Alpha-synuclein assay

Abstract

Methods of immunoassay are described for detecting in a patient sample (such as for example a blood, serum or plasma sample) a biomarker originating from α-Synuclein, and to the use of said methods of immunoassay for detecting and / or monitoring Parkinson's Disease or a level of severity thereof. Monoclonal antibodies and assay kits for use in said methods of immunoassay are also described.

Description

[0001] a-Synuclein Assay

[0002] Field of the invention

[0003] The present invention relates to methods of immunoassay for detecting in a patient sample (such as for example a blood, serum or plasma sample) a biomarker originating from a-Synuclein, and to the use of said methods of immunoassay for detecting and / or monitoring Parkinson’s Disease or a level of severity thereof. The present invention also relates to monoclonal antibodies and assay kits for use in said methods of immunoassay.

[0004] Background

[0005] Parkinson’s disease (PD) is a progressive neurodegenerative condition, characterized by the cardinal features with presence of bradykinesia, rest tremor and rigidity1. The motor signs are often preceded by non-motor functions including sleep, depression, anxiety, and urinary dysfunction. The motor dysfunction is due to the loss of dopamine containing neurons in the substantia nigra pars compacta (SNc), a part of the midbrain which plays a role in the regulation of movement. There is no clear cause, no cure, and as a result of an aging population, the prevalence of PD is expected to rise steadily to 13 million individuals worldwide in 20402. The onset of PD is diagnosed by a neurological and physical exam, including review of symptoms, genetics, and medical history.

[0006] Clinically, two major subtypes of PD can be defined, namely tremor-dominant PD with relative absence of other motor symptoms and non-tremor dominant PD. However, there is a general understanding that PD is highly heterogeneous upon diagnosis, also in rate of progression3. Generally, 50% of PD patients have reached key milestones, such as either postural instability or dementia within 4 years from diagnosis, where 25% have a good 10-year prognosis. For the progressive group of patients the number of severe symptoms increases, where increased medication is provided to manage the symptoms, leading to increased risk of side effects contributing to increased disability45. Dividing patients into subtypes divided by symptoms and how symptoms evolve, may therefore be a path forward6. To eliminate adverse events for progressive patients, there is a need for better diagnostic, prognostic, and efficacy of treatment tools, reflecting the individual patient. Importantly, there is a lack of blood-based biomarkers for PD, and hence there is an intense search for novel biomarker candidates falling into the mentioned categories, particularly those identifying the subset of patients with a rapidly progressing disease, i.e. those in urgent need of intervention.Alpha-Synuclein (a-Synuclein) is a hallmark of all synucleinopathies, including PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). All these diseases, are determined by a-Synuclein aggregates and deposition7. The a-Synuclein protein is composed by an N-terminal region (amphipathic region), Central region (NAC region necessary for aggregation), and C-terminal region (Acidic tail), comprising a 140 aa sequence. Posttranslational modifications and fragmentation of a-Synuclein, has previously been described in the C-terminal truncation of a-Synuclein8. One of the enzymes who are thought to perform this processing, is Calpain-1 , which is interesting considering its calcium dependence and localization at presynaptic terminal, and interestingly this cleavage has been indicated to be an early event related to aggregation of a-Synuclein, a hallmark of PD910. Cleavage sites a-Synuclein by Calpain-1 , have been previously identified by Duffin et al.12, who discovered a cleavage site between amino acid 122 and 123, resulting in an upstream neoepitope. In vivo, they showed how calpain-1 cleaved a -synuclein lead to formation of calpain-1 aggregates of a -synuclein in PD and dementia with Lewy bodies (DLB), together with the fragments being present in human brains and co-localized with neurons in PD and DLB brains.

[0007] Summary of the Invention

[0008] The inventors have identified a specific fragment of a-synuclein cleaved by Calpain-1 which can act as a blood-biomarker for Parkinson’s Disease. The present application describes a competitive ELISA, targeting the N-terminus of Calpain-1 generated fragment of a-synuclein, named a-SYN-C1, and its association with Parkinson’s disease in comparison to healthy donors. The present application also describes a competitive ELISA, targeting the C-terminus of a Calpain-1 generated fragment of a-synuclein, named O-SYN-C2, and it’s association with Parkinson’s disease in comparison to healthy donors.

[0009] The present inventors have developed and validated an enzyme-linked immunosorbent assay (ELISA) targeting a biomarker consisting of a N-terminus sequence of a Calpain-1 generated fragment of a-synuclein (“a -SYN-C1”), and have demonstrated that said biomarker is present and can be detected by said assay in patient serum samples. The present inventors have moreover demonstrated that said biomarker is present in elevated levels in serum samples from patients with synucleinopathies such as Parkinson’s Disease and that the assay can be used to distinguish between synucleinopathy patientsand healthy controls, thereby demonstrating the biological relevance and utility of the biomarker and assay.

[0010] Accordingly, in a first aspect the present invention provides a method of immunoassay comprising;

[0011] i) contacting a patient sample with a monoclonal antibody that specifically binds to a neoepitope generated by the cleavage of a-synuclein by Calpain-1 and

[0012] ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

[0013] Calpain-1 cleaves a-synuclein between amino acid 121 and 122 of the human sequence (Uniprot sequence P37804-1). The neoepitopes generated are the C-terminal sequence LEDMPVDPDN (SEQ ID NO: 1) and the N-terminal sequence EAYEMPSEEG (SEQ ID NO: 2).

[0014] The invention therefore provides a method of immunoassay comprising;

[0015] i) contacting a patient sample with a monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2); also referred to herein as “a -SYN-C1”, the “a -SYN-C1 target sequence”, or simply the “ first target sequence”); and

[0016] ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

[0017] The invention also provides a method of immunoassay comprising;

[0018] i) contacting a patient sample with a monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1); also referred to herein as “a -SYN-C2”, the “a -SYN-C2 target sequence”, or simply the “second target sequence”); and

[0019] ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

[0020] In a preferred embodiment, the method is a method of immunoassay for detecting and / or monitoring a synucleinopathy or a level of severity thereof in a patient, the method further comprising;iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.

[0021] As used herein the term “C-terminus sequence” refers to a C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the C-terminal end of the polypeptide and is not to be construed as meaning in the general direction thereof. As used herein the term “N-terminus sequence” refers to an N-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide and is not to be construed as meaning in the general direction thereof.

[0022] As used herein, the terms “peptide” and “polypeptide” are used synonymously.

[0023] As used herein the term “monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab’)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light" and one "heavy" chain. The N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity.

