WO2013020914A1

WO2013020914A1 - Peg-conjugated peptides

Info

Publication number: WO2013020914A1
Application number: PCT/EP2012/065242
Authority: WO
Inventors: Alf Hamann
Original assignee: Celares Gmbh
Priority date: 2011-08-10
Filing date: 2012-08-03
Publication date: 2013-02-14

Abstract

The invention pertains to a peptide comprising a peptide with a sequence according to SEQ ID NO 1 to 7 covalently coupled to a polyethylene glycol (PEG) molecule for treating multiple sclerosis.

Description

PEG-conjugated Peptides

The invention relates to a method for inducing Foxp3+ regulatory T cells using peptides are covalently conjugated to PEG and to a composition of polyethylene glycol (PEG)-conjugated peptides to induce antigen-specific tolerance against multiple sclerosis (MS).

Background of the invention

Multiple sclerosis (MS) is the most common non-traumatic disorder of the central nervous system (CNS) in young adults, with around 120.000 patients in Germany. Several subtypes, or patterns of progression, have been described: relapsing remitting, secondary progressive, primary progressive and progressive relapsing (Duddy M, Haghikia A, Cocco E, Eggers C, Drulovic J, Carmona O, Zephir H, Gold R. Managing MS in a changing treatment landscape. J Neurol. 201 1 ).

Clinical manifestation is usually characterized by recurrent attacks with neurological deficits and periods of remission. Relapsing-remitting disease can turn into progressive disease leading to disability. Due to its chronicity and the lack of effective therapies, this disorder is associated with substantial socio-economic costs. Current therapies are based on unspecific immunomodulation, do not address underlying immune defects, and require lifelong treatment; in fact, a cure of the disease seems difficult to achieve. Present immunosuppressive therapies are only moderately effective and associated with significant risks of infection, moreover not all patients are responsive to these therapies (Buck D, Hemmer B. Treatment of multiple sclerosis: current concepts and future perspectives J Neurol. 201 1 Jun 3).

Common understanding assumes that MS is an autoimmune disorder characterized by T cell responses against proteins of the central nervous system (CNS) whereby myelin sheath of neurons and even neurons themselves become destroyed. CD4+ T cells seem to be the key contributors to the underlying pathogenic mechanisms. Direct targeting of these autoreactive cells is therefore a promising approach to cure the disease without affecting overall immunity. Especially treatments aiming to enhance the immune system^'s own self-control mechanisms and reestablishing self-tolerance appear attractive. One major mechanism of self-control and tolerance that acts in an antigen-specific manner is based on the existence of self-reactive regulatory T cells (Tregs).

Several experimental strategies to induce antigen-specific tolerance and Tregs have already been tested in MS: Altered peptide ligands (APL), mucosal (oral/nasal) tolerization, DNA vaccination, soluble peptide-induced tolerance and ECDI-coupled-cell induced tolerance. All are based on the use of myelin-derived autoantigens or peptides. However, most of the approaches failed so far (Bielekova B, Goodwin B, Richert N, Cortese I, Kondo T, Afshar G, Gran B, Eaton J, Antel J, Frank JA, McFarland HF, Martin R. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat Med. 2000; 6(10): 1 167-75).

APLs, peptide analogues that bear different binding characteristics compared to the native autoantigen-peptides have been shown to either interfere with T cell activation or to result in qualitatively different outcomes, e.g. preferential induction of anergy or activation of Tregs. There is only one drug approved acting as a surrogate for peptide specific tolerance: glatiramer acetat (GA or Copaxone, Teva Pharmaceuticals). GA is a random mixture of synthetic polypeptides of various lengths, assumed to act as an APL by competition with a MBP epitope for MHCII binding. GA modulates MBP specific T cells and has neuroprotective capacity. However, treatment is beneficial to only 30 % of patients, requires daily subcutanous (s.c.) injections and is associated with high risk of post-injection reaction (Johnson KP Risks vs benefits of glatiramer acetate: a changing perspective as new therapies emerge for multiple sclerosis. Ther Clin Risk Manag. 2010 Apr 15; 6:153-72).

A modified version of the peptide MBP83-99 used as APL has failed in a clinical trial due to safety concerns after a few patients developed exacerbation of disease soon after treatment (Bielekova B, Goodwin B, Richert N, Cortese I, Kondo T, Afshar G, Gran B, Eaton J, Antel J, Frank JA, McFarland HF, Martin R. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat Med. 2000; 6(10): 1 167-75).

Mucosal (oral/nasal) immunization to induce tolerance: Since long, oral administration of antigen is known to induce tolerance in animals. While high dose oral antigen leads to anergy or deletion of antigen-specific T cells, low doses act via induction of regulatory T cells. However, clinical trials in several autoimmune diseases have failed to show significant benefit so far (Faria AM, Weiner HL. Oral tolerance: therapeutic implications for autoimmune diseases. Clin Dev Immunol. 2006; 13(2- 4): 143-57.

DNA vaccination: Instead of administration of whole peptides, a plasmid encoding four myelin proteins was injected intra muscular into patients by Steinman and colleagues. This new approach is called "inverse vaccination" and seems to be quite promising in first clinical trials: In half of MS patients the number of new brain lesions was reduced (Steinman L. Inverse vaccination, the opposite of Jenner's concept, for therapy of autoimmunity. J Intern Med. 2010; 267(5): 441-51 ). Phase 3 trial will start soon. A drawback is that DNA vaccination still is associated with regulatory hurdles.

