EP1476461A2

EP1476461A2 - Immune-modulating peptide made of s. aureus enterotoxin b

Info

Publication number: EP1476461A2
Application number: EP03704622A
Authority: EP
Inventors: Karsten Neuber
Original assignee: AgeLab Pharma GmbH
Current assignee: AgeLab Pharma GmbH
Priority date: 2002-02-15
Filing date: 2003-02-14
Publication date: 2004-11-17
Also published as: US20060127411A1; AU2003206900A1; JP2005531289A; WO2003068812A2; WO2003068812A3

Abstract

The invention relates particularly to peptides which are specifically capable of binding IgE antibodies and can be obtained from naturally occurring S. aureus enterotoxin B (SEB), for example. The immune-modulating properties thereof are substantially different from those of bacterial SEB. Surprisingly, the inventive peptides do not induce proliferation of T cells, as opposed to SEB. Due to their properties, said peptides are suitable for treating diseases that are characterized by an increased serum IgE level and/or an increased production of interferon gamma and for treating diseases that are characterized by an imbalance in the Th1 and Th2 response, e.g. atopic eczema, lupus erythematosus, Crohn's disease, multiple sclerosis, psoriasis, and rheumatoid arthritis.

Description

Immunomodulating peptide from S. aureus enterotoxin B

The invention relates in particular to peptides which are capable of specifically binding IgE antibodies and which are obtainable, for example, from the naturally occurring S. aureus enterotoxin B (SEB). Their immunomodulatory properties differ significantly from those of the bacterial SEB. In contrast to SEB, the peptides according to the invention surprisingly do not induce proliferation of T cells. Due to their properties, the peptides are suitable for the therapy of diseases which are characterized by an increased serum IgE level and / or an increased production of interferon-gamma, and for the therapy of diseases which are characterized by an imbalance of Thl and Th2 immune response are characterized, such as atopic eczema, lupus ery- thematodes, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis.

Staphylococcus aureus is a widespread pathogenic microorganism that can trigger a variety of serious infectious diseases. S. aureus produces enterotoxins (enterotoxin A, B, C, D, E and TSST-1) and endotoxins (coagulase, staphylokinase, lipase, hyaluronidase, DNAse). Due to the development of multiple resistance to various antibiotics, S. aureus has become very important in recent years as a causative agent of hospital infections.

The enterotoxins (SE) produced by S. aureus can be Trigger food poisoning and septic shock [Marrack, P. & Kappler, J. (1990), Science 248, 705].

They are medium-sized proteins (20-30 kD) that are very similar in terms of their amino acid sequences. The agreement between staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin E (SEE) is over 90% [Marrack, P. & Kappler, J. (1990), Science 248, 705].

Interest in the immunological properties of these toxins increased considerably after it was discovered that they were already among the strongest inducers of lymphocyte proliferation in concentrations of 10 ^r13 M - 10 ^"16 M and this proliferation was restricted to the T cells [Fleischer , B. & Schrezenmeier, H. (1988), J. Exp. Med. 167, 1697].

The elucidation of the exact stimulation mechanisms began with the observation that SE's highly purified T cells do not stimulate, but that the toxin-induced proliferation depends on the presence of accessory cells, monocytes or B-lymphocytes. In contrast to ordinary antigens, however, the enterotoxins are not processed and presented in the antigen binding site of the MHC-II molecule, but rather cross-link the MHC-II complex on monocytes or B cells with the T cell receptor [Herrmann, T. et al. (1992), Eur. J. I Munol. 22, 1935].

The binding affinity of enterotoxins to different MHC-II proteins in humans is different. Most SEs prefer ^ bind HLA = DR, - less strongly to DQ and practically not at all to DP [Fleischer, B. (1991), Jmmun. Infection. 19, 8]. The reason for the massive, in contrast to lectins like Concanavalin A, however limited T cell stimulation is the specific binding of the SEs to the outside of the V _ß chain of the T cell receptor (TZR). The other components of the T cell receptor, the so-called Vg, J or D segments, are not binding sites for SEs. Since only T lymphocytes with a certain VG chain of the TZR are stimulated by certain SEs, the toxins are called antigens and not mitogenes.

However, since the number of circulating T cells that can be stimulated by a certain toxin is significantly larger than the number of T lymphocytes that are specific for a conventional antigen, SEs are referred to as "superantigens" [Marrack, P. & Kappler, J. (1990) Science 248, 705].

