CZ329596A3 - Preparation for treating scattered sclerosis - Google Patents

Abstract

The present invention provides compositions and methods for treating multiple sclerosis. Compositions of the invention comprise at least one peptide or modified peptide of myelin basic protein (MBP) which comprises at least one T cell epitope, and IFN- beta in an appropriate pharmaceutically acceptable medium for either oral, subcutaneous or intravenous administration. Methods of the inven include treatment of individuals who either have MS or are suspected of being susceptible to MS by administering a therapeutically effective amount of a composition of the invention or by administering in a therapeutic regimen, a composition comprising at least one peptide or modified peptide of MBP which comprises at least one T cell epitope and further administering IFN- beta .

Description

Technical field

The invention relates to compositions and methods of treatment for multiple sclerosis.

BACKGROUND OF THE INVENTION

Autoimmune diseases are a significant human health problem and are relatively poorly understood. Since there is no apparent directly responsible microbial or viral agent, the treatment and diagnosis of these diseases must be based on their etiology. This always involves a complex series of reactions of endogenous metabolic intermediates, structural components, cells, etc. However, the nature of the autoimmune disease is always associated with the fact that at least one autoantigen must be involved in the sequence of events that lead to symptoms. Autoimmune demyelinating diseases such as multiple sclerosis are no exception.

A commonly used animal model for human multiple sclerosis is experimental allergic encephalomyelitis (EAE), a demyelinating central nervous system disease that can be induced in susceptible strains of mice by immunization with myelin basic protein (MBP) or its immunodominant determinants of T cells (ie T cell epitopes). . MBP is one of the predicted autoantigens in multiple sclerosis (MS) and has been epitope-mapped both in human (Ota et al., Nature 346: 183-187 (1990)) and in rodent (Zamvil et al., Nature 324) : 258-260 (1986)) of the system. Ota and d. Have identified a major T cell epitope recognized by MS patients, ie, 84-102 MBP amino acid residues. Smaller epitopes (amino acid residues 143-168, 61-82, 124-142 and 31-50 MBP) recognized by T cells of MS patients have also been described. Zamvil et al. Have shown that amino acid residues 1-11 of MBP contain the major T cell epitope (s) causing EAE in susceptible rodent strains.

Over the last decade, it has become apparent that peptides corresponding to major T cell epitopes of native protein antigen can be used to induce T cell non-reactivity on themselves or on native protein antigen. Studies by Gaur et al., Science 258: 1491-1494, have shown that MBP T cell epitopes induce reduced T cell reactivity to MBP in adult mice. These studies describe the tolerance to synthetic peptides corresponding to the major immunodominant determinants of MBP (aa111 and 35-47) of whole MBP using a peptide or protein emulsion in a non-immunogenic form (incomplete Freund's adjuvant) described previously for inducing tolerance to other proteins or peptides ( F. Rio et al., Nature 343: 381 (1990)). This reduced T cell reactivity, which was induced by administration of MBP T cell epitopes, was able to prevent MBP-induced EAE formation. However, these studies have shown that aa 1-11, the more dominant epitope of both, causes only about a 50% reduction in subsequent T cell response to MBP and a less immunogenic peptide, and a 35-47 produces only about 20% reduction in subsequent response to MBP. Mixing the two peptides together resulted in a greater reduction in the subsequent T cell response to MBP.

In an entirely different approach to MS treatment, clinical studies have shown that interferons (EFN) are somewhat promising in MS treatment. Administration of IFN-γ has been shown to promote exacerbation of MS, while recombinant EFN-β suppressed it in controlled clinical trials (Hillel and Bever, J. Neuroimmunology 46: 155-164 (1993)). Despite evidence that administration of EFN-β to patients afflicted with MS reduces the frequency and severity of MS seizures, EFN-β does not appear to be a long-awaited drug against MS as hoped by the medical community (Amason, Neurology 43: 641-643 (1993)). In the Arnason clinical study, almost all patients treated with EFN-β had occasional seizures and one in four worsened.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treating multiple sclerosis. Compositions of the invention include at least one peptide or modified myelin basic protein (MBP) peptide that comprises at least one T cell epitope, and EFN-β in a suitable pharmaceutically acceptable medium for oral, subcutaneous or intravenous administration. The methods of the invention include treating a subject either having or suspected of having a susceptibility to MS by administering a therapeutically effective amount of a composition of the invention or administering a composition comprising at least one peptide or modified MBP peptide that includes at least one T cell epitope. and further administering IFN-β.