[0024] In the present invention, a monoclonal antibody comprising any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chains are classified as either kappa or lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to lgG1, lgG2, lgG3, and lgG4 in the case of humans and lgG1, lgG2a, lgG2b, lgG2c and lgG3 in the case of mice. The monoclonal antibody may preferably be of the IgG isotype, including any one of the IgG subclasses.The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al. Antibodies can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific for human or murine IgM, IgG or IgA isotype, or human lgG1, lgG2, lgG3 or lgG4 subclasses or murine IgGI, lgG2a, lgG2b, lgG2c and lgG3. Other suitable methods can be used to identify the isotype.

[0025] The amino acid sequence of the antibodies generated can be determined using standard techniques. For example, RNA can be isolated from the cells and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody. For example, primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the 3’ end of the Kappa or Lamda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced.

[0026] As used herein the term “amount of binding” refers to the quantification of binding between the antibody and peptides in the patient sample. Said quantification may for example be determined by comparing the measured values of binding in the patient sample against a calibration curve produced using measured values of binding in standard samples containing known concentrations of a peptide to which the antibody specifically binds, in order to determine the quantity of peptide to which the antibody specifically binds in the patient sample. Any suitable analytical method can be used for measuring the amount of binding. For example, an ELISA method can be used in which spectrophotometric analysis is used to measure the amount of binding both in the patient samples and when producing the calibration curve.

[0027] As used herein the term “predetermined cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of a disease (i.e. a synucleinopathy such as Parkinson’s disease) or a particular severity thereof in a patient, in that a measured value of the target peptide in a patient sample that is (as appropriate) above or below the statistical cut-off value corresponds to at least a 70% probability, preferably at least an 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, andmost preferably at least a 95% probability of the presence of said disease or particular severity thereof.

[0028] As used herein, the term “values associated with normal healthy subjects” means standardised quantities of binding determined by the method described supra for samples from subjects considered to be healthy, i.e. without disease (i.e. a without a synucleinopathy such as Parkinson’s disease); and the term “values associated with known disease severity” means standardised quantities of binding determined by the method described supra for samples from patients known to have disease (i.e. synucleinopathy such as Parkinson’s disease) of a known severity.

[0029] In certain embodiments, the method may be a method for detecting and / or monitoring a synucleinopathy, such as Parkinson’s disease or a level of severity thereof in a patient.

[0030] As used herein “synucleinopathy” refers to any disease characterized by the presence of aggregates and deposition of a-Synuclein, particularly in neurons, nerve fibers or glial cells. Examples of synucleinopathies include Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), pure autonomic failure (PAF), neuroaxonal dystrophies and Alzheimer's Disease with Amygdalar Restricted Lewy Bodies (AD / ALB). Preferably the synucleinopathy is Parkinson’s disease.

[0031] In preferred embodiments, the patient sample is a human biofluid sample. Preferably the sample is selected from urine, blood (whole blood), plasma or serum. Preferably the sample is a blood-based sample, such as blood (whole blood), plasma or serum.

[0032] The monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) preferably does not specifically bind to a peptide having the N-terminus amino acid sequence NEAYEMPSEEG (SEQ ID NO: 3), i.e. an elongated version of the first target sequence extended at its N-terminus by the addition of an asparagine residue. Preferably, the ratio of the affinity of said antibody for the first target sequence to the affinity of said antibody for the elongated version of the target sequence is at least 10 to 1 , and more preferably is at least 20 to 1 , at least 30 to 1 , at least 40 to 1 , at least 50 to 1 or at least 100 to 1.

[0033] The monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) preferably does not specifically bind to a peptide havingthe N-terminus amino acid sequence AYEMPSEEG (SEQ ID NO: 4), i.e. a truncated version of the first target sequence truncated by removal of the first glutamic acid residue. Preferably, the ratio of the affinity of said antibody for the first target sequence to the affinity of said antibody for the truncated version of the first target sequence is at least 10 to 1 , and more preferably is at least 20 to 1 , at least 30 to 1 , at least 40 to 1 , at least 50 to 1 or at least 100 to 1.

[0034] The monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) may for example be raised against a synthetic peptide having the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2). For example, the monoclonal antibodies may be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminus sequence EAYEMPSEEG (SEQ ID NO: 2), which peptide may optionally be linked at its C-terminus to an immunogenic carrier protein (such as keyhole limpet hemocyanin); (b) isolating and cloning a single antibody producing cell; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0035] The monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) does not specifically bind to a peptide having the C-terminus amino acid sequence LEDMPVDPDNE (SEQ ID NO: 5), i.e. an elongated version of the second target sequence extended at its C-terminus by the addition of a glutamic acid residue. Preferably, the ratio of the affinity of said antibody for the second target sequence to the affinity of said antibody for the elongated version of the second target sequence is at least 10 to 1, and more preferably is at least 20 to 1, at least 30 to 1, at least 40 to 1 , at least 50 to 1 or at least 100 to 1.

[0036] The monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) preferably does not specifically bind to a peptide having the C-terminus amino acid sequence LEDMPVDPD, (SEQ ID NO: 6) i.e. a truncated version of the second target sequence truncated by removal of the last asparagine residue. Preferably, the ratio of the affinity of said antibody for the second target sequence to the affinity of said antibody for the truncated version of the second target sequence is at least 10 to 1 , and more preferably is at least 20 to 1 , at least 30 to 1 , at least 40 to 1 , at least 50 to 1 or at least 100 to 1.The monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) may for example be raised against a synthetic peptide having the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1). For example, the monoclonal antibodies may be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the C-terminus sequence LEDMPVDPDN, which peptide may optionally be linked at its N-terminus to an immunogenic carrier protein (such as keyhole limpet hemocyanin); (b) isolating and cloning a single antibody producing cell; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0037] In preferred embodiments the immunoassay is a competition assay or a sandwich assay. The immunoassay may, for example, be a radio-immunoassay, fluorescence immunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to the person skilled in the art. Most preferably the immunoassay is a competitive ELISA.

[0038] In a second aspect, the present invention provides a method of treating a synucleinopathy in a patient in need thereof, the method comprising:

[0039] (a) carrying out a method of immunoassay for detecting a synucleinopathy or a level of severity thereof in accordance with the first aspect of the present invention on a sample from a patient; and

[0040] (b) administering to the patient a therapy for the treatment of said synucleinopathy if it is determined in step (a) that the patient has said synucleinopathy or a particular level of severity thereof.

[0041] Preferred embodiments of the method in accordance with the second aspect will be apparent from the foregoing discussion of preferred embodiments of the methods according to the first aspect. For example, step (a) may in particular comprise carrying out the method of immunoassay to detect a synucleinopathy or a particular level of severity thereof.

