Alpha-synuclein antibodies for use in diagnostic assays and treatment of alpha-synucleinopathies

Abstract

The present invention relates to alpha-synuclein antibodies which are useful in diagnostic assays for detecting aggregates, oligomers and fibrillar forms of alpha- synuclein in patient samples. These antibodies enable diagnosis and efficient treatment of alpha-synucleinopathies such as Parkinson's Disease (PD) or Multiple System Atrophy (MSA) patients by assessing and monitoring the pathological alpha-synuclein levels in patients.

Description

[0001] Novel Alpha-synuclein antibodies for use in diagnostic assays and treatment of alpha-synucleinopathies

[0002] Field of the invention

[0003] The present invention relates to alpha-synuclein antibodies which are useful in diagnostic assays for detecting and visualizing alpha-synuclein in patient samples. These antibodies enable diagnosis and efficient treatment of alpha-synucleinopathies such as Parkinson’s Disease (PD) or Multiple System Atrophy (MSA) patients by assessing and monitoring the alpha-synuclein levels in patient samples.

[0004] Background of the invention

[0005] Parkinson's disease (PD), Parkinson’s disease with dementia (PDD), dementia with Lewy bodies (DLB) and multiple systems atrophy (MSA) are examples of neurodegenerative disorders commonly referred to as alpha-synucleinopathies and characterized by the presence of alpha-synuclein brain pathology.

[0006] Alpha-synuclein is a small 140 amino acid intraneuronal protein, predominantly located presynaptically in healthy brains. In alpha-synucleinopathies, misfolding of alpha-synuclein results in the formation alpha-synuclein aggregates / oligomers inside neurons, such as Lewy bodies and or inside oligodendrocytes, such as glial cytoplasmic inclusion, which promotes neuronal degradation and oligodendrocyte dysfunction, respectively.

[0007] Small amounts of misfolded (oligomeric / aggregated) alpha-synuclein are found extracellularly in interstitial fluid in brain, in Cerebrospinal fluid (CSF) and blood. The source of this alpha-synuclein is not clear, but it is likely released from cells, both from cell bodies and synapses, for example during synaptic release when neuronal cells are activated. Some of it is also likely coming from diseased and dead cells. Alpha-synuclein is also found in many blood cells and platelets, which are a likely source of alpha-synuclein in blood.

[0008] There is evidence that the neurodegenerative process in alpha-synucleinopathies starts years, perhaps 10-20 years before clinical diagnosis. REM sleep behaviour disorder (RBD) is now recognized as the prodromal stage of an a-synucleinopathy. Most peoplewith RDB will convert to PD or DLB within the next 20 years from diagnosis of RBD. Other signs of prodromal stage of alpha-synucleinopathy include loss of olfaction. Today there are no biochemical methods to aid a clinician's diagnosis of an alpha-synucleinopathy before the motor symptoms are evident. At that point, substantial damage to the brain has already occurred. The importance of accurate diagnostic assays will become even greater as new disease modifying therapies emerge, which will hopefully stop or slow the progression of the disease.

[0009] Development of assays for the detection of aggregated / oligomeric forms of alpha-synuclein has been reported (Anastasia Bougea, 2024 Int J Mol Sci, 7;25(19): 10783. doi: 10.3390 / ijms251910783).

[0010] There is a need for improved diagnostic tools with enhanced sensitivity to identify patients suffering from diseases characterized by alpha-synuclein pathology, especially at early stages of disease to initiate treatment as early as possible. Such tools could also be used to detect and monitor alpha-synuclein levels in samples obtained from patients suffering from alpha-synucleinopathies.

[0011] Summary of the invention

[0012] The present invention provides antibodies with high sensitivity for fibrillar (oligomeric / aggregated) forms of alpha-synuclein which is useful in developing assays to diagnose patients suffering from alpha-synucleinopathies or to monitor the aggregated alpha-synuclein levels over time in samples obtained from such patients. Accordingly, the antibodies of the present invention can be used in assays to diagnose or monitor the disease progression of alpha-synucleinopathies. Alternatively, the antibodies can be used in assays to monitor or follow the treatment response of ongoing treatments of a patient suffering from an alpha-synucleinopathy.

[0013] The antibodies of the invention may also be used in methods of treating alpha-synucleinopathies comprising administering said antibodies to a patient in need thereof.

