3
Modulation der Immunantwort gegen ein bakterielles Aπtlgβn.Modulation of the immune response against a bacterial attack.
In MultiScreeen™-IP 96 well ELISPOT Platten (Milllpore, Bedford, MA, USA), beschichtet mit Antikörpern, spezifisch für humanes Interferon-y (Diaclone Research, Besancon, Frankreich), wurden pro Napf 2 x 103/well PBMC von Tetanustoxoid geimpften Personen eingesät und mit Tetanustoxoid (TT, Chlron Behring GmbH, Marburg, Deutschland; Titrationsreihe von 10 LF/ml bis 0,001 Lf/ml) alleine oder in verschiedenen Kombination mit dem bispβzifischβn Antikörper OE-1 (Titrationsreihe 100 ng/ml - 0,006 ng/ml) über 4 Tage inkubiert. Ale Medium diente AIMV (LifeTechnologies. Egganhθim, Deutschland, 200 icrollter pro Nap - Zur Posltivkontrollβ diente ein Antikörper gegen die CD3ε Kette von T-Lymphozytβn (Klon OKT3, 1 μg/ l), der bispezlfischβ Antikörper OE-1 (1 μg/ml) oder eine hohe Dosis Tetanustoxoid (10 Lf/ml). Zur Negativkontrolle diente ein anti-CD56 Antikörper (Klon ERIC-1, 1 μg/ml), bzw. eine βdiumkontrolle ohne den Zusatz von Antigenen oder Antlkö βrn. Die Menge des gebildeten INF-y (Anzahl der Spots) wurde mit Hilfe eines lFN-γ ELISPOT Kits (Diaclone Research) bestimmt.In MultiScreeen ™ -IP 96 well ELISPOT plates (Milllpore, Bedford, MA, USA), coated with antibodies specific for human interferon-y (Diaclone Research, Besancon, France), 2 × 10 3 / well PBMC from tetanus toxoid were used per well vaccinated and sown with tetanus toxoid (TT, Chlron Behring GmbH, Marburg, Germany; titration series from 10 LF / ml to 0.001 Lf / ml) alone or in various combinations with the bispecific antibody OE-1 (titration series 100 ng / ml - 0.006 ng / ml) incubated for 4 days. AIMV (LifeTechnologies. Egganhθim, Germany, 200 icrollter per nap) served as medium - an antibody against the CD3ε chain of T-lymphocytes (clone OKT3, 1 μg / l), the bispecific β antibody OE-1 (1 μg / ml ) or a high dose of tetanus toxoid (10 Lf / ml). An anti-CD56 antibody (clone ERIC-1, 1 μg / ml) or a βdium control without the addition of antigens or antibodies was used for the negative control INF-y (number of spots) was determined using an IFN-γ ELISPOT kit (Diaclone Research).
In drei unterschiedlichen Versuchsansätzen wurde übereinstimmend nach Gabe von TT in Kombination mit OE-1 (Titerstufe 1 ng/ml) eine Steigerung der INF-γ Ausschüttung um das 2- bis 4- fache im Vergleich zur alleinigen Tetanusantwort bei gleicher Tetanuskonzentratlon gesehen. Die Steigerung der IFN-γ Ausschüttung war dabei abhängig von der Konzentration des TT, wobei die Kombination aus TT plus OE-1 auch der stärksten Induzlerbaren alleinigen TT Antwort (die etwa bei 10 Lf/ml erreicht wird) trotz der in Kombination mit OE-1 eingesetzten niedrigen TT Konzentration (1 Lf/ml) mehrfach überlegen war. Die Negativkontrollβπ zeigten keine spontane IFN-γ Ausschüttung. Die OE-1 und OKT3 Posltivkontroliβn zeigten eine überschießende IFN-y Ausschüttung unter Verlust der Spazrfität in der T Zeil Reaktion für das Tetanustoxoid.In three different test approaches, after the administration of TT in combination with OE-1 (titer level 1 ng / ml), an increase in the INF-γ release by 2 to 4 times compared to the sole tetanus response with the same tetanus concentrate was seen. The increase in IFN-γ release was dependent on the concentration of the TT, whereby the combination of TT plus OE-1 also the strongest inducible sole TT response (which is achieved at around 10 Lf / ml) despite the combination with OE- 1 used low TT concentration (1 Lf / ml) was several times superior. The negative control βπ showed no spontaneous IFN-γ release. The OE-1 and OKT3 positive controls showed an excessive IFN-y release with loss of functionality in the T line reaction for the tetanus toxoid.