The invention relates to methods of treating multiple sclerosis, comprising administering a therapeutically effective amount of at least one MBP antigen peptide in a treatment regimen comprising a therapeutically effective amount of IFN-β. The term "peptide" as used herein refers to an amino acid sequence (aa) having fewer amino acid residues than the protein from which the peptide is derived. The term "antigenic peptide" as used herein refers to any peptide comprising at least one epitope of T cells or any portion of such a peptide comprising at least one epitope of T cells.

With respect to the epitope, it is the essential element or smallest unit recognized by the receptor, particularly immunoglobulins, histocompatibility antigens and T cell receptors, wherein the epitope comprises amino acids of a native protein, such as an autoantigen, that are necessary for receptor recognition. T cell epitopes are believed to be involved in the initiation and duration of the autoimmune response. It is believed that these T cell epitopes trigger early events at the helper T cell level by providing the appropriate HLA molecule on the cell surface containing the antigen, thereby stimulating T cell subpopulations with the relevant T cell receptor for this epitope. These events lead to T cell proliferation, lymphokine secretion, local inflammatory response, mobilization of other immune cells to the antigen / T cell interaction site, and activation of the B cell cascade leading to antibody production.

By exposing a subject to a peptide or protein that comprises at least one T cell epitope of an autoantigen, the T cell subpopulation may be modified so that it becomes unreactive to the autoantigen and does not participate in stimulating the immune response. Furthermore, administration of a protein or peptide that includes at least one T cell epitope may modify the lymphokine secretion profile (e.g., cause a decrease in IL-4 and / or an increase in IL-2, causing a modification of TH1 and TH2 populations compared to exposure to naturally occurring autoantigen). ). In addition, exposure to this peptide may affect T cell subpopulations that normally participate in the autoantigen response, such that the T cells are drained from sites of normal autoantigen exposure (e.g., central nervous system (CNS) tissues) to therapeutic sites of peptide derived therefrom. This redistribution of T cell subpopulations can enhance or reduce the ability of an individual's immune system to stimulate the usual immune response at the site of normal autoantigen exposure, thereby reducing symptoms.

Any MBP-derived peptides that moderate a subject's response to MBP autoantigen may be included in the methods and compositions of the invention. Such peptides can be identified, for example, by detecting the structure and selecting appropriate regions to be produced as peptides (using recombinant expression systems, synthetically or otherwise) that are tested for their ability to influence T cell response to MBP, then selecting peptides containing epitopes recognized by these cells. . Many human MBP peptides including T cell epitopes are described in the art.

Modified antigenic peptides are useful within the scope of the invention. for example, the peptide may be modified to retain the ability to induce T cell anergy and bind MHC protein without having the ability to elicit a strong proliferative response or, optionally, no proliferative response when administered in immunogenic form. In this case, critical T cell receptor binding residues can be determined by known methods (for example, by substituting each residue and determining the presence or absence of T cell reactivity). Residues that prove necessary to interact with the T cell receptor may be modified by replacing the essential amino acid with another, preferably similar, amino acid residue (conservative substitution) whose presence has been shown to promote, decrease but do not eliminate or affect T cell reactivity . In addition, amino acid residues that are not necessary for interaction with the T cell receptor may be modified by replacement with another amino acid, the incorporation of which may promote, reduce but not eliminate or affect T cell reactivity. Furthermore, amino acid residues that are not necessary for interaction with the T cell receptor may be modified by replacement with another amino acid, the incorporation of which may promote, decrease or not affect T cell reactivity but do not eliminate binding to the relevant MHC.

The peptides of the invention may be further modified by substituting an amino acid that has been shown to be necessary for interaction with the MHC protein complex by another, preferably similar amino acid residue (conservative substitution), which has been shown to support, decrease, but not eliminate or affect T cell activity. Further, amino acid residues that are not necessary for interaction with the MHC protein complex may be modified by replacement with another amino acid, the incorporation of which may promote, influence or reduce but not eliminate T cell reactivity. Preferred amino acid substitutions for non-essential amino acids include, but are not limited to, alanine, glutamic acid, or methylamino acid substitutions.

