DE10161738B4 - Fusion proteins against T-cell tumors - Google Patents
Fusion proteins against T-cell tumors Download PDFInfo
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- DE10161738B4 DE10161738B4 DE2001161738 DE10161738A DE10161738B4 DE 10161738 B4 DE10161738 B4 DE 10161738B4 DE 2001161738 DE2001161738 DE 2001161738 DE 10161738 A DE10161738 A DE 10161738A DE 10161738 B4 DE10161738 B4 DE 10161738B4
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Abstract
Fusionsproteine, dadurch gekennzeichnet, dass sie mindestens eine erste Komponente ausgewählt aus CD 58, CD 72, CD 29, Kollagen, oder Laminin und mindestens eine zweite Komponente ausgewählt aus GTPase, GTP Hydrolase, des Endotoxins Vibrio Cholera –Toxin, Protease Subtilisin, HLA –B7, CD 81, CD 86, oder einer Mikrotubulibindedomäne von Gephyrin, Tau, oder MID – 1 enthalten.Fusion proteins characterized in that it comprises at least a first component selected from CD 58, CD 72, CD 29, collagen, or laminin and at least one second component selected from GTPase, GTP hydrolase, the endotoxin Vibrio cholera toxin, Protease Subtilisin, HLA -B7, CD 81, CD 86, or a microtubule binding domain of gephyrin, tau, or MID-1.
Description
Die Erfindung betrifft die Bereiche der Tumorphysiologie und der Biotechnologie.The The invention relates to the fields of tumor physiology and biotechnology.
In der Tumortherapie stellen Operationen, Bestrahlung und Chemotherapie nach wie vor die entscheidenden Maßnahmen zur Therapie der Erkrankung dar. Bei der chemischen Tumortherapie (Chemotherapie) werden je nach Tumortyp meist Zytostatika unterschiedlicher Wirkungsart verwendet, so etwa Alkylantien, Nitrosoharnstoffverbindungen, Folsäureantagonisten, Pyrimidin- und Purinanaloga wie Fluorouracil, Antibiotika mit Wirkung auf die DNA-abhängige RNA-Polymerase oder Enzyme wie L-Asparaginase. Eine Gruppe von Cytostatika für die Chemotherapie sind die Mitosehemmstoffe wie etwa Taxol und Vinca-Alkaloide.In of tumor therapy provide surgery, radiation and chemo as before, the decisive measures for the treatment of the disease In chemical tumor therapy (chemotherapy) are ever mostly used cytostatic agents of different types of effect according to tumor type, such as alkylating agents, nitrosourea compounds, folic acid antagonists, Pyrimidine and purine analogues such as fluorouracil, antibiotics with effect on the DNA-dependent RNA polymerase or enzymes such as L-asparaginase. A group of cytostatics for the Chemotherapy is the mitotic inhibitors such as taxol and vinca alkaloids.
Auf Grund ihrer sehr guten Antitumor-Aktivität haben besonders die Mitosehemmstoffe in letzter Zeit verstärkte Beachtung gefunden. Die Mitosehemmer beeinflussen den Aufbau oder Abbau der aus Mikrotubuli bestehenden Teilungsspindel – und greifen somit an der Zellteilung an. Das bekannte Colchicin oder Vinca-Alkaloide binden an spezifischen Bindestellen des α- oder β-Tubulins – als Baustein der Miktotubuli – und bewirken z.B. eine Hemmung des Aufbaus der Mikrotubuli. Andere Mitiosegifte – beispielsweise das Taxol – bewirkt deren Destabilisierung.On The reason for their very good anti-tumor activity is especially the mitosis inhibitors reinforced recently Attention found. The mitosis inhibitors influence the construction or Degradation of microtubule dividing spindle - and attack thus at the cell division. The well-known colchicine or vinca alkaloids bind to specific binding sites of α- or β-tubulin - as a building block of the microtubules - and cause e.g. an inhibition of the structure of the microtubules. Other Mitiosegifte - for example the taxol - causes their Destabilization.
Mitose- oder Spindelgifte sind hochgradig toxisch und sind daher für therapeutisches Zwecke problematisch. Die Toxizität von Colchicin ist sogar so hoch, daß diese Substanz bislang gar nicht therapeutisch verwendet wird. Das aus Eiben (Taxus) isolierte Alkaloid Taxol ist derzeit Gegenstand intensiver Forschung.mitosis or spindle toxins are highly toxic and are therefore therapeutic Purposes problematic. The toxicity of colchicine is even so high, that these Substance has not been used therapeutically so far. The end Yew (Taxus) isolated alkaloid Taxol is currently the subject of intensive Research.