Depending on the frequency of the V _ß populations, 5-30% of the circulating T cell repertoire can be stimulated in vivo by bacterial superantigens [Choi, Y. (1990), ". Exp. Med. 172, 981]. These T- Cell stimulation induces an abundance of complex pathophysiological mechanisms that have not yet been fully elucidated. In animal models, but also in humans, intoxication with superantigens leads to sy- stemmic reactions, which can range from fever to cardiovascular shock [Fleischer, B. (1991), Immun. Infection. 19, 8]. Table 1 shows the immunological reactions induced by S. aureus enterotoxins. These reactions are likely to be triggered by complex immunological processes. The binding of the toxins to the MHC-II complex and the TZR leads to proliferation, release of cytokines and other mediators and to antibody production (Table 1). In addition, the "cutaneous lymphocyte-associated antigen" (CLA), which controls the recirculation of T cells into the skin, is increasingly expressed [Zöllner, - TM- (1996), Immunol .. Letters- 49, 111] ,

Table 1:

Toxin immunological effect

SEB activation of suppressor cells

SEA-SEE antibody production

SEA, SEB, TSST-1 cytotoxicity and anti-tumor effects

cytokine:

SEB, TSST-1 IL-1

SEA, SEB, SEC, TSST-1 IL-2

SEA, SEB IL-4

SEA, SEB, TSST-1 IFNγ

SEA, SEB, SEC1, TSST-1 TNF

SEA, SEB stimulation of NK cells

SEA, SEB, SED T cell anergy

SEB apoptosis

Tab. 1: Immunological reactions induced by S. aureus enterotoxins. After this hyperactive phase, the superantigen-stimulated T cells change into an anergic state, so that massive immunosuppression can occur. On the other hand, autoreactive but anergic T cells can be activated, which then trigger autoimmune diseases. With multiple sclerosis [Hafler, DA (1988), J. Exp. Med. 167, 1313] and rheumatoid arthritis [Palliard, X. (1991), Science 253, 325], an oligoclonal selection of certain V _E chains has already been made detected, which indicates a superantigen-mediated pathomechanism of these diseases.

Furthermore, it could be shown that SEB increases the IgE synthesis and the production of interleukin-4 in patients with atopic eczema [Neuber, K, (1993), dermatologist 44, 135; Ring, J. et al. (1992) Allexgy 47, 265; Neuber, K. et al. (1995), Int. Arch. Allergy Im unol. 107, 179]. In addition, an eczema-inducing effect for SEB was demonstrated in the patch test [Hofer, M.F. (1999), J. Invest. Dermatol. 112, 171].

Like allergic rhinoconjunctivitis and allergic bronchial asthma, atopic eczema is one of the diseases in which pathologically increased IgE synthesis occurs. So far, these diseases can only be treated symptomatically, ie the suppression of IgE synthesis takes place unspecifically through the suppression of the T lymphocytes involved in the synthesis. The most common systemically used drugs are cortisone or cyclosporin A. These substances often cause very serious systemic side effects (skin atrophy, diabetes, hypertension, kidney and hepatotoxicity, etc.). Such specific side effects would be avoided by a specific modulation of the excessive IgE synthesis. The same also applies to diseases which are accompanied by an increased interferon-gamma synthesis of the lymphocytes. A good example of such a condition is psoriasis vulgaris. reduce T-cells more interferon-gamma and thereby induce the hyperproliferation of the epidermis. Immunosuppressants such as cortisone or cyclosporin A are also used to treat psoriasis. In this case, the selective inhibition of cytokine production would also significantly reduce the rate of side effects.

A pathological Thl or a weakened Th2 reaction has been causally proven or is being discussed in a number of diseases (see also Table 2). Experimental animal findings suggest that these.

Diseases a change in the immune response towards Th2 has a protective effect. Organ-specific autoimmune diseases such as multiple sclerosis are to be mentioned here [Shevach, EM et al. (1999), Springer Semin. Immunopathol. 21, 249-262; Leonard, JP et al. (1997), Grit. Rev. Immunol. 17, 545-553], autoimmune uveitis [Singh, VK et al. (1999), Immunol. Res. 20, 147-161; Sun, B. et al. (1999), Int. Immunol. 11, 1307-1312; Egwuagu, CE et al. (1999), J. Immunol. 162, 510-517], insulin-dependent diabetes mellitus [Rabinovitch, A. et al. (1998), Biochem. Phar acol. 55, 1139-1149], rheumatoid arthritis [Muller, B. et al. (1998), Springer Semin. Immunopathol. 20, 181-196], Behcet's syndrome [Frassanito, MA et al. (1999), Arthritis Rheum. 42, 1967-1974] further the Helicobacter pylori infection (the cause of gastric ulcer and atrophic gastritis, among others) [Smythies, LE et al. (2000), J. Immunol. 165, 1022-1029; Fox, JG et al. (2000), Nat. Med. 6, 536-542; Mattapallil, JJ et al. (2000), Gastroenterology 118, 307-315], inflammatory bowel diseases such as Crohn's disease and others [Romagnani, P. et al. (1997), Curr. Opin. Immunol. 9, 793-799; MacDonald, TT (1999), Curr. Top. Microbiol. Immunol. 236, 113-135], the acute organ transplant rejection [Morelli, AE et al. (2000), Transplantation 69, 2647-2657] and spontaneous recurrent abortions [Jenkins, C. et al. (2000), Fertil. Sterile. 73, 1206-1208]. The object of the present invention is therefore to provide a new active ingredient which is suitable for the treatment of diseases which are characterized by an increased IgE level and / or an increased interferon gamma production and which do not have the disadvantages known in the prior art , In particular, it is an object of the invention to provide active substances which are suitable for the therapy of diseases which are characterized by an imbalance in the ratio of the Thl and Th2 immune response.

