Assays, methods and treatments for alpha-synucleinopathies

Abstract

A method of detecting alpha-synuclein in a sample of a subject as well as assays, methods and treatments of alpha-synucleinopathies are disclosed. The method of the alpha-synuclein comprises the steps of: a) obtaining a sample from a subject; 5b) using the sample for a luminescence assay; and c) optionally comparing the result to a control.

Description

[0001] This application is a divisional application of the application with the filing date of December 19, 2018, application number 2018800830206, and invention title "Assays, Methods, and Treatments for Alpha-Synucleinopathies". Technical Field

[0002] The present invention relates to an assay that can diagnose and effectively treat alpha-synucleinopathies such as Parkinson's disease by evaluating and monitoring the level of alpha-synuclein in a patient. Background Art

[0003] Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are all examples of neurodegenerative diseases with alpha-synuclein brain pathology. PD is the most common movement disorder, characterized by tremors, bradykinesia or difficulty initiating movement, rigidity, and balance disorders (also known as postural instability). It is believed that approximately 4 to 6 million people worldwide are affected by PD. Approximately 80% of PD patients will develop dementia, leading to PDD. Dementia usually occurs in the late stage of Parkinson's disease. In DLB, dementia is the first symptom, but movement symptoms may appear in the first year, so dementia is the clinical distinction between these diseases. DLB may account for up to 15%-20% of all dementias.

[0004] Alpha-synuclein is a small molecule protein composed of 140 amino acids located within nerve cells, mainly in the presynaptic region. The accumulation of alpha-synuclein within nerve cells leads to the formation of various aggregates within neurons, such as Lewy bodies and globoid bodies (larger round cytoplasmic inclusions), Lewy neurites (filamentous inclusions within axons), and small synaptic inclusions.

[0005] The processes leading to α-synuclein aggregation and toxicity are not well understood. Mutations or duplications of the α-synuclein gene are rare causes of PD and DLB. Pathogenic mutations A30P, A53T, E46K (Kruger et al., 1998) (Polymeropoulos et al., 1998) (Zarranz et al., 2004) and duplications and triplications of α-synuclein (Chartier-Harlin et al., 2004) (Singleton et al., 2003) have been reported to cause PD or DLB. Most of these mutations are associated with an increased rate of protofibrillar formation and ultimately fibril formation, and these protofibrils or fibrils may have different toxicities. Aggregation is associated with phosphorylation of α-synuclein at serine 129. Normal α-synuclein is phosphorylated only to a low extent (4%), but the phosphorylation of fibrillar α-synuclein is estimated to be as high as 80%, and antibodies to pS129α-synuclein can detect the smallest aggregates.

[0006] Small amounts of α-synuclein have been found in extracellular interstitial fluid, cerebrospinal fluid (CSF), and blood in the brain. The source of these α-synucleins is not clear, but they may be released from cells, including from cell bodies and synapses (e.g., synaptic release during neuronal cell activation). Some of them may also come from diseased and dying cells. α-Synuclein has also been found in many blood cells and platelets, which may be the source of α-synuclein in the blood. A small fraction of α-synuclein is secreted in exosomes, while most of the α-synuclein in extracellular fluid is free protein and is not related to exosomes.

[0007] Improved diagnostic tools are needed to identify patients in the early stages of neurodegenerative diseases with α-synuclein pathology. There is evidence that the neurodegenerative process may begin 10 - 20 years before clinical diagnosis. REM sleep behavior disorder (RBD) is currently considered to be the prodromal stage of α-synucleinopathies. Most patients with RDB will convert to PD or DLB within 20 years after the diagnosis of RBD. Other signs of the prodromal stage of α-synucleinopathies include olfactory loss.

[0008] Today, there is no biochemical test to help clinicians diagnose before motor symptoms are obvious. By the time motor symptoms are obvious, substantial damage may have occurred in the brain. With the emergence of new and improved disease treatments, accurate diagnostic tests will become even more important, which will hopefully prevent or slow down the progression of the disease.

