WO2006074891A2

WO2006074891A2 - Proteins containing regulatory t-cells for the therapy and diagnosis of illnesses

Info

Publication number: WO2006074891A2
Application number: PCT/EP2006/000122
Authority: WO
Inventors: Petra Lutter; Helmut Jonuleit; Petra Weingarten; Edgar Schmitt; Helmut E. Meyer
Original assignee: Protagen Ag
Priority date: 2005-01-17
Filing date: 2006-01-10
Publication date: 2006-07-20
Also published as: DE102005002110A1; WO2006074891A3

Abstract

The invention relates to a novel proteins containing regulatory T-cells corresponding to the amino acid sequences (see SEQ ID No. 1-27), in particular, to the use thereof as markers and for the therapy and diagnosis of illnesses, in particular, allergies and auto immune illnesses, in particular, rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation, asthma, immune-deficiency diseases, AIDS, transplant rejection, cancer diseases and diabetes.

Description

Title; Regulatory T cells containing proteins for

Therapy and diagnosis of diseases

description

The present invention relates to regulatory T cells containing proteins, in particular their use as markers and for the therapy and diagnosis of diseases, in particular allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation, asthma, immunodeficiency diseases, AIDS, transplant rejection and cancer and diabetes.

Furthermore, the invention relates to suitable binders and a test system (diagnostic agent) based on one or a combination of said proteins.

The immune system is able to differentiate between foreign proteins and structures of the own body, but also between harmless and pathogenic antigens and thus to avoid unnecessary and autoaggressive immune responses. The maintenance of immunological tolerance towards endogenous structures, while the development of protective immune responses against pathogens, is essentially based on the formation of antigen-specific effector cells to the ^'immune system and the formation of antigen-specific suppressor cells to preserve the immunological tolerance.

Sakaguchi et al. described for the first time a subpopulation of CD4 + T helper cells characterized by constitutive expression of the alpha chain of the IL-2 receptor (CD25), which is essential for the control of autoaggressive immune responses in mice (Sakaguchi, S., Sakaguchi, N. , Asano, M., Itoh, M., and Toda, M. (1995) Immunology self-tolerance maintained by activated T cells expressing IL-2 reeeptor alpha-chains (CD25) various autoimmune diseases .J. Immunol. 155, 1151-1164). In the meantime, these CD4 + CD25 + T cells have been identified in various species, including humans, as CD25 + regulatory T cells (Treg for short, characterized by the co-expression of the surface proteins CD4 + and CD25 +), which are considered resident populations. 10% of human peripheral CD4 + T cells. Freshly isolated, CD25 + Tregs are anergic, ie they do not proliferate after allogeneic or polyclonal stimulation, but suppress the proliferation and cytokine production of conventional CD4 + and CD8 + T cells. This suppression is cell-contact and activation-dependent, but antigen-nonspecific [Jonuleit, H., Schmitt, E. , Stassen, M., Tuettenberg, A., Knop, J. , and Enk, AH (2001) Identification and functional characterization of human CD4 (+) CD25 (+) T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193, 1285-1294; Dieckmann, D. , Plottner, H., Berchtold, S. , Berger, T., and Schuler, G. (2001) Ex vivo isolation and characterization of CD4 (+) CD25 (+) T cells with regulatory properties from human blood. J. Exp. Med. 193, 1303-1310; Ng, W.F. , Duggan, P. J., Ponchel, F. , Matarese, G., Lombardi, G. , Edwards, AD, Isaacs, J. D. , and Lechler, R. I. (2001) Human CD4 + CD25 + cells: a naturally occurring population of regulatory T cells. Blood 98, 2736-2744; Seddon, B. and Mason, D. (2000) The third function of the thymus. Immunol. Today 21, 95-99; Seddon, B. and Mason, D. (1999) Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J. Exp. Med. 189, 877-882; Thornton, AM and Shevach, E. M. (1998) CD4 + CD25 + immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287-296; Suri-Payer, E. , Mar, A.Z. , Thornton, AM, and Shevach, E. M. (1998) CD4 + CD25 + T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160, 1212-1218; Piccirillo, C. A., and Shevach, E. M. (2001) Cutting Edge: control of CD8 + T cell activation by CD4 + CD25 + immunoregulatory cells. J. Immuno1. 167, 1137-1140].

The depletion of Tregs in vivo results in a number of autoimmune diseases, but also in improved tumor defense (Sakaguchi (supra)). This finding supports the thesis of an ambiguous function of the Tregs. On the one hand, they prevent the development of autoaggressive immune reactions, but on the other hand, at the same time, they make it more difficult for them to effectively fight off tumor, since tumor cells i. a. represent an immunological "self" and therefore their elimination by Tregs effector T cells is suppressed.The increase in suppressing function of Tregs is considered helpful for the therapy of, in particular, autoimmune diseases, while transient inhibition of their suppressive properties support tumor defense can.

The fact that the suppressive properties are cell-dependent, makes it clear that Treg-specific molecules (markers, targets) have a decisive influence on the functionality of the cells and form the basis for the targeted exploitation of these properties for therapeutic and diagnostic purposes in the field Allergies, Autoimmune Diseases, Chronic Inflammation, Immunodeficiency Diseases,

Transplant rejection and cancers, as well as AIDS, diabetes.

The object of the present invention is therefore to provide new Treg.

The object is achieved in that the new Treg characteristically express one or more proteins. Therefore, the invention relates to Treg-containing proteins and their isolation. Furthermore, the proteins mentioned in Tregs are suitable markers or targets.

According to the invention, the protein composition of the individual T cell subpopulations, in particular the Treg (ie CD4 + CD25 + and CD4 + CD25 + β7 + subpopulations), was specifically investigated according to the invention and specific Treg-specific proteins were identified as an expression product (Table 1 and Table 2). ,

In the course of proteome analysis, proteins were identified as expression products that differ significantly in their expression rate in Treg from those in conventional T cells and therefore lead to surprising properties of Treg, namely either an increase in the suppressing function of Tregs or a transient inhibition of their suppressive Properties that can support the tumor defense.

