RELATED APPLICATIONS

This application is a national stage application filed under 35 U.S.C. § 371 of PCT/EP2017/077968, which claims priority from GB1618432.7, the content of which is hereby expressly incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Mar. 22, 2021, as a text file named “10480-004US1 2021_03_22 Sequence_Listing.txt,” created on Mar. 22, 2021, and having a size of 19 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to methods of detecting, diagnosing and distinguishing between demyelinating diseases such as Multiple Sclerosis. The present invention also relates to methods of treating, and compositions for use in the treatment of, demyelinating diseases such as Multiple Sclerosis.

BACKGROUND OF THE INVENTION

Demyelinating diseases are conditions in which the myelin sheath of neurons is damaged. In some demyelinating diseases, termed inflammatory demyelinating diseases (IDDs), autoimmune reactions are thought to be involved in the myelin damage associated with disease. Devic's disease (neuromyelitis optica) is one example of inflammatory demyelinating diseases, which manifests as an inflamed and demyelinated optic nerve.

In contrast, leukodystrophies, also known as dysmyelinating diseases, are genetic disorders characterized by degeneration of myelin. One example is Pelizaeus-Merzbacher disease (PMD), in which a mutation in a myelin protein called proteolipid protein 1 (PLP1) cause the inability of the myelin formation. In some forms of these diseases, part of PLP is not produced.

Multiple Sclerosis (MS) is a demyelinating disease which manifests as a lifelong neurological condition. An estimated 2.5 million people suffer from MS worldwide, with more than 127,000 people in the UK in 2010, and this number is growing by approximately 2.4% per year. The MS incidence is reported as 165, 140, 174, and 212 (per 100,000) in England, Wales, Northern Ireland, and Scotland, respectively (1,2). MS is also widely considered to be an IDD, although several theories exist as to the mechanism undelying demyelination and inflammation in MS. The damage to the central nerves system (CNS) in MS seems to be due to the immune system mistakenly attacking the myelin sheath (i.e., an autoimmune attack), thereby affecting the brain and spinal cord. Other theories including damage to, or deficiency of, the myelin-producing cells. The cause of the demyelination seen in MS is unknown.

The diagnosis of demyelinating diseases such as MS often takes many years, there are many variables involved and there is no single pattern to the disease. The underlying cause of MS is believed to be a combination of genetic, immunological and environmental factors. However, as disease progression and the course of the disease is unpredictable, until now it has been impossible to determine a specific cause or trigger.

The following observations have been made about the symptoms and incidence of MS:The number and position of lesions on a patient's central nervous system does not necessarily correlate with their relapse occurrence or level of disabilityThe most common MS symptoms are fatigue, pain, bladder and bowel issues, sexual dysfunction, movement and coordination problems, visual problems and cognition and emotional changesMS is about two times more prevalent in women than in menMS is the most common disease of the CNS in young adults, mostly diagnosed in their 20-30sMS is not directly hereditary, although genetic susceptibility plays a part in its developmentMS is not contagious or infectiousThe incidence of MS increases in countries further from the equatorEveryone's MS is different, hence no two individuals will have the same range and severity of symptoms, even if they are closely related.

Currently, there are no symptoms, physical findings or laboratory tests that can conclusively prove or rule out MS. The diagnostic process currently involves a combination of examinations:Neurological examination: the neurologist looks for evidence of two or more areas of scarring in different parts of the central nervous system that have occurred at different points in time.Blood test: while there is no definitive blood test for MS, blood tests are often used to rule out other conditions that cause symptoms similar to those of MS.Magnetic resonance imaging (MRI): the scars caused by MS show up as white patches, giving a clear picture of the effects of MS on the brain and spinal cord.Lumbar puncture: inserting a hollow needle into the base of the spine and drawing off a sample of cerebrospinal fluid (CSF) for analysis.Evoked potentials: to measure the speed of nerve messages along sensory nerves to the brain. Delays in messages, which are often not apparent to the individual being tested, can indicate that there is damage to the nerve pathway.

No individual symptom is unique to the condition, which means that in order to reach a diagnosis, a range of other possible explanations have to be ruled out, resulting in a lengthy diagnosis process.