[0009] There are several assays that can detect total α-synuclein, some of which have been validated or are being validated in large-scale clinical studies (Goldman et al., 2017). These studies have shown an association between a small decrease in total α-synuclein in CSF and Parkinson's disease. There are also several exploratory assays that can detect certain oligomeric forms of α-synuclein and certain phosphorylated forms at serine 129 (Majbour et al., 2016a), and preliminary data suggest that these forms of α-synuclein may be increased in PD CSF. However, the differences between patient and control levels remain small, and there is a large inter-individual variability. All current assays suffer from small differences and large inter-individual variability, making these assays unable to be used to help diagnose diseases with α-synuclein aggregation. In summary, current evidence suggests a decrease in total CSF α-synuclein in PD, and α-synuclein oligomers or phosphorylated α-synuclein subspecies can distinguish PD from controls.

[0010] Results from measuring α-synuclein in peripheral biological fluids, plasma, and saliva are even more variable than in CSF. In the most recent large studies, α-synuclein levels in plasma and saliva did not significantly distinguish PD patients from healthy control participants, and there was also no significant correlation between α-synuclein levels in peripheral biological fluids (plasma and saliva) and α-synuclein levels in CSF (Goldman et al., 2017). Thus, currently CSF α-synuclein has higher diagnostic value for PD than peripheral α-synuclein. However, CSF sampling is a more invasive method of sample acquisition, and it would be beneficial to detect PD using plasma biomarkers if possible. SUMMARY OF THE INVENTION

[0011] Monitoring the aggregation of α-synuclein is crucial for studying the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), disseminated Lewy body disease (DLBD), and multiple system atrophy (MSA). Thus, the methods of the present invention can be used to diagnose or monitor the disease progression of the above diseases. Alternatively, the method can be used to monitor or track treatment responses and have discussions related to patient treatment. In certain embodiments, such treatment can be active or passive immunotherapy against α-synuclein, such as antibody treatment or vaccination with α-synuclein.

[0012] In one embodiment, the present invention is an in vitro method that includes the following steps:

[0013] a) Obtaining a sample from a patient

[0014] b) Using the sample in a luminescence assay of the present invention; and

[0015] c) Optionally, compare the result with a control.

[0016] The sample is preferably a blood sample, a plasma sample or a serum sample.

[0017] The antibody used is an α-synuclein antibody conjugated to a fluorophore. Two different fluorophores can be used in the method of the present invention, which can be conjugated to two antibodies that bind α-synuclein. One fluorophore (donor) has a longer fluorescence lifetime than the other fluorophore (acceptor).

[0018] The donor is preferably selected from Lumi4-Tb (Tb2+ cryptate) or europium cryptate (Eu3+ cryptate), and the acceptor is preferably selected from XL665 or fluorescein or d2.

[0019] The level of energy transfer is detected by measuring fluorescence emission (preferably at two different wavelengths, such as 665 nm and 620 nm) in a compatible reading instrument, and the proximity between the donor and the acceptor is evaluated thereby. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shows measurements made using a PHERAstar FSX device, which can simultaneously measure emissions at 620 nM (Tb cryptate donor) and 665 nm (d2 acceptor). Calculate the ratio 665 / 620 * 10000 for each well. Use this ratio to calculate the relative energy transfer rate ΔF%, and it can be normalized to the protein concentration. The bar graph shows the ΔF% normalized to protein for each plasma sample (diluted 8-fold) measured using Kit 1 and Kit 2 (detecting aggregated α-synuclein). There is a significant difference between three PD patients and three healthy controls. The figure shows that the level of aggregated α-synuclein in the plasma of PD patients is significantly higher, so this method can be used to identify, diagnose and monitor the disease progression of PD patients based on blood samples. DETAILED DESCRIPTION

[0021] The object of the present invention is to provide an assay for use in a diagnostic method for α-synuclein-related diseases, which are α-synucleinopathies, in which aggregated insoluble α-synuclein accumulates in the brain in the form of Lewy bodies, Lewy neurites and small synaptic inclusions. Such diseases include but are not limited to one or more neurodegenerative diseases such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) and REM sleep behavior disorder (RBD) and other neurodegenerative diseases with α-synuclein pathology.

[0022] The present invention provides two new uses for assays, one based on aggregated α-synuclein and one based on phosphorylated α-synuclein. Both assays showed a large increase in the plasma of PD patients compared to the plasma of control individuals ( Figure 1 ).

[0023] According to one aspect of the invention, the invention relates to a method of treating a patient diagnosed with an α-synucleinopathy such as Parkinson's disease by using the luminescence assay of the invention and using an effective amount of an α-synuclein antibody to treat said patient.