Another example of peptide modification is the substitution of cysteine residues preferably by serine, threonine, leucine or glutamic acid to reduce dimerization prior to disulfide bonding. The peptides can be further modified to increase the solubility of the peptide for use in buffered aqueous solutions such as pharmaceutically acceptable carriers or diluents, by adding functional groups to the peptide, terminal portions of the peptide, or by not including hydrophobic T cell epitopes or regions containing hydrophobic epitopes in peptides or hydrophobic protein or peptide regions. For example, a charged amino acid or charged amino acid pairs or triplets may be added to the carboxyterminal or amino terminal terminus of the peptide to increase solubility. Examples of charged amino acids include arginine (R), lysine (K), histidine (H), glutamic acid (E) and aspartic acid (D).

Furthermore, peptides containing "cryptic epitopes" that are also useful in the methods and compositions of the invention can be determined. Cryptic epitopes are those determinants in a protein antigen that are not normally detected by the immune system due to processing and delivery of the native protein antigen to the respective MHC molecule. However, a peptide containing a cryptic epitope is capable of inducing T cell unreactivity, and when a peptide is used as a primer in a subject, T cells obtained from the subject will proliferate in vitro in response to the antigen of the peptide or protein from which the peptide is derived. include at least one cryptic epitope derived from a protein antigen, referred to herein as "cryptic peptides". To confirm the presence of cryptic epitopes, a known T cell proliferation assay can be used in which antigen-initiated T cells are cultured in vitro in the presence of each peptide separately to detect peptide-reactive T cell cell lines. A peptide is believed to comprise at least one cryptic epitope if the T cell line can be determined for the peptide and the T cells are capable of proliferating upon exposure to the peptide and antigen of the protein from which the peptide is derived.

Antigenic peptides useful in the compositions and methods of the invention include the following peptides or portions thereof with residue numbers that correspond to the amino acid residues of the human MBP shown in Figure 3: a peptide comprising all or a portion of the amino acid residues 84-106 of human MBP, a peptide comprising all or part of amino acid residues 84-102 of human MBP, peptide comprising all or part of amino acid residues 89-101 of human MBP, peptide comprising all or part of amino acid residues 140-172 of human MBP, peptide including all amino acid residues 143-168 of human human MBP or a portion thereof, a peptide comprising all or a portion of amino acid residues 142-167 of human MBP, and a peptide comprising all or a portion of amino acid residues 13-25 of a human MBP. The sequences of the selected peptides are shown in FIG. 4. Any of these peptides may be modified as described, or extended beyond the carboxyterminal or amino-terminal end by such a region that they still retain the antigenic characteristics of the contents of at least one T cell epitope.

As a result of the work described herein, it was found that the combination of MBP-derived antigenic peptide and IFN-β administered in a therapeutic regimen has a synergistic effect (Fig. 1c), which surprisingly reduces clinical EAE symptoms in mice to a far greater extent than of these compounds in alleviating the symptoms of EAE when administered alone (Figs. 1a, 1b), and which is greater than would be expected with the mere additive effect of the peptide plus IFN-β.

Since EAE serves as a mouse model of human MS and is induced by MBP, it is expected that a similar effect will be observed in humans. Accordingly, the present invention provides a method of treating individuals having or susceptible to multiple sclerosis by administering an effective amount of an MBP-derived antigenic peptide in a non-immunogenic form (i.e., without adjuvant) in a therapeutic regimen that also includes IFNβ.

Administration of a composition comprising at least one MBP antigen peptide in a therapeutic regimen that includes administration of IFN-β may be accomplished by known procedures at dosages and for a period of time that effectively reduces, eliminates or prevents the symptoms associated with multiple sclerosis. An effective amount of either an antigenic peptide or EFN-β when administered together in a therapeutic regimen is dependent on factors such as the degree of

Ί individual sensitivity and susceptibility to MS, age, sex and weight of the individual and ability of the MBP antigen peptide to elicit an antigenic response in the individual. The active compounds (i.e., the MBP peptide or a mixture thereof with EFN-β) can be administered in an appropriate manner, for example, by injection (subcutaneously, intravenously, etc.), orally, by inhalation, transdermally or rectally. Depending on the mode of administration, the active compound may be coated with some material to protect against the action of enzymes, acids, and other natural conditions that could inactivate it.