[0014] The antibodies of the invention (HLu 12_14, HLu 10_16, HLu 13_11, HLu 14_21, HLu 15_15 and HLu 11_51) all demonstrate high specificity and affinity for alpha-synucleinfibrils (oligomeric / aggregated forms of alpha-synuclein), with minimal binding to monomeric alpha-synuclein. In addition, the inventors of the present invention have demonstrated that these novel antibodies are particularly well suited for use in high sensitivity immunoassays for detecting oligomeric / aggregated / fibrillated alpha-synuclein in human samples. Such assays are preferably in vitro assays to quantify or visualize oligomeric / aggregated forms of alpha-synuclein in human samples.

[0015] Drawings

[0016] Figure 1A shows Exemplary data on specificity of HLu10_16. in binding to alpha-synuclein pre-formed fibrils (PFFs) over monomeric alpha-synuclein assessed by ELISA.

[0017] Figure IB shows Exemplary data on specificity of HLu10_16. in binding to pathological insoluble alpha-synuclein isolated from PD and MSA brain derived brain material over control assessed by ELISA

[0018] Figure 2A shows Exemplary data illustrating the linearity and lower-limit-of-quantifi cation (LLOQ) of quantitative immunoassay. Low variability, wide linear range and clear difference between background and lowest dilution point.

[0019] Figure 2B shows Exemplary data on the immunoassay measuring recovery in CSF samples spiked with high (HQC), medium (MQC) and low (LQC) concentration of alpha-synuclein preformed fibrils (PFFs). Sample recovery shows good accuracy at high (HQC), medium (MQC) and low (LQC) concentration of PFFs spiked into human CSF.

[0020] Figure 3 shows Exemplary data on the dose dependent inhibition of alpha-synuclein pathology (aggregation) development induced by alpha-synuclein preformed fibrils (PFFs) in primary neuronal cultures in vitro. The same inhibition is achieved in the absence and presence of high concentration of monomeric alpha-synuclein. HLu10_16 displays dose dependent inhibition of seeded alpha-synuclein aggregation in primary neuronal cultures in vitro. Minimal effects are observed upon challenge with highconcentrations of monomeric alpha-synuclein supporting the selective binding of of HLu10_16 to fibrillar alpha-synuclein species.

[0021] Figure 4A shows Exemplary data on the efficacy of HLu 12_14 and HLu 10 16 in depleting aggregated species of alpha-synuclein in brain samples from cases of PD and MSA to the level of a positive control, thus targeting all aggregated species in the sample. Aggregation-specific Cisbio analysis of HLu 10 14 and HLu 10 16 immunodepletion of alpha-synuclein aggregates from PD (putamen), MSA (putamen) brain homogenates, compared to control antibodies (Negative control, non-binding IgG and Positive control alpha-synuclein antibody). HTRF ratio is plotted along y-axis as measure of aggregated alpha-synuclein over experimental background. Dashed line represents experimental background.

[0022] Figure 4B shows Exemplary data of western blot analysis of immunoprecipitates using HLul2-14 and HLu10_16 against a negative and positive control. Western blots were stained for both phospho-alpha-synuclein (pS129) and total alpha-synuclein. Western blotting analysis of immunoprecipitates from Fig 4A. Alpha-synuclein is visualized using antibodies against total alpha-synuclein or its phosphorylated (pS129) species. Full-length and truncated species are indicated. Densitometry analysis was performed to quantify full-length total and pS129 alpha-synuclein species. Values below signal background were set to 0.

[0023] Figure 5 – shows Exemplary data of immunohistochemistry performed with HLu 10 16 on brain sections from patients with MSA in the pons. Staining demonstrates that the antibody specifically stains pathological alpha-synuclein aggregates in human brain. A) Entire imaged area of the pons, showing the region of quantification (grey square) for each concentration of the antibody used; 0.0007-0.2 mg / ml.

[0024] B) zoomed in image of the micrographs in A showing positively identified alpha-synuclein staining.C) Quantification of the number of specific puncta detected in the region of quantification for each concentration of the antibody used.

[0025] D) Further zoomed in micrograph showing alpha-synuclein staining with HLu 10 16 at a concentration of 0.07 mg / ml in black. White arrows point to pathological inclusions being included in the analysis (examples only, not comprehensive). Horizontal bars indicate limits of the area of quantification.

