ES2370957T3 - COMPOSITIONS THAT INCLUDE PEPTIDES FROM THE BASIC PROTEIN OF MYELINE AND ITS MEDICAL USES. - Google Patents

Abstract

Composition comprising the following myelin basic protein peptides: MBP 30-44; MBP 83-99; MBP 131-145; and MBP 140-154.

Description

Compositions comprising peptides of the myelin basic protein and its medical uses.

The present invention relates to a composition comprising peptides of the myelin basic protein. The composition can be used for the treatment of multiple sclerosis.

Introduction

MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is the most common disabling neurological condition that affects young adults. In the United Kingdom, approximately 85,000 people have MS.

In multiple sclerosis (MS), inflammation of nerve tissue causes the loss of myelin, a fatty material that acts as a protective isolation for nerve fibers in the brain and spinal cord. The loss of myelin, or demyelination, leaves multiple areas of scar tissue, or sclerosis, along nerve cells. Consequently, sclerosis results in multiple and varied symptoms and signs, usually with repeated remissions and recurrences.

The usual symptoms of MS include loss of vision, hesitant and uneven passage, confusing pronunciation, as well as urinary frequency and incontinence, and MS can also cause mood swings and depression, muscle spasms and severe paralysis.

Currently, it is accepted that MS is an autoimmune disease mediated by autoreactive T cells.

The usual treatments for MS generally inhibit the immune system. For example, one treatment includes bone marrow transplantation along with the administration of cytostatics and immunoinhibitory medications. This treatment is effective for some patients, but it is expensive and generally high risk. Additionally, the administration of cytostatics is considered a matter of controversy to treat MS, because its effects are unclear and the potential side effects are severe.

Interferon-beta (lFNβ) treatment reduces the symptoms of MS in some patients and is therefore widely used. However, the mechanism of action of interferon beta is unclear and treatment with IFNβ is not effective for many patients. In addition, treatment with lFN� is combined with the development of anti-lFN� antibodies in most patients (Giovannoni, G., Munschauer, FE, 3rd edition, and Deisenhammer, F (2002) " Neutralizing antibodies to interferon beta during the treatment of multiple sclerosis J. Neurol Neurosurg Psychiatry 73,465-469.

Usually, there is no effective treatment for MS. Treatment focuses solely on reducing symptoms, usually by general inhibition of the immune system. There is, therefore, the urgent need for a therapy specifically aimed at local immune responses that are associated with the onset and progression of the disease.

SYNTHETIC PEPTIDES

Metzler and Wraith (Int. Immunol. 5: 1159-1165 (1993)) were the first researchers to describe the use of synthetic peptides to induce the inhibition of an autoimmune response in the autoimmune encephalomyelitis (EAE) model, a model of MS Usually used in vivo. In this study, peptides derived from MBP were administered intranasally, finding that the level of disease inhibition correlated with the antigenic strength of the peptide used.

Later, in 1995, Liu and Wraith (Int Immunol. 7: 1255-1263) showed that it was also possible to induce EAE inhibition in mice by intraperitoneal administration of soluble MBP derived peptides. In this study, inhibition of both Th1 and Th2 responses was obtained, and it was shown that administration of peptides after the start of an immune response could lead to inhibition of the ongoing immune reaction.

However, it was discovered that not all peptides that could act as epitopes of C cells could induce tolerance. Peptide 89-101 of the myelin basic protein (MBP) is an immunodominant antigen after immunization and is also a very effective immunogen both in terms of sensitization for T cell reactivity and EAE induction. However, this peptide has proved ineffective in inducing tolerance when administered in solution (Anderton and Wraith (1998) Eur. J. Immnol 28: 1251-1261).

The present inventors have previously demonstrated that there is a relationship between the ability of a peptide to bind to an MHC molecule of type l or ll and to be presented to a T cell without antigenic processing.

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posterior, and its ability to induce tolerance in vivo. Peptides that are independent antigenic processors, (i.e., do not require antigenic processing after MHC binding), can be predicted to be tolerogenic in vivo. These peptides have been called "apitopes" by Antigen Processing Independent epiTOPES.

WO 02/16410 discloses the following apitopes of the myelin basic protein (MBP): 30-44, 8094, 83-99, 81-95, 82-96, 83-97, 84-98, 110- 124, 130-144, 131-145, 132-146 and 133-147.

WO 03/064464 identifies the following MBP peptides as apitopes: 134-148; 135-149; 136-150; 137-151; 138-152 and 140-154.

Summary of the invention

In the context of the present invention it has been discovered that a "cocktail" of four MBP peptides, all of which are apitopes, is particularly effective in treating MS. It is believed that the four peptides can exert a synergistic effect when combined with each other.

In a first aspect, therefore, the present invention relates to a composition that includes the following peptides of the myelin basic protein:

MBP 30-44 MBP 83-99 MBP 131-145; and MBP 140-154.

The composition may consist essentially of MBP 30-44, 83-89, 131-145 and 140-154.

The composition can be used to treat or prevent a disease, particularly multiple sclerosis.

The composition can be used to treat or prevent optic neuritis associated with multiple sclerosis.

In a second aspect, the present invention relates to a method for treating or preventing multiple sclerosis, and / or optic neuritis associated with multiple sclerosis, administering to a subject a composition according to the first aspect of the invention.