Die meisten Mitosehemmer binden an das β-Tubulin der Mikrotubuli. Dazu weisen sie Bindungsstellen auf, deren unterschiedliche hohe Spezifität für eine Klassifizierung der Mitosehemmer herangezogen wird. So werden verschiedene Gruppen wie der Colchizin-Typ, der Taxan-Typ, der Vinca Alkaloid Typ oder der Rhyoxin Typ unterschieden.The Most mitotic inhibitors bind to the microtubule β-tubulin. To they have binding sites whose different high specificity for a classification the mitosis inhibitor is used. So are different groups such as the colchicine type, the taxane type, the vinca alkaloid type or the rhyoxin type distinguished.
Auf Grund der hohen Toxizität der Zytostatika ist eine Therapie mit diesen Substanzen mit vielen Nebenwirkungen verbunden, die für die betroffenen Patienten oft kaum erträglich sind. Daher wird seit vielen Jahren an der Verbesserung der Therapien mit der Zielsetzung der Vermeidung oder Reduzierung der Nebenwirkungen gearbeitet. Ein Ansatz dazu stellt der Versuch dar, die Wirkstoffe gezielt nur zu den zu therapierenden Zellen – d.h. zu den Zielzellen – zu lenken.On Reason of high toxicity The cytostatics is a therapy with these substances with many Side effects associated with the affected patients are often hardly bearable. Therefore, since many years to improve the therapies with the objective worked to avoid or reduce the side effects. One Approach to this is the attempt to target the active ingredients only the cells to be treated - i. to the target cells - too to steer.
Eine Möglichkeit zur Verwirklichung dieses Ansatzes basiert im wesentlichen darauf, zelltypspezifische Epitope zu identifizieren, einen Epitop-spezifischen monoklonalen Antikörper zu erzeugen und den derart gewonnen Antikörper oder Antigen-bindende Fragmente davon mit einem therapeutisch wirksamen Molekül zu koppeln. Ein derartiger Ansatz ist Gegenstand eines Forschungsprojekts der Universität von Kalifornien mit dem Ziel einer spezifischen Therapie von Brustkrebs (Sherie L. Morrison, Ph.D.: "Antibody Fusion Proteins for the Therapy of Breast Cancer", University of California, Los Angeles, 1997-1999). Hierbei wurden Antikörper gegen die brustkrebsspezifischen Moleküle her2/neu und CEA verwendet und mit immunstimulierenden Molekülen verbunden, welche die Aktivität der T-Zellen stimulieren.A possibility to implement this approach is essentially based on to identify cell type-specific epitopes, an epitope-specific monoclonal antibody to produce and the thus obtained antibody or antigen-binding To couple fragments thereof with a therapeutically active molecule. Such an approach is the subject of a research project of the university from California with the aim of a specific therapy of breast cancer (Sherie L. Morrison, Ph.D .: "Antibody Fusion Proteins for the Therapy of Breast Cancer, University of California, Los Angeles. 1997-1999). This antibody was used against the breast cancer specific molecules her2 / new and CEA used and linked to immunostimulatory molecules, which the activity stimulate T cells.
Obwohl dieser Ansatz mit dem Vorteil einer hohen therapeutischen Selektivität einhergeht, ist er in der Praxis nur unter großen Anstrengungen bei hohem Aufwand und langer Entwicklungsdauer umzusetzen, da zahlreiche Entwicklungsschritte zu seiner Realisierung erforderlich sind. Hierzu müssen zunächst für den jeweiligen Zelltyp spezifische Antigene isoliert werden. Da es sich bei diesen in der Regel um Proteinantigene handelt, werden im folgenden zellspezifische Epitope des Antigens ermittelt, die möglichst geringe Ähnlichkeiten zu Epitopen der Proteine anderer Zelltypen aufweisen. Dies ist erforderlich zur Vermeidung von Kreuzreaktivitäten der therapeutisch eingesetzten Antikörper. Anschließend erfolgt die Herstellung monoklonaler, gegen das jeweilige Antigen gerichteter Antikörper, die im weiteren aufwendigen Selektions- und/oder Mutageneseverfahren wie etwa Phage Display unterzogen werden müssen, um zu einem Antikörper möglichst hoher Spezifität, bzw. möglichst geringer Kreuzreaktivität zu gelangen.Even though this approach is associated with the benefit of high therapeutic selectivity, he is in practice only with great effort at high To implement effort and long development time, since numerous development steps necessary for its realization. For this you must first for each Cell-type specific antigens are isolated. As it is with these usually protein antigens, will be cell-specific in the following Epitopes of the antigen determines the lowest possible similarities to epitopes of the proteins of other cell types. This is required to avoid cross-reactivity of the therapeutically used Antibody. Subsequently the production of monoclonal, against the respective antigen directed antibody, in the further elaborate selection and / or mutagenesis Such as phage display must be subjected to an antibody as possible high specificity, or as possible low cross-reactivity to get.