[0042] The therapy may be any therapy suitable for treating the synucleinopathy in question. The therapy may for example comprise or consist of one or more medicaments, an nonpharmaceutical interventions such as deep brain stimulation, speech therapy or physical therapy or combinations thereof. Medicaments may be formulated for topical or systemic administration. Topical medicaments may for example be formulated as creams, foams,gels, lotions, or ointments for administration. Systemic medicaments may for example be formulated for enteral or parenteral administration.

[0043] For example, suitable pharmaceutical therapies include Levodopa or dopamine agonists such as cabergoline, co-careldopa, foslevodopa-foscarbidopa, Apomorphine, bromocriptine, pramipexole, ropinirole and rotigotine; catechol-O-methyltransferase inhibitors (COMT inhibitors) such as Entacapone, Opicapone, Tolcapone, Nebicapone, Neluxicapone or Nitecapone; aromatic L-amino acid decarboxylase inhibitors such as Benserazide, Carbidopa, Methyldopa, alpha-Difluoromethyl-DOPA, 3', 4', 5,7-Tetrahydroxy-8-methoxyisoflavone, Epigallocatechin gallate or Epigallocatechin;

[0044] Monoamine oxidase inhibitors (MAOIs) such as selegiline, or moclobemide, or Pargyline; Fludrocortisone or Midodrine. Medicaments that reduce the accumulation or aggregation of a-Synuclein such as immunotherapy against a-Synuclein optionally with agents that reduce neuroinflammation are also envisaged.

[0045] In a third aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2).

[0046] In a fourth aspect, the present invention provides a monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1).

[0047] The antibody according to the third and fourth aspect of the invention are, in particular, suitable for use in carrying out the methods of immunoassay according to the first aspect of the invention. Preferred embodiments and features of the antibody according to the third and fourth aspect will therefore be apparent from the above discussion of the preferred embodiments of the methods according to the first aspect.

[0048] In a fifth aspect, the present invention provides an immunoassay kit comprising a monoclonal antibody in accordance with the third aspect of the present invention, and at least one of:

[0049] a streptavidin coated well plate

[0050] a biotinylated peptide EAYEMPSEEG-L-Biotin (SEQ ID NO: 7), wherein L is an optional linker

[0051] a biotinylated peptide Biotin-L-LEDMPVDPDN (SEQ ID NO: 8), wherein L is an optional linker

[0052] a secondary antibody for use in a sandwich immunoassaya calibrator protein comprising the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2)

[0053] - a calibrator protein comprising the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1)

[0054] an antibody biotinylation kit

[0055] an antibody HRP labelling kit

[0056] an antibody radiolabelling kit

[0057] The immunoassay kit according to the fourth aspect of the invention is, in particular, suitable for use in carrying out the method of immunoassay according to the first aspect of the invention. Further preferred embodiments and features of the immunoassay kit according to the fourth aspect will therefore be apparent from the above discussion of the preferred embodiments of the methods according to the first aspect.

[0058] Monoclonal antibodies that specifically bind to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) can be generated via any suitable techniques known in the art. For example, the monoclonal antibody may be raised against a synthetic peptide comprising or consisting of the amino acid sequence LEDMPVDPDN (SEQ ID NO: 1), such as, for example, by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence LEDMPVDPDN (SEQ ID NO: 1) which may be optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning a single antibody-producing cell, and assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0059] Monoclonal antibodies that specifically bind to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) can be generated via any suitable techniques known in the art. For example, the monoclonal antibody may be raised against a synthetic peptide comprising or consisting of the amino acid sequence EAYEMPSEEG (SEQ ID NO: 2), such as, for example, by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence EAYEMPSEEG (SEQ ID NO: 2), which may be optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning a single antibody-producing cell, and assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.The monoclonal antibody that specifically binds to the N-terminal target sequence of a-Synuclein (i.e., EAYEMPSEEG (SEQ ID NO: 2)) may preferably comprise one or more complementarity-determining regions (CDRs) selected from:

[0060] CDR-H1: SSWIF (SEQ ID NO: 9)

[0061] CDR-H2: DIHPKNGKTNYNEKFKG (SEQ ID NO: 10)

[0062] CDR-H3: YPY (SEQ ID NO: 11)

[0063] CDR-L1: RSSQTIVHGNGNTYLE (SEQ ID NO: 12)

[0064] CDR-L2: KVSNRFS (SEQ ID NO: 13)

[0065] CDR-L3: FQGSHVPCT (SEQ ID NO: 14).

[0066] Preferably, the monoclonal antibody may comprise at least 2, 3, 4, 5, or all 6 of the above listed CDR sequences.

[0067] Preferably, the monoclonal antibody has a light chain variable region comprising the CDR sequences:

[0068] CDR-L1: RSSQTIVHGNGNTYLE (SEQ ID NO: 12)

[0069] CDR-L2: KVSNRFS (SEQ ID NO: 13)

[0070] CDR-L3: FQGSHVPCT (SEQ ID NO: 14).

[0071] Preferably, the monoclonal antibody may have a light chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics).

[0072] RSSQTIVHGNGNTYLEH / YLQ PGQSP LL / YKVSNRFSGVPDRFSGSGSGTDFTL / / VR VEAEDLG VYYCFQGSHVPCT (SEQ ID NO: 15)

[0073] Preferably, the monoclonal antibody has a heavy chain variable region comprising the CDR sequences:

[0074] CDR-H1: SSWIF (SEQ ID NO: 9)

[0075] CDR-H2: DIHPKNGKTNYNEKFKG (SEQ ID NO: 10)

[0076] CDR-H3: YPY (SEQ ID NO: 11).

[0077] Preferably, the monoclonal antibody may have a heavy chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantiallyidentical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics).

[0078] SSN\FWTKQKPGQGLEWIGD\HPKNGKJNYNEK.FKGKATLTVDTSSSTAYVDLGSLTSE DSGVYYCAGYPY (SEQ ID NO: 16)

[0079] As used herein, the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences Between the CDRs of another antibody if they have at least 70%, 80%, 90% or at least 95% similarity or identity. The similar or identical amino acids may be contiguous or noncontiguous.

[0080] The framework sequences may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gin; Group 3 His, Arg, Lys; Group 4 Met, Leu, lie, Vai, Cys; Group 5 Phe, Thy, Trp.

[0081] A program such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences.

[0082] In certain preferred embodiments, the monoclonal antibody that specifically binds to a -SYN-C1 may comprise the light chain variable region sequence:

[0083] DVLMTQSPLSLTVSLGDQAS / SCRSSQTIVHGNGNTYLEI / I / YLQ PGQSP LL / YKVSNRF SGVPDRFSGSGSG TDFTLKINR VEAEDLGVYYCFQGSHVPCTFGAGTKLELK (SEQ ID NO: 17)and / or the heavy chain variable region sequence:

[0084] QVQLQQPGSVLVRPGASVKLSCKTSGYTFTSS\N\FWTKQKPGQGLEWIGD\HPKNGKT

[0085] (SEQ

[0086]

[0087] ID NO: 18) (CDRs in bold and underlined; framework sequences in italics).