Soluble peptide immunization to induce tolerance: Intravenous injection of high doses of soluble peptides leads to an initial burst of proliferation and, on repeated application, to induction of anergy or deletion of peptide-specific T cells by activation induced cell death. In addition, especially at low doses, induction of Tregs can be observed. In a clinical placebo-controlled phase II trial, an intra venous (i.v.) injection of 500 mg MBP82-98 peptide (called MBP8298) every six months reduced autoantibody titers in the cerebrospinal fluid of the patients drastically and delayed progression of MS by around five years in patients with HLA haplotype DR2 compared to placebo treated patients (Warren KG, Catz I, Ferenczi LZ, Krantz MJ. Intravenous synthetic peptide MBP8298 delayed disease progression in an HLA Class ll-defined cohort of patients with progressive multiple sclerosis: results of a 24-month double-blind placebo-controlled clinical trial and 5 years of follow- up treatment. Eur J Neurol. 2006; 13(8): 887-95). However, a phase III clinical trial including 600 patients showed no significant benefit for patients treated with this drug.

One possible explanation is that tolerization based on a single peptide must fail because epitope spreading leads to the involvement of several epitopes in autoreactivity. To address this issue David Wraith used a cocktail of four myelin basic protein (MBP) epitopes. A current clinical trial shows encouraging results with up to 40 % reduction of MBP-specific T cell proliferation (Dolgin E. The inverse of immunity, Nat Med. 2010; 16(7): 740-3). Smith et al., 2006, (Smith CE, Miller SD. Multi-peptide coupled-cell tolerance ameliorates ongoing relapsing EAE associated with multiple pathogenic autoreactivities. J Autoimmun. 2006; 27(4): 218-31 ) used 7 different peptides that represent the major known T-cell epitopes of myelin proteins for tolerization, an approach being presently in phase I trial.

Animal studies have demonstrated that there is a significant risk of exacerbation of the disease or induction of anaphylactic responses by injection of soluble peptides (Genain CP, Abel K, Belmar N, Villinger F, Rosenberg DP, Linington C, Raine CS, Hauser SL. Late complications of immune deviation therapy in a nonhuman primate. Science. 1996; 274(5295): 2054-7; Smith CE, Eagar TN, Strominger JL, Miller SD Proc Natl Acad Sci U S A. Differential induction of IgE-mediated anaphylaxis after soluble vs. cell-bound tolerogenic peptide therapy of autoimmune encephalomyelitis. 2005; 102(27): 9595-600).

Peptides coupled to ECDI-fixed autologous cells: A promising approach is based on coupling several myelin-peptides to ethylen carbodiimide (ECDI)-fixed spleenocytes isolated from patient^'s blood as proposed by Stephen Miller et al. (The induction of cell-mediated immunity and tolerance with protein antigens coupled to syngeneic lymphoid cells. J Exp Med. 1979; 149(3):758-73). ECDI fixation induces apoptosis of donor cells that are taken up by immature/tolerogenic host antigen presenting cells (APCs). Coupled auto-antigen is re-processed and presented by these cells under non-immunogenic conditions whereby T cells specific for the auto-antigen are targeted and become tolerized. Tolerance is induced and maintained by regulatory T cells expressing Foxp3 or secreting anti-inflammatory cytokines as Ιί-10/ΤΘΡβ (Smith CE, Miller SD. Multi-peptide coupled- cell tolerance ameliorates ongoing relapsing EAE associated with multiple pathogenic autoreactivities. J Autoimmun. 2006; 27(4): 218-31 ). Compared to soluble peptide administration, there seems to be a reduced risk of anaphylactic responses (Smith CE, Eagar TN, Strominger JL, Miller SD Proc Natl Acad Sci U S A. Differential induction of IgE-mediated anaphylaxis after soluble vs. cell-bound tolerogenic peptide therapy of autoimmune encephalomyelitis. 2005; 5; 102(27): 9595-600).

In 2010, a combined phase I and Ma clinical trial has started at the Institute for Neuroimmunology and Clinical MS Research (inims) in Hamburg, where autologous peripheral blood leukocytes collected from patient^'s blood are ECDI fixed and coupled with a cocktail of seven immunodominant myelin-peptides to induce long-term tolerance in effector autoreactive T cells as well as prevention of future relapses by tolerizing naive T cells (Turley DM, Miller SD. Prospects for antigen-specific tolerance based therapies for the treatment of multiple sclerosis. Results Probl Cell Differ. 2010; 51 : 217-35). A drawback of this approach is that it requires very sophisticated and expensive infrastructures compared to production of standardized vaccines.

Vaccination strategies with immunodominant peptides to induce tolerance are still highly attractive and promise considerable benefits compared to conventional immunomodulatory drugs. However, efficacy and/or practicability are rather unsatisfactory so far. One reason is that peptides have unfavorable pharmacokinetic properties (short half-life in circulation) and might cause immune stimulation due to poor solubility/aggregation problems. The strategy of coupling to autologous cells is elegant, but not feasible for large-scale application.