Some of these proteins have already been described in their function or involvement in the regulation of the immune response.

However, the specific suitability of these proteins for the manipulation and modification of Treg is not recognized, particularly with regard to the regulation of an immune response.

Treg containing proteins or their corresponding

Amino acid sequences according to Table 1:

Human expressed proteins according to proteome analysis:

Protein expression

SEQ ratio (n =

ID protein genes 3) CD25 +

No. name Acc. No. Gene locus Tregs / activated

CD4 + T cells)

Treg containing proteins or their corresponding

Amino Acid Sequences Table 2:

Murine expressed proteins according to proteome analysis:

(The last column in Tab. 1 and 2 is explained in the examples)

The mentioned proteins in Table 1 or 2 are hereafter

«Proteins or their ^• corresponding amino acid sequence» called.

Therefore, the invention relates to an isolated regulatory

CD4 ⁺ CD25 ⁺ T cell and / or CD4 ⁺ CD25 ⁺ β7 ⁺ containing at least one protein corresponding to the amino acid sequences SEQ ID No. 1-

16 and / or SEQ ID No. 17-27 or with SEQ ID No. 1-16 and / or SEQ ID No. 17-27 one

Have sequence identity or homology of 70% or more.

Furthermore, it is preferred that the proteins or their corresponding amino acid sequence are expressed and modified compared to conventional T cells

Have expression rate (see Figure 1, and Table 1 or 2) that sufficiently indicate an altered immunological response.

Likewise, these proteins of the invention or their corresponding amino acid sequences may be modified, for. B. by post-translational modifications, such as glycosylation ^■.

These modified proteins are included according to the invention.

In the context of this invention, "Treg" is understood as meaning those T cell subpopulations which are of human origin or can be derived from mammals (eg mouse) However, according to the invention, the subpopulations Treg-CD4 + CD25 + are preferred. "Isolated Treg" are ex -vivo cells (outside the living body) and if necessary separated from other T cells. By means of isolation it is also possible to accumulate Treg cells which contain said proteins (see examples).

The term "native Treg" describes "in vivo" (within the living body) Treg found, e.g. B. in human blood or thymus or mammals.

In a further embodiment, the Tregs according to the invention containing one or more of the proteins according to SEQ ID No. 1-16 and SEQ ID No. 17-27, recombinantly modified in such a way that they contain an amino acid sequence according to the invention, or can be obtained from a corresponding nucleic acid sequence, specifically to the amino acid sequences SEQ ID No. 1-16 the nucleic acid sequences SEQ ID No. 28-43 and to the amino acid sequences SEQ ID No. 17-27 the nucleic acid sequences SEQ ID No. 44-54. Therefore, the invention also relates to those amino acid sequences which have a sequence identity or homology of 70% and more, preferably 80% and more, more preferably 90-95% or more with SEQ ID No. 1-16 and SEQ ID No. 17-27. Also included are also such analogous amino acid sequences, ^• ensure due to the exchange of one or more amino acid (s) in these sequences, but the desired function of the respective protein.

In another embodiment, fusion proteins are also involved, containing an amino acid sequence of the invention or a named protein as a partial sequence. Examples of recombinant fusion proteins are given in EP 282 042 Bl (His-Tag).

Furthermore, the invention relates to nucleic acids which code for one of the said proteins and which are preferably contained in Tregs for one or more of the proteins or code for the amino acid sequences according to the invention, in particular SEQ ID No. 28-43 and nucleic acid sequences SEQ ID No. 44-54.

In a further preferred embodiment, the nucleic acid according to the invention contains one or more non-coding sequences and / or a poly (A) sequence, one or more recognition sequences and, if necessary, one or more potential N-glycosylation parts. The non-coding sequences are regulatory sequences, such as promoter or enhancer sequences, for the controlled expression of the coding gene containing the nucleic acids according to the invention. Furthermore, such nucleic acids can be the subject of customary expression vectors, customary host cells or customary gene therapy vectors (for example J. Sambrook, E. F. Fritsch, T. Maniatis (1989), Molecular cloning: A laboratory manual, 2nd Edition, CoId Spring Habor Laboratory Press, CoId Spring Habor, USA or Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989)).

The term "nucleic acid" (synonym: polynucleotide) has the meaning in the sense of DNA or RNA or chemical analogs and the like.

The proteins of the invention or their corresponding amino acid sequences can secrete and bind to membrane-bound proteins on Treg or effector cells. In addition, they can cross-link such membrane-bound proteins and therefore influence and regulate their functions. This property can be used according to the invention to influence the interaction between Treg and T-effector cells, e.g. For the treatment of diseases associated with Treg or effector cells.

Furthermore, the proteins of the invention or their corresponding amino acid sequences may be present in the cytosol or other organelles of the Tregs, e.g. B. present in the mitochondria. Therefore, the invention relates to such Treg, wherein at least one of the proteins according to the invention or their corresponding amino acid sequences is secreted, membrane-bound or presented on the surface or in the cytosol.

By means of recombinant methods, at least one protein according to the invention or its corresponding amino acid sequence can be enriched in Treg or on the surface of Treg. For this purpose, an amino acid sequence or nucleic acid according to the invention can be introduced into Treg. For this purpose, the skilled person uses the usual methods for transfection.

In a further embodiment, the "Tregs containing proteins or their corresponding amino acid sequences" according to the invention are recombinantly altered in that an amino acid sequence according to the invention, preferably SEQ ID No. 1-16 or-amino acid sequences SEQ ID No. 17-27 or nucleic acid sequences according to the invention SEQ ID No. 28-43 and SEQ ID No. 44-54 included.

The invention further relates to binders for at least one isolated regulatory T cell or native regulatory T cell containing at least one of the proteins or their corresponding amino acid sequences.

The binders can not be finally selected from the group: inhibitor, agonist, antagonist, probe, antibody or immunomodulator.