Current Treatments

There is currently no cure for MS. A limited number of treatments are available to manage MS symptoms and their impacts on a patient's life by modifying the course of the disease. Disease modifying drugs can reduce the frequency and severity of relapses, but are not effective in treating primary progressive MS. The only available treatments aim to alleviate symptoms, for instance medication for seizures. Although research into genetic and stem cell treatments of demyelinating diseases is ongoing, no effective treatments of the underlying diseases are currently available.

SUMMARY OF THE INVENTION

The inventors have identified peptide antigens from diverse proteins that play an important role in MS development. Based on these findings, the present invention provides assays, which can quantify the level of particular antibodies in blood samples and which can be used to distinguish demyelination disease patients from subjects that do not have a demyelinating disease, such as patients that have a dysmyelinating disease. Based on these findings, the present inventors also provide novel animal models of demyelinating diseases and novel therapies for demyelinating diseases.

The central feature of the invention is a 4-amino acid (aa) motif. In some of the proteins that play an important role in MS development, the 4-aa motif is present as an “AHSL” motif. In other proteins that play an important role in MS development, the 4-aa motif is present as an “ADSL” or a “DDSL” motif.

Accordingly, in a first aspect, the invention provides an oligopeptide comprising the sequence “ADSL” (SEQ ID NO: 4).

The 4-aa motif of the invention (ADSL) belongs to a broader class of oligopeptides comprising the sequence “Z1Z2SL” (SEQ ID NO: 1). In most embodiments, residue “Z1” represents “A” (alanine), so the oligopeptide of the invention comprises the sequence “AZ2SL” (SEQ ID NO: 2). In other embodiments, residue “Z1” represents “D” (aspartic acid), so the oligopeptide of the invention comprises the sequence “DZ2SL” (SEQ ID NO: 3). In some embodiments, residue “Z2” is “D” (aspartic acid), so the oligopeptide of the invention comprises the sequence “ADSL” (SEQ ID NO: 4). In other embodiments, residue “Z2” is “H” (histidine), so the oligopeptide of comprises the sequence “AHSL” (SEQ ID NO: 5).

The 4-aa motif of the invention may be provided as a tetrapeptide, having only 4 amino acid residues (and not comprising any further amino acid residue). Alternatively, the 4-aa motif may be provided as part of longer a oligopeptide, e.g. 12-mer, 17-mer, or an oligopeptide of any length as described herein.

In preferred embodiments of the invention, the oligopeptide comprises an arginine residue at a position that is towards the N-terminus of the oligopeptide from the “Z1Z2SL” motif, and separated by 7 intervening amino acids from the “Z1” residue of the “Z1Z2SL” motif. In other words, some embodiments of the invention comprise the sequence “RXXXXXXXZ1Z2SL” (SEQ ID NO: 7) wherein “X” represents any natural or unnatural amino acid residue. In some embodiments, the amino acid sequence of the oligopeptide consists of SEQ ID NO: 7, although it may include other (non-peptide) chemical or physical moieties.

In some embodiments of the invention, the amino acid sequence “Z1Z2SL” (SEQ ID NO: 1) is part of the sequence “HZ1Z2SL” (SEQ ID NO: 8) (such that the oligopeptide comprises the amino acid sequence “HZ1Z2SL”). In other words, some embodiments of the invention comprise the sequence HADSL (SEQ ID NO: 48). In other embodiments, the amino acid sequence “Z1Z2SL” (SEQ ID NO: 1) or “AZ2SL” (SEQ ID NO: 2) is part of the sequence “QZ1Z2SL” (SEQ ID NO: 9) (such that the oligopeptide comprises the amino acid sequence “QZ1Z2SL”). In some embodiments, the amino acid sequence of the oligopeptide consists of SEQ ID NO: 8 or 9, although it may include other (non-peptide) chemical or physical moieties.

The skilled person will appreciate that the embodiments of the invention can be combined. For instance, the sequence of SEQ ID NO: 7 can be combined with of SEQ ID NOs: 8 or 9. The oligopeptides of those embodiments respectively comprise the sequence “RXXXXX HZ1Z2SL” (SEQ ID NO: 10) or “RXXXXXXQZ1Z2SL” (SEQ ID NO: 11).