In the process of the second aspect of the invention, the composition can be administered according to a dose increase protocol.

It has been discovered that two of the peptides of the "cocktail" join HLA-DQ6 (MBP 30-44 and 131-145) and two (MBP 140154 and 83-99) join HLA-DR2. The joint use of these apitopes provides a wider coverage of the different haplotypes of the Major Histocompatibility Complex (MHC) that are seen in MS patients than single peptide therapy.

The method of the second aspect of the invention may involve administration of the composition to an HLA-DQ6 or HLA-DR2 positive individual.

In a third aspect, the present invention provides a kit that includes the following myelin basic protein protein peptides:

MBP 30-44 MBP 83-99 MBP 131-145; and MBP 140-154

for simultaneous administration, separately or sequentially.

Introduction to the figures

Figure 1: Presentation of MPB 30-44 by HLA-DQ6 to the EM 49: D3 T cell clone.

Figure 2: Presentation of MPB 130-144 by HLA-DQ6 to T cell clone EM 17: A2.

Figure 3: Presentation of MPB 139-153 by HLA-DR2a and HLA-DR2b L-cells transfected to the N5: 19 T cell clone

Figure 4: Proliferative response of lymph nodes after induction of tolerance with the

EM6 apitope.

Figure 5: Proliferation of splenocytes in response to the MS7 apitope. Splenocytes were obtained from mice treated by intranasal administration of PBS or the EM7 apitope (83-99).

5 Figure 6: Proliferation of splenocytes in response to the MS7 apitope. Splenocytes were obtained from mice treated by subcutaneous intradermal administration of PBS or the EM7 apitope (83-99).

Figure 7: Proliferation of lFNy and IL-2 by splenocytes obtained from mice treated by subcutaneous intradermal administration of PBS or the EM7 apitope (83-99).

Figure 8: Example of Snellens scheme.

Figure 9: PBMC responses of patients to the basic myelin protein (MBP). 15 Figure 10: PBMC responses of patients to ATX-EM-1467

Figure 11: Comparison of the proliferative response of T cells to MBP before (visit 1) and after (visit 8) of treatment with ATX-EM-1467 20

Detailed description

The first aspect of the invention relates to a composition that includes various peptides of the myelin basic protein. 25

Basic Myelin Protein

The myelin basic protein (MBP) is an 18.5 kDa protein that can be isolated from white matter in the human brain. The mature protein has 170 amino acids and the sequence is very available in the literature.

30 (see, for example, Chou et al (1986) J. Neurochem. 46: 47-53, Figure 1; Kamholz et al (1986). PNAS 83: 49624966, Figure 2; US Patent No. 5,817,629, SEQ ID No. 1; Roth et al) (1987), J. Neurosci. Res. 17: 321-328, Figure 4; Medeveczky et al (2006), FEBS Letters 580: 545-552, Figure 3B)

MBP PEPTIDES

35 The term " peptide " it is used in the normal sense to mean a series of residues, typically L-amino acids, typically linked to each other by peptide bonds between the a-amino group and the carboxy groups of adjacent amino acids. The term includes modified peptides and synthetic synthetic peptides.

The peptides used in the composition and kit of the present invention can be prepared using chemical methods (Peptide Chemistry, Apractical Textbook. Mikos Bodansky, Springer Verlag, Berlin). For example, the peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), fragmented from the resin, and purified by preparative high performance liquid chromatography (for example, Creighton ( 1983), Proteins Structures And Molecular Principles, WH Freeman and Co,

45 New York, NY). Automated synthesis can be obtained, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

Peptides can alternatively be prepared by recombination or fragmentation procedures of a longer polypeptide. For example, the peptide can be obtained by fragmentation from the full length MBP. The composition of a peptide can be confirmed by amino acid analysis or sequencing (for example, the Edman degradation procedure)

The peptides used in the composition and kits of the present invention are the following:

55 MBP 30-44:

H-Pro-Arg-His-Arg-Asp-Thr-Gly-Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-NH2

MBP 83-99: 60 H-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro-NH2

MBP 131-145:

65 H-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Phe-Lys-Gly-Val-Asp-NH2 MBP 140-154:

H-Gly-Phe-Lys-Gly-Val-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ile-Phe-NH2

5 The terms " MBP 30-44 ", " MBP 83-99 ", " MBP 131-145 " and "MBP 140-154" They comprise modified peptides. For example, the peptides may be mutated, by insertion, deletion or substitution of amino acids, provided that the MHC binding specificity of the unmodified peptide is preserved, together with its ability to be presented to a T cell. The peptide may, for example , have 5, 4, 3, 2, or 1 or 0 mutations of the unmodified sequence.

Alternatively (or in addition), modifications can be made without changing the amino acid sequence of the peptide. For example, D-amino acids or other unnatural amino acids may be included, the normal amide linkage can be replaced by bonds of ester or alkyl structure, N- or C-alkyl substituents, side chain modifications, restrictions such as disulfide bridges may be included or ester or side chain joints

15 amidics Such changes can lead to greater stability in vivo of the peptide, and to a longer duration of its life.