The binder can also induce a signal, such as a color reaction, radioactive label, which suffices to identify and modify a Treg containing proteins. Therefore, in the broadest sense, the binder is also an addressed molecule according to the invention which binds to a suitable signal-promoting receptor to Treg containing proteins and generates feedback in Treg due to the above-mentioned protein in Treg.

For example, ^'can by means of an inhibitor or modulator of one or more of the proteins or their corresponding amino acid sequences are enriched advantageous in Treg. With the help of a probe can also z. B. Further . Treg cells containing one of the proteins or their corresponding amino acid sequences can be identified. Such a probe is, for example, an antibody which specifically contains one or more epitopes present on the amino acid sequences according to the invention (preparation, for example, according to Kohler). For example, by bivalent binders having multiple epitopes, one against one of said proteins, the other against z. B. CD25, CD44, CD45, GITR, CTLA-4, galectin, Fox P3 or one of the proteins according to the invention and corresponding amino acid sequences is used to cross-link the Treg invention containing proteins or their corresponding amino acid sequences via these surface proteins with other Treg or effector cells, with the aim according to the invention of influencing the activity of these effector cells.

Functionally, the binders have the function of activating or deactivating the isolated Treg or native Treg containing at least one of the proteins or their corresponding amino acid sequences.

Therefore, the Treg containing proteins or their corresponding amino acid sequences or binders are suitable as drugs, preferably for the treatment and therapy of diseases namely allergies, autoimmune diseases, especially rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation, asthma, immunodeficiency diseases, AIDS , Transplant rejection and cancer as well as diabetes.

In particular, such autoimmune diseases selected from the group: Alopecia Areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac sprue, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), polyneuropathy, Churg-Strauss syndrome (granulomatosis), CREST Syndrome (Raynaud Syndrome), CoId Agglutinin Disease, Cryoglobulinemia, Fibromyalgia, Fibromyositis, Graves' Disease, Guillain-Barre Syndrome, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia, IgA Nephropathy, Liehen Planus, Meniere's Disease, Polyarteritis Nodosa, Polychondritis, Polyglandular - Syndrome, polymyalgia rheumatica, primary agammaglobulinemia, biliary cirrhosis, psoriasis, Reiter's disease, sarcoidosis, Sj ögren's disease, Takayasu's arteritis, vasculitis, vitiligo, Wegener's granulomatosis.

Isolated Treg containing one or more of the proteins or their corresponding amino acid sequences, modified according to the invention, can be administered to the body to be treated. On the other hand, suitable binders can be administered to the patient in sufficient dosage. The Treg-containing proteins or their corresponding amino acid sequences and / or binders are this if necessary. formulated with other excipients.

Furthermore, the invention relates to the use of said proteins or their corresponding amino acid sequences in Treg as markers or targets.

In particular, the proteins can serve as target for the manipulation or modulation of the suppressive properties of the Tregs. This can be done for example by means of a binder or a substance. Further, the binder or substance may be an inhibitor which inhibits, inhibits or promotes the expression of one or more of said proteins.

Furthermore, the Treg-specific proteins can serve singly or in combination as markers to identify Treg with (enhanced) suppressive properties.

Furthermore, the invention relates to a test system comprising at least one binder and at least one Treg containing proteins or their corresponding amino acid sequences, for the identification of suitable binders or Treg, preferably those with increased suppressive properties. Therefore, the invention also relates to a ^Testsys' tem comprising at least one Treg containing one or more of the proteins or their corresponding amino acid sequence and at least one target cell, particularly T-cell, B-cell, macrophage, Prädendritische cell, dendritic cell, embryonic cell, and / or Fibroblast, which are incubated with at least one Treg for in vitro detection of suppressive properties, in particular cellular immune response of effector cells of the immune system, in particular B cells, NK cells, preferably T cells, T helper cells.

Due to the particular cell-contact-dependent suppressive properties of Treg containing one or more of the proteins or their corresponding amino acid sequence, the cellular immune response of the target cells can be tested in the test system according to the invention.

An immune response, for example, by the synthesis of cytokines such. B. gamma interferon, interleukins are detected. The corresponding cytokine accumulates intracellularly in this test system and can be detected by fluorescence-coupled antibodies (eg ELISA). Further, by expression of surface molecules, lysis of the target cell or cell proliferation. In a fluorescence activated cell sorter (FACS), it is possible to determine the proportion of immune cells that can be stimulated or not stimulated or activated or deactivated. Further detection methods are not conclusive cytokine assay, ELISPOT, proliferation tests or 5lCr release tests (see General: Current Protocols of Immunology (1999), Coligan J E, Kruisbeek AM, Margulies D H, Shevach E M and Strober W ., John Wiley & Sons). In a further embodiment, the effector cells are mammalian cells, in particular human or murine cells or immune cell line and / or cultured primary immune cell.

In a further embodiment, the test system is incubated with at least one further substance which can trigger an immune response, such as, for example, proteins, epitopes, protein fragments, antigens.

Furthermore, such a test system is suitable for the identification of binders of the invention.

The invention further relates to a diagnostic agent (synonym: array or assay) for carrying out the test systems according to the invention and optionally a pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers are glass, polystyrene, polypropylene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamides, agarose, alumium hydroxide or magnitide. Further, the carrier may consist of 96 corrugated plates and higher.

The diagnostic agent may be in solution, bound to a solid matrix and / or added with an adjuvant.

Furthermore, the diagnostic agent can be applied to a patient as desired in vivo (eg capsule, tablet).

A diagnostic kit according to the invention is therefore suitable for the diagnosis of diseases namely allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation, asthma, immunodeficiency diseases, AIDS, transplant rejection and cancers and diabetes. In particular, autoimmune diseases such as alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac sprue, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), polyneuropathy, Churg-Strauss syndrome (granulomatosis), CREST syndrome (Raynaud's Syndrome), CoId Agglutinin Disease, Cryoglobulinemia, Fibromyalgia, Fibromyositis, Graves' Disease, Guillain-Barre Syndrome, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia, IgA Nephropathy, Liehen Planus, Meniere's Disease, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Primary Agammaglobulinemia, Biliary Cirrhosis, Psoriasis, Reiter's Disease, Sarcoidosis, Sjögren's Disease, Takayasu's Arteritis, Vasculitis, Vitiligo, Wegener's Granulomatosis.