In the context of the present invention, it has now surprisingly been found that peptides can be isolated from the naturally occurring S. aureus enterotoxin B (SEB) which achieve the above-mentioned objects.

In particular, a peptide can be isolated which is capable of specifically binding IgE antibodies. Surprisingly, its immunomodulatory properties differ considerably from those of the bacterial SEB. In contrast to SEB, the peptide according to the invention, the amino acid sequence of which is shown in SEQ ID NO: 10, surprisingly does not induce proliferation of T cells, but on the other hand inhibits the interferon-gamma synthesis of stimulated T lymphocytes. Because of its properties, the peptide is suitable for the therapy of diseases which are characterized by increased serum IgE levels and / or increased production of interferon gamma. The diseases are preferably allergic rhinoconjunctivitis, allergic bronchial asthma and psoriasis vulgaris.

In addition to the peptide according to the invention, further peptides according to the invention were produced which have the same or essentially the same biological and / or immunological properties. Because of their special properties, ie binding of IgE antibodies and inhibition of interferon gamma Synthesis without induction of T cell proliferation, the peptides according to the invention are particularly suitable for the treatment of diseases which are associated with increased IgE synthesis and increased interferon-gamma synthesis, as is the case, for example, with atopic eczema (neurodermatitis) , This condition has been described in the chronic course of atopic eczema. Interferon gamma-producing T lymphocytes are found in the skin and there are increased serum IgE values. The therapeutic effect of the peptides could either be achieved directly by an inactivating binding to the IgE, or indirectly by modulating the cytokine production of the T lymphocytes. In this context, inhibition of other cytokines such as IL-4 and IL-13 are also considered, which are known to stimulate IgE synthesis.

The present invention therefore relates to a polypeptide which has the N-terminal amino acid sequence as shown in SEQ ID NO: 15 and which can be obtained by trypsin cleavage of S. aureus enterotoxin B as a 12 kD cleavage fragment.

In particular, the invention relates to a polypeptide (hereinafter referred to as peptide P1) which has the amino acid sequence given in SEQ ID NO: 10 and which inhibits the synthesis of interferon-gamma by stimulated T lymphocytes in vitro.

The present invention further includes homologs and derivatives of the aforementioned polypeptides that bind to IgE. The homologous polypeptides are substances which, however, differ from the aforementioned amino acid sequences, in particular from the sequence according to SEQ ID NO: 10, as a result of the exchange of one or more amino acids or by insertion or deletion of one or more amino acids, wherein they have a sequence homology of about 50 to 99%, preferably at least about 75%. A sequence homolo gie of at least 85% is particularly preferred. According to the invention, a derivative is understood to mean those polypeptides in which one or more amino acids of the aforementioned sequences, in particular the sequence according to SEQ ID NO: 10, are replaced by a stereoisomer (ie replacement of one or more L-amino acids by D-amino acids) or a corresponding one , modified amino acid are exchanged. Modified amino acids are understood to mean those amino acids whose side chains are chemically changed compared to naturally occurring amino acids, for example by glycosylation,

Phosphorylation, —- methylation-, etc.—. Such modifications are well known to those skilled in the art. According to a preferred embodiment of the invention, the homologs and derivatives which bind IgE have the same or essentially the same properties as the aforementioned peptide according to SEQ ID NO: 10, i.e. they inhibit the synthesis of interferon-gamma stimulated T lymphocytes in vitro and / or they do not induce the proliferation of T lymphocytes.

According to a preferred embodiment of the invention, the homolog (hereinafter referred to as peptide P2) has the amino acid sequence given in SEQ ID NO: 11.

The invention also includes polypeptides with a length of more than 9 amino acids which contain the amino acid sequence given in SEQ ID NO: 10 or 11 and which inhibit the synthesis of interferon-gamma by stimulated T-lymphocytes in vitro.

The invention further relates to a nucleic acid molecule which contains a nucleic acid sequence coding for a aforementioned polypeptide. It preferably has the nucleic acid sequence given in SEQ ID NO: 9. The nucleic acid molecule can be used to convert the sequence into an expression vector to clone in and to produce the peptide according to the invention recombinantly. These methods are well known to those skilled in the art.

In the context of the present invention it was surprisingly found that the peptides according to the invention, in particular Pl and P2, are able to influence the ratio between Thl and Th2 cells and thus also the ratio of the corresponding cytokines.