In einem weiteren Versuch wurden PBMC von Tetanustoxoid geimpften Personen für drei Tage in RPMM640 Medium (PAA, Linz, Österreich) ♦ 10 % fetalem Kalbersβrum (FCS, Biochrom AG) unter Verwendung der zuvor ermittelten optimalen Stimulationsbedlngungeπ (TT 1 Lf/ml + OE- 2 ng/ml; bzw. zur Kontrolle antl-CD5θ Antikörper 2 ng ml + TT 1 Lf/ml; bzw. kein Antikörper + TT 1 Lf/ml) vorsijmuliert. Die Zellen wurden nach der dreitägigen Prästimulationsphase zweimal mit Medium gewaschen und für 24 Stunden im Inkubator In Medium (RPMI1Θ40 + 10% FCS) ohne Zusatz von Tetanustoxoid oder Antikörper inkubiert. Danach erfolgte eine ELISPOT Test (3 Tage Iπkubatioπsdauer) auf humanes IFN^y (Diaclone Reβearch) unter Verwendung von serumfreiem AIMV Medium und Zusatz (einer hohen Dosierung) von TT (10 Lf/ml), bzw. unter Weglassung von TT (Mβdiumkontrolle).In a further experiment, PBMC from persons vaccinated with tetanus toxoid were exposed for three days in RPMM640 medium (PAA, Linz, Austria) ♦ 10% fetal calf serum (FCS, Biochrom AG) using the previously determined optimal stimulation conditions (TT 1 Lf / ml + OE- 2 ng / ml; or to control antl-CD5θ antibody 2 ng ml + TT 1 Lf / ml; or no antibody + TT 1 Lf / ml). After the three-day pre-stimulation phase, the cells were washed twice with medium and incubated for 24 hours in an incubator in medium (RPMI1Θ40 + 10% FCS) without the addition of tetanus toxoid or antibody. This was followed by an ELISPOT test (3 days incubation period) on human IFN ^ y (diaclone re-search) using serum-free AIMV medium and addition (a high dosage) of TT (10 Lf / ml), or omitting TT (medium control) ,
Im IFN-7 ELISPOT Test zeigte sich, dass nur die mit TT + OE-1 stimulierten PBMC 24 Stunden nach der Prästimulation refraktär waren gegen eine erneute Stimulation mit TT (10 Lf/ml), d.h. nur sehr geringe IFN-γ Mengen sekretierten. während die Kontrollen (PBMC stimuliert mit TT alleine oder in Kombination mit anti-CD56 Antikörpern) eine normale TT Antwort zeigten. Die TT + OE-1 prästlmullerten Zellen waren interessanterweise nicht refraktär gegen eine unspezifiache Stimulation mit beispielsweise OKT3 Antikörpern 100 ng/ml.The IFN-7 ELISPOT test showed that only the PBMC stimulated with TT + OE-1 were refractory 24 hours after the pre-stimulation against renewed stimulation with TT (10 Lf / ml), ie only very small amounts of IFN-γ were secreted. during controls (PBMC stimulated with TT alone or in Combination with anti-CD56 antibodies) showed a normal TT response. Interestingly, the TT + OE-1 premilled cells were not refractory to non-specific stimulation with, for example, OKT3 antibodies 100 ng / ml.