[0088] Figures

[0089] Figure 1. Overview of a-SYN-C1 assay specificity. A) Structure of a-Synuclein, indicating the a-SYN-01 assay targeting the N-terminal region of a-Synuclein. B) Sequence alignment of the targeted a-Synuclein sequence in human, mouse, bovine, and rat species (black box). The sequence was aligned using Uniprot. C) Specificity of the a -SYN-C assay. Reactivity towards the standard peptide (EAYEMPSEEGYQDYEPEA; SEQ ID NO: 19), truncated peptide (AY EM PS EEG; SEQ NO ID: 4), elongated peptide (NEAYEMPSEEG; SEQ NO ID: 3) and non-sense standard peptide (ELPARITPSQ; SEQ ID NO: 20). No background signal was detected when coating with a non-sense coating peptide (Biotin- ELPARITPSQ (SEQ ID NO: 21)). Signals are shown as relative luminescence (RLU) per second, as a function of standard peptide.

[0090] Figure 2. Specificity of a-SYN-C1 towards Calpain-1 fragments of a-Synuclein. a-SYN-C fragments were cleaved after 1 hour, 3 hours and 6 hours of incubation with a-Synuclein and Calpain-1 (n=3 for all settings). The negative controls included cleavage buffer, the enzyme Calpain-1, and intact a-Synuclein. No statistical differences were found between the full-length a-synuclein and a-synuclein Cleaved by Calpain-1 (p=0.100 for all three times). Data are depicted as mean± 95%CI.

[0091] Figure 3 - Discovery Cohort: Serum levels of a -SYN-C1 is upregulated in patients with Parkinson’s Disease.

[0092] healthy donors (n=16), and Parkinson’s disease (n=16). Statistical differences between the healthy donors and patients with Parkinson’s Disease were calculated using a Mann-Whitney ll-test. The diagnostic potential was calculated by the area under the Receiver Operating Curve (ROC). Graphs are shown as mean±95% Cl. ***p<0.001.Figure 4 - Validation Cohort: Serum levels of a -SYN-C1 is upregulated in patients with Parkinson’s Disease.

[0093] healthy donors (n=15), and Parkinson’s disease (n=30).. Statistical differences between the healthy donors and patients with Parkinson’s Disease were calculated using a Mann-Whitney ll-test. The diagnostic potential was calculated by the area under the Receiver Operating Curve (ROC). Graphs are shown as mean±95% Cl. ****p<0.0001.

[0094] Examples

[0095] The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0096] Materials and methods

[0097] Materials and Methods

[0098] Synthetic peptides used for generation of monoclonal antibodies, assay development and assay validation were purchased from Genscript (Piscataway, NJ, US) (Table 1).

[0099] Table 1. Sequence of the N terminus synthetic peptides used for monoclonal antibody production, assay development, and validation. *Keyhole Limpet Hemocyanin Peptides Sequence

[0100] Immunogenic peptide EAYEMPSEEG-GGC-KLH (SEQ ID NO: 22)

[0101] Coating peptide EAYEMPSEEGK-Bio (SEQ ID NO: 23)

[0102] Selection Peptide EAYEMPSEEGYQDYEPEA (SEQ ID NO: 19)

[0103]

[0104] Elongated selection NEAYEMPSEEG (SEQ ID NO: 3)

[0105] peptide

[0106] Truncated selection AYEMPSEEG (SEQ ID NO: 4)

[0107] peptide

[0108] Non-sense coating Bio-ELPARITPSQ / ELPARITPSQ (SEQ ID NO: 21 / SEQ ID and standard peptide NO: 20)

[0109]

[0110] Monoclonal antibody development, production, and characterization - N terminus sequence, a-SYN-C1

[0111] Monoclonal antibodies were generated by immunization of the following amino acid sequence122' J.EAYEMPSEEG'132(SEQ ID NO: 2), targeting a Calpain-1 cleaved N-terminal a-synuclein sequence. Immunization was initiated by subcutaneous injection of 200 l emulsified antigen and 100 g of immunogenic peptide (EAYEMPSEEG-GGC-KLH (SEQ ID NO: 22)) in 4- to 6-week-old Balb / C mice using Stimmune (Thermo Fisher). Immunizations were repeated every second week. The mouse with the highest stable serum titer was selected for fusion and boosted intravenously with 50 pg immunogenic peptide in 100 pl 0.9% NaCI solution 3 days before isolation of the spleen for cell fusion. Hybridoma cells were produced by fusion of the mouse spleen cells with SP2 / 0 myeloma cells as described by Gefter et al.18. The generated clones were plated into a 96-well microtiter plates for further growth, and the limiting dilution method was applied to promote monoclonal growth. Reactivity to the supernatant were tested by an indirect ELISA performed on streptavidin-coated plates. EAYEMPSEEGK-Biotin(SEQ ID NO: 23) was used as screening peptide, while the standard peptide EAYEMPSEEGYQDYEPEA (SEQ ID NO: 19) was used to further test the specificity of the newly developed antibody clones. Supernatant was collected from the hybridoma cells and purified using HiTrap affinity columns (GEHealthcare Life Science, Little Chaifront, Buckinghamshire, UK) according to manufacturer’s instructions and antibody isotype was determined using Rapid ELISA Mouse monoclonal antibody Isotyping Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

[0112] The generated antibodies were sequenced and the CDRs determined.

[0113] The sequence of the chains are as follows (Signal peptide in bold and italic; CDRs underlined and in bold; framework sequences in italics; constant region in normal font):

[0114] Heavy chain: Amino acid sequence (461 aa)MG WSSIILFL VA TA TGVHSQ VQLQQPGSVL VRPGA S VKLSCKTSG YTFTSS\N\FWTKQK PGQGLEWIGD HPKNGKTNYNEKFKGKATLTVDTSSSTAYVDLGSLTSEDSGVYYCAGY PYI / I / GQGTLVTV'SAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL SSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCWVDVSEDDPDVQISWFVNNVEVH TAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR APQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGS YFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 24)

[0115] Light chain: Amino acid sequence (238 aa) MKLPVRLLVLMFWIPASTSDVLMTQSPLSLTVSLGDQASISCRSSQTNHGNGNTYLEW YLQKPGQSPKLLIYKVSNRFSG VPDRFSGSGSG TDFTLKINR VEAEDLGVYYCFQGSHV PCTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE

[0116] C (SEQ ID NO: 25)

[0117] Reactivity towards human serum was tested with purchased samples from a commercial supplier (Valley Biomedical, Winchester, VA). The monoclonal antibodies were selected to specifically recognize the standard peptide (EAYEMPSEEGYQDYEPEA; SEQ ID NO: 19), and not an elongated or truncated sequence of the peptides (NEAYEMPSEEG (SEQ ID NO: 3) and AYEMPSEEG (SEQ ID NO: 4), respectively).