The modification of epitopes can be carried out in predictions for a more efficient induction of T cells, obtained using the program "Peptide Binding Predictions" conceived by K. Parker (NIH), which can

20 can be found at http://www-bi-mas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform (see also Parker. K.C et al, 1994. J. Immunol. 152: 163).

MBP peptides can be formulated in the composition in the form of salts or in a neutral form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the peptide) and

Which are formed with inorganic acids, such as, for example, hydrochloric acid, or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or maleic acid. The salts formed with the free carboxylic groups can also be obtained from inorganic bases such as, for example, sodium, potassium, calcium ammonium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine

30 COMPOSITION

The composition of the present invention may be for therapeutic or prophylactic use.

The composition can be prepared as an injectable, either as a liquid solution or as a suspension; an appropriate solid form can also be prepared for the solution in, or the liquid suspension before injection. The preparation can also be emulsified, or the peptides encapsulated in liposomes. The active substances can be mixed with the excipients that are pharmaceutically acceptable and compatible with them. Appropriate excipients are, for example, water, saline solutions, (for example, phosphate buffered saline),

40 dextrose, glycerol, ethanol, or the like and their combinations.

In addition, if desired, the composition may contain smaller amounts of auxiliary substances, such as wetting or emulsifying agents, and / or pH buffering agents. Buffer salts include phosphate, citrate, acetate. Hydrochloric acid and sodium hydroxide can be used for pH adjustment. For stabilization,

Disaccharides such as sucrose or trehalose can be used.

In the composition, the relative proportion of the peptides (MBP 30-44; MBP 83-99, MBP 131-145; MBP 140-154) may be approximately 1: 1: 1: 1. Alternatively, the relative proportions of each peptide can be altered, for example, to center the tolerogenic response in a particular subset of the T cells.

50 self-reactive, or if one peptide is found to exert its function better than the others, in particular HLA types.

After formulation, the composition can be incorporated into a sterile container that is then sealed and stored at low temperature, for example 4 ° C, or it can be lyophilized.

Conveniently, the composition is prepared as a lyophilized powder (freeze-dried). Freeze drying allows long-term storage in a stabilized manner. Freeze-drying procedures are well known in the art, see, for example http://www.devicelink.com/ivdt/archive/97/01/006.html. Dough-producing agents are commonly used before lyophilization such as mannitol, dextran or glycine.

The composition can be administered in a convenient manner such as by the oral, intravenous (if water soluble), intramuscular, subcutaneous, sublingual, intranasal, intradermal or rectal routes, or using an "implant" (for example, using slow release molecules).

The composition can be advantageously administered intranasally, subcutaneously or intradermally.

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The method and pharmaceutical composition of the invention can be used to treat a human individual. Typically, a doctor will determine the most appropriate actual dose for an individual, which will vary with the age, weight and response of the particular patient.

In a preferred embodiment, a "dose increase" protocol can be followed if various doses are administered to the patient in ascending concentrations. Such an approach has been used, for example, for phospholipase A2 peptides in immunotherapeutic applications against bee venom allergy (Muller et al, (1998), J. Allergy Clin Immunol. 101: 747-754, and Akdis et al (1998), J. Clin. Invest. 102: 98-106.

KITS

Conveniently, the four MBP peptides can be administered together, in the form of a mixed composition or cocktail. However, it may be circumstantially preferred to provide the peptides separately, in the form of a kit, for simultaneous, separate, sequential or combined administration.

For example, the kit may include all four peptides in separate containers, or two containers, each comprising two peptides. The contents of the containers may or may not be combined before administration.

The kit may also include mixing and / or administration means (for example, a vaporizer for intranasal administration, or a syringe and needle for subcutaneous / intradermal dosing. The kit may also include instructions for use.

The pharmaceutical composition of the kit of the invention can be used to treat and / or prevent a pathology.

In particular, the composition / kit can be used to treat and / or prevent multiple sclerosis and / or optic neuritis.

MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is the most common neurological disorder among young adults (20 to 40 years old), which affects approximately 385,000 people in Europe and 300,000 in the USA. It is a chronic degenerative disease of the central nervous system, in which there is a gradual destruction of myelin in places through the brain and / or spinal cord, interfering with neural connectivity and causing muscle weakness, loss of coordination and alterations. Visual and speech.

After 10 years, approximately half of the individuals who were initially diagnosed with multiple sclerosis with relapses-EM remissions (RREM) found that the frequency of recurrences decreases, but that the disability increases. This is known as progressive secondary MS (SPEM). Some estimate that within 25 years, approximately 90% of the population with RREM will progress to the progressive secondary type. As with RREM, progressive secondary MS can vary greatly. For some patients, the increase or progression of disability is very gradual, and for others, it can take place more quickly. In general, however, recovery from attacks is more and more incomplete, and symptoms tend to increase, increasing disability. Clinical attacks become less pronounced, and remissions tend to disappear, but at this time more CNS tissue has been destroyed and the damage that has accumulated is more clearly seen in MRIs.

The composition of the invention can be used to treat a patient having a recent onset of MS, RREM or SPEM.

The composition of the present invention can reduce or improve one or more of the symptoms of MS, including loss or reduction of vision, hesitant and uneven passage, confusing pronunciation, urinary frequency and incontinence, mood swings and depression. . SPEM may be associated with muscle spasms and severe paralysis.