In einem weiteren Experiment wurden wie zuvor bereits beschrieben PBMC von Tetanustoxoid geimpften Personen für drei Tage in RPMI1640 Medium + 10 % fetalem Kälbβrserum unter Verwendung der optimalen Stlmulationsbβdlngungen (TT 1 Lf/ml + OE-1 2 ng/ml; bzw. zur Kontrolle aηtl-CDS6 Antikörper 2 ng/ml + TT 1 Lf/m); bzw. kein Antikörper + TT 1 Lf/ml) prästimuliert, danach letfoch nicht (wie im vorherigen Versuch) für 24 h in Medium ohne TT oder Antikörper Inkubiert, sondern Jetzt für weitere drei Tage mit (einer optimalen Dosierung von) 10 Lf/ml TT (in RPM11640+10% FCS) inkubiert, um zu prüfen, ob sich so die Anergie In Bezug auf die TT Antwort durchbrechen lässt. Im anschließend durchgeführten ELISPOT Test (3 Tage Inkubation, AIMV Medium) zeigte sich trotzdem keine Normalisierung der IFN-γ Produktion dort, wo die Zellen Initial mit OE-1 und TT prästimuliert worden waren, sondern Immer noch eine ca. 50-70 % Refraktärität der TT Antwort.In a further experiment, as previously described, PBMC from persons vaccinated with tetanus toxoid were exposed for three days in RPMI1640 medium + 10% fetal calf serum using the optimal stimulation conditions (TT 1 Lf / ml + OE-1 2 ng / ml; or as a control as a control) -CDS6 antibody 2 ng / ml + TT 1 Lf / m); or no antibody + TT 1 Lf / ml), then letfoch not (as in the previous experiment) incubated for 24 h in medium without TT or antibody, but now for another three days with (an optimal dosage of) 10 Lf / ml TT (in RPM11640 + 10% FCS) incubated to check whether the anergy related to the TT response can be broken. In the subsequent ELISPOT test (3 days incubation, AIMV medium), however, there was no normalization of IFN-γ production where the cells were initially pre-stimulated with OE-1 and TT, but still approximately 50-70% refractory the TT answer.
Die Ergebnisse belegen, dass ein erflndungsgemälißr Wirkstoff In der Lage Ist, die Immunrβaktion auf ein Antigen zu verstärken und zugleich auch In der Lage ist, je nach Verwendung, eine antigeπspezifische Anergie auszulösen. Die Auslosung einer Anergie ist von besonderer therapeutischer Bedeutung bei der Behandlung von Autoimmunerkrankungen, Allergien und Organabstαßuπgen. The results show that an active ingredient according to the invention is able to increase the immune response to an antigen and, at the same time, is also able, depending on the use, to trigger an antigen-specific anergy. The triggering of anergy is of particular therapeutic importance in the treatment of autoimmune diseases, allergies and organ rejection.

Claims

Patentansprüche :Claims:
1. Wirkstoff mit1. Active ingredient with
einer ersten molekularen Komponente, welche an ein erstes Molekül bindet, welches ausgewählt ist aus der Gruppe bestehend aus "NCAM, KIR2DL, KIR2DS, KIR3DL, CD94/CD159, CD57, CD85, CDlβ, NKp30, NKp44, NKp4β, NKG2D und CD244", unda first molecular component which binds to a first molecule which is selected from the group consisting of "NCAM, KIR2DL, KIR2DS, KIR3DL, CD94 / CD159, CD57, CD85, CDlβ, NKp30, NKp44, NKp4β, NKG2D and CD244", and
einer zweiten molekularen Komponente, welche an ein zweites Molekül bindet, welches ausgewählt ist aus der Gruppe bestehend aus "CD2, CD4, CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD26, CD27, CD28, CD30, CD31,a second molecular component which binds to a second molecule which is selected from the group consisting of "CD2, CD4, CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD26, CD27, CD28, CD30, CD31 .