Here, a novel approach to improve efficacy of peptide tolerization is described that is adaptable to large-scale production and application.

Description of the invention

The inventor developed a novel strategy to induce protective tolerogenic responses involving Tregs that can be used to treat autoimmune diseases, notably MS, by peptides coupled to the high molecular weight hydrophilic polymer polyethylene glycol (PEG).

The invention refers in a first aspect to a molecule for preventing or treating multiple sclerosis. Such a molecule is also referred to herein as a "PEG-conjugated peptide" and comprises or consists of:

- a peptide moiety with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7, and

- a PEG moiety, wherein the peptide moiety is covalently coupled to the PEG moiety. The peptide moiety of the molecule is an autoantigenic peptide. The molecules of the invention show higher efficacy compared to previous attempts to treat MS. Preferably, the peptide moiety, derived from a peptide with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7, is covalently coupled to the polyethylene glycol (PEG) molecule via a functional group of the peptide. In a preferred embodiment of the invention, the covalent coupling to an activated PEG occurs at the N- Terminus or at an additional amino acid, which can be a naturally occurring (e.g. Cysteine) or an artificial amino acid (e.g. azido-Tyrosine or Para-acetyl-phenylalanine). The activation group of the activated PEG comprises at least one functional group that allows for the covalent coupling to a functional group of the (naturally occurring or artificial) amino acid. Activation groups for activating PEG can be, for example, carboxyl, NHS-ester, p-nitrophenylcarbonate, hydroxybenzotriazolylcarbonate, amine, aldehyde, ketone, carboxylhydrazide, iodoacetamide, aminoxy , thiol, alkine, azide (e.g. for making use of the so-called "click" chemistry), and preferably maleimide or vinyl sulfone groups. The activated polyethylene glycol (PEG) used in the described invention is polydisperse, a distribution of polyethylene glycol molecules with different chain length and molecular weights, respectively. However, in another embodiment the activated polyethylene glycol (PEG) can be non-dispersed and discrete, respectively (WO 0809601 A1 ; EP0608538; EP04006315).

The peptides comprised by the molecule of the invention can be peptides with a sequence of SEQ ID NO 1 to 7, but wherein the sequence according to SEQ ID NO 1 to 7 has been changed by amino acid exchange, addition, and/or deletion. In particular, at least one amino acid can be changed, preferably two, three, four, or five amino acids are changed when compared to the sequences of SEQ ID NO 1 to 7. The molecule of the invention comprising such a peptide moiety with changed amino acids still shows the same in vitro or in vivo effect with regard to inducing antigen-specific tolerance against MS as a molecule without changes in the amino acids, but the effect may be qualitatively different, i.e. of a different strength.

A molecule of the invention is useful in medicine, and may be used as a medicament against multiple sclerosis.

The term "PEG" is used here synonymously for polyethylene glycol and methoxy-polyethylene glycol/polyethylene glycol monomethylether. It is known to the skilled artisan that these compounds may both be used for the modification of drugs and differ in the end-group at one end of the PEG chain where the hydroxyl group may be modified with a methoxy group. Furthermore, it is known to a person of skill in the art that the other end of the PEG needs to be modified with specific "activation groups" in order to couple the PEG specifically to a peptide. An overview about such activation groups is given in Zalipsky, Bioconjugate Chem. 1995, 6, 150-165. The PEG molecule might be either linear or branched, the latter allowing multivalent peptide constructs, as described below and herein. The coupling of PEG to a biologically active agent, commonly known as PEGylation, has been applied to numerous proteins and antibody fragments in order to reduce their immunogenicity and increase their circulation time in plasma (Bailon P, Won CY. PEG-modified biopharmaceuticals, Expert Opin. Drug Deliv. 2009 Jan; 6: 1 -16). PEGylation is known to shield the surface of biologic drugs and in this way to reduce their activity and immunogenicity. Thus, it was expected that PEGylation of small autoantigenic peptides would reduce their tolerization effect by masking antigenic epitopes. However, the inventor demonstrated that the capacity of T cells to react to the PEG-coupled peptide was not significantly reduced compared to free peptide.

This allows for the use of PEG-conjugated peptides as effective agents for tolerance induction by vaccination. The inventor provides evidence that coupling of peptides and PEG results in superior properties relevant for therapy:

1. Coupled peptides are longer available in the organism and recognized by the immune system (Fig. 1 , bioavailability). This leads to an increased number of protective Tregs under these conditions. Also, the de novo induction of Tregs from na^'ive T cells is improved by using PEG- conjugates (Fig. 2, de novo induction). The increase in cell number and frequency of Tregs after injection of PEG-conjugated peptide, compared to free peptide, is especially pronounced after repeated application (Fig. 3).

2. The application of PEG-conjugated peptides has a surprising down-regulatory effect on the generation or survival of potentially harmful effector cells, as identified by intracellular staining of the inflammatory cytokine TNF. Most importantly, this occurs even when an inflammatory stimulus (in the form of LPS) is applied simultaneously (Fig. 4, cytokines). This indicates that PEG- conjugated peptides are safer than non-conjugated peptides when being applied to humans, where ongoing disease or simultaneous infection could lead to adverse reactions upon vaccination with conventional peptides. In addition to the increase in Tregs, the simultaneous reduction of dangerous effector cells shifts the immunological balance in a favorable way only when the peptide is PEG-conjugated. This shows that treatment of autoimmune patients with PEG-coupled peptide is an efficient and safe approach.