In particular, the composition can improve optic neuritis.

OPTICAL NEURITIS

Optic neuritis (ON) is an inflammation, accompanied by demyelination, of the optic nerve that innervates the retina of the eye. It constitutes a variable condition and can present with some of the following symptoms: blurred vision, loss of visual acuity, loss of some or all of the colored vision, total or partial blindness and pain behind the eye.

Optic neuritis is one of the most frequent symptoms of multiple sclerosis and is the most common symptom of the onset of MS. ON can be attributed, however, to causes other than MS, such as ischemic optic neuropathy.

ON occurs unilaterally (only in one eye) in 70% of cases.

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Very typically, optic neuritis first affects people between 15 and 50 years of age. In this age group, studies indicate that more than 50% of patients will suffer from multiple sclerosis within 15 years. As with MS, women have approximately twice as much as men, more likely, ON, and the prevalence among Caucasian peoples is higher than in other ethnic groups.

The main symptoms of optic neuritis are:

-

Loss of visual acuity (blurred vision)

-Eye pain. -Dyschromatopsia (reduced color vision)

-

Phosphenes of movement and sound (sensations of visual lightning obtained by eye movements from side to side or sound).

-

Uhthoff symptom, worsening of symptoms with heat or exhaustion.

Treatment of ON with a composition according to the present invention can prevent, reduce or improve any of these symptoms. To control the progression of ON, visual acuity can be conveniently measured using a Snellens scheme.

Examples

The following examples are provided by way of illustration and not limitation of the present invention. The invention particularly relates to the specific embodiments described in these examples.

For the purpose of the examples, the following names may be used for MBP peptides:

MBP peptide

Name in the Examples (interchangeable)

30-44

ATX-EM-01 apitope EM 1

83-99

ATX-EM-07 apitope EM7

131-145

ATX-EM-04 apitope EM4

140-154

ATX-EM-06 apitope EM6

The composition of the four peptides is called ATX-EM-1467.

Example 1: Identification of T cell epitopes with the myelin basic protein limited to HLA-DQ6.

Type II MHC genes confer susceptibility to multiple sclerosis (Nepom and Ehrlich (1991), Ann.Rev.Immunol. 9: 493-525). In Caucasians in North and Central Europe, Australia and North America, the association is related to the HLA-DR2 molecule (DR2a [DRB5 * 0101] and DR2b [DRB1 * 1501] of MHC. Haines et al (1998) , Human Mol. Genet. 7: 1229-1234). Although the DR2 and DQ6 alleles of MHC are found in different loci, there is a significant unstable equilibrium between the two alleles. The concordance between the two alleles is 99%, much greater than expected if the alleles had been randomly reassociated. Therefore, the possibility arises that certain T-cell epitopes with MS may be limited to HLA-DQ6, rather than HLA-DR2.

The objective of this study was to identify whether the MBP epitopes of human T cells are presented by the HLA-DQ6 molecule to the T cell clones isolated from the HLA-DR2 EM positive patients. T cell activation was detected by proliferation, using the incorporation of [3H] radioactive thymidine.

Peptide antigens

MBP peptides 30-44, 130-144 and 139-153 were synthesized using L-amino acids and standard F-moc chemistry in an Abimed AEM 422 multiple peptide synthesizer.

Antigen presenting cells

L-cells transfected with HLA-DQ6, HLA-DR2a or HLA-DR2b, or Mgar cells (EACC, Porton Down, UK), which express 1 HLA-DQ, DP and DR, were used as APC.

T cell clones

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The EM-49: D3 and EM 17: A2 clones of T cells were generated from the EM patients and the N5: 19 clone was generated from a normal individual. All three clones were isolated from DR2 positive individuals.

Antigenic presentation test

Antigen presenting cells were incubated with the different concentrations of the peptide and the appropriate T cells. Proliferation and, therefore, the activation of T cells, was measured by the incorporation of [3 H] thymidine, expressed as the stimulation index (SI = counts corrected per minute (ccpm) of the culture containing the peptide / ccpm of the culture without peptide).

Results

When MBP 30-44 peptide was presented by L cells transfected with HLA-DQ6, the EM 49: D3 clone of T cells resulted in a very intense proliferative response (1.5 times higher than Mgar control cells) to the highest peptide concentration (Figure 1). When MBP 130-144 was presented by L cells transfected with HLA-DQ6, an even greater response was induced in EM 17: A2-T cells (a 3.24 fold increase in proliferation, compared to control cells Mgar-Figure 2).

A third T cell clone isolated from an individual DR2 N5: 19 responded to the main epitope MBP: 140-154 and to a series of peptides with 15 polymer units that overlap and cover this region (138-156). Peptide 139-153 stimulated the proliferation of the N5: 19 T cell clone when it was presented by L cells by HLA-DR2a, but not by T cells transfected by HLA-DR2b (Figure 3).

conclusion

T-cell clones isolated from HLA-DR2 positive individuals respond to the MBP epitopes limited to HLA-DQ6 of T cells. This implies that the association of HLA-DR2 with multiple sclerosis is not limited to DR2 restriction. of the MBP epitopes of T cells, but also that DQ6 is limited.