CD37, CD38, CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDwβO, CD621, CD70, CD73, CD75, CD76, CD83, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD101, CD107a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152,CD37, CD38, CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDwβO, CD621, CD70, CD73, CD75, CD76, CD83, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD99, CD101, CD107a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152,
CD153, CD154, CD155, CD160, CD161, CD166, CD226, CD245, CD246, CD24 und einem Bestandteil des T-Zell-Rezeptor-Komplexes" ,CD153, CD154, CD155, CD160, CD161, CD166, CD226, CD245, CD246, CD24 and a component of the T cell receptor complex ",
wobei die erste und die zweite molekulare Komponente covalent miteinander verbunden sind.wherein the first and second molecular components are covalently bonded together.
2. Wirkstoff nach Anspruch 1, wobei die erste molekulare Komponente ausgewählt ist aus der Gruppe bestehend aus "H-2K, H-2D, HLA-C, HLA-BW4, HLA-4, HLA-E, UL40, HLA- Klasse I, ULlβ, UL18, ICAM, IgG, Influenza-Virus- Haemagglutm, Haemagglutin des Paramfluenza Virus, MIC, ULBP, CD48, Antikörper gegen das erste Molekül, Antikorperfragmente gegen das erste Molekül, Fusionsprotein enthaltend einen Antikörper oder ein Antikor- perfragment gegen das erste Molekül, bindende Peptido-Mimetika der vorstehenden Stoffe, und bindende Mutanten, Homologe oder Fragmente vorstehender Stoffe, insbesondere vorstehender natürlicher Liganden".2. Active ingredient according to claim 1, wherein the first molecular component is selected from the group consisting of "H-2K, H-2D, HLA-C, HLA-BW4, HLA-4, HLA-E, UL40, HLA class I." , ULlβ, UL18, ICAM, IgG, influenza virus hemagglutm, hemagglutin of the Paramfluenza virus, MIC, ULBP, CD48, antibodies against the first molecule, antibody fragments against the first molecule, fusion protein containing an antibody or an antibody fragment against the first molecule, binding peptido-mimetics of the above substances, and binding mutants, homologs or fragments of the above substances, especially the above natural ligands ".
3. Wirkstoff nach Anspruch 1 oder 2, wobei die zweite molekulare Komponente ausgewählt ist aus der Gruppe bestehend aus "Antigene, Cytokine, Chemokine, Wachstumsfaktoren, Antikörper gegen das zweite Molekül, Antikorperfragmente gegen das zweite Molekül, Fusion- sprotein enthaltend einen Antikörper oder ein Antikor- perfragment gegen das zweite Molekül, bindende Peptido-Mimetika der vorstehenden Stoffe, und bindende Mutanten, Homologe oder Fragmente vorstehender Stoffe".3. Active ingredient according to claim 1 or 2, wherein the second molecular component is selected from the group consisting of "antigens, cytokines, chemokines, growth factors, antibodies against the second molecule, antibody fragments against the second molecule, fusion protein containing an antibody or a Antibody fragment against the second molecule, binding peptido-mimetics of the above substances, and binding mutants, homologues or fragments of the above substances ".
4. Pharmazeutische Zusammensetzung enthaltend einem4. Pharmaceutical composition containing one
Wirkstoff nach einem der Ansprüche 1 bis 3 in physiologisch wirksamer Dosis sowie, optional, Hilfs- und/oder Tragerstoffe zur Herrichtung für eine definierte ga- lenische Darreichungsform.Active ingredient according to one of claims 1 to 3 in a physiologically effective dose and, optionally, auxiliaries and / or carriers for preparation for a defined galenic dosage form.
