DE10161738B4 - Fusion proteins against T-cell tumors - Google Patents

Abstract

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

The The invention relates to the fields of tumor physiology and biotechnology.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

at of acute lymphocytic leukemia (7 - A LL) CD 1 is a characteristic surface marker.

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.

The The object of the invention is T cell tumors effectively and selectively to cure.

The The object of the invention is novel fusion proteins T cell tumors or to improve the treatment of this disease.

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.

The Fusion proteins according to the invention can their effect by means of cell-damaging Exercise domains.

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.

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 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.

proteases and toxins can induce apoptosis through their cleavage and binding functions.

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 cell specific Regions are CD 58, CD 72, CD 29, collagen, laminin or their regions.

CD 58 or LFA - 3 weighs 55 - 70 kd, binds CD 2 and is an adhesion molecule.

CD 72 is a C-type lectin weighs 42 kd and ligand for CD 5, collagen and laminin also bind CD 2.

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.

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.

phosphorylation by kinases can increase energetic level of molecules.

The Fusion proteins are produced recombinantly. This procedure are known in the art.

Fusion Proteins According to the Invention and Comparison Proteins Against T Cell Tumors Table 1

Table 2

Claims (3)

Fusion proteins, characterized in that they contain at least one 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. Fusion proteins according to claim 1, characterized in that that they are His day for purification, Ser / Tyr phosphorylation day or joint domains in particular five Glycine residues contain. Nucleic acid sequences, Vectors, cloning and expression systems for all fusion proteins the claims 1 and 2.