.Tabelle_2-:

Overweight TH1 Overweight TH2 Psoriasis vulgaris Atopic eczema

Autoimmune uveitis Allergic asthma

Allergic contact eczema Allergic rhinoconjunctivitis

Behcet's ulcerative colitis syndrome

Diabetes Mellitus Graves' Disease

Hashimoto's thyroiditis multiple sclerosis (Myelinreak¬

Heliobacter pylori infection (autoantibodies)

Lupus erythematosus mycosis fungoides

Crohn's disease

Multiple sclerosis (activation of astrocytes)

Organ transplant rejection reaction

Arthropathic psoriasis

Rheumatoid arthritis

Spontaneous, recurrent abortions

Tab. 2: Diseases with an imbalance in the TH1 / TH2 ratio, which are suitable for therapy with the peptides.

Because of the immunological properties of the proteins and polypeptides according to the invention, these are particularly suitable for the production of a pharmaceutical composition for inducing or enhancing a Thl or Th2 immune response by increasing the Th2 or lowering the Thl response and vice versa, in particular for producing a pharmaceutical composition for the treatment of diseases associated with a (predominant) Th2 or Thl immune response. Preferably in these diseases are diseases selected from the group consisting of multiple sclerosis, autoimmune uveitis, diabetes mellitus, rheumatoid arthritis, Behcet ^λ s syndrome, helicobacter pylori infection, inflammatory bowel diseases (Crohn's -insbesondere- -Crohn) - akuter- Organ transplant rejection and spontaneous recurrent abortions.

The invention furthermore relates to the use of a polypeptide which has or contains the amino acid sequence according to SEQ ID NO: 10 for the production of a pharmaceutical composition for immunomodulation and for inhibiting the cytokine production of lymphocytes, in particular T-lymphocytes. Also included is the use of such a polypeptide for the manufacture of a pharmaceutical composition for the treatment of diseases associated with an increased production of cytokines (such as interferon-gamma) and / or an increased level of IgE antibodies. The diseases are, in particular, atopic eczema (especially in the chronic form), bronchial asthma, allergic rhinoconjunctivitis, psoriasis, rheumatoid arthritis or multiple sclerosis. Other diseases are listed in Table 2. According to a special embodiment, the cytokine is interferon-gamma, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), interleukin-12 (IL- 12) and / or interleukin-13 (IL-13).

The present invention includes the use of a polypeptide described above which has an amino acid sequence homologous to or derived from SEQ ID NO: 10. outward Regarding the homologous or sequences derived therefrom, reference is made to the above definition of the "homologues" and "derivatives", including in particular those IgE-binding polypeptides which do not induce the proliferation of T lymphocytes and / or in vitro the synthesis of Inhibit interferon gamma by stimulated T lymphocytes. According to a particular embodiment of the invention, the polypeptide used has the sequence according to SEQ ID NO: 11. As far as the invention relates to the use of polypeptides which contain the sequence according to SEQ ID NO: 10 or SEQ ID NO: 11 or sequences homologous therefrom or derived therefrom. with “the same or essentially the same biological or immunological properties as part of a longer amino acid sequence, polypeptides with a length of up to 30 amino acids, in particular with a length of up to 15 amino acids, are preferred.

Due to its special biological properties, the polypeptides according to the invention are suitable for in vitro inhibition of interferon gamma production in lymphocytes, in particular in human, peripheral T-lymphocytes of the blood.

The peptides according to the invention modulate both the stimulated and the spontaneous cytokine production of TH1 and TH2 lymphocyte subpopulations. The endogenous activation status of the lymphocytes seems to play an important role in the direction of the peptide-induced effects. In patients with atopic eczema, an inhibition of the endogenously increased synthesis of all cytokines by the peptide P1 according to the invention is observed. In the meantime it is known that not only the TH2 cytokines are increased in these patients, but that an increased IFNγ production is observed especially in the chronic inpatient form. The peptide P1 according to the invention in particular is able not only to inhibit the TH2 cytokine synthesis of the lymphocytes of these patients, but also the IFNγ production, which is of considerable therapeutic relevance, for example by alleviating the symptoms until healing.

Of particular interest to psoriatics is the induction of a massively increased IL-10 release by the peptides according to the invention. There are various indications that IL-10 can have a beneficial effect on the clinical appearance of psoriasis. The targeted effect of the peptides according to the invention on the IL-10 production of the lymphocytes of psoriasis is of particular therapeutic importance in the interaction with the effects on the other cytokines, e.g. by alleviating the symptoms until healing.

The differentiated effects of the peptides according to the invention on the spontaneous or endogenous cytokine synthesis in various pathological activation states of the immune system show that the peptides also show immunomodulatory activity against other diseases caused by changes in the TH1 / TH2 balance (Table 2).