[0118] Monoclonal antibody development, production, and characterization - C terminus sequence, a-SYN--C2

[0119] Monoclonal antibodies are generated by immunization of the following amino acid sequence113' LEDMPVDPDNJ.'122(SEQ ID NO: 1), targeting a C-terminal neoepitope of the Calpain-1 cleaved a-synuclein sequence. Immunization is initiated by subcutaneous injection of emulsified antigen and immunogenic peptide (KLH-GGC-LEDMPVDPDN (SEQ ID NO: 26)) in 4- to 6-week-old Balb / C mice using Stimmune (Thermo Fisher).

[0120] Immunizations are repeated every second week. The mouse with the highest stable serum titer is selected for fusion and boosted intravenously with immunogenic peptide in NaCI solution 3 days before isolation of the spleen for cell fusion. Hybridoma cells are produced by fusion of the mouse spleen cells with SP2 / 0 myeloma cells as described by Gefter et al.18. The generated clones are plated into a 96-well microtiter plates for further growth, and the limiting dilution method is applied to promote monoclonal growth.

[0121] Reactivity to the supernatant is tested by an indirect ELISA performed on streptavidin-coated plates. Biotin-LEDMPVDPDN (SEQ ID NO: 27) is used as screening peptide, while the standard peptide with the C-terminus XLEDMPVDPDN (SEQ ID NO: 28) where X is 1 or more preceding amino acids from the human a-synuclein sequence (Uniprot Sequence P37840) is used to further test the specificity of the newly developed antibody clones. Supernatant is collected from the hybridoma cells and purified using HiTrap affinity columns (GEHealthcare Life Science, Little Chaifront, Buckinghamshire, UK) according to manufacturer’s instructions and antibody isotype is determined using Rapid ELISA Mouse monoclonal antibody Isotyping Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

[0122] Reactivity towards human serum is tested with purchased samples from a commercial supplier (Valley Biomedical, Winchester, VA). The monoclonal antibodies are selected to specifically recognize the standard peptide (XLEDMPVDPDN; SEQ ID NO: 28), and not an elongated or truncated sequence of the peptides (LEDMPVDPDNE (SEQ ID NO: 5) and LEDMPVDPD (SEQ ID NO: 6), respectively).

[0123] a-SYN-C1 assay development

[0124] The development of the competitive colorimetric immunoassay included preliminary optimizing experiments to identify the right reagents, concentrations, incubation-time and -temperature. The final a-synuclein competitive ELISA procedure was as follows: A 96-well streptavidin-coated microplate (Greiner Bio-One, Kremsmunster, Austria) was coated with 2 ng / mL biotinylated synthetic peptide (EAYEMPSEEG-K-Biotin (SEQ ID NO: 23)) dissolved in assay buffer (25 mM phosphate buffered saline (PBS), 1% bovine serum albumin, 0.1% Tween-20, 0.36% Bronidox, 8 g / L NaCI, adjusted to pH 7.4 at 20 °C) and incubated for 30 min at 20 °C with constant shaking (300 rpm) in darkness. Next, 20 pL / well of standard peptide (50 ng / mL) and samples were added to the appropriate wells, followed by the addition of 100 pL / well of HRP-labelled antibody diluted in assay buffer to a concertation of 281 ng / mL and incubated for 1 h at 20 °C with constant shaking (300 rpm) in darkness. After each incubation step, wells were washed five times with the washing buffer (20mM Tris, 50mM NaCI, pH 7.2). The TMB solution was placed at room temperature 60 min prior to use and 100 pL / well were added to plate and incubated for 15 min at 20 °C with constant shaking (300 rpm) in darkness. The reaction was stopped by adding 100 pl / well of 1% H2SO4. The absorbance was measured at 450 nm with 650 nm as a reference within 30 min on a microplate absorbance reader (VersaMax, Molecular Devices, CA, USA). A standard curve was plotted using a 4-parameter logistic curve fit Y= (A - D) / (1 + (x / C)AB) + D, where R > 0.9. Data were analyzed using the SoftMax Pro version 7.0.3 software.

[0125] a-SYN-C2 assay development

[0126] Similarly, the development of the competitive colorimetric immunoassay includes preliminary optimizing experiments to identify the right reagents, concentrations, incubation-time and -temperature. The typical a-synuclein competitive ELISA procedure is as follows: A 96-well streptavidin-coated microplate (Greiner Bio-One, Kremsmunster, Austria) is coated with biotinylated synthetic peptide (Biotin-K-LEDMPVDPDN (SEQ ID NO: 29)) dissolved in assay buffer and incubated at 20 °C with constant shaking (300 rpm) in darkness. Next, standard peptide and samples are added to the appropriate wells, followed by the addition of HRP-labelled antibody diluted in assay buffer and incubated at 20 °C with constant shaking (300 rpm) in darkness. After each incubation step, wells are washed five times with the washing buffer.The TMB solution is placed at room temperature 60 min prior to use and added to plate and incubated at 20 °C with constant shaking (300 rpm) in darkness. The reaction is stopped by adding 1% H2SO4. The absorbance was measured at 450 nm with 650 nm as a reference within 30 min on a microplate absorbance reader (VersaMax, Molecular Devices, CA, USA). A standard curve was plotted using a 4-parameter logistic curve fit Y = (A - D) / (1 + (x / C)AB) + D, where R > 0.9. Data were analyzed using the SoftMax Pro version 7.0.3 software.