5. Pharmazeutische Zusammensetzung nach Anspruch 4, weiterhin enthaltend Blutzellen, insbesondere Leukozyten und/oder Lymphozyten. β. Pharmazeutische Zusammensetzung nach Anspruch 5, wobei die Blutzellen humanen Ursprungs sind.5. Pharmaceutical composition according to claim 4, further comprising blood cells, in particular leukocytes and / or lymphocytes. β. A pharmaceutical composition according to claim 5, wherein the blood cells are of human origin.
7. Verwendung eines Wirkstoffes nach einem der Ansprüche 1 bis 3 zur Herstellung einer pharmazeutischen Zusammensetzung zur Stimulierung des Immunsystems zwecks Behandlung oder Prophylaxe einer Erkrankung oder zur Behandlung einer Erkrankung mit Beeinträchtigung des Immunsystems, beispielsweise aus der Gruppe, bestehend aus "Tumorerkrankungen, T-Zell-Mangelerkrankungen, Erkrankungen mit T-Zell-Unterfunktion, Erkrankungen mit benigner oder maligner NK-Zell Vermehrung, Autoimmunerkrankungen, im Besonderen, jedoch nicht ausschließlich solche mit NK-Zell Überfunktion, Allergien und immunologisch mediierte Entzundungs- und Abstoßungsreaktionen" .7. Use of an active ingredient according to one of claims 1 to 3 for the manufacture of a pharmaceutical composition for stimulating the immune system for the purpose of treatment or prophylaxis of a disease or for the treatment of a disease with impairment of the immune system, for example from the group consisting of "tumor diseases, T-cell Deficiency diseases, diseases with T-cell underfunction, diseases with benign or malignant NK-cell proliferation, autoimmune diseases, in particular, but not exclusively, those with NK-cell overfunction, allergies and immunologically mediated inflammation and rejection reactions ".
8. Verwendung nach Anspruch 7, wobei eine pharmazeutische Zusammensetzung nach einem der Ansprüche 4 bis β hergestellt wird.8. Use according to claim 7, wherein a pharmaceutical composition according to one of claims 4 to β is produced.
9. Verfahren zur Stimulierung des Immunsystems, insbesondere zur Behandlung einer Erkrankung mit Beeinträchtigung des Immunsystems, in einem Saugetier, insbesondere einem Menschen, wobei dem Saugetier eine pharmazeutische Zusammensetzung nach einem der An- spruche 4 bis β dargereicht wird. 9. A method for stimulating the immune system, in particular for the treatment of a disease with impairment of the immune system, in a mammal, in particular a human, wherein the mammal is given a pharmaceutical composition according to one of claims 4 to β.
10. Verfahren zur Steigerung der Immunantwort beim Menschen, wobei die Funktion von Natürlichen Killer- (NK-) Zellen durch mindestens einen Wirkstoff, insbesondere nach einem der Ansprüche 1 bis 3, gehemmt wird.10. A method for increasing the immune response in humans, the function of natural killer (NK) cells being inhibited by at least one active ingredient, in particular according to one of claims 1 to 3.
11. Verfahren oder Wirkstoff nach Anspruch 10, wobei der Wirkstoff an die Zellmembran von menschlichen NK- Zellen bindet.11. The method or active ingredient according to claim 10, wherein the active ingredient binds to the cell membrane of human NK cells.
12. Verfahren oder Wirkstoff nach Anspruch 10 oder 11, wobei der Wirkstoff an einen inhibit-orischen Rezeptor, an einen aktivierenden Rezeptor, an ein Adhäsionsmolekül, an einen Chemokin-Rezeptor oder an ein Zytokin-Rezeptor auf NK-Zellen bindet.12. The method or active ingredient according to claim 10 or 11, wherein the active ingredient binds to an inhibitory receptor, to an activating receptor, to an adhesion molecule, to a chemokine receptor or to a cytokine receptor on NK cells.
13. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 12, wobei der Wirkstoff an ein Molekül auf der Zellmembran von NK-Zellen bindet, wobei dieses Moleküle ausgewählt sind aus einer Gruppe umfassend NCAM, KIR2DL, KIR2DS, KIR3DL, CD94/CD159, CD57, CD85, CDlβ, NKp30, NKp44, NKp46, NKG2D und CD244.13. The method or active ingredient according to any one of claims 10 to 12, wherein the active ingredient binds to a molecule on the cell membrane of NK cells, these molecules being selected from a group comprising NCAM, KIR2DL, KIR2DS, KIR3DL, CD94 / CD159, CD57 , CD85, CDlβ, NKp30, NKp44, NKp46, NKG2D and CD244.
14. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 13, ausgewählt aus einer Gruppe, umfassend H-2K, H-2D, HLA-C, HLA-BW4, HLA-4, HLA-E, UL40, HLA-Klasse I, UL18. 14. The method or active ingredient according to any one of claims 10 to 13, selected from a group comprising H-2K, H-2D, HLA-C, HLA-BW4, HLA-4, HLA-E, UL40, HLA class I, UL18.
15. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 14, wobei der Wirkstoff einen Antikörper, ein Antikorperfragment oder ein Fusionsprotein enthaltend mindestens einen Antikörper oder mindestens ein Antikorperfragment darstellt, welches an Membranbestand- teile von NK-Zellen bindet.15. The method or active ingredient according to any one of claims 10 to 14, wherein the active ingredient is an antibody, an antibody fragment or a fusion protein containing at least one antibody or at least one antibody fragment which binds to membrane components of NK cells.
16. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 15, wobei der Wirkstoff ein Peptido-Mimetikum eines der Wirkstoffe nach einem der Ansprüche 13 bis 15 darstellt.16. The method or active ingredient according to any one of claims 10 to 15, wherein the active ingredient is a peptido-mimetic of one of the active ingredients according to one of claims 13 to 15.
17. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 13, dadurch charakterisiert, dass der Wirkstoff an einen aktivierenden Rezeptor auf NK-Zellen bindet, aber diesen Rezeptor nicht aktiviert.17. The method or active ingredient according to any one of claims 10 to 13, characterized in that the active ingredient binds to an activating receptor on NK cells, but does not activate this receptor.
18. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 13, wobei der Wirkstoff Mutanten oder Fragmente oder Peptido-Mimetika eines Liganden darstellt, welcher an NK-Zellen bindet.18. The method or active ingredient according to any one of claims 10 to 13, wherein the active ingredient is mutants or fragments or peptido-mimetics of a ligand that binds to NK cells.
19. Verfahren oder Wirkstoff nach Anspruch 18, ausgewählt aus einer Gruppe, umfassend ICAM, IgG, Influenza- Virus- Haemagglutinin, Haemagglutinin des Parainflu- enza Virus; HLA-C; HLA-E, H-2D, MIC, ULBP, CD48. 19. The method or active ingredient according to claim 18, selected from a group comprising ICAM, IgG, influenza virus hemagglutinin, hemagglutinin of the Parainfluenza virus; HLA-C; HLA-E, H-2D, MIC, ULBP, CD48.
20. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 13, wobei der Wirkstoff einen Liganden, welcher an einen aktivierenden Rezeptor auf NK-Zellen bindet, inaktiviert .20. The method or active ingredient according to any one of claims 10 to 13, wherein the active ingredient inactivates a ligand which binds to an activating receptor on NK cells.
55
21. Verfahren oder Wirkstoff nach Anspruch 20, ausgewählt aus einer Gruppe umfassend einen Antikörper, ein An- tikorperfragment, ein Fusionsprotein enthaltend minde- 10 stens einen Antikörper oder mindestens ein21. The method or active ingredient according to claim 20, selected from a group comprising an antibody, an antibody fragment, a fusion protein containing at least one antibody or at least one
Antikorperfragment, das ULlβ Protein, ein Homolog des ULlβ Proteins oder ein Peptidimimetikum des ULlβ Proteins .Antibody fragment, the ULlβ protein, a homologue of the ULlβ protein or a peptide imimetic of the ULlβ protein.