In some embodiments of the invention, the oligopeptide comprises the amino acid sequence “Z1Z2SLE” (SEQ ID NO: 12). In some embodiments of the invention, the oligopeptide comprises the amino acid sequence “Z1Z2SLXR” (SEQ ID NO: 13). Some embodiments of the invention comprise the amino acid sequence “Z1Z2SLER” (SEQ ID NO: 34) and some embodiments of the invention comprise the amino acid sequence “RXXXXXXXZ1Z2SLER” (SEQ ID NO: 35). Those oligopeptides of the inventions that include glutamic acid “E” at ‘position 13’ and include arginine “R” at ‘position 14’, e.g. SEQ ID NOs: 34 and 35, are particularly important in the context of the therapeutic aspects of this invention.

In some embodiments of the invention, the oligopeptide comprises amino acid sequence “SXXXXXZ1Z2SL” (SEQ ID NO: 14). In some embodiments, the oligopeptide comprises amino acid sequence “PXXXXXZ1Z2SL” (SEQ ID NO: 15). In other words, the oligopeptide of the invention may comprise amino acid sequence SXXXXXADSL (SEQ ID NO: 49) or amino acid sequence PXXXXXADSL (SEQ ID NO: 50). In some embodiments, the oligopeptide has between 12-17 amino acids. For instance, the oligopeptide of the invention may have 12 amino acids, or the oligopeptide of the invention may have 17 amino acids. In some embodiments, the oligopeptide comprises or consists of amino acid sequence “RXXXXXXXZ1Z2SLXXXXX” (SEQ ID NO: 17). Residue “X” represents any natural amino acid residue or an unnatural amino acid residue.

In some embodiments, the amino acid sequence of the oligopeptide of the invention consists of any one of SEQ ID NOs: 8-17, although it may include other (non-peptide) chemical or physical moieties.

In some embodiments of this disclosure, the amino acid sequence “DZ2SL” (SEQ ID NO: 3) is “DDSL” (SEQ ID NO: 6). In other embodiments, the amino acid sequence “DZ2SL” is “DHSL” (SEQ ID NO: 16).

As also shown inFIG. 13, one oligopeptide of the invention has the sequence “RLTLSPEQAHSLILQHL” (SEQ ID NO: 18), which is denoted antigen “S”. Another oligopeptide of the invention has the sequence “MPRTQENAHSLERCWL” (SEQ ID NO: 19), which is denoted antigen “H”. Another oligopeptide of the invention has the sequence “RGSRGQHQAHSLERVCH” (SEQ ID NO: 20), which is denoted antigen “P”. Another oligopeptide of the invention has the sequence “RVPGCAHHADSLYTYPV” (SEQ ID NO: 21), which is denoted antigen “F”. Another oligopeptide of the invention has the sequence “RHSDEHHHDDSLPHPQQ” (SEQ ID NO: 22), which is denoted antigen “E”. Another oligopeptide of the invention has the sequence “RDSANIYHADSLKGRFT” (SEQ ID NO: 23), which is denoted antigen “A”. Another oligopeptide of the invention has the sequence “RLGTHVLEAHSLDKVSH” (SEQ ID NO: 24). Another oligopeptide of the invention has the sequence “RMSTPNPHADSL” (SEQ ID NO: 25). Another oligopeptide of the invention has the sequence “SSPEHRLAHSLERDYG” (SEQ ID NO: 26).

In some embodiments, the oligopeptide of the invention has the sequence RLTLSPEQAHSL (SEQ ID NO: 27), RGSRGQHQAHSL (SEQ ID NO: 28), RVPGCAHHADSL (SEQ ID NO: 29), RHSDEHHHDDSL (SEQ ID NO: 30), RDSANIYHADSL (SEQ ID NO: 31), RLGTHVLEAHSL (SEQ ID NO: 32) or RMSTPNPHADSL (SEQ ID NO: 33).

In another aspect, the invention provides nucleic acids encoding the oligopeptides of the invention. The nucleic acid may be a vector, which itself may be comprised within a host cell. The oligopeptide of the invention may be expressed by the host cell comprising the nucleic acid of the invention. The nucleic acid of the invention is capable of expressing the oligopeptide in a host cell. This distinguishes the nucleic acid of the invention from genomic nucleic acid sequences which may express the amino acid motifs of the invention, but as part of larger proteins, not as part of an oligopeptide.