[0024] The therapeutic effect of antibody treatment can be evaluated by measuring the level of α-synuclein in the blood, plasma or serum of the patient. This evaluation can be performed before antibody treatment (e.g., at the time of patient diagnosis) or after 1, 2, 3, 4 or more treatments. For example, according to one embodiment, the method can be used to monitor the therapeutic effect of antibody treatment and disease progression.

[0025] The assay may comprise the following steps:

[0026] a. Under appropriate binding conditions, using a blood, plasma or serum sample of the patient for the luminescence assay; and

[0027] b. If applicable, comparing the data with control data of a person not suffering from Parkinson's disease, or for example, comparing the data with data obtained from the same person at different (earlier) time points, thereby monitoring disease progression.

[0028] Based on the results, it can be determined whether the patient should continue treatment or, if newly diagnosed, whether the patient should start treatment. For example, if the data obtained by the detection shows more α-synuclein compared to the control, it can be recommended to continue treatment or start treatment.

[0029] The difference between the control and the patient may exceed 2-fold, 3-fold, 4-fold or higher. As Figure 1 shown, the control level is very low, so a positive result can be obtained by ignoring the background.

[0030] The assay is a luminescence-based assay that measures luminescence based on HTRF (Homogeneous Time-Resolved Fluorescence). This technique combines Fluorescence Resonance Energy Transfer (FRET) technology with time-resolved measurement (TR) (Degorce et al., 2009, Current Chemical Genomics, 3, 22-32). In a TR-FRET assay, a signal is generated by fluorescence resonance energy transfer between a donor and an acceptor molecule (e.g., conjugated to an antibody) when they are very close to each other.

[0031] The HTRF technology can use europium cryptates as fluorescent donors to monitor reactions between biomolecules such as antibodies or terbium cryptates (Tb). Examples include europium cryptates (Eu3+ cryptates) and Lumi4-Tb (Tb2+ cryptates).

[0032] The acceptor developed for HTRF can be XL665, which is a phycobiliprotein pigment purified from red algae. XL665 is a huge heterologous hexameric large structure with a molecular weight of 105 kDa, which is crosslinked after isolation to obtain better stability in the HTRF assay and maintain its photophysical properties. A class of acceptors with a series of photophysical properties very similar to XL665 but characterized by an organic structure 100 times smaller than XL665 are such as earth chelates or cryptates. Using smaller entities can solve the steric hindrance problems sometimes suspected in XL665-based TR-FRET systems. These near-infrared acceptors are also particularly suitable for homogeneous assays because their emission is less likely to be interfered by the intrinsic medium or the autofluorescence of compounds that occur during typical compound screening processes. The properties of these red acceptors also make them suitable for coupling with terbium cryptates. In addition, due to the presence of other peaks within its emission spectrum, terbium cryptates can be coupled with green acceptors such as fluorescein, emitting fluorescence in the range of 520 nm, which can, for example, allow the design of multiplex detection with two readings.

[0033] In particular, it is advantageous to use fluorescent compounds such as rare earth chelates or cryptates, especially terbium, europium, dysprosium, samarium or neodymium chelates or cryptates. Terbium or europium cryptates are preferably used.

[0034] In the fluorescence method for detection and / or determination using the measurement method of the present invention, the rare earth cryptates described in European patent applications EP 180 492 and EP 321 353 will be advantageously selected.

[0035] Preferably, the terbium cryptate Tb trisbipyridine or europium cryptate Eu trisbipyridine described in European patent application 180 492 or the cryptates Eu trisbipyridine diamine and Tb trisbipyridine diamine described in European patent application EP 321353 are used.

[0036] According to an advantageous feature, the fluorescent donor compound is an europium cryptate, and the fluorescent acceptor compound is selected from d2, allophycocyanin, allophycocyanin B, phycocyanin C or phycocyanin R.

[0037] It is also possible to use a phosphorescent compound such as eosin or erythrosine as the luminescent donor. In this case, it is advantageous to use a fluorescent acceptor compound selected from chlorophylls such as those mentioned in European patent applications EP 71 991 and EP 314 406 or porphyrins such as those mentioned in European patent application EP 71 991 or phthalocyanines such as those mentioned in international patent application WO 88 04777.