[0026] Detailed description of the invention

[0027] The object of the present invention is to provide alpha-synuclein antibodies for use in diagnostic methods for alpha-synuclein related disorders, i.e., alpha-synucleinopathies wherein accumulation of aggregated insoluble alpha-synuclein in the form of Lewy bodies or Glial cytoplasmic inclusions, are present in the brain. Such disorders include, but are not limited to, one or more neurodegenerative disorders such as Parkinson's disease (PD), Parkinson’s disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and REM sleep behavioural disorder (RBD) and other neurodegenerative disorders with alpha-synuclein pathology - combined these disorders are referred to as alpha-synucleinopathies.

[0028] The present invention provides highly sensitive antibodies for use in assays for detecting aggregated / oligomeric alpha-synuclein in human samples. Such sample may have been obtained from a subject suffering from an alpha-synucleinopathy. In one aspect the antibodies are employed in assays for diagnostic purposes. In an embodiment of this aspect the level of aggregated / oligomeric alpha-synuclein in the patient sample will be evaluated and compared to healthy control levels of aggregated / oligomeric alpha-synuclein, and following this evaluation the diagnosis can be established based on the ratio between the aggregated / oligomeric alpha-synuclein levels in the patients versus healthy controls. In another embodiment of this aspect the level of aggregated / oligomeric alpha-synuclein in the patient sample can be evaluated and compared to the patients baseline levels of aggregated / oligomeric alpha-synuclein and following this evaluation the diagnosis can be established based on the ratio between the patient’s current level of aggregated / oligomeric alpha-synuclein levels compared to thebaseline levels. In another aspect the antibodies are employed in assays for monitoring or detecting alpha-synuclein, which may be for the purpose of following treatment response or disease progression over time. In another aspect the antibodies of the invention may be used for treating alpha-synucleinopathies in a patient suffering from such disease.

[0029] According to one aspect of the invention, the invention relates to a method of treating a patient diagnosed with an alpha-synucleinopathy, such as Parkinson Disease or MSA, said method comprise using an antibody of the invention in such method by treating said patients with an effective amount of a therapeutic alpha-synuclein antibody and monitoring the treatment response with assays employing antibodies of the invention to detect aggregated alpha-synuclein in samples from said patient. In certain embodiments the antibodies of the invention may be therapeutic antibodies for treating alpha-synucleinopathy.

[0030] The treatment effect of the therapeutic antibody treatment may be evaluated measuring the level of aggregated / oligomeric alpha-synuclein in but not limited to CSF, blood, plasma, serum, tissue sections from any organ, urine and tears. The evaluation may be made prior to treatment initiation (e.g. when diagnosing the patient or anytime thereafter) or after at least about 1, 2, 3, 4 or more administrations of the therapeutic antibody. Following this the measurement of aggregated / oligomeric alpha-synuclein in the patient samples may be performed continuously on a regular basis, such as every 1 month, every 2 months, every 3 month, every 4 months, every 5 month, every 6 month or yearly. Based on the result of this evaluation of treatment response it may be determined whether the patient shall continue treatment, or if just diagnosed if the patient is to initiate treatment.

[0031] The present invention relates to alpha-synuclein antibodies for use in methods of diagnosis or following alpha-synucleinopathy progression in patients comprising the steps of

[0032] a) Obtaining a sample from a patientb) Applying the sample on an assay for detecting aggregated / oligomeric alpha-synuclein, which assay employs an antibody of the invention,

[0033] c) Optionally, comparing the results with a control

[0034] The control is preferably a sample from a healthy control or a previous sample obtained from the same patient.

[0035] The sample is preferably a CSF, blood sample, a serum sample, a plasma sample, a tissue section or a tissue sample.

[0036] The antibodies of the invention are alpha-synuclein antibodies which bind to human alpha-synuclein aggregates / oligomers.

[0037] The amino acid numbering of alpha-synuclein is given with respect to the sequence as shown below, with methionine (M) being amino acid residue 1 (SEQ ID NO: 1):

[0038] MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0039] Antibodies of the invention

[0040] Antibodies of the invention include HLu 10 16, HLu 11 51, HLu 12_14, HLu 13 11, HLu 14 21 and HLu 15 15. These antibodies are characterized by binding aggregated / oligomeric alpha-synuclein and having the following amino acid sequences. All CDRs are determined using the Kabat system.