In the MBP composition of the present invention, two of the peptides bind HLA-DQ6 (MBP 30-44 and 131145) and two bind HLA-DR2 (MBP 140-154 and 83-99), therefore, Peptide therapy with these apitopes can be well targeted to HLA-DR2 or HLA-DQ6 individuals.

Example 2: Induction of tolerance with the EM6 apitope in an HLA: DR2 transgenic mouse.

The study was designed to demonstrate that an EM6 apitope, when presented by an MHC type II molecule, can induce immune tolerance in a murine humanized model of multiple sclerosis. The EM6 apitope is an apitope selected from the epitopes of the MBP T cells corresponding to MBP 140-154, and is presented by MHC type ll in the presentation cells, being able to stimulate the T cells without being processed (see WO 03/064464). The murine model used was transgenic for the human MHC HLA: DR2 molecule (DRB1 * 1501) (Madsen et al (1999), Nature Genetics 23: 343-347).

The induction of energy or changes in the CD4 + T cell population in a mouse after administration of an apitope can be controlled by a reduction in the proliferation of T cells when stimulated in vivo with an antigen.

Peptide synthesis

The EM6 apitope (MBP peptide 140-154) was synthesized using L-amino acids and standard F-moc chemistry in an Abimed AEM 422 multiple peptide synthesizer.

Mice and tolerance induction

In the study, HLA DR2 transgenic mice with an age of 8 to 12 weeks were used. Tolerance was induced in mice by pretreatment with 100 1g of apitope EM6 in 25 1l of phosphate buffered saline (PBS) or 25 1l of PBS only intranasally, on days -8, -6 and -4, before of immunization on day 0.

After tolerance induction, the mice were immunized subcutaneously with 100 µl of an emulsion containing an identical volume of Freund's complete adjuvant (CFA) and PBS containing 200 µg of EM6 apitope and 400 µg of heat-removed Mycobacterium tuberculosis. A control group of the animals, which were previously induced intranasally tolerance with PBS, were immunized without the peptide.

Ten days after the subcutaneous immunization, the draining popliteal and inguinal lymph nodes were removed, determining the activation of the T cells by verifying the proliferation of the T cells in response to various concentrations of the EM6 apitope. Proliferation was determined by incorporating [3 H] thymidine, which was expressed as a stimulation index (SI = counts corrected per minute (ccpm) of the culture containing the peptide / ccpm of the culture

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without peptide).

Results

Group A of mice that was induced to tolerance with PBS and then immunized with apitope EM6, responded to antigenic stimulation when they were re-stimulated with apitope EM6 in a dose-dependent manner (Figure 4). With an increase in the concentration of the peptide, Sl increased an average of 2.5 to 10. All mice in this group demonstrated that intranasally administered PBS could not induce tolerance for the EM6 apitope.

Contrary to this, intranasal pretreatment with the EM6 apitope had a profound effect on the proliferative response of lymphocytes stimulated with this peptide. The lymphocytes of group B mice could not respond to any significant degree, even at the high peptide concentration of 150 1g / ml (mean 3 of Sl, figure 4). These data suggest that apitope EM6 induced lymphocyte tolerance in HLA-DR2 mice.

Lymphocytes extracted from mice that had been pretreated and immunized with PBS (Group C), showed no response to the EM6 apitope (Figure 4). This lack of response to the peptide within Group C confirms that the proliferative response seen in Group A truly constituted a response to immunization with the EM6 apitope.

conclusion

These data support the hypothesis that an MBP (apitope EM6) peptide that does not require processing and binds to HLA molecules: DR2 MHC type ll, can induce tolerance when administered intranasally.

Example 3: Induction of tolerance with the EM7 apitope (MBP 83-99) in doubly transgenic mice showing HLA: DR2 and the T-cell receptor (MBP) 82-100

This study investigates the ability of the apitope EM7 (MB 83-99 peptide) to induce tolerance in doubly transgenic mice expressing HLA: DR2 together with the T-cell receptor for peptide (MBP) 82100 of the myelin basic protein bound to HLA: DR2.

The splenocytes of these doubly transgenic animals proliferate in vitro in response to the EM7 apitope. The reduction of the cancellation of the splenocytic response in vitro to an apitope after repeated administration in vivo indicates that a state of tolerance was achieved.

The induction of tolerance has been achieved through the use of both the nasal and the subcutaneous-intradermal routes of apitope administration.

Tolerance induction

Groups of 6 or 7 double transgenic mice matched according to sex (8-12 weeks old) were used. In the first experiment, a group was treated intranasally ten times with 100 µg of the EM7 apitope, in 25 µl of PBS at regular intervals for three weeks. The control group received only PBS. In the second experiment, the same amount of the peptide was provided by subcutaneous / intradermal administration in 100 µl of PBS. The control group received the same volume of PBS.

Proliferation test

Three days after the last administration of the peptide or PBS, the spleens were collected and cell suspensions were prepared. Splenocytes were cultured with 0.5 or 5 1g / ml of the EM7 apitope, and were evaluated for proliferation by incorporation of [3 H] thymidine after 48, 72 and 96 hours of culture. The results are expressed as stimulation indices (SI = counts per minute (cpm) of the culture containing antigen / counts per minute without antigen).