Darüber hinaus ergeben sich häufig Schwierigkeiten bei der Herstellung des gebrauchsfertigen Therapeutikums, da ein nicht-humaner Antikörper modifiziert werden muß, um ohne hohes allergenes Potential eingesetzt werden zu können. Dazu können die variablen Regionen, insbesondere jedoch die Complementary determining regions (CDR) in ein humanes Antikörpergerüst eingesetzt, wobei im fertigen Therapeutikum unterschiedlich große antigenspezifische Elemente des therapeutischen Antikörpers, so etwa die antigenbindenden Fragmente (Fab) zum Einsatz kommen. Dabei handelt es sich in aller Regel um antigenspezifische Elemente, die mindestens aus zwei separaten Polypeptidketten bestehen. Die Herstellung dieser komplexen antigenspezifischen Elemente und ihre Verknüpfung mit dem eigentlich therapeutischen Molekül ist in der Praxis oft aufwendig und erfordert komplexe Expressionskonstrukte und entsprechend geeignete Wirtszellen.In addition, difficulties often arise in the preparation of the ready-to-use therapeutic agent, since a non-human antibody must be modified in order to be able to be used without high allergenic potential. For this purpose, the variable regions, but in particular the complementarity determining regions (CDR) used in a human antibody scaffold, wherein the finished therapeutic different sized antigen-specific elements of the therapeutic antibody, such as the antigenbinden the fragments (Fab) are used. These are usually antigen-specific elements that consist of at least two separate polypeptide chains. The preparation of these complex antigen-specific elements and their linkage with the actual therapeutic molecule is often expensive in practice and requires complex expression constructs and correspondingly suitable host cells.
T – Zell – Tumoren repräsentieren monoklonale Auswüchse normaler Zellpopulationen. Jeder einzelne T – Zell – Tumor hat ein normales Äquivalent und behält viele der Eigenschaften der Zelle bei, von er abstammt. Einige von diesen Tumoren repräsentieren massive Auswüchse eines seltenen Zelltyps wie z.B. die allgemeine akute lymphatische Leukämie, die von einer lymphatischen Vorläuferzelle abstammt.T cell tumors represent monoclonal outgrowths normal cell populations. Every single T cell tumor has a normal equivalent and keeps many of the characteristics of the cell, derived from it. Some of represent these tumors massive outgrowths of a rare cell type, e.g. the general acute lymphatic Leukemia, that of a lymphoid progenitor cell descended.
Es gibt unterschiedliche T – Zell – Tumoren, die durch entsprechende charakteristische Oberflächenmarker gekennzeichnet sind. Bei der allgemeinen akuten lymphatischen Leukämie sind die Moleküle CD 10, CD 19 und CD 20 charakteristische Oberflächenmarker. Thymome leiten sich von Thymusstroma – oder Thymusepithelzellen ab, und ihre charkateristische Oberflächenmarker sind Cytokeratine.It There are different T cell tumors, which are characterized by corresponding characteristic surface markers. In general acute lymphoblastic leukemia, the molecules are CD10, CD 19 and CD 20 characteristic surface markers. Conduct thymomas from Thymusstroma - or Thymic epithelial cells, and their charkateristic surface markers are cytokeratins.
Bei der akuten lymphatischen Leukämie (7 – A LL) ist CD 1 ein charakteristischer Oberflächenmarker.at of acute lymphocytic leukemia (7 - A LL) CD 1 is a characteristic surface marker.
Bei Sezary – Syndrom, adulten 7 – Zell – Leukämie und chronischer lymphocytischen Leukämie (CLL) sind CD 3/ TZR, CD 4 oder CD 8 charakteristische Oberflächenmarker. (C.A.Janeway und P.Travers, 1995 Immunologie, Spektrum akademischer Verlag, 5.273). Leukämien sind allgemein schwer zu heilen. Die Chemotherapie ist erstens nicht effektiv und zweitens nicht selektiv, d.h. die Anzahl und Härte der Nebenwirkungen und Gegenanzeigen ist gross.at Sezary syndrome, adult 7 - cell leukemia and chronic lymphocytic leukemia (CLL) are CD 3 / TZR, CD 4 or CD 8 characteristic surface markers. (C.A. Janeway and P. Travers, 1995 Immunology, Spectrum of Academic Publisher, 5,273). leukemia are generally difficult to cure. First of all, chemotherapy is not effectively and secondly non-selective, i. the number and hardness of Side effects and contraindications are great.