[0041] All fully human IgGl antibodies of the invention have the following heavy chain constant domain (SEQ ID NO: 2): IgGl HC constant domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKAll VHH Fc Fusion antibodies of the invention have the following heavy chain Fc domain (SEQ ID NO: 3): Fc domain of VHH fusion antibodies DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0042] In certain embodiments the antibodies may be subject to modifications during antibody production, which may for example include truncation of the C-terminal Lysine in the constant domain of the heavy chain or the Fc domain of the VHH fusion antibody.

[0043] All fully human IgGl antibodies of the invention have the following light chain constant domain (SEQ ID NO: 4): IgGl LC constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0044] Antibody HLu 11 51:

[0045] Full length heavy chain (HC): SEQ ID NO: 5 EVHLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLDWVGRIRSKANSYAT AYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRQSGPFDYWGQGTLVAVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK CDRH1: GSAMH (SEQ ID NO: 9)

[0046] CDRH2: RIRSKANSYATAYAASVKG (SEQ ID NO: 10)

[0047] CDRH3: QSGPFDY (SEQ ID NO: 11)

[0048] Variable domain of the heavy chain (VH): SEQ ID NO: 6 EVHLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLDWVGRIRSKANSYAT AYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRQSGPFDYWGQGTLVAVSS

[0049] Full length light chain (LC): SEQ ID NO: 7 DIQMTQSPSSLSASVGDRVTITCRASQGISNFLAWYQQKAGKVPKLLIYTASTLLSGVPS RFSGSGSGTYFTLTISSLQPEDVATYYCQKYNSAPLTFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCDRL1: RASQGISNFLA (SEQ ID NO: 12)

[0050] CDRL2: TASTLLS (SEQ ID NO: 13)

[0051] CDRL3: QKYNSAPLT (SEQ ID NO: 14)

[0052] Variable domain of the light chain (VL): SEQ ID NO: 8 DIQMTQSPSSLSASVGDRVTITCRASQGISNFLAWYQQKAGKVPKLLIYTASTLLSGVPSRFSG SGSGTYFTLTISSLQPEDVATYYCQKYNSAPLTFGGGTKVEIK

[0053] Antibody HLu 10 16:

[0054] Full length heavy chain (HC): SEQ ID NO: 15 EVQLVESGGGLVQPGGSLKVSCAASGFTFSDSAIHWVRQASGKGLDWVGRIRSKANSYAK AYGASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSPYGFDIWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK CDRH1: DSAIH (SEQ ID NO: 19)

[0055] CDRH2: RIRSKANSYAKAYGASVKG (SEQ ID NO: 20)

[0056] CDRH3: GGSPYGFDI (SEQ ID NO: 21)

[0057] Variable domain of the heavy chain (VH): SEQ ID NO: 16 EVQLVESGGGLVQPGGSLKVSCAASGFTFSDSAIHWVRQASGKGLDWVGRIRSKANSYAK AYGASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSPYGFDIWGQGTMVTVSS

[0058] Full length light chain (LC): SEQ ID NO: 17 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTIPWTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CDRL1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 22)

[0059] CDRL2: WASTRES (SEQ ID NO: 23)

[0060] CDRL3: QQYYTIPWT (SEQ ID NO: 24)

[0061] Variable domain of the light chain (VL): SEQ ID NO: 18 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTIPWTFGQGTKVEIK

[0062] Antibody HLu 12_14:

[0063] Full length fusion heavy chain (HC): SEQ ID NO: 25 QLQLVESGGGLVQAGGSLRLSCAASGGTFRTYTMGWFRQRPGKERRLLSRISRTGLTTTY ADSVKGRFTISRDNAKNTVHLQMNSLKLEDTAVYYCAARPCAVLDSCEWNYWGPGTQVTV SSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CDRH1: TYTMG (SEQ ID NO: 27)

[0064] CDRH2: RISRTGLTTTYADSVKG (SEQ ID NO: 28)

[0065] CDRH3: RPCAVLDSCEWNY (SEQ ID NO: 29)