[0127] Technical evaluation

[0128] Assay linearity was determined by two-fold dilutions of four human serum samples and calculated as percentage of recovery of the undiluted sample. To determine the assay accuracy, human serum samples were spiked with standard peptide and a human serum sample with a known high a-SYN-CI concentration and calculated as the percentage recovery of the measured value and the expected concentration of the peptide or human serum sample. Specificity of the generated monoclonal antibody was calculated as percentage of signal inhibition by two-fold diluted standard peptide (EAYEMPSEEGYQDYEPEA; SEQ ID NO: 19), elongated peptide (NEAYEMPSEEG; SEQ ID NO: 3), truncated peptide (AYEMPSEEG; SEQ ID NO: 4), and non-sense peptide (ELPARITPSQ; SEQ ID NO: 20). Interference of substances present in blood, was tested by adding a low / high content of hemoglobin (2.50 / 5 mg / mL), lipemia / lipids (1.50 / 5 mg / mL) and biotin (5-100 ng / mL) to a serum sample with a known concentration of a-SYN-CI. The normal reference levels for hemoglobin, lipemia / lipids and biotin were 0-10 mg / dL (0-0.0016 mmol / L), <150 mg / dl (<1.69 mmol / L) and 0.22-3.00 ng / ml, respectively. Thestability of the analyte was examined through temperature tests (0, 2-, 4-, 24-, and 48-hours of incubation at either 4 °C or 20 °C) and five freeze-thaw cycles of human serum samples. The recovery was calculated with 0 hours / 0 cycle of the sample as a reference. The intra- and inter-assay variation was determined by 10 independent runs of eight quality controls and two kit control runs in double determinations, while the measurement range was defined as the range between lower limit of measurement range (LLMR) and the upper limit of measurement range (ULMR) on the 10 runs. The standard curves from the 10 independent runs were used to determinate the IC50 (half-maximal inhibition concentration). All samples for the technical validation and biological measurements were run in double determination.

[0129] The O-SYN-C2 assay is similarly assessed.

[0130] In vitro cleavage

[0131] Human recombinant a-Synuclein (cat no. AG938, Sigma-Aldrich, Darmstadt, Germany), was cleaved with Native human Calpain-1 (cat. No. ab91019, abeam, Cambridge, UK). The a-Synuclein was reconstituted in MilliQ water and mixed in a 10:1 concentration with Calpain-1 in cleavage buffer (100 mM HEPES, 100 mM NaCI, 10 mM CaCI2, 2 mM Zn acetate, pH 7.5). As controls, the cleavage buffer, and the cleavage buffer with a-Synuclein or Calpain-1 were mixed alone. Cleavage was performed for 1hour, 3hours, and 6 hours at 37 °C, and subsequently stopped by 50 pM EDTA. Samples were analyzed in the a-SYN-CI assay.

[0132] Biological evaluation of a-SYN-C1 in a discovery and validation cohort

[0133] The biological capability of a-SYN-CI was evaluated in two independent cohorts (Discovery and Validation) in serum samples obtained from the commercial vendors Proteogenex (Culver City, CA, USA), and was approved by the local ethical committee (Russian Oncological Research Center, Blokhin Rams Ethics Committee review form, Protocol no. PG-ONC 2003 / 1), and BiolVT (Westerbury, NY, USA). Informed consent was obtained from all participants. The discovery cohort included patients with Parkinson’s disease (n=16) and healthy donors (n=16), while the validation cohort included patients with Parkinson’s disease (n=30) and healthy donors (n=15). The study was executed in compliance with the Helsinki Declaration of 2013. Serum samples were obtained and stored at -80°C until biomarker analysis.

[0134] AnimalsAll animals were treated according to the guidelines for animal welfare. Monoclonal antibody production in mice was approved by the Danish National Authority (The Animal Experiments Inspectorate) under approval number 2013-15-2934-00956. The study is reported in accordance with ARRIVE guidelines. Mice were purchased from Brogaarden, Denmark, and sacrificed by cervical dislocation.

[0135] Statistical analysis

[0136] Patient characteristics are presented as a number (frequency) and percentage for categorical variables and either mean with standard deviation or mean with range for continuous variables. Statistical differences between the full-length a-synuclein and a-synuclein cleaved by Calpain-1 during the cleavage experiments (1 hours, 3 hours, and 6 hours), together with healthy donors and patients with Parkinson’s Disease were calculated using a Mann-Whitney ll-test. The diagnostic potential was calculated by the area under the Receiver Operating Curve (ROC). Graphs are shown as mean±95% Cl. For all statistical analyses performed, a P-value below 0.05 was considered significant. Statistical analysis and graphs were performed using GraphPad Prism version 9 (GraphPad Software, Inc., La Jolla, CA) and MedCalc version 19.3 (MedCalc Software, Ostend, Belgium).

[0137] Results

[0138] Specificity, accuracy, and precision of the a-SYN-C1 assay

[0139] The a-SYN-C1 assay targets a neo-epitope fragment located in the C-terminal region (Figure 1A). The human sequence was aligned using UNIPROT, and the corresponding sequence in mouse, rat, and bovine are 100% aligned (Figure 1B). The hybridomas producing the best mAbs were screened for reactivity towards the standard peptide (synthetic peptide for calibration curve) and native material (serum). The clone NBH-499-1 was chosen for assay development and determined as an lgG1 subtype.

[0140] To evaluate the specificity of the a-SYN-C1 assay, the mAb was tested towards the elongated peptide, truncated peptide, non-sense standard peptide non-sense coater, and full-length a-Synuclein protein, and showed no reactivity towards those peptides (Figure 1C). In vitro cleavage experiments confirmed that cleavage of a-SYN by calpain-1 resulted in the generation of a-SYN-C fragments within 1 hour of cleavage after which they decreased. No reactivity was found towards the non-cleaved a-Synuclein or Calpain-1 (Figure 2) confirming very high specificity of the assay for the cleavage epitope.Technical validation was performed to evaluate the novel a -SYN-C1 assay. Overall, the a-SYN-01 assay demonstrated good technical performance (Table 2), including accepted inter- and intra-variations, analytes stability and limited interference from endogenous.

[0141] Table 2. Summary of technical parameters for a-SYN-C1 assay

[0142] TECHNICAL VALIDATION RESULTS (Accepted recovery: 100 ± 20%) IC50 3.44 ng / mL

[0143] Measurement range (LLMR-ULMR) 0.27-30.30 ng / mL

[0144] Inter-assay variation, mean (range) 5.8 % (2.7-9.6)

[0145] Intra-assay variation, mean (range) 12.8 % (4.6-21.0)

[0146] Dilution recovery of human serum, mean 105 % (87.0-106)

[0147] (range)

[0148] Spiking recovery (selection peptide in 105 % (88.0-122.0)

[0149] serum), mean (range)

[0150] Analyte stability and recovery, 48h at 92.8% (86.2-100.4) 192.8% (81.9-102.9) 4°C / 20°C, mean (range)

[0151] Freeze-thaw stability, five cycles, mean 81.7 % (68.0-89.9)

[0152] (range)

[0153] Hemoglobin interference (low / high), mean 101.3 % (100.0-107.4) / 95.1% (88.3-101.6) (range)

[0154] Lipemia interference (low / high), mean 106.5 % (100.9-108.0) / 110.4 % (108.0-113.3) (range)

[0155] Biotin interference <100 ng / mL

[0156] a-SYN-C1 is elevated in patients with Parkinson’s Disease compared to healthy controls - results from the discovery and evaluation cohort

[0157] Two study cohorts were included to test the biological utility of the assay. The discovery cohort included patients with Parkinson’s disease (mean age: 73.7 years, 50.0% male), and healthy donors (mean age: 59.5 years, 56.3% male), Table 3.