As described herein, the oligopeptide of the invention can be used in applications related to the diagnosis and treatment of demyelinating diseases such as MS. The inventors have demonstrated that the oligopeptides of the invention can be used in methods of measuring the levels of certain antibodies in a sample from a subject. The inventors have also found that these antibodies are of diagnostic significance for demyelinating diseases such as multiple sclerosis (MS).

Accordingly, in a second aspect, the invention provides in vitro methods for measuring the level, in a sample, of antibody that binds to the oligopeptides of the invention. This method comprises providing the oligopeptide immobilized on a substrate, bringing the sample and the substrate into contact with each other, and measuring the level of antibody bound to the substrate.

In some embodiments, the oligopeptide is conjugated to a first member of a specific binding pair and the substrate comprises the second member of the specific binding pair, such that the oligopeptide has been immobilized on the substrate by bringing the first member and the second member of the specific binding pair into contact with each other. The specific binding pair may be comprised of biotin and streptavidin or a protein that is functionally equivalent to streptavidin because it is also capable of binding biotin. In some embodiments, the first member of the specific binding pair (that is conjugated to the oligopeptide of the invention) is biotin. The substrate may be an ELISA plate. The substrate may be a magnetic bead. The sample may be a serum sample that has been obtained from a human subject.

The oligopeptides of the invention (e.g. SEQ ID NO: 27-32) can induce MS, to various severity, in both male and female gender. Among these sequences, those having ‘D’ amino acid at position Z2are more effective. Amongst antigen sequences with ‘D’ amino acid at Z2, antigens F and A (SEQ ID NOs: 21 and 23) are particularly effective in the in vitro method for measuring the level of antibody when the sample is taken from a male subject. Oligopeptides with SEQ ID NO: 29 and 31 are also preferred in the in vitro method for measuring the level of antibody when the sample is taken from a male subject.

Therefore, in the in vitro method for measuring the level of antibody that binds to the oligopeptides of the invention in a sample, the subject may be a male subject and the oligopeptide may have a sequence that is selected from the group consisting of (SEQ ID NO: 21), (SEQ ID NO: 23), (SEQ ID NO: 29), and (SEQ ID NO: 31).

In the in vitro method for measuring the level of antibody that binds to the oligopeptides of the invention in a sample, the subject may be a female subject and the oligopeptide antigens P and E (SEQ ID NOs: 20 and 22) are the best sequences. Oligopeptides with SEQ ID NO: 28 and 30 are also preferred in the in vitro method for measuring the level of antibody when the sample is taken from a female subject. Therefore in the in vitro method for measuring the level of antibody that binds to the oligopeptides of the invention in a sample, the subject may be a female subject and the oligopeptide may have a sequence that is selected from the group consisting of (SEQ ID NO: 20), (SEQ ID NO: 22), (SEQ ID NO: 28), and (SEQ ID NO: 30).

In some embodiments, the in vitro method further comprises the step of comparing the level of antibody that binds the oligopeptide of the invention with a control level which is representative of the level of antibody that binds the oligopeptide in a healthy subject. In some embodiments, a lower level of antibody that binds the oligopeptide in the sample, compared with the control level of antibody that binds the oligopeptide, is indicative of a demyelinating disease such as multiple sclerosis (MS). The level of antibody may be used to distinguish between a leukodystrophy and a demyelinating disease such as multiple sclerosis (MS) in a patient with a disease of the myelin sheath.

In some embodiments, the part of the in vitro method involving binding the antibody to the oligopeptide is performed at a pH of approximately 7.1 (e.g. between pH 7.05 to pH 7.15). In other embodiments, the part of the in vitro method involving binding the antibody to the oligopeptide is performed at a pH of approximately 7.4 (e.g. between pH 7.35 to pH 7.45). In embodiments where the binding the antibody to the oligopeptide is performed at a pH of approximately 7.1 (i.e., the sample is buffered to a pH of approximately 7.1, e.g. pH 7.05 to pH 7.15), the level of antibody in MS patient samples is often found to be higher than the level of antibody in control samples. In contrast, in embodiments where the binding the antibody to the oligopeptide is performed at a pH of approximately 7.4 (i.e., the sample is buffered to a pH of approximately 7.4, e.g. pH 7.35 to pH 7.45), the level of antibody in MS patient samples is often found to be lower than the level of antibody in control samples.