For example, about 1 µg to 3 mg, especially about 20 to 500 µg, of MBP-derived antigenic peptide per dosage unit may be administered by injection. Preferably, a dosage unit of 100 to 10,000 units of IFN-β may be injected. The dosage regimen of the two compounds may be adjusted to provide an optimal therapeutic response. For example, EFN-β and the preparation comprising the MBP-derived antigenic peptide may be administered simultaneously or preferably may be administered with a difference of at least six hours, preferably at least 12 hours, especially with a difference of at least 24 hours. over a period of days or weeks and may be shortened or extended as required by the therapeutic situation.

The invention also relates to a novel composition comprising a physical mixture of an antigenic peptide derived from MBP and EFN-β and a pharmaceutically acceptable carrier or diluent. The composition may be used as part of a therapeutic regimen for treating or preventing multiple sclerosis in an individual.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. la graphically shows an experiment showing the effect of IFN-β on EAE in two groups of 10 adult female mice F _t (SJL x TLP), which was induced EAE using guinea pig MBP in complete adjuvant plus pertussis toxin on day 0 and receive either only incomplete Freund's adjuvant (control) or administered on day 9, 12, 16 and 20 (indicated by arrows on the x-axis) 2000 EFN-β units interperitoneally (ip), with the y-axis showing the mean clinical record (MCS) for each group : 0 - no clinical signs of EAE, 1 - flabby tail, 2 - partial hind limb paralysis, 3 - total hind limb paralysis, 4 - partial to total hind limb paralysis, 5 - death.

Giant. 1b is a graphical representation of an experiment showing the effect of aa 1-11 MBP peptides in two groups of 10 adult female Fi mice (SJL x TLP) in which EAE was induced with guinea pig MBP in complete adjuvant plus pertussis toxin on day 0 and administered either with PBS (control) or administered on days 10, 13, 17 and 21 (indicated by arrows on the x-axis) 250 nmol a and 1-11 intravenously, with the y-axis showing the median clinical record for each group as in Fig. 1a.

Giant. 1c is a graphical representation of an experiment showing the effect of aa 1-11 MBP peptides, administered in combination with EFN-β, on EAE in two groups of 10 adult female Fi mice (SJL x TLP) in which EAE was induced by guinea pig MBP in complete adjuvant plus pertussis toxin on day 0 and administered either with PBS (control) or given on days 10, 13, 17 and 21 (indicated by open arrows on the x-axis) 250 nmol a and 1-11 intravenously and treated on day 9, 12, 16 20 (indicated by solid arrows on the x-axis) 2000 IFN-β units interperitoneally (ip), with the y-axis showing the median clinical record as in Fig. 1a.

Giant. 2 is a graphical representation of an experiment showing the effect of different doses of IFN-β (10000 units and 2000 units, respectively) on EAE in two groups of 10 adult female Fi mice (SJL x TLP) in which EAE was induced by guinea pig MBP in complete adjuvant plus pertussis toxin on day 0 and administered either with PBS (control) or administered on days 9, 13, 16 (indicated by solid arrows on the x-axis) 10000, respectively. 2000 units of EFN-β interperitoneally (i.p.), with the y-axis showing the median clinical record as in Fig. 1a.

Giant. 3 shows the full-length amino acid sequence of human MBP.

Giant. 4 depicts the amino acid sequences of selected MBP peptides useful in the invention.

DETAILED DESCRIPTION OF THE INVENTION

A non-limiting example is given to illustrate the invention.

Synthesis of mouse MBP aa 1-1 peptide

Mouse MBP aa 1-11 peptide was synthesized using standard Fmoc / tBoc synthesis and purified by HPLC.

EAE induction

EAE was induced in 6 to 8 'week old female mice (SJL x PL) by Fi (Jackson Labs, Bar Harbor, ME) by immunizing mice with 100 µg of purified guinea pig MBP in CFA (GIBCO Lab., Grand Island, NY) containing 400 pg of H37RA M. tuberculosis strain (DIFCO Lab., Detroit, MI), subcutaneously at the base of the tail. 200 ng of pertussis toxin (JHL BIOSCIENCE, Lenexa, Kansas) was administered twice intravenously (i.v.) on the day of immunization and 48 h later. Mice were monitored daily for disease symptoms and scored on a scale of 0 - no clinical signs of EAE, 1 - flaccid unresponsive tail, 2 - partial hind limb paralysis, 3 - total hind limb paralysis, 4 - partial to complete forelimb limb and 5 - death. Data are expressed as the mean of the disease severity scores for each day, including all animals in the group. Mice were followed for 26 days. Once the mouse died on EAE, a score of 5 was included in the calculations for all subsequent days.

