SK145896A3 - Medicaments containing therapeutically effective quantity of antigen peptid of human mbp and ifn-beta - 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

The present invention provides compositions and methods for treating multiple sclerosis.

The 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 IFN-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 MS susceptibility 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 comprising at least one T cell epitope, in a therapeutic regimen and further administering IFN-β.

The invention relates to methods of treating multiple sclerosis, comprising administering a therapeutically effective amount of at least one antigenic MBP peptide in a treatment regimen comprising a therapeutically effective amount of IFN-β. The term "peptide" refers here to an amino acid sequence (aa) with 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 T cell epitope or any portion of such a peptide comprising at least one T cell epitope.

As far as the epitope is concerned, it is an essential element or the smallest unit recognized by the receptor, especially immunoglobulins, histocompatible antigens and T cell receptors, where the epitope includes amino acids of the native protein, such as autoantigen, which are essential for receptor recognition. T cell epitopes are thought 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 surface of the antigen-containing cell, 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.

Exposing a subject to a peptide or protein that includes at least one T cell epitope of an autoantigen can modify the T cell subpopulation so that they become unreactive to the autoantigen and do not participate in stimulating the immune response. Furthermore, administration of a protein or peptide that comprises 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 autoantigen response, such that T cells are drained from sites of normal autoantigen exposure (e.g., central nervous system (CNS) tissues) to therapeutic peptide delivery sites derived therefrom. This redistribution of T cell subpopulations can enhance or decrease the ability of an individual's immune system to stimulate a conventional immune response at the site of normal autoantigen exposure and thus reduce 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. Also, 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 affect the response of T cells to MBP, 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 to bind MHC protein without having the ability to elicit a strong proliferative response or, optionally, no proliferative response when administered in an immunogenic form. In this case, it is possible to determine critical binding residues for the T cell receptor 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 . 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 support, decrease, 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, another, preferably similar amino acid residue (conservative substitution) that has been shown to support, decrease, but not eliminate or affect its presence. T cell activity. Furthermore, 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 support, not affect 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 limit dimerization through disulfide bonds. The peptides may 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, to increase solubility, a charged amino acid or charged amino acid pairs or triplets may be added to the carboxyterminal or amino terminal of the peptide. 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 in the immune system due to the 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 if a peptide is used as a primer in a subject, the 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. Peptides that comprise at least one cryptic epitope derived from a protein antigen are 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 cell line can be determined for a given 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 protein shown in FIG. 3: peptide comprising all or part of amino acid residues 84-106 of human MBP, 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 amino acid residues 140 -172 human MBP or a portion thereof, a peptide comprising all or a portion of amino acid residues 143-168 of human MBP, a peptide comprising all or a portion of amino acid residues 142-167 of a human MBP, and a peptide comprising all or a portion of amino acid residues 13-25 of human MBP. The sequences of selected peptides are shown in FIG. 4. Any of these peptides may be modified as described, or may be extended beyond the carboxyterminal or aminoterminal end by a region that still retains the antigenic characteristic of the contents of at least one epitope of T cells.

As a result of the work described herein, it has been found that the combination of MBP-derived antigenic peptide and IFN-β administered in a therapeutic regimen has a synergistic effect (Fig. 1c), surprisingly reducing the clinical symptoms of EAE in mice to a far greater extent than the effects of each of these agents in alleviating the symptoms of EAE when administered alone (Fig. 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 a subject having or susceptible to multiple sclerosis by administering an effective amount of an MBP-derived antigenic peptide in a non-immunogenic form (i.e., no adjuvant) in a therapeutic regimen that also includes IFN-β.

Administration of a composition comprising at least one antigenic MBP peptide in a therapeutic regimen that includes administration of IFN-β may be accomplished by known procedures at dosages and over a period of time that effectively reduces, eliminates or prevents symptoms associated with multiple sclerosis. An effective amount of either an antigenic peptide or IFN-β when administered together in a therapeutic regimen is dependent on factors such as the degree of susceptibility and susceptibility of the individual to MS, the age, sex and weight of the individual and the 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 IFN-β) can be administered by a suitable route, for example, by injection (subcutaneously, intravenously, etc.), orally, by inhalation, transdermally, or rectally. Depending on the route of administration, the active compound may be coated with certain material to protect against the action of enzymes, acids, and other natural conditions that could inactivate it.

For example, about 1 gg to 3 mg, especially about 20 to 500 µg of MBP-derived antigenic peptide per dosage unit may be injected. 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 the optimal therapeutic response. For example, IFN-β and the composition comprising an antigenic peptide derived from MBP 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. The therapeutic regimen of administration of the antigenic peptide and IFN-β may last for days or weeks and may be shortened or extended as required by the therapeutic situation.

The invention also resides in a novel composition comprising a physical mixture of an antigenic peptide derived from MBP and IFN-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

Fig. 1a graphically depicts an experiment showing the effect of IFN-β on EAE in two groups of 10 adult female F1 mice (SJL x TLP) that were induced by EAE with guinea pig MBP in complete adjuvant plus pertussis toxin on day 0 and either treated only with incomplete Freund's adjuvant (control) or administered on days 9, 12, 16 and 20 (indicated by arrows on the x-axis) 2000 IFN-β units interperitoneally (ip), with the y-axis showing the mean clinical record (MCS) for each group: 0 no clinical signs of EAE, 1 - sagging non-responsive tail, 2 - partial hind limb paralysis, 3 - complete hind limb paralysis, 4 - partial to complete hind limb paralysis, S - death.

Fig. 1b is a graphical representation of an experiment showing the effect of aa 1-11 MBP peptide in two groups of 10 adult female F 1 mice (SJL x TLP) in which EAE was induced by guinea pig MBP in complete adjuvant plus pertussis toxin at 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 and 1-11 intravenously, with the y-axis showing the median clinical record for each group as in FIG. Ia.