In some embodiments, the in vitro method comprises measuring the level of antibody that binds a first oligopeptide of the invention, and the method further comprises measuring the level of antibody that binds a second oligopeptide of the invention.

In a related (third) aspect, kits for performing the in vitro methods for measuring antibody levels in a sample as described herein are also provided. The kit will contain required components to perform the described methods, and may further comprise additional components such as syringes, test tubes, and/or an instruction page.

In a fourth aspect, the invention provides a method of inducing a disease of the myelin sheath in a non-human animal, the method comprising administering to the non-human animal both an oligopeptide of the invention and an adjuvant. In some embodiments, the adjuvant is Freund's complete adjuvant. The oligopeptide and the adjuvant may be administered at the same time. The oligopeptide and the adjuvant may be administered by intradermal injection. In some embodiments, the induced disease of the myelin sheath is characteristic of a demyelinating disease such as multiple sclerosis (MS). In most embodiments, plaque formation is induced in the brain of the non-human animal. In some embodiments, plaque formation is induced without substantial damage to the blood brain barrier (BBB). In some embodiments, the method also comprises administering Pertussis toxin to the non-human animal. The non-human animal may be a mouse, a rat, a guinea pig, a pig, a cow, a cat, a chicken or any other animal with PLP sequence (for the sequence alignment of PLP and other lipophilins in various species seeFIG. 16.2of reference (20)). In some embodiments, the weight of the non-human animal is reduced following administration of the oligopeptide of the invention. In some embodiments, the animal is sacrificed following disease induction and the number of plaques in a mid-brain section the animal (which is taken as representative of number of plaques in the rest of the brain) is higher than the number of plaques in a mid-brain section of equivalent size in a control animal of the same type that has not undergone disease induction as described herein. In some embodiments, the animal exhibits lowered muscular function compared with the muscular function of a control animal of the same type that has not undergone disease induction as described herein, as measured by an objective muscle function test such as the fall-from-ring test or the fall-from-inverted-cage test.

In a related (fifth) aspect, the invention provides an animal that is a model of a demyelinating disease in humans, wherein the animal has a demyelinating disease that has been induced according to the fourth aspect of the invention. The fourth and fifth aspects of the invention provide an invaluable tool for the research of diseases of the myelin sheath that will in likelihood lead to yet further substantial benefits to man.

In a sixth aspect, the invention provides an oligopeptide of the invention for use as a medicament. In a seventh aspect, the invention provides an oligopeptide of the invention for use in diagnostics.

In an eighth aspect, the invention provides an oligopeptide of the invention for use in a method of treating a demyelinating disease in a subject, the method comprising administering the oligopeptide to the subject.

The disease of may be an inflammatory demyelinating diseases (IDDs) such as multiple sclerosis (MS). In some embodiments, the disease is primary progressive MS. Preferably, the oligopeptide is administered intravenously, orally, intranasally or via an aerosol.

Preferably the subject is a human subject. The oligopeptide of the invention may be administered to the subject more than once. For instance, the oligopeptide of the invention may be administered every day, e.g. once per day or twice per day. Alternatively, the oligopeptide of the invention may be administered weekly or twice per week or three times per week. Alternatively, oligopeptide of the invention may be administered once or twice per month.

A dosage of the oligopeptide of the invention may be in the range of 1 mg per dose to 200 mg per dose. For instance, one dose of the oligopeptide of the invention may be 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, or 150 mg per dose.

In a ninth aspect, the invention provides an antibody that specifically binds to the oligopeptide of the invention.

The antibody may be identified by eluting antibody that binds to the substrate of the second and third aspects, and characterising the antibody. The antibody may be a polyclonal antibody. Alternatively, the antibody may be a monoclonal antibody. For instance, an antibody may be identified as described herein, and then cloned and expressed in a hybridoma.