15 22. Verfahren oder Wirkstoff nach einem der Ansprüche 10 bis 21, verbunden mit einem Molekül, welches an einen T-Lymphozyten bindet.22. The method or active ingredient according to any one of claims 10 to 21, connected to a molecule which binds to a T lymphocyte.
20 23. Verfahren oder Wirkstoff nach Anspruch 22, bei welchem das Molekül an CD2, CD3, CD4,CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD2β, CD27, CD28, CD30, CD31, CD37, CD38, CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDwβO, CD621, CD70, CD73, CD75, CD76, CD83,23. The method or active ingredient according to claim 22, in which the molecule comprises CD2, CD3, CD4, CD44, CD69, CD5, CD6, CD7, CD8, CD9, CD25, CD2β, CD27, CD28, CD30, CD31, CD37, CD38 , CD45RA, CD45RB, CD49a, CD49d, CD49f, CD50, CD52, CDwβO, CD621, CD70, CD73, CD75, CD76, CD83,
25 CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD101, CDl07a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152, CD153, CD154, CD155, CD160, CD161, CDlββ, CD226, CD245, CD246, CD24 oder an einen Bestandteil des T-25 CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD101, CDl07a, CD107b, CD109, CD121a, CD121b, CD122, CD124, CD127, CDwl28, CD132, CD134, CDwl37, CD148, CD152, CD153, CD153, , CD155, CD160, CD161, CDlββ, CD226, CD245, CD246, CD24 or to a component of the T-
30 Zell-Rezeptor-Komplexes bindet. 30 cell-receptor complex binds.
24. Verfahren oder Wirkstoff nach Anspruch 10, wobei der Wirkstoff in einem Organismus Antikörper erzeugt, welche die Funktion von NK-Zellen inhibieren.24. The method or active ingredient according to claim 10, wherein the active ingredient produces antibodies in an organism which inhibit the function of NK cells.
25. Verfahren oder Wirkstoff nach Anspruch 24, ausgewählt aus einer Gruppe, umfassend KIR2DL, KIR3DL, CD94/NKG2A, NCAM, CD85, CD57, HLA-E, MIC, ULBP, CD48, HLA-C.25. The method or active ingredient according to claim 24, selected from a group comprising KIR2DL, KIR3DL, CD94 / NKG2A, NCAM, CD85, CD57, HLA-E, MIC, ULBP, CD48, HLA-C.
26. Verwendung eines Wirkstoffes nach einem der Ansprüche 10 bis 25 für die Inhibition von NK-Zellen in einer Zubereitung von Blutzellen, Leukozyten oder Lym- phozyten, wobei der Wirkstoff der Zell-Zubereitung hinzugesetzt wird.26. Use of an active substance according to one of claims 10 to 25 for the inhibition of NK cells in a preparation of blood cells, leukocytes or lymphocytes, the active substance being added to the cell preparation.
27. Verwendung einer Zell-Zubereitung nach Anspruch 26 für die Stimulierung des Immunsystems.27. Use of a cell preparation according to claim 26 for stimulating the immune system.
28. Verwendung eines Wirkstoffes nach Anspruch 10 bis 27 für die Herstellung eines Arzneimittels zur Stimulierung des Immunsystems.28. Use of an active ingredient according to claims 10 to 27 for the manufacture of a medicament for stimulating the immune system.
29. Arzneimittel nach Anspruch 28 für die Vorbeuge oder Behandlung einer Erkrankung, welche mit einer Beeinträchtigung des Immunsystems einhergeht. 29. Medicament according to claim 28 for the prevention or treatment of a disease which is associated with an impairment of the immune system.
PCT/DE2003/004268 2002-12-20 2003-12-19 Increase of the immune response by substances influencing the function of natural killer cells WO2004056873A1 (en)

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