The invention further relates to a pharmaceutical composition which contains one or more polypeptides according to the invention as an active ingredient and optionally also one or more pharmaceutically acceptable auxiliaries and / or carriers.

The peptide isolated from SEB can be obtained by digesting enterotoxin B from S. aureus with trypsin and then isolating the 12 kD fragment from the reaction mixture. Alternatively, genetic engineering methods of production are of course also possible by introducing the nucleic acid sequence shown in SEQ ID NO: 9 into an expression vector, introducing this into suitable host cells, cultivating the host cells under conditions which lead to protein expression. are suitable and the expressed polypeptide is isolated, which is encoded by the nucleic acid sequence.

The invention is illustrated below using examples, a sequence listing and figures.

EXAMPLES

Example 1; Immunoblot for the detection of specific IgE antibodies against SEB.

The gel electrophoretic separation of proteins (SDS-PAGE) was carried out according to the method of Lughtenberg et al. [Lughtenberg, B. (1975), FEBS Lett. 58, 254]. 12.5% polyacrylamide release gels were used. Standard protein mixtures (Biorad, MW standard, 203 kD - 7.5 kD) were separated in parallel to determine the molecular weights. The proteins were stained with Coomassie-Blue G-250. 10 bags were available in the collection gel for sample collection, the amount of protein used being between 10 and 100 μg. The protein solution to be separated (20 μl) was mixed with 20 μl sample buffer, filled into the collecting egg pockets and then covered with an electrode buffer. The electrophoresis was carried out in electrode buffer and with a constant current flow of 20 mA. When the bromophenol blue band had reached the end of the separating gel (running time up to 2 h), the electrophoresis was stopped. The protein mixtures to be examined were separated by gel electophoresis (SDS-PAGE) and transferred to nitrocellulose (pore size 0.45 μm) using the Western blot method. The transfer took place with cooling in an electro-blot chamber (BioRad, Munich) immediately after the gel electrophoresis for 18-20 h at a constant current of 0.2-0.3 A. The nitrocellulose blots were incubated with human serum for 4 h on a swivel table at RT (Dianova, Hamburg). The blots were then washed five times and then again incubated for 4 h at RT with a secondary antibody against human IgE (anti-human IgE antibody, from Serva, Heidelberg) and the binding was visualized by an enzymatic color reaction.

The result of the immunoblot is shown in FIG. 1. With the help of the immunoblot, “specific IgE antibodies against SEB could be detected in sera from patients with atopic eczema.

Peroxidase-labeled antibodies were used in the immunoblots. It should be noted that the result of the blot (in scanned form) shown in FIG. 1 does not correspond to the actual color gradations that can be seen with the naked eye. The bands to be recognized as shadows on the original blot are so weak compared to the bands incubated with atopic sera that they have to be considered as non-specific reactions.

Example 2; Cleavage of SEB with trypsin

SEB (100 μg / 100 μl) was digested enzymatically with 10 μl trypsin (4 mg / ml aqua dest.) At 37 ° C. for 18 h. The solution was then applied to SDS gels and the corresponding cleavage products separated. The conditions for the gel electrophoresis corresponded to the conditions given in Example 1. The cleavage products were incubated in the immunoblot with sera from the patients with atopic eczema mentioned in Example 1. Serum from healthy control persons served as a control. The result of the trypsin digestion is shown in Figure 2. After trypsin cleavage, an approximately 12 kD cleavage product was found, with which the IgE from the serum of the patients with atopic eczema entered into a bond which could be detected in the immunoblot (FIG. 2). The detection was carried out as described in Example 1 with the anti-human IgE antibody (from Serva, Heidelberg).

Example 3: Sequencing of the IgE-binding cleavage product

The cleavage product, identified as IgE binding, with a molecular weight of approximately -12 kD was eluted from the gel and then mechanically according to the method of P. Edman [Edman, P. (1950), Acta Chem. Scand , 4, 283], the N-terminal sequence KVTAQELDYL being determined. The sequence analysis thus revealed a sequence that begins at position 181 (from the N-terminal end) of the SEB molecule. Figure 3 shows the result of the sequence analysis.

Example 4; Synthesis of short peptides

Starting from the sequenced sequence of the 12 kD cleavage product, several peptides with overlapping sequences were produced before and after the sequenced area:

(a) H N G N Q L D K Y R S I T V R V F E (SEQ ID NO: 1)

(b) V R V F E D G K N L L S F D V Q T N K (SEQ ID NO: 2)

(c) V Q T N K K K V T A Q E L D Y L T R H (SEQ ID NO: 3)

(d) Y L T R H Y L V K N K K L Y E F N N S (SEQ ID NO: 4)

(e) E F N N S P Y E T G Y I K F I E N E N (SEQ ID NO: 5)

(f) I E N S F W Y D M M P A P G D K F D (SEQ ID NO: 6)

(g) GDKFDQSKYLMMYNDNKMVD (SEQ ID NO: 7) (h) NKMVDSKDVKIEVYLTTKKK (SEQ ID NO: 8) [Note: the "SEQ ID NO:" used corresponds to the sequence number <400> according to WIPO standard ST.25.]