[0038] According to another feature of the present invention, a luminescence method for detecting and / or determining α-synuclein in a medium (such as blood, plasma or serum) comprises the following steps:

[0039] 1) adding a primary anti-α-synuclein antibody coupled to a luminescent donor to the medium containing α-synuclein from a patient or subject;

[0040] 2) adding a secondary α-synuclein antibody coupled to a luminescent receptor;

[0041] 3) incubating the medium after adding the reagents;

[0042] 4) exciting the resulting medium at the excitation wavelength of the luminescent donor; and

[0043] 5) measuring the signal of the luminescent donor at a certain wavelength (this measurement serves as a reference), and the signal generated by energy transfer at different wavelengths.

[0044] According to one embodiment, the assay is expected to be used to detect α-synuclein aggregation using HTRF technology. Specific α-synuclein monoclonal antibodies labeled with a donor or receptor such as a Tb cryptate are used to detect aggregated α-synuclein. When the dyes are very close, the donor is excited with a light source (laser or flash lamp), triggering fluorescence resonance energy transfer (FRET) towards the receptor, which then fluoresces at a specific wavelength (665 nm). The antibody labeled with the receptor or Tb binds to α-synuclein. When α-synuclein aggregates, the antibody labeled with the receptor or Tb will be very close, resulting in FRET. The signal intensity is proportional to the number of aggregates formed.

[0045] Monitoring the aggregation of α-synuclein is very important for studying the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), disseminated Lewy body disease (DLBD) and multiple system atrophy (MSA). Therefore, the method of the present invention can be used for diagnosing or monitoring the above diseases. Alternatively, the method can be used to monitor or track treatment responses and make decisions related to the treatment of patients. In one embodiment, the treatment is α-synuclein antibody treatment.

[0046] Accordingly, the present invention relates to a method for diagnosing or tracking synucleinopathies such as Parkinson's disease in a patient, the method comprising the steps of

[0047] a) obtaining a sample from the patient

[0048] b) applying the sample to the luminescence assay as described above; and

[0049] c) optionally comparing the result with a control.

[0050] The sample is preferably a blood sample, a serum sample or a plasma sample.

[0051] The antibody used can be an α-synuclein antibody conjugated to a fluorophore. The fluorophore (donor) has a longer fluorescence lifetime than another fluorophore (acceptor) used. The donor is preferably selected from Lumi4-Tb (Tb2+ cryptate) or europium cryptate (Eu3+ cryptate), and the acceptor is preferably selected from d2, XL665 or fluorescein.

[0052] The level of energy transfer is detected by measuring fluorescence emission (preferably at two different wavelengths, such as 665 nm and 620 nm) in a compatible reading instrument, thereby assessing the proximity between the donor and the acceptor.

[0053] As used herein, the term "α-synuclein" is synonymous with "α-synuclein protein" and refers to any α-synuclein protein subtype (e.g., identified as P37840, 1-3 in UniProt). The amino acid numbering of α-synuclein with respect to the sequence is shown below, where methionine (M) is amino acid residue 1 (SEQ ID NO:1):

[0054] MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA

[0055] GKTKEGVLYV GSKTKEGVVH GVATVAEKTK

[0056] EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA

[0057] ATGFVKKDQL GKNEEGAPQE GILEDMPVDP

[0058] DNEAYEMPSE EGYQDYEPEA

[0059] In some embodiments, the α-synuclein antibody can bind to the C-terminal residues between 120-140 of α-synuclein or pS129α-synuclein.

[0060] The present invention also relates to a method for treating a patient with Parkinson's disease by administering an effective amount of an antibody that binds to an epitope on α-synuclein, wherein the patient has been diagnosed or monitored by the assay of the present invention.

[0061] Examples

[0062] The assay can be used to identify, diagnose, and monitor patients with α-synucleinopathies (such as Parkinson's disease) using blood samples.

[0063] Assay principle:

[0064] The assay is based on the use of time-resolved fluorescence - resonance energy transfer (TR-FRET) technology.

[0065] FRET is based on the energy transfer between two dyes (donor and acceptor). When the donor is excited, it transfers energy to the acceptor, and then the acceptor emits fluorescence that is measured. This can only occur when the dyes are very close to each other. Conventional FRET studies are limited by background fluorescence and are very short-lived. This problem can be solved by combining time-resolved measurements with FRET to avoid background and non-specific fluorescence. At the same time, the acceptor in TR-FRET is designed to emit long-lived fluorescence when participating in FRET.