[0158]

[0159] Discovery Cohort Validation Cohort

[0160]

[0161] Healthy donors Parkinson's p value Healthy Parkinson's p value (n=16) Disease donors Disease

[0162] (n=16) (n=15) (n=30)

[0163] Age, mean (SD) 59.5 (6.35) 73.7 (2.89) 0.004 60.3 (5.6) 64.2 (6.7) 0.065

[0164] Sex, Male, n (%) 9 (56.3%) 8 (50%) 0.727 9 (60.0%) 15 (50%) 0.531

[0165] BMI, mean (SD) NA 25.2 (3.7) 0.592 NA 26.1 (3.2)

[0166] Caucasian, n (%) 16 (100%) 16 (100%) 1.000 15 (100%) 30 (100%) 1.000

[0167] Years since diagnosis, NA 1.5 (2.0) NA 1.1 (0.4)

[0168] mean (SD)

[0169] Signs

[0170] Hypokinesia, n (%) 5 (31.3%) 8 (26.7%)

[0171] Postural instability, n (%) 9 (56.3%) 10 (33.3%)

[0172] Muscle rigidity, n (%) 6 (37.5%) 14 (46.7%)

[0173] Tremor, n (%) 9 (56.3%) 16 (53.3%)

[0174] Current treatment

[0175]

[0176] Levodopa, n (%) 8 (50%) 10 (33.3%)Abbreviations: BMI: Body Mass Index. Categorical variables are written as number (percentage), while continuous variables are mean (standard deviation). Statistical differences between the clinical and patient demographics were calculated by a Mann-Whitney test.

[0177] Here a -SYN-C1 was significantly elevated in serum from patients with Parkinson’s disease compared to healthy donors (p=0.0007, Figure 3A) and presented an AU ROC of 0.83 (95%CI: 0.69-0.98, p=0.0014, Figure 3B). The validation cohort included patients with Parkinson’s disease (mean age: 64.2 years, 50% male), and healthy donors (mean age: 60.3, 60% male), Table 3. Here a -SYN-C1 was significantly elevated in serum from patients with Parkinson’s disease (p=0.0001, Figure 4A), and presented an AUROC of 0.85 (95% Cl: p=0.0001 Figure 4B).

[0178] Discussion

[0179] In this study, a competitive ELISA for detection of a-SYN-CI was developed, validated, and evaluated using a monoclonal antibody targeting the N-terminal cleavage site of Calpain-1 cleaved a-Synuclein. The main findings of this study were as follows:

[0180] 1) development of a robust and specific a-SYN-CI assay towards the sequence EAYEMPSEEG (SEQ ID NO: 2);

[0181] 2) a-SYN-CI is detectable in human serum;

[0182] 3) a -SYN-C1 is upregulated in patients diagnosed with Parkinson’s disease compared to healthy donors.

[0183] This is the first study to develop and technically validate a blood-based biomarker quantifying the levels of a specific fragment targeting a-Synuclein degraded by Calpain-1, followed by biological evaluation in serum from patients with Parkinson’s disease.

[0184] The a -SYN-C1 assay is characterized as a technically robust and accurate assay by showing acceptable dilution recovery, interference, and stability tests. The intra- and intervariation was accepted with values of 5.8% and 12.8%, respectively. The assay was further characterized as being specific towards the N-terminal cleavage site of a-synuclein after cleavage by Calpain-1 , with highest activity after 1 hour of incubation, which confirmed that calpain-1 cleaves and generates the targeted fragment.

[0185] Calpain-1 has previously known to be involved in formation and secretion of A in Alzheimer’s disease, indicating its role in neurological disorders13. Cleavage sites ofCalpain-1 cleaved a-synuclein were originally identified by Mishizen-Ebert et. al. in 200314, while the site targeting in this assay was identified by Dufty et. al. in 2007, who indicated that this cleavage could contribute to the initiation of the synucleinopathy, i.e. trigger aggregation and thereby play an important role in the initiation of PD12. How fragmented a-Synuclein contributes to maintaining tissue homeostasis and function needs further evaluation, but studies have showed truncated a-Synuclein fragments are linked to neurotoxicity in healthy individuals and PD patients1516.

[0186] Previously developed biomarkers targeting a-Synuclein has been focused on targeting the intact protein, by immunohistochemistry, ELISA, western blot, Luminex, and mass spectrometry17. Fragments of a-Synuclein could aid achieve the unmet need of finding patients with early PD, monitoring progressive patients, and identifying those who are likely to benefit from a given treatment.

[0187] In conclusion, this study quantitatively measures a specific fragment of a-Synuclein degraded by Calpain-1 in serum PD patients and demonstrates the clinical utility of the fragment as a diagnostic tool. Since a-Synuclein is well-established to have an important role in PD pathology, easily accessible tools to quantify changes in serum, could provide valuable information for assessing patients' suitability for targeted treatments, thereby addressing the current gap in biomarkers for clinical management and trials.

[0188] In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used to mean ‘including or consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.References

[0189] 1. Port, R. J. et al. People with Parkinson’s Disease: What Symptoms Do They Most Want to Improve and How Does This Change with Disease Duration? J. Parkinsons. Dis.

[0190] 11, 715-724 (2021).

[0191] 2. Ray Dorsey, E. et al. Global, regional, and national burden of Parkinson’s disease, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet. Neurol. 17, 939 (2018).

[0192] 3. Greenland, J. C., Williams-Gray, C. H. & Barker, R. A. The clinical heterogeneity of Parkinson’s disease and its therapeutic implications. Eur. J. Neurosci. 49, 328-338 (2019).

[0193] 4. Kalia, L. V. & Lang, A. E. Parkinson’s disease. Lancet (London, England) 386, 896-912 (2015).

[0194] 5. Vu, T. C., Nutt, J. G. & Holford, N. H. G. Progression of motor and nonmotor features of Parkinson’s disease and their response to treatment. Br. J. Clin. Pharmacol.

[0195] 74, 267-283 (2012).

[0196] 6. Lawton, M. et al. Developing and validating Parkinson’s disease subtypes and their motor and cognitive progression. J. Neurol. Neurosurg. Psychiatry 89, 1279-1287 (2018).

[0197] 7. Meade, R. M., Fairlie, D. P. & Mason, J. M. Alpha-synuclein structure and Parkinson’s disease. Mol. Neurodegener. 14, 1-14 (2019).