The following examples serve to illustrate the invention without limiting the invention to these. • In addition ^' figures and sequences are explained.

Examples

Example 1: Isolation and Stimulation of Human T Cell Populations

Conventional CD4 + CD25-T effector cells (hereafter referred to as CD4 + T cells) and CD4 + CD25 + T cells (hereinafter referred to as CD25 + Treg cells) were isolated from buffy coats and leukapherizates from healthy human donors.

Example Ia: CD4 + CD25 + regulatory T cells (CD25 + Tregs)

The starting material is the leukapheresis of voluntary, healthy donors, which is from the Transfusion Center Mainz produced and contains on average 7-10 x 10 ⁹ leukocytes.

In the first step, the mononuclear cells are isolated by Ficoll gradient centrifugation and then washed extensively with PBS + 1 mM EDTA.

The isolated leucocytes are then taken up in PBS + 0.5% HSA (human serum albumin) + 1 mM EDTA and incubated with anti-CD25 microbeads (2 μl microbeads / 107 leukocytes, Microbeads: Miltenyi GmbH, Bergisch-Gladbach, Germany) for 15 min , incubated at 4 ⁰ C. After incubation, the leukocytes are washed 2x with PBS + 1 mM EDTA.

To isolate the CD25 + leukocytes, the cells are then applied to a separation column (LS Columns, Miltenyi) and separated in the permanent magnet (Miltenyi). The average yield of CD25 + leukocytes is 1.2-2% (purity> 97%).

To deplete CD4-negative contamination CD25 + leukocytes subsequently with CD8, ^'CD19-, CD14-Dynabeads (Dynal, Hamburg, FRG, 3 beads / cell) and mouse IgGl anti-human CD45RA monoclonal antibodies (Coulter / Immunotech , Hamburg, Germany, 1 μg mAb / 10 ⁶ leukocytes) 20 min. incubated in X-VIVO-15. The bound CD8 +, CD19 + and CD14 + contaminations can be removed directly using a permanent magnet (Dynal), the CD45RA + cells are removed with anti-mouse IgG Dynalbeads (Dynal) in the permanent magnet. This depletion step is then repeated again (purity of CD4 + CD25 + leukocytes> 95%).

Example Ib: oc4β1 + and oc4β7 + subpopulations of human regulatory T cells Human CD25 + Tregs contain two functionally distinct subpopulations that differ in the expression of integrins. Approx. 20% of the Tregs express the α4ß7 integrin, 80% the

Isolation of these subpopulations requires the following changes to the isolation protocol:

In the first step, the isolated leukocytes are 15 min. at 4 ⁰ C with mouse IgG anti-human CD25-FITC mAb (2 ul mAb / 107 leukocytes M-A251, BD Pharmingen, San Diego, USA) and incubated then washed extensively with PBS + 1 mM EDTA.

The FITC-positive cells are isolated using anti-FITC multisort microbeads (Miltenyi). The method is carried out analogously to the direct isolation of CD25 + leukocytes with CD25 microbeads. Subsequently, the microbeads are removed by enzymatic digestion, according to the manufacturer (Miltenyi), from the surface of the leukocytes. The CD4 - negative contaminations are depleted as described above with CD8, CD19 and CD14 Dynabeads, CD45RA + cells are not depleted (purity CD4 + CD25 + T cells> 95%).

In the next step, the α ⁴ ß7 + subpopulation is isolated. For this purpose, the CD4 + CD25 + T cells are incubated with a rat IgG anti-human β7 integrin PE mAb (BD-PharMingen, 2 μl / 107 cells) for 15 min. incubated at 4 ° C and then washed extensively with PBS + 1 mM EDTA. The isolation of the β7 + T cells is analogous to the isolation of CD25 + T cells using anti-PE microbeads (Miltenyi) resulting in a purity of CD4 + CD25 + (χ4β7 + cells> 90%.) The negative fraction expresses the integrin _α 4ßl (purity CD4 + CD25 + _α 4ßl + cells> 80%).

Example 2: Characterization of human CD4 + CD25 + regulatory T cells CD25 + Tregs are characterized by their inhibitory effect on the activation of CD4 + and CD8 + T cells in vitro.

The functional characterization of CD25 + Tregs in vitro is analyzed in coculture assays with CD4 + T-leader lines. For this purpose, the T cells are stimulated either with allogeneic, mature dendritic T cells or polyclonal with anti-CD3 + anti-CD28 mAbs.

Example 3: Multisort positive selection of CD4 + CD25 + and CD4 + CD25 + β7 + T cells

The isolation of Treg took place in several steps. First, CD4 + T cells were isolated using the CD4 MACS multisort kit (Miltenyi, Bergisch-Gladbach, Germany) and extracted therefrom with anti-CD25-FITC (M-A251, BD PharMingen, San Diego, USA) and anti-CD25. FITC multisort beads (Miltenyi, Bergisch-Gladbach, Germany) the CD4 + CD25 + T cells. Subsequently, B cells, macrophages and CD8 + T cells were depleted using CD19, CD14 and CD8 Dynabeads (Dynal, Hamburg, Germany). For the isolation of

CD4 + CD25 + β74 Treg (subpopulation of CD4 + CD25 + Treg) were used β7-PE and anti-PE beads (Miltenyi, Bergisch-Gladbach, Germany). The purity of the isolated cells was monitored by FACS analysis.