The unexpected effect on T effector cell generation/survival might point to hitherto not known specific interference mechanisms of PEG-coupled peptides modulating the immune response.

3. The overall superiority of the peptides of the invention to treat the autoimmune disease MS is demonstrated by the efficient prevention of the disease in a mouse model with PEG-conjugated, but not conventional peptide treatment (Fig. 5, EAE-prevention).

The low efficacy of previous attempts to treat MS by peptide tolerization caused by antigen spreading is overcome here by providing a peptide of table 1 (i.e. with a sequence of SEQ ID NO 1 to 7) covalently bound to a PEG. In a preferred embodiment of the invention, the peptide with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7 (listed in table 1 ) is bound to a PEG molecule.

In another preferred embodiment of the invention, more than one peptide with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7 (listed in table 1 ) is bound to one PEG. In this embodiment, the PEG used is a branched molecule. For example, two, three, or four peptides with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7 can be covalently bound to one branched PEG molecule. Preferably, all peptides bound to the branched PEG are peptides with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7. However, it is also possible to bind other peptides or other substances than peptides to a PEG.

Accordingly, in one embodiment, the invention refers also to a molecule comprising a branched PEG with more than one peptide covalently bound to it, wherein at least one of these covalently bound peptides is a peptide of table 1 . It is possible that the branched PEG in bound to peptides of the same or different amino acid sequence.

The synthesis of such a branched PEG is described, e.g., in WO 0809601 A1 .

The molecule of the invention may comprise a peptide moiety with a sequence that varies from the sequence chosen from the group of sequences according to SEQ ID NO 1 to 7 ("derivative"), but is recognized by the same T cell receptors.

Such a derivative is a peptide molecule differing in length or containing exchanges in not more than five amino acids (altered peptides), preferably in 1 or 2 amino acids. The reactivity of such a derivative peptide with T cells of defined specificity is not changed with respect to the unaltered peptide, but may show a changed quantitative effect. The suitability of a derivative is tested by its capacity to stimulate T cells reactive to the original peptide in appropriate assay systems such as thymidine incorporation, proliferation of CFSE-marked cells, cytokine-production or upregulation of activation markers upon incubation with the specific peptide and antigen presenting cells, but not with control peptides.

In another embodiment of the invention, it is preferred to use monovalent conjugates of the peptide with PEG, where preferably only one linear or branched PEG is coupled 1 :1 to a peptide; this monovalency precludes crosslinking of antibodies or cellular receptors, thus avoiding immune activation that could lead to adverse effects including aggravation of the disease.

Alternatively, multivalent conjugates of peptide with PEG can be used, where at least two peptide moieties are coupled to one branched PEG. Here, activating effects of a multivalent presentation are prevented by the inherent physicochemical properties such as hydrophilicity of the PEG molecule.

In another embodiment of the invention, it is preferred that the PEG moiety of the molecule has a molecular weight between 2 and 100 kDa, preferably between 5 and 100 kDa, more preferably between 10 and 60 kDa and most preferably between 20 and 40 kDa (high molecular weight PEG); the use of a high molecular weight PEG ensures an overall size of the conjugate that leads to increased bioavailability. The PEG may be linear or branched. Coupling lower molecular weight PEG (< 2 kDa) to a peptide of the invention is less efficient (Fig. 6).

Indeed, the results obtained prove an increased efficacy of these conjugates in inducing regulatory T cells and suppressing inflammatory diseases as compared to free peptide. In the present invention, molecular weight of PEG given represents the average molecular weight. It is known in the art that PEG is polydispersed and therefore a mixture of PEG with different molecular weights and chain lengths. Thus, an average molecular weight of, for example, 40 kDa will also include PEG species with molecular weights of 35 kDa or 45 kDa.

In another embodiment of the invention, it is preferred to use the molecule of the invention for the induction of Foxp3+ regulatory T cells.

In another embodiment of the invention, it is preferred to use the conjugation of peptides to PEG as a treatment for MS with increased clinical efficacy, and fewer side effects. The term "treatment", as used herein, may refer to completely of partially curing the disease or to alleviating a symptom of the disease.

The invention also relates to a pharmaceutical composition ("vaccine") for preventing or treating multiple sclerosis, wherein the composition comprises at least one molecule according to the invention as described above and herein.

The pharmaceutical composition of the invention may additionally comprise a pharmaceutically acceptable carrier, diluents, and/or adjuvant.

The pharmaceutical compositions of the molecules of the invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. The present invention therefore includes pharmaceutical formulations comprising the molecules described herein, including pharmaceutically acceptable salts thereof, in pharmaceutically acceptable carriers for parenteral administration. Also, the present invention includes such compounds, or salts thereof, which have been lyophilized and which may be reconstituted to form pharmaceutically acceptable formulations for administration, as by intravenous, intramuscular, or subcutaneous injection. Administration may also be intradermal or transdermal.