[0066] Variable domain of the heavy chain (VHH): SEQ ID NO: 26 QLQLVESGGGLVQAGGSLRLSCAASGGTFRTYTMGWFRQRPGKERRLLSRISRTGLTTTY ADSVKGRFTISRDNAKNTVHLQMNSLKLEDTAVYYCAARPCAVLDSCEWNYWGPGTQVTV SS

[0067] Antibody HLu 13 11:

[0068] Full length heavy chain (HC): SEQ ID NO: 30 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDDAAVYYCARAPYNSMDVWGKGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK CDRH1: SYGIS (SEQ ID NO: 34)

[0069] CDRH2: WISAYNGNTNYAQKLQG (SEQ ID NO: 35)

[0070] CDRH3: APYNSMDV (SEQ ID NO: 36)

[0071] Variable domain of the heavy chain (VH): SEQ ID NO: 31 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKL QGRVTMTTDTSTSTAYMELRSLRSDDAAVYYCARAPYNSMDVWGKGTTVTVSS

[0072] Full length light chain (LC): SEQ ID NO: 32 DVVLTQSPLSLPVTLGQPASISCRSSHSLVYSNGNTYLNWFRQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQGTHWPLTFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CDRL1: RSSHSLVYSNGNTYLN (SEQ ID NO: 37)

[0073] CDRL2: KVSNRDS (SEQ ID NO: 38)

[0074] CDRL3: MQGTHWPLT (SEQ ID NO: 39)

[0075] Variable domain of the light chain (VL): SEQ ID NO: 33 DVVLTQSPLSLPVTLGQPASISCRSSHSLVYSNGNTYLNWFRQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQGTHWPLTFGGGTKVEIK

[0076] HLu 14 21:

[0077] Full length heavy chain (HC): SEQ ID NO: 40

[0078] EVQLVESGGGLVQPGGSLKLSCAASGFIFSGSAMHWVRQASGKGLEWVGRIRTKANSYATAYAASVKGRFTISRDDSKDTAYLQMNSLKTEDTAVYYCTRPLMSTLTTFDFWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CDRH1: GSAMH (SEQ ID NO: 44)

[0079] CDRH2: RIRTKANSYATAYAASVKG (SEQ ID NO: 45)

[0080] CDRH3: PLMSTLTTFDF (SEQ ID NO: 46)

[0081] Variable domain of the heavy chain (VH): SEQ ID NO: 41 EVQLVESGGGLVQPGGSLKLSCAASGFIFSGSAMHWVRQASGKGLEWVGRIRTKANSYATAYAA SVKGRFTISRDDSKDTAYLQMNSLKTEDTAVYYCTRPLMSTLTTFDFWGQGTLVTVSS

[0082] Full length light chain (LC): SEQ ID NO: 42 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKVLIYAASTLQSGAPS RFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSVPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CDRL1: RASQGISNYLA (SEQ ID NO: 47)

[0083] CDRL2: AASTLQS (SEQ ID NO: 48)

[0084] CDRL3: QKYNSVPWT (SEQ ID NO: 49)

[0085] Variable domain of the light chain (VL): SEQ ID NO: 43 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKVLIYAASTLQSGAPS RFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSVPWTFGQGTKVEIK

[0086] Antibody HLu 15 15:

[0087] Full length heavy chain (HC): SEQ ID NO: 50 EVQLVESGGGLVQPGGSLKISCAASGFTFSGSAMHWVRQASGKGLEWVGRIRSKPNSYAT AYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSPYGFDIWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK CDRH1: GSAMH (SEQ ID NO: 54)

[0088] CDRH2: RIRSKPNSYATAYAASVKG (SEQ ID NO: 55)

[0089] CDRH3: GGSPYGFDI (SEQ ID NO: 56)

[0090] Variable domain of the heavy chain (VH): SEQ ID NO: 51 EVQLVESGGGLVQPGGSLKISCAASGFTFSGSAMHWVRQASGKGLEWVGRIRSKPNSYAT AYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRGGSPYGFDIWGQGTMVTVSS

[0091] Full length heavy chain (LC): SEQ ID NO: 52DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC CDRL1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 57)

[0092] CDRL2: WASTRES (SEQ ID NO: 58)

[0093] CDRL3: QQYYSTPWT (SEQ ID NO: 59)

[0094] Variable domain of the light chain (VL): SEQ ID NO: 53 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPWTFGQGTKVEIK

[0095] The antibodies of the invention (HLu 12_14, HLu 10_16, HLu 13_11, HLu 14_21, HLu 15_15 and HLu 11_51) demonstrate high specificity and affinity for alpha-synuclein fibrils, aggregates and oligomers, with minimal binding to monomeric a-synuclein.