The peptides with the sequences (a) to (h) were then tested as described in Example 2. In addition to the 12 kD cleavage product, the peptide (c) was the only one to show IgE binding.

The sequenced amino acid sequence of the peptide (c) was compared using a database (SwissProt) for possible homologies with other known molecules. It was found that - between the “IgE-binding sequence Peptide and an amino acid sequence of the extracellular domain of the human low-affinity IgE receptor (CD23) has a homology of 89% (see FIG. 4). Using the Smith-Waterman algorithm, the homology between SEB and CD23 could be calculated as significant (p = 0.03).

Thereupon two further peptides were synthesized which had the sequence QELDYLTRH (PI; SEQ ID NO: 10) and QEEDFLTLH (P2; SEQ ID NO: 11; corresponding to the homologous sequence section from the CD23 molecule).

These two peptides were then used in the further experiments to stimulate the lymphocytes (cf. Examples 6 and 7).

Example 5; Dotblot to detect the binding of serum IσE to the peptides

With the aid of a dot blot, it was possible to show that the two peptides according to the invention can bind IgE antibodies from the serum of patients with atopic eczema. For this purpose, the sera from 5 different patients with atopic eczema were first pre-cleaned using protein G columns (1 ml, Pharmacia). The sera were prefiltered through 0.8 μm syringe heads. The column was then coupled with 10 ml of binding buffer (20 mM PBS, pH 7.0) “drop to drop” and washed at a flow rate of 1 ml / min. Then 1 ml of serum was filtered in each case and the filtrate was collected as soon as there was a It was washed with 5 ml PBS and the remaining filtrate was collected until it became clear The filtered serum was diluted 1: 2 in 20 mM PBS.

To carry out the dot blot, 20 μg each of the peptide-Pl-and-P-2 and 1 μg of SEB were placed on a membrane and dried overnight. The membrane was blocked with 5% skim milk / PBS Tween. Then the sera and a positive control (Sheep anti-SEB-IgG, 10 μl / ml, SERVA) were used and incubated for 1 h at RT. The membranes were then washed three times. To detect the serum IgE antibodies, biotin-labeled anti-human IgE (Pharmingen) was incubated in a concentration of 4 μg / 5 ml for 1 h at RT and the membrane was then washed three times. After a further incubation for 1 h at RT with the streptavidin-labeled second antibody and washing three times, the color reaction was triggered using BCIP and NBT as the substrate. The results of the dot blot are shown in FIG. 5. It can be seen that both peptides according to the invention with sera 4, 5 and 6 show a weak but clearly detectable IgE binding. All sera showed IgE binding to SEB.

Example 6; Immunomodulatory properties of the peptides

Peripheral blood lymphocytes (PBMC) from healthy voluntary donors were isolated via a Ficoll gradient and cultured in a concentration of 10 ⁶ cells / ml in RPMI 1640 medium + 10% FCS (fetal calf serum). SEB (1 μg / 10 ⁶ cells) and the peptide (5 μg / 10 ⁶ cells) were used as stimuli. The proliferation of the cells and the intracellular production of interferon-gamma and interleukin-4 were measured by flow cytometry after 2 days of incubation at 37 ° C. and 5% CO ₂ . After the peripheral blood lymphocytes were isolated by a Ficoll-Paque gradient centrifugation, the cell number was adjusted to 2 × 10 ⁶ cells / ml. The cells were taken up in 15 ml conical polypropylene tubes and then incubated in the incubator for 2 days. For proliferation, the PBMC were incubated with 60 μM bromodeoxyuridine (BrdU) in the last 5 h of the incubation period. For the measurement of the intracellular — cytokines — the — last — 5-h-Brefeidin-A __ (. BFA., _ 20_ μl / 1 ml PBMC) were added. Then 20 mM EDTA (100 μl / l ml PBMC) was added and then 10 ml cold PBS. After centrifugation, 3 ml of "FACS Lysing Solution" (BD Bioscience, Heidelberg) were added to the cells and, after a washing step, the "FACS Permeabilization Solution" (BD Bioscience, Heidelberg). Anti-BrdU-FITC antibodies with DNase (proliferation) or anti-cytokine antibodies (intracellular cytokine determination) were then added to the cell pellet and incubated for 30 min. The cells were then washed and fixed with 200 μl of 1% paraformaldehyde. The fluorescence was measured and evaluated on the flow cytometer (FACScan, BD Bioscience).