[0066] In the α-synuclein aggregation kit, specific monoclonal antibodies are labeled with donor or acceptor molecules. In the phospho-synuclein kit (S129P), two antibodies are used, one is an α-synuclein antibody, and the other is a phospho-synuclein-specific antibody (S129P), where one antibody is labeled with the donor and the other is labeled with the acceptor dye. When the antibodies (labeled with donor and acceptor dyes) bind to human α-synuclein aggregates, they become very close to each other and produce FRET upon excitation.

[0067] Patients

[0068] Patients were recruited from the outpatient neurology department of Bispebjerg-Frederiksberg Hospital. The clinical diagnosis of PD was defined according to the clinical diagnostic criteria of the UK Parkinson’s Disease Society Brain Bank. Patients were consecutively enrolled, and all patients were clinically followed up. At the time of follow-up, only patients who met the clear PD diagnostic criteria were included in the study. Subjects in the control group did not have diseases that could affect the central nervous system.

[0069] Plasma samples

[0070] Venous blood was drawn during each outpatient visit and processed on the same day at the Bispebjerg Movement Disorders Biobank (Copenhagen, Denmark). All plasma samples were collected at the time of inclusion. Samples were collected in EDTA-coated polypropylene tubes and centrifuged at 2000×g for 10 minutes at 4 °C; then the supernatant plasma was aliquoted and stored at -80 °C in 400 μL polypropylene (PP) tubes until the day of analysis, on which day it was thawed on ice for 30 minutes.

[0071] Example 1

[0072] Samples from patients (3 control samples and 3 PD patient samples) were analyzed. Two commercial kits from Cisbio, namely the α-synuclein aggregation kit (Kit 1) and the phospho-synuclein kit (Kit 2), were used to compare the samples. Each sample was tested at three different dilutions. The samples were diluted using the buffer supplied with the kit and pipetted into 386-well plates in duplicate. The antibody mixture was added to the wells and incubated at room temperature. The plates contained positive and negative controls.

[0073] Measurements were made using a PHERAstar FSX instrument, which can simultaneously measure emissions at 620 nM (Tb cryptate donor) and 665 nm (d2 acceptor). The ratio 665 / 620*10000 was calculated for each well. The relative energy transfer rate ΔF% was calculated using this ratio and could be normalized to the protein concentration.

[0074] The bar graph shows the ΔF% normalized to protein for each sample (diluted 8-fold) measured using Kit 1 and Kit 2. There was a clear difference between three PD patients and three healthy controls. This example shows that the levels of aggregated α-synuclein and phospho-synuclein in the plasma of PD patients were significantly higher, and this method can be used to identify and diagnose PD cases based on blood samples.

Claims

1. A method for detecting α-synuclein in a subject's sample, the method comprising the following steps: a) Obtaining a sample from the subject; b) Using the sample for a luminescence assay; and c) Optionally comparing the result with a control.

2. The method according to claim 1, wherein the sample is a blood sample, a plasma sample or a serum sample.

3. The method according to claim 1, wherein the luminescence assay is performed using an α-synuclein antibody that binds within residues 120-140 on α-synuclein or pS129α-synuclein.

4. The method according to claim 1, wherein the luminescence assay is performed using two different α-synuclein antibodies that bind to different epitopes within residues 120-140 on α-synuclein or pS129α-synuclein.

5. The method according to claim 3 or 4, wherein one fluorophore (donor) used has a longer fluorescence lifetime than the other fluorophore (acceptor) used.

6. The method according to claim 5, wherein the donor fluorophore is selected from Lumi4-Tb (Tb2+ cryptate), europium cryptate (Eu3+ cryptate).

7. The method according to claim 5, wherein the acceptor fluorophore is selected from d2, XL665 or fluorescein.

8. The method according to claim 1, wherein the energy transfer level is detected by measuring fluorescence, thereby assessing the proximity between the donor and the acceptor.

9. The method according to any one of the preceding claims, wherein if the method shows that the α-synuclein in the sample is higher than the control sample of a healthy subject, the subject is treated with an effective amount of an anti-α-synuclein antibody.

10. The method according to any one of the preceding claims, wherein the method is used for diagnosing or monitoring α-synucleinopathy.

11. The method according to any one of the preceding claims, wherein the method is used for monitoring the therapeutic response of a drug for treating α-synucleinopathy.

12. The method according to any one of the preceding claims, wherein the subject is human.

13. A method for treating a patient with Parkinson's disease by administering an effective amount of an antibody that binds to an epitope on α-synuclein, wherein the patient has been diagnosed or monitored by the assay of claims 1-9.

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