[0096] All antibodies of the invention are usable in high sensitivity immunoassays for detecting biomarkers in human samples. In the context of the present invention such biomarker is aggregated / oligomeric alpha-synuclein.

[0097] In some embodiments the antibodies of the invention are used in pairs, where one is used as a capture antibody and one is used as a detection antibody. In certain embodiments the capture antibody is biotin-labeled, and the detection antibody is labeled with a marker. Such marker may be a fluorescent marker, such as a fluorophore. In certain embodiments the capture antibody is immobilized, such as bound to a surface, such as bound to Magnetic Particles.

[0098] In a particular embodiment, HLu 13 11 and HLu 14 21 is utilized as such antibody pair to quantify oligomeric / aggregated alpha-synuclein. In a preferred embodiment the detection antibody is HLu 14 21 and the capture antibody is HLu 13 11. In another embodiment the capture and / or detection antibody is HLu 10 16.

[0099] In a further embodiment the detection antibody is labeled with 647-fluorophore and the capture antibody is biotin-labeled.In certain embodiments the detection antibody is selected from the group consisting of: HLu 12_14, HLu 10_16, HLu 13_11, HLu 14_21, HLu 15_15 and HLu 11_51 and the capture antibody is selected from the group consisting of: HLu 12_14, HLu 10_16, HLu 13_11, HLu 14_21, HLu 15_15 and HLu 11_51.

[0100] In further embodiments of the invention the antibodies bind specifically to both Preformed fibrils (PFFs) and material derived from MSA and PD patient samples, but not to monomeric alpha-synuclein and control material from healthy controls. In a preferred embodiment antibody HLu 10 16 display specific binding to both PFFs, MSA and PD insoluble material, but not to monomeric alpha-synuclein and control material. In preferred embodiments, the MSA and PD insoluble material are detergent-insoluble fractions extracted from brain samples from patients suffering from these diseases.

[0101] In further embodiment of the invention the antibodies retain their ability to inhibit PFF-induced aggregation of pSer129 alpha-synuclein in cortical primary neurons despite being pre-incubated with a molar excess of monomeric alpha-synuclein and exposure to monomer during the initial incubation period. In a particular embodiment, the presence of excess monomeric alpha-synuclein does not substantially diminish the ability of the antibodies of the invention to exhibit functional neutralization of fibril-induced seeding. In a preferred embodiment HLu 10 16 retain it’s ability to inhibit PFF-induced aggregation of pSer129-positive alpha-synuclein aggregates in cortical primary neurons despite pre-incubation with a molar excess of monomeric alpha-synuclein and exposure to monomer during the initial incubation period. In a particular embodiment the presence of excess monomeric alpha-synuclein does not substantially diminish the ability of HLu 10 16 to exhibit functional neutralization of fibril-induced seeding.

[0102] In preferred embodiment of the invention the antibodies selectively binds aggregated alpha-synuclein species and exhibits functional selectivity for pathological alpha-synuclein conformations in a cellular model of seeded aggregation. In an even more preferred embodiment HLu 10 16 selectively binds aggregated alpha-synuclein speciesand exhibits functional selectivity for pathological alpha-synuclein conformations in a cellular model of seeded aggregation.

[0103] In further embodiments of the invention the antibodies binds specifically to pathological alpha-synuclein in brain sections from MSA and PD patients. In a preferred embodiment antibody HLu10_16 specifically stains pathological alpha-synuclein aggregates in human brain samples.

[0104] Specific embodiments of the invention

[0105] The following embodiments describes the invention in further detail. The embodiments are numbered consecutively, starting from number El.

[0106] El. An alpha-synuclein monoclonal antibody or a fragment thereof comprising:

[0107] a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

[0108] b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:10; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:11; d. a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

[0109] e. a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and f. a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

[0110] E2. The monoclonal antibody or a fragment thereof according to embodiment El, wherein said antibody comprises a heavy chain variable domain consisting of SEQ ID NO:6 and a light chain variable domain consisting of SEQ ID NO:8.