The proliferation was determined by the incorporation of BrdU in lymphocytes on the flow cytometer. The incubation of the lymphocytes with different concentrations of the peptides P1 and P2 according to the invention showed no significant effects of the peptides on the proliferation (FIG. 6A). The SEB-induced proliferation was neither significantly modified by the peptides alone (FIG. 6A) nor by the costimulation with SEB + peptide PI (FIG. 6B). The SEB-induced lymphocyte proliferation was inhibited only in high concentrations (FIG. 6B). In the next step, the intracellular IFNγ (FIG. 7) and IL-4 production after stimulation with SEB and the peptides P1 and P2 according to the invention were examined (FIG. 7). It was shown that Pl stronger than P2 suppresses the production of IFNγ, a cytokine which mediates a variety of immunological functions, in a dose-dependent manner (FIG. 7A) and that IL-4 (FIG. 7B) is stimulated in a dose-dependent manner. The lymphocytes examined in these experiments were from healthy volunteers. Accordingly, the ratio between IFNγ and II-4 was changed by the peptides in a dose-dependent manner in favor of the IL-4-producing TH2 lymphocytes (FIG. 7C).

Example 7: Effect of the peptides on secretory cytokine production

Peripheral blood lymphocytes from healthy voluntary donors were isolated using a Ficoll gradient and taken up in a concentration of 10 ^s cells / ml in RPMI 1640 medium + 10% FCS (fetal calf serum). The cells were seeded in 24-well plates and with the beiden- - incubating peptides (200 ^ ug / 10 ⁶ _cells.) For 2 days in an incubator (37 ° C, 5% C0 _2). The supernatants were then collected and first frozen at -20 ° C. The human TH1 cytokines IL-2, IFNγ and TNFα as well as the TH2 cytokines IL-4, IL-5 and IL-10 were in the cell culture supernatant with the "Cytometric Bead Array (CBA)" ^{~ from} the company B&D (Heidelberg) The test was carried out exactly according to the manufacturer's instructions.

In a first series of experiments, the effects of the peptides P1 and P2 according to the invention on the spontaneous cytokine secretion of peripheral blood lymphocytes in healthy subjects (n = 3) were investigated. Figure 8 shows the effects on the THl cytokines (IL-2, IFNγ, TNFα) and on the TH2 cytokines (IL-4, IL-5, IL-10).

It was shown that Pl mainly inhibits the spontaneous production of the TH1 cytokines IL-2 and IFNγ, while P2 inhibits TNFα more strongly. Peptide 2 induces the synthesis of TH2 cytokines (IL-4, IL-5, IL-10) in healthy volunteers, while PI also completely inhibits the synthesis of IL-4 and IL-5.

The results of these studies show that Pl and P2 influence the ratio of TH1 and TH2 cytokms.

Therefore, in the next step the same experiment was carried out with peripheral blood lymphocytes from patients with severe atopic eczema (TH2> TH1) or severe psoriasis vulgaris (TH1> TH2). The results are shown in FIGS. 9 and 10.

It was found that in patients with atopic eczema or psoriasis vulgaris partly offsetting effects on the already endogenously altered spontaneous cytokine secretion _^ th occurring defects. In patients with atopic dermatitis (Fig. 9) was the spontaneous production of all cytokines, ie both the TH1 - and the TH2 cytokines increased significantly. Pl inhibits the increased cytokine production of all TH populations in these patients, but especially here also the IFNγ synthesis, which is increased in chronic atopic eczema.

In patients with psoriasis vulgaris there was a massive stimulation of IL-10 and IL-5 synthesis by PI (FIG. 10).

Claims

claims;

1. Polypeptide with the amino acid sequence given in SEQ ID NO: 10.

2. Polypeptide, characterized in that it has the N-terminal amino acid sequence as shown in SEQ ID NO: 15 and is obtainable by trypsin cleavage of S. aureus enterotoxin B as a 12 kD cleavage fragment.

3. polypeptide, characterized in that it is a homologue or a derivative of the polypeptide according to claim 1 or 2, which binds to IgE.

4. Polypeptide according to claim 3, characterized in that one or more L-amino acids are replaced by D-amino acids in the derivative.

5. Polypeptide according to claim 3 and 4, characterized in that it does not induce the proliferation of T lymphocytes.

6. Polypeptide according to claims 2 to 5, characterized in that it inhibits the synthesis of interferon-gamma stimulated T lymphocytes in vitro.

7. Polypeptide after. claims 2 to 6, characterized in that it modulates both the stimulated and the spontaneous cytokine production of Thl and Th2 lymphocyte subpopulations in vi tro.

8. Polypeptide according to claims 2 to 7, characterized in that it has the amino acid sequence given in SEQ ID NO: 11.

9. nucleic acid molecule, characterized in that it has a nucleic acid sequence coding for a polypeptide according to claims 1 to 8.

10. Nucleic acid molecule according to claim 9, characterized in that it has the nucleic acid sequence given in SEQ ID NO: 9.