Appropriate dosage levels may be determined by any suitable method known to one skilled in the art. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific molecule employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the condition to be treated. Dosage levels preferably lie in the range of 0.01 to 100 mg/kg body weight, more preferably in the range of 0.1 to 50 mg/kg body weight of the patient to be treated.

For a pharmaceutical composition of the invention that comprises more than one active agent, the respective active agents may be formulated together in a single dosage form. Alternatively, they may be formulated separately and packaged together, or they may be administered independently. In certain cases, a patient may be receiving one drug for the treatment of another indication; this invention then comprises administering the other drug.

It may be advantageous to combine or co-administer a pharmaceutical composition of the invention with other classes of drug. Drugs which may be co-administered with a peptide of the invention include, but are not limited to, Rapamycin (Sirolimus; Rapamune^®, Pfizer), anti CD3 (Muromonab-CD3; Orthoclone OKT3^®), low-dose Cyclosporin A (Ciclosporin), or other agents suppressing or eliminating selectively pathogenic effector cells. The respective drugs may be administered simultaneously, separately or sequentially.

The composition of the invention is preferably administered to a patient in a phase of remission to prevent relapse.

In a further aspect, the invention relates to a use of a single peptide or to the composition of peptides as described above and herein, or of the PEG-conjugated peptide as described above and herein for treating multiple sclerosis.

In a another aspect, the invention relates to a method for preventing or treating a chronic inflammatory disease of the central nervous system (CNS) with a therapeutically effective amount of a composition of peptides as described above and herein or with a peptide as described above and herein. In a further aspect, the invention also relates to a method (in vitro or in vivo) for inducing/expanding regulatory T- (Treg) cells (CD4+CD25+Foxp3+). Such a method comprises at least the step of presenting applied peptide composition as described above and herein by antigen-presenting cells to naive T cells or regulatory T- (Treg) cells to induce or expand Foxp3+ Tregs. The Treg cells are induced/expanded for auto-tolerance.

In another aspect, the invention relates to a method (in vitro or in vivo) to reduce inflammatory cell numbers or production of inflammatory cytokines causing disease by treating a chronic inflammatory disease of the central nervous system with a therapeutically effective amount of a composition or of a molecule as described above and herein or with a peptide as described above and herein.

In yet another aspect, the invention relates to methods for the preparation of PEG-conjugated peptides. Preferably, these methods are suited to modify the peptides at a specific site of the peptide in a molar ratio of 1 :1 . Alternatively, several peptide molecules might be bound to one branched PEG molecule. In preferred embodiments, the PEG is specifically coupled to an amino group (e.g. N-Terminus or ε-amino group of Lysine), a thiol group, a carboxyl group, a guanidine group, a carbonyl group, a hydroxyl group, a hydrazine group, an alkyne group, or an azido group present in the peptide. Ways of performing such reactions are known to a person of skill in the art. In most preferred embodiments, the reactive group is present in the peptide only once. For this purpose, for example an extra cystein residue or an artificial amino acid such as azido- phenylalanine can be introduced into the peptide.

Accordingly, the invention also pertains to a method for synthesizing a molecule as described above and herein, with a peptide moiety and a PEG moiety, comprising

- a PEG molecule with an activation group (as described above and herein), and

- coupling the activated PEG covalently to a peptide comprising or consisting of a sequence of SEQ ID NO 1 to 7.

In a further aspect, the invention relates to a method (in vitro or in vivo) for reducing the number of proinflammatory T-cells in a population of cells, comprising applying a molecule of SEQ ID NO 1 to 7 or a pharmaceutical composition containing such a molecule to the population. Proinflammatory T cells are defined as (autoantigen-specific) T cells capable to produce inflammatory cytokines such as TNF, IFNy or GM-CSF. In a preferred embodiment of this method, the reduction of the number of proinflammatory T-cells in the population of cells is achieved through cell death.

In another aspect, the treatment with PEG-peptides is combined with short-term immunosuppression, notably selective suppression of effector cells, such as Rapamycin treatment or use of other agents not inhibiting regulatory T cells or their induction, to suppress the ability of pre-existing effector/memory cells to inhibit Treg formation. Success of therapy may be evaluated by quantification of lesions by MRI, clinical relapse, or other diagnostic means that are state of the art.

Table 1 : Peptides that are to be conjugated to PEG to form molecules for preventing or treating multiple sclerosis according to the invention. For this purpose, all peptides are PEG conjugated at the N-terminus. The PEG molecule and the manner of binding to PEG are described herein.

SEQ ID NO Peptide

SEQ ID NO 1 MBP 13-32

SEQ ID NO 2 MBP 83-99

SEQ ID NO 3 MBP 1 1 1-129

SEQ ID NO 4 MBP 146-170

SEQ ID NO 5 MOG 1-20

SEQ ID NO 6 MOG 35-55

SEQ ID NO 7 PLP 139-154

SEQ ID NO 8 pOVA 323-339

Tables and Figures

Table 1 shows a list of 7 myelin peptides that are, preferably N-terminally, monovalently PEG- conjugated, and the experimentally used OVA peptide. The respective amino-acid sequences are listed in the sequence listing.