Die Aufgabe der Erfindung ist T – Zell – Tumoren effektiv und selektiv zu heilen.The The object of the invention is T cell tumors effectively and selectively to cure.
Die Aufgabe der Erfindung besteht in neuartigen Fusionsproteinen gegen T – Zell – Tumoren oder zur Verbesserung der Behandlung dieser Krankheit.The The object of the invention is novel fusion proteins T cell tumors or to improve the treatment of this disease.
Die Aufgabe der Erfindung wird durch Fusionsproteine umfassend T – Zell – spezifische Liganden und zellschädigenden Domänen gelöst. T – Zell – spezifische Liganden erkennen Rezeptoren, die sich auf der Oberfläche der T – Zellen befinden. Wenn diese Zellen sich krankhaft verändern, dienen diese Rezeptoren als spezifische Marker.The The object of the invention is characterized by fusion proteins comprising T cell - specific Ligands and cell damaging domains solved. T cell specific Ligands recognize receptors that are located on the surface of the T cells are located. When these cells change pathologically, these receptors serve as specific markers.
Die Fusionsproteine gemäß der Erfindung können ihre Wirkung mittels zellschädigenden Domänen ausüben.The Fusion proteins according to the invention can their effect by means of cell-damaging Exercise domains.
Zellschädigende Regionen umfassen sowohl zytotoxische oder zytostatische Proteine oder ihre Regionen, sowie Immunzellreaktionfördernde Proteine oder ihre Regionen. Zytostatische Regionen können direkt in den Zellen bzw. im Zellinneren oder Zytosol schädigend wirken. Sie umfassen unter anderem Mikrotubulibindedomänen der Proteinen Tau, Gephyrin, MID – 1, sowie GTP Hydrolasen, Protease Subtilisin, und Endotoxinen wie Vibrio Cholera – Toxin.cytotoxic Regions include both cytotoxic or cytostatic proteins or their regions, as well as immune cell-promoting proteins or theirs Regions. Cytostatic regions can be directly in the cells or damaging in the cell interior or cytosol Act. They include, among others, microtubule binding domains of the Proteins Tau, Gephyrin, MID - 1, as well as GTP hydrolases, protease subtilisin, and endotoxins like Vibrio Cholera - toxin.
Tau und Gephyrin werden in Neuronen überexprimiert und Neuronen teilen sich nicht oder wenig. Mikrotubulibindedomänen sind in der Lage Mikrotubuli – als Bestandteile des Zytoskeletts zu binden bzw. zu fesseln und somit die Zellteilung und Wachstum zu hemmen. Wenn eine Tumorzelle sich nicht mehr teilen kann, stirbt sie ab. Die Wirkung ist also ähnlich den chemotherapeutisch wirksamen kleinen Molekülen, die sich ebenfalls an Mikrotubuli binden. Die Nachteile dieser chemotherapeutisch wirksamen Molekülen bestehen darin, dass sie nicht abgebaut werden können und dass sie wegen ihrer Grösse schnell mit dem Kreislauf verbreitet werden, wodurch mehrere unerwünschte Nebenwirkungen hervorgerufen werden. Fusionsproteine gemäß dieser Erfindung können durch Proteasen abgebaut werden und sind, im Unterschied zu den kleinen chemotherapeutischen Molekülen, selektiv bzw. zellspezifisch. Daraus folgt bessere Wirkung und Reduzierung der Nebenwikrungen.dew and gephyrin are overexpressed in neurons and neurons do not divide or little. Are microtubule binding domains capable of microtubules - as To bind or bind components of the cytoskeleton and thus to inhibit cell division and growth. When a tumor cell turns can no longer share, she dies. The effect is similar to that chemotherapeutically effective small molecules that also bind to Bind microtubules. The disadvantages of this chemotherapeutically effective molecules are that they can not be mined and that they are because of their Size be spread rapidly with the circulation, eliminating several unwanted side effects be caused. Fusion proteins according to this invention can by Proteases are degraded and are, unlike the small ones chemotherapeutic molecules, selective or cell-specific. From this follows better effect and reduction of Nebenwikrungen.
GTP Hydrolasen bauen GTP ab und verwandeln diese Moleküle in GDP und Phosphate. GTP bildet Komplexe mit Tubulinen und ist nötig für die Tubulin – Polymerisation. Also kann durch GTP – Abbau bzw. durch Senkung der GTP – Konzentration, die Tubulin – Polymerisation verlangsamt oder sogar unterbunden werden. Dies führt zur Hemmung der Zellteilung und des Wachstums.GTP Hydrolases degrade GTP and turn these molecules into GDP and phosphates. GTP forms complexes with tubulins and is required for tubulin polymerization. So can through GTP - degradation or by lowering the GTP concentration, the tubulin polymerization slowed down or even stopped. This leads to Inhibition of cell division and growth.