11. Use of a polypeptide which contains the sequence according to SEQ -I-D NO: 10, a sequence which is homologous or derived therefrom, the polypeptide binding to IgE, for the production of a pharmaceutical composition for immunomodulation.

12. Use of a polypeptide according to claims 1 to 8 for the manufacture of a pharmaceutical composition for immunomodulation.

13. Use of a polypeptide which contains the sequence according to SEQ ID NO: 10, a sequence homologous to or derived therefrom, the polypeptide binding to IgE, for the production of a pharmaceutical composition for inhibiting the cytokine production of lymphocytes.

14. Use of a polypeptide according to claims 1 to 8 for the production of a pharmaceutical composition for inhibiting the cytokine production of lymphocytes.

15. Use according to claim 13 and 14, characterized in that the lymphocytes are T-lymphocytes.

16. Use according to claim 15, characterized in that the cytokine interferon-gamma, interleukin-4 (IL-4), in- terleukin-5 (IL-5), interleukin-10 (IL-10), interleukin-12 (IL-12) and / or interleukin-13 (IL-13).

17. Use of a polypeptide which contains the sequence according to SEQ ID NO: 10, a sequence homologous to or derived therefrom, wherein the polypeptide binds to IgE, for the production of a pharmaceutical composition for the treatment of diseases associated with an increased production of cytokines and / or accompanied by an elevated level of IgE antibodies.

18. Use of a polypeptide according to claims 1 to 8 for the manufacture of a pharmaceutical composition for the treatment of diseases which are associated with an increased production of cytokines and / or an increased level of IgE antibodies.

19. Use according to claim 17 and 18, characterized in that the cytokine interferon-gamma, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), interleukin 12 (IL-12) and / or interleukin-13 (IL-13).

20. Use according to claim 17 and 18, characterized in that the disease is atopic eczema, bronchial asthma, allergic rhinoconjunctivitis, psoriasis, rheumatoid arthritis or multiple sclerosis.

21. Use of a polypeptide which contains the sequence according to SEQ ID NO: 10, a sequence homologous to or derived therefrom, the polypeptide binding to IgE, for producing a pharmaceutical composition for inducing or enhancing a Thl or Th2 immune response.

22. Use of a polypeptide according to claims 1 to 8 for the manufacture of a pharmaceutical composition for inducing or enhancing a Thl or Th2 immune response

23. Use according to claims 21 and 22 for the production of a pharmaceutical composition for the treatment of diseases which are associated with a Thl or Th2 immune response.

-24. — Use — according to claims 21 to 23, characterized in that the disease psoriasis vulgaris, autoimmune uveitis, allergic contact dermatitis, Behcet's syndrome, diabetes mellitus, Hashimoto's thyroiditis, helio- bacter pylori infection, lupus erythematosus, Crohn's disease, multiple sclerosis, organ transplant rejection, psoriasis arthropathica, rheumatoid arthritis and spontaneous, recurrent abortions.

25. Use according to claim 24, characterized in that the inflammatory bowel disease is Crohn's disease.

26. Use of a polypeptide which contains the sequence according to SEQ ID NO: 10, a sequence homologous to or derived therefrom, the polypeptide binding to IgE, for in vitro inhibition of interferon gamma production in human, peripheral T-lymphocytes of the blood.

27. Use of a polypeptide according to claims 1 to 8 for the in vitro inhibition of interferon gamma production in human peripheral T-lymphocytes of the blood.

28. Use of a polypeptide which contains the sequence according to SEQ ID NO: 10, a sequence homologous to or derived therefrom, the polypeptide binding to IgE for the in vitro modulation of the stimulated and spontaneous cytokine production of Thl and Th2 lymphocyte subpopulations.

29. Use of a polypeptide according to claims 1 to 8 for in vitro modulation of the stimulated and spontaneous cytokine production of Thl and Th2 lymphocyte subpopulations.

30. Use according to claims 11 to 29, characterized in that the polypeptide is a polypeptide with a sequence derived from SEQ ID NO: 10, in which one or more L-amino acids by D-amino acids are exchanged.

31. Pharmaceutical composition, characterized in that it contains a polypeptide according to claims 1 to 8.

32. Pharmaceutical composition according to claim 31, characterized in that it further contains one or more pharmaceutically acceptable auxiliaries and / or carriers.

33. A method for producing a polypeptide according to claim

1, characterized in that the nucleic acid sequence shown in SEQ ID NO: 9 is introduced into an expression vector, this is introduced into suitable host cells, the host cells are cultivated under conditions which are suitable for protein expression and the expressed polypeptide is derived from the nucleic acid sequence is encoded, isolated.

34. A method for producing a polypeptide according to claim

2, characterized in that enterotoxin B from S. digested aureus with trypsin and then isolated the 12 kD fragment from the reaction mixture.