Cytokine measurement

In the second experiment, where tolerance induction was induced by the subcutaneous / intradermal route, the cell culture supernatants of the splenocytes were recovered at 48 and 72 hours of the culture, and were evaluated for the presence of lFN- and e interleukin-2 (lL-2), using an adsorption enzyme immunoassay assay (ELISA).

Results

In the three moments that were investigated, the splenocytes of mice treated with PBS both intranasally and subcutaneously-intradermally, showed an intense proliferation of the EM7 apitope, increasing the magnitude of the response with the peptide concentration. A surprising reduction in proliferation was observed in splenocyte cultures of mice treated with the EM7 apitope by both routes of administration. As shown in Figure 5, after 72 hours the average Sl that was observed with cultures containing 5 1g / ml of the EM7 apitope was 17 in the mice intranasally treated with the EM7 apitope. In the corresponding cultures of the control mice treated with PBS, an average SI of 89 was observed. Similarly, such

5 as shown in Figure 6, administration of the EM7 apitope by the subcutaneous-intradermal route resulted in a decrease in the average Sl, from 124 in animals treated with PBS to 49 in animals treated with the EM7 apitope.

The levels of IFNy and IL-2 that were detected in the culture supernatants of 48 hours, are shown in the

10 Figure 7. It is appreciated that repeated treatment in vivo with the peptide results in an impressive reduction in the reduction of both cytokines, accompanying the reduction in the proliferative response.

conclusion

15 These results indicate that tolerance to the EM7 apitope can be successfully induced in a murine model designed to mimic the human system, supporting its potential for use in multiple sclerosis therapy. In addition, its effectiveness is maintained when administered by the subcutaneous / intradermal route, the route of choice for man.

Although the in vivo efficacy of the EM6 and EM7 apitopes has been demonstrated, it is not currently possible to demonstrate the

20 efficacy of apitopes EM1 and EM4. This is due to the lack of expression of the HLA-DQ6 molecule by transgenic mice. Mice expressing the molecule have been created, but it has been found that they cannot generate CD4-T cells in the thymus, and therefore do not organize an immune response to the antigen in the context of HLA-DQ6.

Example 4: Study of dose increase with a composition of four MBP peptides.

The combination of four peptides (MBP 30-44, 131-145, 140-154 and 83-99) was used as an open dose increase study of ATX-EM-1467 in patients with secondary progressive multiple sclerosis.

30 Patients were monitored at four weeks and three months after the final dose of the study for visual acuity (ie, reduction in optic neuritis), immunological parameters and CNS inflammation.

Example 4A - Visual acuity control

35 Optic neuritis (ON) is an inflammation, with accompanying demyelination, of the optic nerve that supplies the eye's retina. ON constitutes one of the most common symptoms in multiple sclerosis, and is the most common initial symptom in this disease.

Typical symptoms of ON include: blurred vision, loss of visual acuity, loss of some or all of the color vision 40, partial or total blindness and pain in the back of the eye.

The effect of treatment with ATX-EM-1467 on the optic neuritis resulting from the neuroinflammatory process involved in MS was investigated. ATX-EM-1467 was administered following the dose increase protocol indicated above, thus determining the patient's visual acuity one month after

45 treatment, using a standard Snellens scheme (Figure 8).

The results show a clinically significant improvement in visual acuity one month after treatment. This was demonstrated in the analysis of the initial visual acuity test that was performed by screening with the test that was performed the following month. The initial screening of the 6/24 and 6/9 sight measurements (in the

50 right and left eye respectively) improved to 6/9 (right eye) and 6/6 (left eye) after treatment. The patient's eye vision had not previously changed during the past two years.

Example 4B-Control of immunological parameters.

55 As explained above, the effect of peptide therapy with a cocktail containing four apitopeTM peptides of the myelin basic protein (ATX-EM-1467) was investigated in patients with MS. Each patient underwent a screening at the beginning of the test, up to 14 days before the final dose (visit 1). The first dose of 25 1g of ATX-EM-1467 was administered at visit 2, 50 1g at visit 3, 100 1g at visit 4, 400 1g at visit 5 and 800 1g at visits 6 and 7. carried out a further examination after one month, and three monthly follow-ups (visits

60 8 & 9). The following table summarizes the protocol and shows the number of visits to the clinic for each patient, along with the dose of the peptide and the blood sample.

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TABLE 1

VISIT NUMBER

DOSE OF ATX-EM-1467 (μg) COMMENTARY

one

- Pre-visit 50 ml of the blood sample collected for immunology

2

25 No sample was collected for immunology

3

fifty 50 ml of the blood sample for immunology

4

100 50 ml of the blood sample for immunology

5

400 50 ml of the blood sample for immunology

6

800 50 ml of the blood sample for immunology

7

800 50 ml of the blood sample for immunology

8

- 50 ml of the blood sample for immunology taken four weeks after the visit 7.

Blood samples were collected at visits 1 and 3 to 9, and tested for immune responses to different antigens that included the purified protein derivative (PPD) of Mycobacterium tuberculosis, as a positive control, the protein Basic purified human myelin (MBP) and ATX-EM-1467. The immune responses that should be determined included in vitro proliferation of T cells, cytokine secretion in tissue culture supernatants and cytokine RNA generation.