Fig. 1c is a graphical representation of an experiment showing the effect of aa 1-11 MBP peptide, administered in combination with IFN-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 given either PBS (control) or given on days 10, 13, 17 and 21 (indicated by open arrows on the x-axis) 250 nmol and 1-11 intravenously and treated on day 9, 12, 16 20 (indicated by solid arrows on the x-axis) 2,000 IFN-β units interperitoneally (ip), the y-axis showing the median clinical record as in FIG. Ia.

Fig. 2 is a graphical representation of an experiment showing the effect of different doses of IFN-β (10,000 units and 2,000 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 given either PBS (control) or given on day 9, 13, 16 (indicated by solid arrows on the x-axis) 10,000, respectively. 2,000 IFN-β units interperitoneally (i.p.), the y-axis showing the median clinical record as in FIG. Ia.

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

Fig. 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 murine peptide MBP aa 1-11

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

EAE induction

EAE was elicited in 6-8 week old female mice (SJL x PL) by Fj (Jackson Labs, Bar Harbor, ME) by immunizing mice with 100 µg of purified turkey MBP in CFA (GIBCO Lab., Grand Island, NY) containing 400 µg. H37RA M. tuberculosis strain (DIF CO Lab., Detroit, MI), subcutaneously at the tail base. 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 - flabby non-responding tail, 2 - partial hind limb paralysis, 3 - total hind limb paralysis, 4 - partial to complete forelimb paralysis, and With death. Data are expressed as the mean of disease severity scores for each day, including all animals in the group. Mice were followed for 26 days. Once the mouse died on EAE, an S rating was included in the calculations for all subsequent days.

Effect of IFN-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 2 000 IFN-β units 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, which is advantageous, will be appropriate for IFN-β experiments, as the chances of toxicity due to higher IFN-β are less likely. 2,000 units of IFN-β were then used in the remaining experiments shown in FIG. 1a to 1c, since 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. on days 9, 12, 16 and 20. As shown in FIG. It was evident that the IFN-β-treated group had only slightly milder symptoms over time than the control group.

Effect of murine MBP aa 1-11 peptide on EAE

The effect of the murine MBP aa 1-11 peptide was determined and the results are shown in FIG. lb. 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. administered 250 nmol of peptide aa 1-11 on days 10, 13, 17 and 21. Mice were monitored as described above. As shown in FIG. 1b, mice treated with α 1-11 had less severe symptoms than the control group.

Effect of EAE treatment with aa 1-11 peptide and IFN-β combination therapy

The effects of treatment with the combination of aa 1-11 peptide and IFN-β are shown in FIG. c. One group of mice was i.p. administered PBS (control) after EAE induction and one group of mice was i.v. 250 nmol of peptide aa 1-11 administered on days 10, 13, 17 and 21 (open arrows) and 2,000 units of IFN-β on days 9, 12, 16 and 20. As shown in FIG. Obviously, a group of mice treated with a combination of peptide and IFN-β showed a significant reduction in symptom strength compared to both the control group and the IFN-β alone or the peptide alone shown in FIG. 1a and 1b, indicating a synergistic effect of the combination. Thus, a treatment regimen that includes a combination of a peptide and IFN-β provides enhanced efficacy in alleviating the strength 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 covered by the appended claims and are within the spirit of the invention.

Claims (15)

What is claimed is: 1. A method of treating multiple sclerosis in an individual comprising administering a therapeutically effective amount of at least one antigenic MBP peptide in a therapeutic regimen comprising administering a therapeutically effective amount of IFN-β. r I The method of claim 1, wherein the antigenic peptide MBP and IFN-β are administered simultaneously. The method of claim 1, wherein the antigenic peptide MBP and IFN-β are administered with a difference of at least 24 h. » The method of claim 1, wherein the at least one 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 or part of amino acid residues 89-101 of human MBP, a peptide comprising all or part of amino acid residues 140172 of human MBP, peptide comprising all or part of amino acid residues 143-168 of human MBP, 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. 5. A composition comprising a therapeutically effective amount of at least one human MBP antigen peptide and a therapeutically effective amount of IFN-β in a pharmaceutically acceptable carrier or diluent. The composition of claim 5, wherein the at least one antigenic peptide is selected from the group consisting of a peptide comprising all or part of an amino acid residue of 84-106 human MBP, a peptide comprising all or part of an amino acid residue of 84-102 human MBP, a peptide comprising all or part of amino acid residues 89-101 of human MBP, a peptide comprising all or part of amino acid residues 140172 of human MBP, peptide comprising all or part of amino acid residues 143-168 of human MBP, 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. A method of preventing multiple sclerosis outbreaks in a subject susceptible to multiple sclerosis, comprising administering a therapeutically effective amount of at least one antigenic MBP peptide in a treatment regimen comprising administering a therapeutically effective amount of IFN-β. The method of claim 4, wherein the at least one antigenic peptide is modified. The method of claim 4, comprising at least two antigenic peptides. The composition of claim 5, wherein the at least one 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 or part of human MBP amino acid residues 143-168, a peptide comprising all or part of human MBP amino acid residues 142167, and a peptide comprising all or part of human MBP amino acid residues 13-25, the sequences of which are depicted in FIG. 4th An isolated MBP peptide comprising amino acid residues 142-167 shown in FIG. 4th 12. A composition comprising the isolated peptide of claim 1 11th An isolated MBP peptide having a carboxyterminal and amino terminal end, comprising amino acid residues 142-167, wherein the peptide is extended by at least one amino acid residue after the carboxy terminal end. The isolated peptide of claim 13, wherein the peptide retains its antigenic character by the presence of at least one epitope of T cells. 15. A composition comprising the isolated peptide of claim 1