Example 4: Functional analysis of human freshly isolated CD4 + CD25 + T cells

CD25 is a typical surface molecule on Treg, but it is not only expressed in this cell type. For this reason, a functional control of the suppressive properties of the isolated cells was carried out before each analysis. Example 5: Polyclonal stimulation with anti-CD3 and anti-CD28 monoclonal antibodies

A constant number of conventional CD4 + T cells (Ix105 / well) can be polyclonally activated with anti-CD3 (1 μg / ml, OKT-3) and anti-CD28 monoclonal antibodies (2 μg / ml, CD28.2) in the presence of a varying number of CD4 + CD25 + T cells (ratio 1: 1 to 1: 4). T-cell proliferation was measured after three days of culture and subsequent pulsed treatment for 3 hours with 3HTdR (37 kBq / well). The cells thus tested were used for the proteome analyzes. Total cell lysates from cultured cells for 2DE The extraction of the proteins from the cells after cell lysis was carried out by a slightly modified Klose method (Klose, J. and Kobalz, U., Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome Electrophoresis 16, 1034-1059 (1995) and Klose, J. Fractionated extraction of total tissue proteins from mouse and human for 2-D electrophoresis, Methods Mol Biol 112, 67-85 (1999)). The cells were mechanically lysed in a phosphate buffer containing protease inhibitors against a variety of different proteases by means of ultrasound and glass beads. The 2D gel electrophoresis disrupting nucleic acids were digested at room temperature within 20 min by addition of the nuclease benzonase. The proteins were dissolved in a buffer containing urea and thiourea containing DTT. For the isoelectric focusing of the proteins Servalyte 2-4 were added.

Example 6: Isolation and Stimulation of Murine T Cell Populations Conventional CD4 + CD25-T effector cells (hereinafter referred to as CD4 + T cells) and CD4 + CD25 + T cells (hereinafter referred to as CD25 + Treg cells) were obtained from mouse spleens of healthy male BALB / C mice.

Example 6a: CD4 + CD25 + regulatory T cells (CD25 + Tregs)

The starting materials are mouse spleens. In the first step, the CD25 + Treg cells are positively selected using MACS. For this purpose, the CD25 + Treg cells are first labeled with a biotinylated anti-CD25 monoclonal antibody and then with a strepavidin-phycoerithrin (PE). To isolate the CD25 + Treg cells, the cells are incubated with anti-PE beads then applied to a MACS separation column (LS Columns, Miltenyi) and separated in the permanent magnet. Contaminations by B cells and macrophages as well as CD8 + T cells were caused by negative. Selection removed. For this wound the CD25 + Treg fraction with Dynabeads against B220, CD8 and MAC-I offset. These cells were removed with a Magnetic Particel Concentrator according to the manufacturer's instructions. The average purity of the obtained CD25 + Treg in this method is> 97%. To obtain a sufficient number of cells for functional characterization and proteome analysis of the cells. An average of 30 mouse sponges are pooled.

Example 6b: CD4 + T cells

The CD25 + depleted fraction is labeled with biotinylated anti-CD4 monoclonal antibody followed by strepavidin beads and then applied to a MACS separation column (LS Columns, Miltenyi) and separated in the permanent magnet.

Example 7: Characterization of murine CD4 + CD25 + regulatory T cells CD25 + Tregs are characterized by their inhibitory effect on the activation of CD4 + and CD8 + T cells in vitro.

The functional characterization of murine CD25 + Tregs is analogous to the characterization of human CD25 + Tregs analyzed in vitro in co-culture assays with CD4 + T helper cells.

The cells tested in this way were used for the proteome analyzes and are prepared analogously to the human T cells for proteome analysis.

Example 8: Protein separation by means of 2DE

The isoelectric focusing (IEF) of the proteins was carried out according to the method of Klose (Klose, J., Protein Mapping by combined isoelectric focusing and electrophoresis of mouse tissues.) A novel approach to testing for induced point mutations in mammals, human genetics, 26, 231- 243 (1975)) with carrier ampholytes in polyacrylamide round gels under reducing conditions. The separations were carried out in a pH range of 2 to 11 with the length of the IEF gel being 40 cm. Protein separation of proteins separated by IEF by SDS-PAGE was carried out in 15% polyacrylamide gels. Prior to loading on the gel for SDS-PAGE, the IEF gel strands were washed twice with running buffer (0, 3% (w / v) Tris base, 1, 44% (w / v) glycine, 0.1% (w / v). v) SDS) to remove excess DTT. Subsequently, the gel strand was placed on the SDS gel without air bubbles and fixed with a 1% agarose solution (with bromophenol blue). The entry of the proteins into the gel was carried out at 65 mA for 15 min and the separation within about 5 h at 100 mA for 0, 75 mm thick analytical gels or at 75 and 200 mA for 1, 0 or 1, 5 mm thick preparative gels. The separation distance was 30 cm.

In order to obtain as high a sensitivity as possible in the protein detection, analytical gels were stained with silver according to a modified method of Heukeshoven and Dernick (Heukeshoven, J and Dernick, R. Impioved silver staining procedure for fast staining in PhastSystem Development Unit. Electrophoresis 9, 28-32 (1988)), modified according to Klose and Kobalz (Klose, J. and Kobalz, U. Two-dimensional electrophoresis of proteins: an updated protocol and iτnr>^" H Γ-ΛH nnB for a functional analysis of the genome, B. 5))

Addition of glutaraldehyde and formaldehyde uses, in order to increase the sensitivity, a subsequent mass spectrometric identification of the proteins is hardly possible. For this reason, if necessary, a modified variant of the mass spectrometry compatible staining according to Blum et al. (Blum, H., Beier, H. and Gross, H. J. Improved silver staining of plant proteins, RNA and DNA in Polyacrylamide gels. Electrophoresis, 8, 93-99 (1987)). Colloidal Coomassie staining according to Neuhoff et al. (Neuhoff V., Arold N., Taube D. and Ehrhardt W., Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255 -262 (1988)) with Coomassie Brilliant Blue G-250 was used for the proteins in preparative 2DE gels, which were analyzed by mass spectrometry. Alternatively, particularly for proteins that could not be stained with colloidal Coomassie staining, silver was used in a modified protocol without the addition of glutaric dialdehyde (Blum, H., Beier, H. and Gross, HJ Improved silver staining of plant proteins. RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93-99 (1987)). Example 9: Differential proteome analysis

The digitization of the polyacrylamide gels was carried out on silver-colored gels for the image evaluation after drying the gels with a transmitted-light scanner. The quantitative evaluation of the relative protein intensities was carried out using a special image analysis software suitable for these analyzes (ProteomWeaver Ver. 2.0, Definiens, Germany).