Figure 1 shows the prolonged bioavailability of a PEG-conjugated peptide (the molecules of the invention) as studied in a transgenic mouse model using the model-peptide pOVA (ovalbumin peptide 323-339). pOVA-PEG but not pOVA is able to stimulate TCR-transgenic DO1 1.10 CD4⁺ T cells transferred 3 days after intravenous peptide vaccination and to induce Foxp3+ Tregs. 5-10⁶- 1x10⁷ OVA-specific CD4⁺ CFSE⁺ cells were transferred into BALB/c mice 6 hours or 3 days after peptide vaccination with 5 pg pOVA or equimolar pOVA-PEG. Foxp3 expression as well as proliferation was assessed by FACS analysis on day 6. Peripheral lymph node cells were gated on OVA-specific CD4⁺ T cells.

Figure 2 shows increased frequency of Foxp3+ cells by pOVA-PEG compared to unmodified peptide. 2.5x10⁶ OVA-specific CFSE-labeled CD4+ CD25- aE- T cells were transferred into BALB/c mice on day 0 and mice were intravenously tolerized on day 1 by 5 pg pOVA or equimolar pOVA-PEG . Foxp3 expression was assessed by FACS analysis on day 6. Lymph node cells were gated on OVA-specific CD4⁺ T cells. cLN=celiac lymph node; mLN=mesenterial lymph nodes; pLN=peripheral lymph nodes; popLN=popliteal lymph nodes.

Figure 3 shows that the frequency (cell numbers) of Tregs is increased stronger when PEG- conjugated peptides are injected twice: In the OVA transgenic mouse model 1 x10⁷ OVA-specific CFSE labeled CD4+ T cells were transferred into BALB/c mice on day 0. The mice were i.v. tolerized on day 1 and 7 by 5 pg pOVA or an equimolar amount of pOVA-PEG. Foxp3 expression was assessed by FACS analysis on day 14. Lymph node cells were gated on OVA-specific CD4+ T cells.

Figure 4 shows tolerance induction by PEG-conjugated myelin-specific T cell epitopes in EAE. Disease was induced in C57BI/6 mice by subcutaneous immunization with 250 pg of the encephalitogenic peptide MOG35-55 in CFA. Mice were treated 7 days prior to induction of disease with a single intravenous injection of PBS, 7.6 pg MOG35-55 or an equimolar amount of MOG35-55-PEG. (A) The mean clinical score (degree of paralysis in tail and legs) was determined in groups of 6 mice. The Mann-Whitney-U-test was used for testing statistical significance of differences between groups. All differences were significant with p<0.01. Figure 5 shows that coupling lower molecular weight PEG than 20 kd is less efficient. Peptides were coupled to PEGs with different size (5kd, 20kd, 40kd) and tested in vivo. Bioavailability was dependent on the size of the PEG, whereby PEGs lower than 20 kd were inefficient to increase bioavailablity compared to unconjugated peptide. In the OVA transgenic mouse model 1x107 OVA- specific CD4+ CFSE+ cells from DO1 1.10 mice were transferred into BALB/c mice 3 days after intravenous (i.v.) peptide vaccination with 5 pg pOVA or an equimolar amount of pOVA-PEG. Proliferation was assessed as dilution of CFSE (reduction of geo mean) by FACS analysis on day 8. Spleen cells were gated on OVA-specific CD4+ T cells.

Figure 6 shows the RP-HPLC analysis of A) PLP peptide and B) 20 kDa PEG-PLP detected by evaporating light scattering detection.

Figure 7 shows the RP-HPLC analysis of A) Cys-Ala-MOG peptide and B) 20 kDa PEG-Cys-Ala- MOG detected by evaporating light scattering detection.

Examples

Materials & Methods

Conjugation of peptides to PEG

Example 1 :

Peptides were N-terminally conjugated with a 20 kDa PEG-acid molecule. First, the acid function of the PEG derivate was activated by Diisopropylcarbodiimid/Hydroxybenzotriazol in DCM (Dichloromethane). A PEG derivate is a PEG molecule as defined herein wherein the terminal OH- group is modified to a COOH-group. Subsequently, the N-terminal amino-acid group of a partial protected peptide was conjugated to this. Splitting of protection groups and purification were performed in standard procedures of peptide chemistry. Purity was analyzed using HPLC and the Pauly test.

Example 2: Conjugation of 20 kDa PEG-Maleimid to a Cystein residue at position 2 of PLP-peptide

125 mg of PEG-Maleimide solved in 500 μΙ_ water were added to a solution of 10 mg PLP peptide in 500 μΙ_ of 20 mmol/l Sodium phosphate buffer pH 7.2. The reaction mixture was stirred at room temperature for 1 hour. Subsequently, the resulting 20 kDa PEG-PLP peptide was purified by cation-exchange chromatography using MacroCap SP with an Akta chromatography system.

Example 3: Conjugation of 20 kDa PEG-Maleimide to an artificial N-terminal Cys-Ala-MOG peptide

150 mg of PEG-Maleimid solved in 500 μΙ_ water were added to a solution of 10 mg Cys-Ala-MOG peptide in 500 μΙ_ of 20 mmol/l Sodium phosphate buffer pH 7.2 containing 500 pmol/l of Tris(2- carboxyethyl)phosphine. The reaction mixture was stirred at room temperature for 2 hours. Subsequently, the resulting 20 kDa PEG-Cys-Ala-MOG peptide was purified by cation-exchange chromatography using MacroCap SP with an Akta chromatography system.