Results

A significant response to MBP was observed, with a peak in proliferation at visit 3 (Figure 9). This was correlated with a peak in gamma interferon secretion. Importantly, however, the level of gamma interferon secretion fell after the third visit and reached the previous level in visit 8. Levels of lL-10 did not change significantly until visit 7, at which time a significant increase in the secretion of this cytokine. The levels of lL-4, lL5 and TNF alpha were approximately the previous one in response to MBP at all times.

No proliferative responses or an increase in cytokine responses to ATX-EM-1467 were observed (Figure 10)

conclusion

At visit three, a response to MBP was observed, demonstrating a climax in the secretion of lL-2 and interferon gamma that correlates with a peak in proliferation at this time. This increase in response is believed to be attributable to the administration of peptides.

However, importantly, the transient increase in cytokine secretion followed a return to gamma interferon base levels. There was also a significant increase in the secretion of IL-10 after the second administration of ATX-EM-1467 at the highest dose. Previously, it has been reported that, in animal models, specific immunotherapy with synthetic peptides is effective, leading to the induction of regulatory T cells secreting lL-10. The induction of regulatory T cells that secrete lL-10 in mice, implies a transient response to peptides, with secretion of gamma interferon at low levels (Burkhart et al, 1999 Int. Immunol 11: 1625-1634; Sundstedt et al ., 2003 J Immunol 170: 1240-1248). This is followed by a decrease in gamma interferon and a concomitant increase in 10. The kinetics of cytokine secretion shown after treatment with ATX-EM-1467 effectively reproduces the pattern previously observed in experimental mice, suggesting that ATX-EM-1467 can induce regulatory T cells that secrete IL-10.

Example 4C- The response to MBP is significantly down-regulated downwards by treatment with ATX-EM-1467.

Six patients with multiple secondary progressive sclerosis (SPEM) signed up for and completed a clinical trial of phase l / lla following the dose increase protocol provided in Example 4B.

The HLA genotyping showed that the six patients who signed up for the clinical trial represented a wide variety of HLA haplotypes, including the HLA-DR15 haplotype associated with MS. The wide distribution of HLA-DRB1 represented by the patients who signed up for this test implies that ATX-EM-1467 will be safe and well tolerated in MS patients regardless of their HLA-DR genotype.

The treatment of patients with ATX-EM-1467 did not generate antipeptide antibodies. This indicates that the use of ATX-EM-1467 is safe and correlates with the lack of complications of the injection site or the allergic manifestations associated with the trial.

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A complete analysis of the T-cell responses showed that:

a) Treatment with ATX-EM-1467 did not have a non-specific immunosuppressive effect. This is clearly shown.

due to the fact that the immune response to the purified protein derivative, an antigen from Mycobacterium

tuberculosis was maintained (data not shown).

b) Treatment with ATX-EM-1467, using the protocol, did not lead to an aggressive immune response to ATX-EM-1467 or myelin basic protein (data not shown).

c) Comparison of the proliferative response of T cells before (visit 1) and after treatment (visit 8) with ATX-EM-1467, revealed a significant reduction in the response to myelin basic protein (Figure 11). ). The three individuals who responded to MBP in V1 all showed a reduction in the response to protein by V8. Considering all patients together, there is evidence for a significant reduction in the response to MBP from V1 to V8 (P = 0.0313).

Materials and methods

Formulations and doses

Each of the four peptides is independently prepared under contract using solid phase peptide synthesis, purified using HPLC. They are freeze dried.

ATX-EM-1467 is a 1: 1: 1: 1 mixture of EM1, EM4, EM6 and EM7 apitopes in phosphate buffered saline for intradermal administration.

Two intensities of ATX-EM-1467, designated ATX-EM-1467A and ATX-EM-1467B, which contained 4 mg / ml and 0.5 mg / ml of the peptide, respectively, were prepared to allow dose increase. The regimen uses five injections of dose increase, (25, 50, 100, 400 and 800 1g of total dose) that are administered 7 to 14 days apart. Patients then receive a second dose of 800 1g, 7 to 14 days after the first dose of 800 1g.

After receiving all six doses of the study medication, the patient is evaluated at four weeks and three months after that final dose.

Visual acuity test

A standard-sized Snellens scheme was used to carry out the trial, with a slight posterior illumination and the patient being located 6 meters away (Figure 8).

Each eye was examined separately with respect to the lowest line in the scheme that the patient could read. This was then noted as visual acuity = 6 / (the line that the patient has been able to read).

Immunoassays

i) Proliferation of T cells

Cryopreserved PBMC was placed in 1 ml cultures containing 1.5 x 106 cells in a-MEM, in 48-well tissue culture plates (Nunc International, Costar, Corning Inc, New York, USA). Responses to MBP and peptide antigens at various concentrations were monitored over a period of 10 days. Control wells did not contain antigens. After 20 hours or 2, 4, 6, 8 and 10 days of culture, duplicate 100 µl aliquots of the cell suspension were removed from each of the 1 ml cultures, in order to measure proliferation in response to the antigen by absorbing [3H] thymidine.

ii) Measurement of secreted cytokines (IL-2 and IL-4, IL-5, IL-10, TNF-a and IFN): Cytometric test of the microsphere set:

The cytokine levels of the culture supernatant were determined by the cytometric microsphere assay (Becton Dickenson Biosciences, Cowley, Oxford, United Kingdom), following the manufacturer's instructions. After obtaining the sample data using the FACS Calibur (BD Biosciences), the results were generated graphically and tabularly using BD CBA software. The minimum quantifiable levels of cytokine were as follows: IL-2 and IL-4, 2.6 pgml-1, IL-5 2.4 pgml-1, IL-10 and TNF-a 2.8 pgml-1, IFN -and 7.1 pgml-1.