The proteins found by the image evaluation were manually cut out of the gels. With the aid of a washing robot, the gel pieces were alternately washed in each case three times alternately with in each case 10 μl of digestion buffer (10 mM NH4HCO3) or digestion buffer / acetonitrile 1: 1 in order to remove the dye and buffer additives. For silver-stained spots, the silver was washed at room temperature within about 10 minutes before washing by addition of 15 μl of decolorizing solution (100 mM potassium hexacyanoferrate (III) / 30 mM sodium thiosulfate, 1: 1). Oxidized for 1 min. Subsequently, the gel pieces were dehydrated in the vacuum centrifuge and mixed with in each case 2 μl of a trypsin solution (0.05 μg / μl trypsin in digestion buffer). The proteolytic cleavage was at 37 ⁰ C for at least 4 h or overnight. The resulting proteolysis products were extracted from the gel matrix within 30 min by addition of 5 μl of 0.1% TFA at room temperature.

Example 10: MALDI-TOF mass spectrometry

The determination of the peptide masses of proteolytically cleaved proteins was carried out with a MALDI-TOF mass spectrometer of the type Ultraflex (Bruker Daltonik, Bremen, Germany). In this method, the analyte molecules (peptides) are cocrystallized in a UV-active matrix. For the matrix solution, a saturated (χ-cyano-4-hydroxy-cinnamic acid solution in 50% Acetonitrile / 0.1% TFA 1: 1 (solution A) diluted with solution A in the ratio 1: 1. Prior to measurements, the peptides were accumulated in ZipTips ™ (activated by 10 μl 0.1% TFA) by accumulating the analyte solution several times to enrich for Cl8 material, washed once with 10 μl of 0.1% TFA and then with 1.2 μl of matrix solution eluted onto the sample tray. The so-called peptide mass fingerprint spectra (PMFs) of the samples dried on the sample plate were measured at the following settings: Acquisition method: reflector, voltage polarity: positive,

Acceleration voltage: 25 kV, Reflector voltage: 26, 3 kV, Lens voltage: 6, 2 kV, Reflector detector voltage: 1, 72 kV and Deflection voltage: 0 kV

The calibration of the mass spectra was carried out by a calibration algorithm of the Proteinscape® database (Bruker Daltonik) automatically Autoproteolyseprodukte of trypsin and on known, recurring in the spectra peptides from contaminants such. B. Keratin.

The peptide mass spectra were analyzed with the aid of a non-redundant NCBI protein database using the meta search engine of ProteinScape ^® and the search algorithms MASCOT and ProFound (version 3.1.2002).

Evaluation:

In the case of the following human proteins, a reduction in the amount of protein in activated Treg cells compared to conventional T cells was found: tumor protein, translationally-controlled 1; Homoebox Prox 1; FK506-binding protein 4; stathmin 1; MO25 protein; Proteasome subunit beta type 7; Nucleoside diphosphate kinase A; ribonucleoside diphosphate reductase; Flap structure-specific endonuclease 1; dUTP pyrophosphatase and eukaryotic translation initiation factor 5A.

In the case of the following human proteins, an increase in the amount of protein in activated Treg cells was found in comparison with conventional T cells: tropomyosin 3; actin, beta, cytoplasmic, - EH domain-containing 1; Actin gamma and heterogeneous nuclear ribonucleoprotein A2 / B1 isoform 2. These results were found in four independent human donors.

Reduction in the amount of protein in activated Treg cells over conventional T cells was found in the following murine proteins: elongation factor 1-delta, elongation factor 1-beta (EF-1-beta), peroxiredoxin 4, LSP1_MOUSE lymphocyte-specific protein 1 (protein pp52) (52 kDa phosphoprotein) (lymphocyte-specific antigen WP34), protein Annexin All, ferritin light chain, ferritin heavy chain and enolase 3

The following murine proteins have been found to increase protein levels in activated Treg cells over conventional T cells: J kappa-recombination signal binding protein, serine (or cysteine) proteinase inhibitor, clade B, member 9b serine (or cysteine) proteinase inhibitor, clade B, member Ib

These results were found in five independent murine T cell pools.

Sequences (see Sequence Listing) and figures:

PROTEIN SEQ ID NO. 1 tropomyosin 3:

Protein alias names: TPM3; alpha tropomyosin 3; alpha- tropomyosin, slow skeletal Gene map locus: Iq22-q23

Protein sequence

Accession: P06753

Organism: Homo sapiens

Size: 284AA

DNA sequence

Accession: X04201.1

Open Reading Frame: 59 to 916

SEQ ID NO. 28 PROTEIN SEQ ID NO. 2 EH domain containinq 1

Protein alias names: EHD1; Calcium binding protein; Calcium ion binding; Signal transduction; Cell communication

Gene Map Locus: Ilql3

Protein sequence

Accession: NP_006786.2

Organism: Homo sapiens

Size: 534AA

SEQ ID NO. 2

DNA sequence

Accession: NM_006795.2

Open Reading Frame: 256 to 1860

SEQ ID NO. 29

PROTEIN SEQ ID NO: 3 actin, beta, cytoplasmic isoform A, B and C:

Protein aliases: ACTB; beta-actin Gene map locus: 7p22 -pl2

Protein sequence

Accession: NP_001092.1

Organism: Homo sapiens

Size: 375AA

SEQ ID NO. 3

DNA sequence

Accession: NM_00110Ϊ .2

Open Reading Frame: 74 to 1201

SEQ ID NO. 30

PROTEIN SEQ ID NO. 4 actin gamma:

Protein aliases: cytoskeletal gamma-actin; actin, cytoplasmic, 2 genes map locus: 17q25