[0198] 8. Li, W. et al. Aggregation promoting C-terminal truncation of a-synuclein is a normal cellular process and is enhanced by the familial Parkinson’s disease-linked mutations. Proc. Natl. Acad. Sci. U. S. A. 102, 2162-2167 (2005).

[0199] 9. Mishizen-Eberz AJ, Norris EH, Giasson Bl, Hodara R, Ischiropoulos H, Lee VM, Trojanowski JQ, L. D. Cleavage of a-Synuclein by Calpain: Potential Role in Degradation of Fibrillized and Nitrated Species of a-Synuclein. Biochemistry 44, 7818-7829 (2005). 10. Mizuta I, Satake W, Nakabayashi Y, Ito C, Suzuki S, Momose Y, Nagai Y, Oka A, Inoko H, F. J. Multiple candidate gene analysis identifies a-synuclein as a susceptibility gene for sporadic Parkinson’s disease. Hum Mol Genet 15, 1151-1158 (2006).

[0200] 11. Karsdal, M. a et al. Biochemical markers and the FDA Critical Path: how biomarkers may contribute to the understanding of pathophysiology and provide unique and necessary tools fordrug development. Biomarkers 14, 181-202 (2009).

[0201] 12. Dufty, B. M. et al. Calpain-cleavage of alpha-synuclein: connecting proteolytic processing to disease-linked aggregation. Am. J. Pathol. 170, 1725-1738 (2007).

[0202] 13. Nixon, R. A. et al. Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer’s disease. Ann. N. Y. Acad. Sci. 747, 77-91 (1994).14. Mishizen-Eberz, A. J. et al. Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro. J. Neurochem. 86, 836-847 (2003).

[0203] 15. Liu, C. W. et al. A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease. J. Biol. Chem. 280, 22670-22678 (2005).

[0204] 16. Murray, I. V. J. et al. Role of alpha-synuclein carboxy-terminus on fibril formation in vitro. Biochemistry 42, 8530-8540 (2003).

[0205] 17. Atik, A., Stewart, T. & Zhang, J. Alpha-Synuclein as a Biomarker for Parkinson’s Disease, doi: 10.1111 / bpa.12370.

[0206] 18. Gefter, M. L., Margulies, D. H. & Scharff, M. D. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 3, 231-6 (1977).

Claims

Claims1. A method of immunoassay comprising;i) contacting a patient sample with a monoclonal antibody that specifically binds to a neoepitope generated by the cleavage of a-synuclein by Calpain-1 andii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

2. Amethod of immunoassay of claim 1 , said method comprising;i) contacting a patient sample with a monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) and / or a monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1);andii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

3. A method of immunoassay of claim 1 or claim 2, said method comprising;i) contacting a patient sample with a monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1);andii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

4. A method as claimed in any preceding claim, wherein the method is a method of immunoassay for detecting and / or monitoring a synucleinopathy or a level of severity thereof in a patient, the method further comprising;iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.

5. A method as claimed in claim 4, wherein the synucleinopathy is selected from Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), pure autonomic failure (PAF), neuroaxonal dystrophies and Alzheimer's Disease with Amygdalar Restricted Lewy Bodies (AD / ALB).

6. A method as claimed in claim 4 or claim 5, wherein the synucleinopathy is Parkinson’s disease (PD).

7. A method as claimed in any preceding claim, wherein the patient sample is selected from blood, plasma or serum.

8. A method as claimed in any one of claims 2 to 7, wherein the monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) does not specifically bind to a peptide having the N-terminus amino acid sequence N EAYEMPSEEG (SEQ ID NO: 3).

9. A method as claimed in any one of claims 2 to 8, wherein the monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) does not specifically bind to a peptide having the N-terminus amino acid sequence AYEMPSEEG (SEQ ID NO: 4).

10. A method as claimed in any one of claims 2 to 9, wherein the monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2) is raised against a synthetic peptide having the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2).

11. A method as claimed in any one of claims 3 to 7, wherein the monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) does not specifically bind to a peptide having the C-terminus amino acid sequence LEDMPVDPDNE (SEQ ID NO: 5).

12. A method as claimed in any one of claims 3 to 7 or 11 , wherein the monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) does not specifically bind to a peptide having the C-terminus amino acid sequence LEDMPVDPD (SEQ ID NO: 6).

13. A method as claimed in any one of claims 3 to 7, 11 or 12 wherein the monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1) is raised against a synthetic peptide having the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1).

14. A method as claimed in any preceding claim, wherein the immunoassay is a competition assay or a sandwich assay.

15. A method as claimed in any preceding claim, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

16. A method of treating a synucleinopathy in a patient in need thereof, the method comprising:(a) carrying out a method of immunoassay of any one of claims 1 to 15; and(b) administering to the patient a therapy for the treatment of said synucleinopathy if it is determined in step (iii) that the patient has said synucleinopathy or a particular level of severity thereof.

17. A monoclonal antibody that specifically binds to the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2).

18. A monoclonal antibody as claimed in claim 17, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminus amino acid sequence N EAYEMPSEEG (SEQ ID NO: 3).

19. A monoclonal antibody as claimed in claim 17 or 18, wherein the monoclonal antibody does not specifically bind to a peptide having the N-terminus amino acid sequence AYEMPSEEG (SEQ ID NO: 4).

20. A monoclonal antibody as claimed in any one of claims 17 to 19, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2).

21. A monoclonal antibody that specifically binds to the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1).

22. A monoclonal antibody as claimed in claim 21 , wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence LEDMPVDPDNE (SEQ ID NO: 5).

23. A monoclonal antibody as claimed in claim 21 or 22, wherein the monoclonal antibody does not specifically bind to a peptide having the C-terminus amino acid sequence LEDMPVDPD (SEQ ID NO: 6).

24. A monoclonal antibody as claimed in any one of claims 21 to 23, wherein the monoclonal antibody is raised against a synthetic peptide having the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1).

25. An immunoassay kit comprising a monoclonal antibody as claimed in any one of claims 17 to 24, and at least one of:a streptavidin coated well platea biotinylated peptide EAYEMPSEEG-L-Biotin (SEQ ID NO: 7), wherein L is an optional linkera biotinylated peptide Biotin-L-LEDMPVDPDN (SEQ ID NO: 8), wherein L is an optional linkera secondary antibody for use in a sandwich immunoassaya calibrator protein comprising the N-terminus amino acid sequence EAYEMPSEEG (SEQ ID NO: 2)a calibrator protein comprising the C-terminus amino acid sequence LEDMPVDPDN (SEQ ID NO: 1)an antibody biotinylation kitan antibody HRP labelling kitan antibody radiolabelling kit.

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