Proteasen und Toxine können durch ihre spaltenden und bindenden Funktionen Apoptose induzieren.proteases and toxins can induce apoptosis through their cleavage and binding functions.
Immunzellreaktion fördernde Regionen können Immunzellen wie z.B. Makrophagen oder zytotoxische T – Zellen gegen Zielzellen und zwar gegen T – Zellen bzw. gegen andere T – Zellen richten. Sie umfassen T – Zell – aktivierende Regionen HLA – B7, CD 81 und CD 82 sowie Fc Regionen zur Aktivierung der Makrophagen.Immune cell response promotional Regions can Immune cells, e.g. Macrophages or cytotoxic T cells against target cells against T cells or against others T cells judge. They include T cell activating Regions HLA - B7, CD 81 and CD 82 as well as Fc regions for activating macrophages.
T – Zell – spezifische Regionen sind CD 58, CD 72, CD 29, Kollagen, Laminin oder ihre Regionen.T cell specific Regions are CD 58, CD 72, CD 29, collagen, laminin or their regions.
CD 58 oder LFA – 3 wiegt 55 – 70 kd, bindet CD 2 und ist ein Adhäsionsmolekül.CD 58 or LFA - 3 weighs 55 - 70 kd, binds CD 2 and is an adhesion molecule.
CD 72 ist ein C – Typ – Lektin wiegt 42 kd und Ligand für CD 5, Kollagen und Laminin binden auch CD 2.CD 72 is a C-type lectin weighs 42 kd and ligand for CD 5, collagen and laminin also bind CD 2.
Als Beispiel für den Wirkungsmechanismus: eine krankhaft veränderte T – Zelle nimmt die CD 58 – MBD oder CD 72 – MBD Fusionen auf. MBD ist die Abkürzung für Mikrotubuli – binderegion. Nach der Internalisierung werden Mikrotubuli der Zielzellen gebunden und gefesselt und dies führt zur Unterbindung der Zellteilung.When example for the mechanism of action: a diseased T - cell takes the CD 58 - MBD or CD 72 - MBD Mergers. MBD is the abbreviation for microtubule binding region. After internalization microtubules of the target cells are bound and tied up and this leads to stop cell division.
Die Fusionsproteine gemäß dieser Erfindung können His – tags zur Reinigung, Ser/Tyr – tag zur Phosphorylierung sowie Gelenkdomänen aus insbesondere Fünf – Glyzin – Resten enthalten.The Fusion proteins according to this Invention can His tags for cleaning, Ser / Tyr - tag for phosphorylation as well as joint domains of in particular five - glycine residues contain.
Phosphorylierung durch Kinasen kann zur Erhöhung des energetischen Niveaus der Moleküle führen.phosphorylation by kinases can increase energetic level of molecules.
Die Fusionsproteinen werden rekombinant hergestellt. Diese Verfahren sind dem Fachmann bekannt.The Fusion proteins are produced recombinantly. This procedure are known in the art.
Erfindungsgemäße Fusionsproteine sowie Vergleichsproteine gegen T – Zell – Tumore Tabelle 1 Fusion Proteins According to the Invention and Comparison Proteins Against T Cell Tumors Table 1
Tabelle 2 Table 2
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WO1997020048A2 (en) * | 1995-11-30 | 1997-06-05 | Bristol-Myers Squibb Company | Modified sfv molecules which mediate adhesion between cells and uses thereof |
DE10160248A1 (en) * | 2001-12-07 | 2003-06-26 | Alexander Cherkasky | New fusion protein, useful for treating e.g. tumors, viral infections and autoimmune disease, comprises an Fc antibody region and at least one other domain and improves the response of antigen-presenting cells |
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WO1997020048A2 (en) * | 1995-11-30 | 1997-06-05 | Bristol-Myers Squibb Company | Modified sfv molecules which mediate adhesion between cells and uses thereof |
DE10160248A1 (en) * | 2001-12-07 | 2003-06-26 | Alexander Cherkasky | New fusion protein, useful for treating e.g. tumors, viral infections and autoimmune disease, comprises an Fc antibody region and at least one other domain and improves the response of antigen-presenting cells |
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Cancer Immunol. Immunother. 1997, 45(3/4), S. 156-158 * |
Chemical Abstracts: Vol.128, No. 9, 1998, 100890v * |
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