Protein sequence

Accession: NP_001605.1

Organism: Homo sapiens

Size: 375AA

SEQ ID NO. 4 DNA Sequence Accession: NM_001614.2

Open Reading Frame: 75 to 1202

SEQ ID NO. 31

PROTEIN SEQ ID NO. 5 Heterogeneous nuclear ribonucleoprotein A2 / B1 - isoform 2:

Protein aliases: heterogeneous nuclear ribonucleoprotein a2; hnrpa2; heterogeneous nuclear ribonucleoprotein bl; HNRPBl; nuclear ribonucleoprotein particle a2 protein

Gene map locus: 7pl5

Protein Sequence Accession: NP_112533.1; gi: 4504447 Organism: Homo sapiens Size: 353AA

SEQ ID NO. 5

DNA sequence

Accession _. : NM_031243.1

Open Reading Frame: 170 to 1231

SEQ ID NO. 32

PROTEIN SEQ ID NO. 6 tumor protein, translationally-controlled 1:

Protein aliases: fortilin; histamine-releasing factor; immunoglobulin e-dependent; HRF gene map locus: 13ql2-ql4

Protein Sequence Accession: NP_003286; gi: 4507669 Organism: Homo sapiens Size: 172 AA

SEQ ID NO. 6

DNA sequence

Accession: NM_003295; gi: 4507668

Open Reading Frame: 1 to 172

SEQ ID NO. 33

PROTEIN SEQ ID NO. 7 Homeobox prox 1

Protein aliases: homeobox gene proxl Gene map locus: Iq32.2-q32.3

Protein Sequence Accession: NP_057123; gi: 7706322 Organism: Homo sapiens Size: 244 AA

SEQ ID NO. 7

DNA Seguence

Accession: NM_016039; gi: 7706321

Open Reading Frame:

SEQ ID NO. 34

PROTEIN SEQ ID NO. 8 FK506-binding protein 4:

Protein alias: t-cell fk506-binding protein, 59 kd;

FKBP59; FKBP52 gene map locus: 12pl3.33

Protein sequence

Accession: NP_002005.1; gi: 4503729

Organism: Homo sapiens

Size: 459 AA

SEQ ID NO. 8th

DNA sequence

Accession: NM_002014.2; gi: 17149846

Open Reading Frame: 1 to 2251

SEQ ID NO. 35

PROTEIN SEQ ID NO. 9 stathmin 1:

Protein aliases: stathmin; smn; leukemia-associated phosphoprotein pl8; LAP18 METABLASTIN Gene map locus: Ip36.1-p35

Protein sequence

Accession: NP_005554

Organism: Homo sapiens

Size: 149AA

SEQ ID NO. 9

DNA sequence

Accession: NM_005563.2

Open Reading Frame: 92 to 541

SEQ ID NO. 36

PROTEIN SEQ ID NO. 10 MO25 protein:

Protein aliases: CGI-66 Gene map locus: 2q37.1

Protein Sequence Accession: NP_057373 Organism: Homo Sapiens Size: 341 AA

SEQ ID NO. 10

DNA Sequence Accession: NM_016289; gi: 19745179

Open Reading Frame: 325 to 1350

SEQ ID NO. 37

PROTEIN SEQ ID NO. 11 Proteasome subunit beta type 7:

Protein aliases: beta-type, 7; PSMB7 Gene map locus: 9q34. Il-q34.12

Protein Sequence Accession: Organism: Homo Sapiens Size: 277AA

SEQ ID NO. 11

DNA Sequence Accession: NM 002799.2

Open Reading Frame: Open Reading Frame: 18 to 851 SEQ ID NO. 38

PROTEIN SEQ ID NO: 12 Nucleoside diphosphate kinase A - isoform A and B:

Protein aliases: metastasis inhibition factor nm23; nonmetastatic protein 23; NM23; nonmetastatic protein 23, homolog 1; nm23hl nucleoside diphosphate kinase-a; ndpka gzma-activated dnase; gaad awd; drosophila, homologue of; awd nm23hlb, included

Gene map locus: 17q21.3

Protein Sequence Accession: NP_000260; gi: 4557797 Organism: Homo sapiens Size: 152 AA

SEQ ID NO. 12 DNA Sequence Accession: NM_000269.1 Open Reading Frame: 85 to 543

SEQ ID NO. 39

PROTEIN SEQ ID NO. 13 ribonucleoside diphosphate reductase - isoform A and B:

Protein aliases: ribonucleotide reductase, large subunit ribonucleotide reductase, rl subunit; rl Gene map locus: Ilpl5.5

Protein sequence

Accession: NP_001024; gi: 4506749

Organism: Homo sapiens

Size: 792 AA

SEQ ID NO. 13

DNA sequence

Accession: NM_001033.2; gi: 21071083

Open Reading Frame: 233 to 2611

SEQ ID NO. 40

PROTEIN SEQ ID NO. 14 Flap structure-specific endonuclease 1

Protein aliases: maturation factor-1; Nose IV; MFl Gene map locus: Ilql2

Protein sequence

Accession: NP_004102; gi: 4758356

Organism: Homo sapiens

Size: 380 AA

SEQ ID NO. 14

DNA sequence

Accession: NM_004111; gi: 19718776

Open Reading Frame: 1 to 2265

SEQ ID NO. 41

PROTEIN SEQ ID NO. 15 dUTP pyrophosphatase isoform A and B:

Protein alias: deoxyuridine triphosphate; nucleotidohydrolase; deoxyuridine triphosphatase Gene map locus: 15ql5-q21.1

Protein Sequence Accession: P33316 Organism: Homo Sapiens Size: AA252AA SEQ ID NO: 15

DNA sequence

Accession: U90223.1

Open Reading Frame: 63 to 821

SEQ ID NO. 42 ^"

PROTEIN SEQ ID NO. 16 Eukaryotic translation initiation factor 5A:

Protein alias names: EIF5A; EIF5A1; eIF 4D

Gene map locus: 17pl3-pl2

Protein sequence

Accession: NP 001961.1

SEQ ID NO. 16

DNA sequence

Accepsr on: NM_001970.3

Op "r - - ,, .. ^■ .. ^• -> _!! Frame: 125 to 589 SEQ ID No. 43

FIG. 1 shows modified proteins in regulatory T cells containing proteins according to the invention or their amino acid sequences in comparison to conventional CD4 + T cells.