Example 4: Conjugation of 20 kDa PEG-Aldehyde to the N-terminal a-amino group PLP peptide

150 mg of PEG-Aldehyde solved in 500 pL water were added to a solution of 10 mg PLP peptide in 500 pL of 50 mmol/l Sodium acetate buffer pH 5.6 containing 40 mmol/l of NaCNBH₃. The reaction mixture was stirred at room temperature for 20 hours. Subsequently, the resulting 20 kDa PEG- PLP peptide was purified by cation-exchange chromatography using MacroCap SP with an Akta chromatography system. Example 5: Analysis of PEG-conjugated peptides by RP-HPLC

Analysis of PEG-conjugated PLP peptide was performed by RP-HPLC on a Waters Alliance System using a Butyl C4 column 4.6 x 250 mm (5 pm). The flow rate was 1.5 mL/min. 10pL sample were injected. Sample was eluted with a linear gradient from 70:15:15 to 50:25:25 water:acetonitrile:isopropanol each with 0.1 % TFA in 10 min. PEG was detected using an evaporating light scattering detector (ELSD).

Coupling of further peptides from the list is carried out in an analogous manner.

Functional tests of the PEG-peptide conjugates in animal models were carried out as described in the figure legends:

Extended bioavailability:

In a transgenic mouse model using the model-peptide pOVA (ovalbumin peptide 323-339) the inventor could show that conjugation of peptides to PEG prolonged the bioavailability of the peptide. To analyze this 5-10⁶ - 1x10⁷ OVA-specific CD4⁺ CFSE⁺ cells from DO1 1 .10 mice were transferred into BALB/c mice 6 hours or 3 days after intravenous (i.v.) peptide vaccination with 5 pg pOVA or an equimolar amount of pOVA-PEG. Foxp3 expression as well as proliferation was assessed by FACS analysis on day 6 (Fig. 1 ).

Increased numbers of Foxp3+ Tregs:

Tolerization with PEG-conjugated peptides i.v. augments the frequency of de novo induced Tregs. This was shown in the same transgenic mouse model as described in figure 1 by transfer of 2.5x10⁶ OVA-specific CFSE labeled CD4+ CD25- aE- T cells into BALB/c mice on day 0. The mice were i.v. tolerized on day 1 by 5 pg pOVA or an equimolar amount of pOVA-PEG. Foxp3 expression was assessed by FACS analysis on day 6. (Fig. 2).

Frequency of Tregs is even stronger increased when PEG-conjugated peptides are injected twice: In the same transgenic mouse model 1 x10⁷ OVA-specific CFSE labeled CD4+T cells were transferred into BALB/c mice on day 0. The mice were i.v. tolerized on day 1 and 7 by 5 pg pOVA or an equimolar amount of pOVA-PEG. Foxp3 expression was assessed by FACS analysis on day 14. (Fig. 3).

Decreased frequencies of T cells producing pro-inflammatory cytokines:

Vaccination with PEG-conjugated peptides decreases the frequency of T cells producing the proinflammatory cytokine TNF. For this study 5-10⁶ - 1 x10⁷ OVA-specific CD4+ T cells were transferred into BALB/c mice on day 0 and mice were i.v. tolerized on day 1 by 5 pg pOVA or an equimolar amount of pOVA-PEG. TNF expression was assessed after 4 h stimulation with PMA/lonomycin in presence of Brefeldin A (to block secretion) and subsequent FACS analysis on day 7 (Fig. 4).

Enhanced protection in EAE:

It was shown that PEGylated conjugates are able to effectively induce tolerance in vivo. The conjugate displays strongly enhanced protection compared to peptides alone in an animal model of MS: EAE (Experimental Autoimmune Encephalomyelitis). The disease was induced in C57BI/6 mice by subcutaneous immunization with 250 pg of the encephalitogenic peptide MOG35-55 in CFA and pertussis toxin injection on day 0 and on day 2. Mice were treated 7 days prior to induction of disease with a single i.v. injection of PBS, 7.6 pg MOG35-55 or an equimolar amount of MOG35-55-PEG (Fig. 5).

Coupling lower molecular weight PEG than 20 kd is less efficient:

Peptides were coupled to PEGs with different size (5 kd, 20 kd, 40 kd) and tested in vivo. Bioavailability was dependent on the size of the PEG, whereby PEGs lower than 20 kd were inefficient to increase bioavailablity compared to unconjugated peptide. In the OVA transgenic mouse model 1x10⁷ OVA-specific CD4+ CFSE+ cells from DO1 1 .10 mice were transferred into BALB/c mice 3 days after intravenous (i.v.) peptide vaccination with 5 pg pOVA or an equimolar amount of pOVA-PEG. Proliferation was assessed as dilution of CFSE (reduction of geo mean) by FACS analysis on day 8 (Fig. 6).

Peptide Sequences

All peptides shown here (with a sequence according to SEQ ID NO 1 to 8) are, according to the invention, PEGylated at the N-terminus, i.e. each peptide is linked to a PEG molecule. All peptides were synthesized as acetate salts; a synthesis as another suitable salt is also possible.