Quantitative real-time PCR to measure cytokine RNA (IL-2, IL-10, IFN-v and TNF-a

For the analysis of IL-2, IL-10, IFN-y and TFN-a, PCR reactions were carried out in a final volume of 20 l which

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contained a mixture of cDNA, PCR buffer, 6.25 mM MgCl2, 0.4 mM dNTP, direct and reverse primers (direct primer concentrations: IL-2 300 nM, IL-10 600 nM, IFN-y 600 Nm, TNF -a 600 nM, reverse primer concentrations: IL-2 600 nM, IL-10 900 nM, IFN-y 900 Nm, TNF-a 600 nM), 200 nM FAM-TAMRA probe, and 0.05 U1l-1 of Platinum Taq polymerase (Invitrogen). Cycling conditions were: an initial stage of denaturation at 94 ° C for 30 seconds, followed by 35 cycles with 15 s at 94 ° C and 1 min at 60 ° C. For -2 microglobulin, the PCR mixture described above was supplemented with 0.1 uM of direct and inverse primers, 3 mM MgCl2, quantified with SYBR Green 1 (Molecular Probes Inc, dilution X30000 of the stock). After a 1 minute denaturation step at 95 ° C, amplification continued with 35 cycles of 15 s at 94 ° C, 1 min at 61 ° C and 1 min at 72 ° C. PCR reactions and fluorescence detection of the generated amplicons was performed in an Opticon 2 system (MJ Research, USA). The base fluorescence was established by making measurements from cycle 1 to 10. Ct values were calculated by determining the point at which the fluorescence exceeds the standard deviation of the baseline value from 8 to 10 times. The samples were tested in duplicate, calculating the number of copies from a standard curve for each target DNA. Differences in the number of RNA copy entries and cDNA synthesis were corrected by normalizing cytokine expression to the expression of -2 microglobulin.

HLA typing

The analysis of the expression of the HLA gene was carried out by a standard single-chain conformation polymorphism, the technique of polymerase chain reaction on DNA extracted from leukocytes from peripheral blood. The HLA type of each patient was used to interpret the results of immunological tests.

Serum Antiseptic Antibody Assays

96-well plates were coated with 1-10 1g / ml of each EM1, EM4, EM6 or EM7 apitope at pH 9.6, overnight, at 4 ° C. The plates were washed four times with phosphate buffered saline, pH 7.2, 0.05% Tween (PBS-Tween), blocking the wells with 5% FCS in PBS for 1 hour at room temperature. Serums were diluted 1: 100 in PBS-Tween and incubated in duplicate wells for one hour at room temperature. After 4 washes, the goat-horseradish peroxidase (Sigma) anti-human IgG conjugate diluted 1: 12000 in PBS was added to each well, the plates were incubated for one hour at room temperature. After 4 washes, 0.4 mg / ml o-phenylenediamine dihydrochloride (Sigma) plus 30% hydrogen peroxide was added, and incubated at room temperature for 15-20 minutes. Color development was stopped with 2.0 M H2SO4 (50 1l) and optical density (OD) values measured at 490 nm with a plate ELISA reader. The results were provided as OD for 1: 100 diluted serum.

MRI scan

Inflammation in the CNS is investigated using a gadolinium-enhanced MRI scan. The volume and number of lesions that underwent enhancement are examined and compared with the base scan.

Claims (8)

1. Composition comprising the following myelin basic protein peptides: 5 MBP 30-44; MBP 83-99; MBP 131-145; Y MBP 140-154. Composition according to claim 1, consisting essentially of the following myelin basic protein peptides MBP 30-44; MBP 83-99; 15 MBP 131-145; and MBP 140-154. 3. Composition according to claim 1 or 2, for use in the treatment or prevention of a disease. twenty 4. Composition according to claim 1 or 2, for use in the treatment or prevention of multiple sclerosis. 5. Composition according to claim 1 or 2, for use in the treatment or prevention of optic neuritis associated with multiple sclerosis. 6. Use of a composition according to claim 1 or 2, in the preparation of a medicament for treating multiple sclerosis. Use of a composition according to claim 1 or 2, in the preparation of a medicament for treating optic neuritis associated with multiple sclerosis. 8. Composition for use according to claim 3 or 4, for administration following a protocol of dose increase 35

9.

 Composition for use according to claim 3 or 4, for administration to an HLA-DQ6 or HLA-DR2 positive individual.

10.

 Kit comprising the following myelin basic protein peptides:

40 MBP 30-44; MBP 83-99; MBP 131-145; and MBP 140-154 45 for simultaneous administration, separately or sequentially