Effect of EFN-β on EAE

Titration experiment to determine the effects of different doses of IFN-β on EAE (Fig. 2). One group of mice was administered intraperitoneally with PBS on days 9, 13 and 16 (control) after EAE induction, one group of mice received 10,000 units of IFN-β on days 9, 13 and 16 (open circle) and one group of mice received 2000 units EFN-β on days 9, 13 and 16 (solid circle). As shown in Fig. 2, the symptoms at the same time points are the same for both doses of IFN-β, indicating that a lower dose is preferred for IFN-β experiments, as the chances of toxicity due to a higher dose of IFN- β are less likely. 2000 units of IFN-β were then used in the remaining experiments shown in Figures 1a to 1c because this dose showed an improvement in the clinical trial.

As shown in Fig. 1a, a control group of mice was administered PBS and another group of 2000 units of IFN-β i.p. at day 9, 12, 16 and 20. As shown in Figure 1a, the LFN-β-treated group had only slightly weaker symptoms over time than the control group.

Effect of murine MBP aa 1-11 peptides on EAE

The effect of murine MBP aa 1-11 peptides was determined and the results are shown in Fig. 1b. One group of mice was administered i.p. PBS on days 10, 13, 17 and 21 (control) after EAE induction and one group of mice was i.v. 250 nmol of aa 1-11 peptides were administered on days 10, 13, 17 and 21. Mice were monitored as described above. As shown in Fig. Ib, mice treated with α 1-11 had less severe symptoms than the control group.

Effect of treatment with aa 1-11 peptide combination and DFN-β on EAE

The effects of aa 1-11 and IFN-β combination therapy are shown in Figure Ic. One group of mice was i.p. administered PBS (control) after EAE induction and one group of mice was i.v. 250 nmol of aa 1-11 peptides were administered on days 10, 13, 17 and 21 (open arrows) and 2000 units of IFN-β on days 9, 12, 16 and 20. As can be seen in Figure Ic, a group of mice treated with a combination of peptides and IFN-β showed a significant reduction in symptom strength compared to both the control group and the treatment with IFN-β alone or with the peptide alone shown in Figures 1a and 1b, indicating a synergistic effect of the combination. Thus, a treatment regimen that includes a combination of peptides and IFN-β provides an enhanced mitigating effect of EAE symptoms.

The invention is described with reference to preferred embodiments, but the same results can be achieved with other embodiments. Variations and modifications of the invention will be apparent to those skilled in the art, which are within the scope of the appended claims and are within the scope of the invention.

Claims (5)

PATENT CLAIMS jj ⁰¹ S ° ^Q 4 '?? 9 ΐ A composition for the treatment of multiple sclerosis, characterized by: comprising a therapeutically effective amount of at least one antigenic peptide of human MBP1 and a therapeutically effective amount of IFN-β in a pharmaceutically acceptable carrier or diluent. The composition of claim 1, wherein the antigenic peptide is selected from the group consisting of a peptide comprising all or a portion of amino acid residues 84-106 of human MBP, a peptide comprising all or a portion of amino acid residues 84-102 of human MBP, a peptide comprising all amino acid residues 89-101 of human MBP or a portion thereof, peptide comprising all or a portion of amino acid residues 140-172 of human MBP, a peptide comprising all or a portion of amino acid residues 143-168 of human MBP, a peptide comprising all amino acid residues 142-167 of human MBP, or a portion thereof and a peptide comprising all or a portion of amino acid residues 13-25 of human MBP. The composition of claim 1, wherein the antigenic peptide is selected from the group consisting of a peptide comprising all or a portion of amino acid residues 84-102 of human MBP, a peptide comprising all or a portion of amino acid residues 89-101 of human MBP, a peptide comprising all amino acid residues 143-168 of human MBP or portion thereof, a peptide comprising all or a portion of amino acid residues 142-167 of human MBP, and a peptide comprising all or a portion of amino acid residues 13-25 of human MBP, the sequences of which are depicted in FIG. An isolated MBP peptide comprising amino acid residues 142-167 shown in Figure 4. The composition of claim 1, comprising the isolated peptide of claim 4.