Sections from 2D gels of human protein spots that are activated in the comparison of conventional CD4 + T cells with activated CD25 + Tregs differed in their ^'protein spot intensity.

Claims

claims

1. Isolated regulatory CD4 ⁺ CD25 ⁺ T cell containing at least one protein corresponding to the amino acid sequences SEQ ID No. 1-16 and / or SEQ ID No. 17-27 or with SEQ ID No. 1-16 and / or SEQ ID No. 17-27 one

Have sequence identity or homology of 70% or more.

2. Isolated T-regulatory cell according to claim 1, consisting of the subpopulation CD4 ⁺ CD25 ⁺ ß7 ⁺ .

3. Isolated regulatory T cell according to any one of claims 1 to 2, characterized in that at least one expressed protein according to claims 1 or 2 secreted, membrane-bound or presented on the surface of the T-regulatory cell or in the cytosol.

4. An isolated regulatory T cell according to any one of claims 1 to 3, characterized in that at least one expressed protein according to claims 1 or 2 is enriched in the regulatory T cell or on the surface of the regulatory T cell.

5. Isolated regulatory T cell according to one of claims 1 to 4, characterized in that at least one nucleic acid encoding at least one protein according to claims 1 or 2 is containing and, if necessary. one or more non-coding sequences and / or a poly (A) sequence and / or

Recognition sequences and / or regulatory sequences, such as promoter or enhancer sequences.

6. Isolated T-regulatory cell according to one of claims 1 to 5, characterized in that the nucleic acid sequence is selected from SEQ ID No. 28-43 and / or SEQ ID No. 44-54.

7. Isolated or native regulatory CD4 ⁺ CD25 ⁺ T cell containing at least one protein according to claims 1 or 2 as target or marker.

8. Binder on at least one isolated regulatory T cell according to any one of claims 1-7 or native regulatory T cell according to claim 7.

9. Binder according to claim 8, selected from the group

^■ inhibitor, agonist, antagonist, probe, antibody or immunomodulator.

10. A binder according to any one of claims 8 or 9, wherein the binder has one or more epitopes against at least one protein according to claims 1 or 2.

The binder of claim 10, wherein the binder additionally has one or more epitopes against a surface protein.

12. A binder according to claim 11, wherein the surface protein is selected from the group CD25, CD44, CD45, GITR, CTLA-4, galectin, Fox P3.

13. A binder according to any one of claims 8 to 12, wherein the isolated regulatory T cell or native regulatory T cell containing at least one protein according to claims 1 or 2 is activated or deactivated.

14. A medicament comprising at least one binder according to any one of claims 8 to 13 or isolated T regulatory cells according to any one of claims 1 to 7.

15. A pharmaceutical composition according to claim 14 for the treatment and therapy of diseases of allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation, asthma, immunodeficiency diseases, AIDS, transplant rejection and cancer and diabetes.

16. Medicament according to claim 15, wherein the autoimmune diseases are selected from the group: Alopecia Areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac sprue, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), polyneuropathy, Churg-Strauss Syndrome (Granulomatosis), CREST Syndrome (Raynaud's Syndrome), CoId Agglutinin Disease, Cryoglobulinemia, Fibromyalgia, Fibromyositis, Graves' Disease, Guillain-Barre Syndrome, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia, IgA Nephropathy, Liehen Planus, Crohn's Disease Meniere, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Primary Agammaglobulinemia, Biliary Cirrhosis, Psoriasis, Reiter's Disease, Sarcoidosis, Sjogren's Disease, Takayasu's Arteritis, Vasculitis, Vitiligo, Wegener's Granulomatosis.

17. Test system comprising at least one binder and at least one regulatory T cell containing protein according to claims 1 or 2, for the identification of suitable binders or regulatory T cells, preferably those with increased suppressive properties.

18. Test system comprising at least one regulatory T cell containing a protein according to any one of claims 1 or 2 and at least one target cell, in particular T cell, B cell, macrophage, predendritic cell, dendritic cell, embryonic cell and / or fibroblast, the be incubated with at least one regulatory T cell for in vitro detection of suppressive properties, in particular cellular immune response of effector cells of the immune system, in particular B cells, NK cells, preferably T cells, T helper lines.

19. Test system according to claim 18, wherein the effector cells are mammalian cells, in particular human or murine cells or immune cell line and / or cultured primary immune cell.

20. Test system according to claim 18 or 19, wherein at least one further substance is incubated, which can trigger an immune response, such as proteins, epitopes, protein fragments, antigens or binders.

21. Diagnostic agent containing a test system according to any one of claims 18 to 20 and optionally a pharmaceutically acceptable carrier.

22. A diagnostic agent according to claim 21 for the diagnosis of diseases namely allergies, autoimmune diseases, in particular rheumatoid arthritis, multiple sclerosis or Crohn's disease, chronic inflammation, asthma, immunodeficiency diseases, AIDS, transplant rejection and cancer and diabetes.

23. A diagnostic agent according to claim 22 for the diagnosis of diseases and that of autoimmune diseases selected from the group Alopecia Areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, Behcet's disease, celiac sprue, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Polyneuropathy, Churg-Strauss Syndrome (Granulomatosis), CREST Syndrome (Raynaud's Syndrome), CoId Agglutinin Disease, Cryoglobulinemia, Fibromyalgia, Fibromyositis, Graves' Disease, Guillain-Barre Syndrome, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia, IgA Nephropathy, Liehen Planus , Meniere's Disease, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndrome, Polymyalgia Rheumatica, Primary Agammaglobulinemia, Biliary Cirrhosis, Psoriasis, Reiter's Disease, Sarcoidosis, Sjogren's Disease, Takayasu's Arteritis, Vasculitis, Vitiligo, Wegener's Granulomatosis.