SEQ ID NO 1: MBP13-32: H-Lys-Tyr-Leu-Ala-Thr-Ala-Ser-Thr-Met-Asp-His-Ala-Arg-His-Gly-Phe- Leu-Pro-Arg-His-OH

Molecular formula (net): CioiH₁₅₇N₃₃02₇S; Relative molecular mass: 2309.6

SEQ ID NO 2: MBP83-99: H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-lle-Val-Thr-Pro-Arg-Thr-

Pro-OH

Molecular formula (net): C93H143N25O24; Relative molecular mass: 1995.3

SEQ ID NO 3: MBP111-129: H-Leu-Ser-Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg-Pro-Gly-Phe- Gly-Tyr-Gly-Gly-OH

Molecular formula (net): C92H₁₂9N₂7026; Relative molecular mass: 2029.2

SEQ ID NO 4: MBP146-170: H-Ala-Gln-Gly-Thr-Leu-Ser-Lys-lle-Phe-Lys-Leu-Gly-Gly-Arg-Asp- Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Arg-Arg-OH

Molecular formula (net): Οι_ΐ2Η₁₉4Ν4ο0₃48; Relative molecular mass: 2677.1

SEQ ID NO 5: MOG1-20: H-Gly-Gln-Phe-Arg-Val-lle-Gly-Pro-Arg-His-Pro-lle-Arg-Ala-Leu-Val-Gly- Asp-Glu-Val-OH

Molecular formula (net): C99H₁₆2N₃2026; Relative molecular mass: 2216.6

SEQ ID NO 6: MOG35-55: H-Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu- Tyr-Arg-Asn-Gly-Lys-OH

Molecular formula (net): C120H179N35O28S, Relative molecular mass: 2592.0

M EVGWYRPPFSRVVHLYRN GK

SEQ ID NO 7: PLP139-154: H-His-Cys-Leu-Gly-Lys-Trp-Leu-Gly-His-Pro-Asp-Lys-Phe-Val-Gly-lle- OH

Molecular formula (net): C85H127N23O19S; Relative molecular mass: 1807.2

HCLGKWLG H PDKFVG I

SEQ ID NO 8: pOVA (ovalbumin peptide 323-339): H-ILE-SER-GLN-ALA-VAL-HIS-ALA-ALA-HIS- ALA-GLU-ILE-ASN-GLU-ALA-GLY-ARG-OH

Molecular formula (net): C₇4H₁₂oN26025; Relative molecular mass: 1773.91

ISQAVHAAHAEI N EAG R

Claims

1. A molecule for preventing or treating multiple sclerosis, comprising

- a polyethylene glycol (PEG) moiety.

wherein the peptide moiety is covalently coupled to the PEG moiety.

2. The molecule of claim 1 , wherein the peptide moiety has a sequence, wherein the sequence of one, several or all peptides according to SEQ ID NO 1 -7 is changed by amino acid exchange, addition or deletion in 1 to 5 positions of the sequence.

3. The molecule of claim 1 or 2, wherein peptide moiety is covalently coupled to the PEG moiety via the N-terminus.

4. The molecule of claims 1 to 3, wherein the PEG moiety has a molecular weight of at least 2 kDa up to 100 kDa, preferably 5 kDa to 100 kDa, preferably of at least 20 kDa.

5. The molecule of claims 1 or 4, wherein the PEG moiety is non-linear.

6. The molecule of claims 1 to 5 for use in medicine, in particular for preventing or treating multiple sclerosis.

7. A pharmaceutical composition for preventing or treating multiple sclerosis, wherein the composition comprises at least one molecule according to claims 1 to 6.

8. The composition of claim 7, comprising a pharmaceutically acceptable carrier, diluent or additive.

9. A method for synthesizing a molecule of claim 1 to 8, comprising

- a PEG molecule comprising an activation group, and

- coupling the activated PEG covalently to a peptide comprising a sequence of SEQ ID NO 1 to 7.

10. A method for preventing or treating a chronic inflammatory disease of the central nervous system (CNS), in particular for preventing or treating multiple sclerosis, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of claim 7, or of a molecule of claims 1 to 6.

1 1 . The method of claim 10, comprising further administering a compound for the selective suppression of effector cells that do not inhibit regulatory T cells or the induction of regulatory T cells, such as Rapamycin, anti CD3 (Muromonab-CD3), low-dose Cyclosporin A (Ciclosporin), or other agents suppressing or eliminating selectively pathogenic effector cells.

12. A method for inducing regulatory T- (Treg) cells, comprising inducing or expanding regulatory T- (Treg) cells by presentation of a molecule of claims 1 to 6 peptide coupled to PEG according to claim 1-5 with a sequence chosen from the group of sequences according to SEQ ID NO 1 to 7 or a part thereof, or of a composition of claims 6 or 7 in the presence of antigen presenting cells.

13. A method for reducing the number of proinflammatory T-cells in a population of cells, comprising applying a molecule of claims 1 to 6 or a composition of claim 7 to the population of cells.

14. The method of claim 13, wherein the reduction of the number of proinflammatory T-cells in the population of cells is achieved through cell death.