[0111] E3. The monoclonal antibody according to any one of embodiment E1-E2, wherein said antibody comprises a heavy chain consisting of SEQ ID NO:5 and a light chain consisting of SEQID NO:7.E4. An alpha-synuclein monoclonal antibody or a fragment thereof comprising:

[0112] a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:19; b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:20; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:21; d. a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:22;

[0113] e. a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:23; and f. a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:24.

[0114] E5. The monoclonal antibody or a fragment thereof comprising according to embodiment E4, wherein said antibody comprises a heavy chain variable domain consisting of SEQ ID NO:16 and a light chain variable domain consisting of SEQ ID NO:18.

[0115] E6. The monoclonal antibody according to any one of embodiment E4-E5, wherein said antibody comprises a heavy chain consisting of SEQ ID NO:15 and a light chain consisting of SEQ ID NO:17.

[0116] E7. A monoclonal VHH-Fc fusion alpha-synuclein antibody comprising:

[0117] a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:27; b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:29.

[0118] E8. The monoclonal antibody or a fragment thereof according to embodiment E7, wherein said antibody comprises a heavy chain variable domain consisting of SEQ ID NO:26.E9. The monoclonal antibody according to any one of embodiment E7-E8, wherein said antibody comprises a heavy chain consisting of SEQ ID NO:25.

[0119] E10. An alpha-synuclein monoclonal antibody or a fragment thereof comprising:

[0120] a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:34; b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:35; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:36; d. a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:37;

[0121] e. a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:38; and f. a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

[0122] Ell. The monoclonal antibody or a fragment thereof according to embodiment E10, wherein said antibody comprises a heavy chain variable domain consisting of SEQ ID NO:31 and a light chain variable domain consisting of SEQ ID NO:33.

[0123] E12. The monoclonal antibody according to any one of embodiment E10-E11, wherein said antibody comprises a heavy chain consisting of SEQ ID NO:30 and a light chain consisting of SEQ ID NO:32.

[0124] El 3. An alpha-synuclein monoclonal antibody or a fragment thereof comprising:

[0125] a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:44; b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:45; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:46; d. a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:47;e. a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:48; and f. a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:49.

[0126] E14. The monoclonal antibody or a fragment thereof according to embodiment E13, wherein said antibody comprises a heavy chain variable domain consisting of SEQ ID NO:41 and a light chain variable domain consisting of SEQ ID NO:43.

[0127] E15. The monoclonal antibody according to any one of embodiment E13-E14, wherein said antibody comprises a heavy chain consisting of SEQ ID NO:40 and a light chain consisting of SEQ ID NO:42.

[0128] El 6. An alpha-synuclein monoclonal antibody or a fragment thereof comprising:

[0129] a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:54; b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:55; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:56; d. a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:57;

[0130] e. a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:58; and f. a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:59.

[0131] E17. The monoclonal antibody or a fragment thereof according to embodiment E16, wherein said antibody comprises a heavy chain variable domain consisting of SEQ ID NO:51 and a light chain variable domain consisting of SEQ ID NO:53.E18. The monoclonal antibody according to any one of embodiment E16-E17, wherein said antibody comprises a heavy chain consisting of SEQ ID NO:50 and a light chain consisting of SEQ ID NO:52.

[0132] E19. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use in treating a patient diagnosed with an alpha-synucleinopathy, such as but not limited to PD or MSA.

[0133] E20. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use in detecting aggregated alpha-synuclein in a sample obtained from a patient diagnosed with an alpha-synucleinopathy.

[0134] E21. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use in detecting oligomeric alpha-synuclein in a sample obtained from a patient diagnosed with an alpha-synucleinopathy.

[0135] E22. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use in detecting alpha-synuclein fibrils in a sample obtained from a patient diagnosed with an alpha-synucleinopathy.

[0136] E23. The monoclonal antibody or a fragment thereof according to any of claims El -El 8 for use in diagnosing a patient with an alpha-synucleinopathy.

[0137] E24. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E19-E23, wherein said use comprise employing said antibody in an assay to detect aggregated or oligomeric alpha-synuclein or alpha-synuclein fibrils in a sample obtained from a patient suffering from an alpha-synucleinopathy.

[0138] E25. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to embodiment E24, wherein said sample is a CSF sample, ablood sample, a serum sample, a plasma sample, a detergent-insoluble fraction extracted from a brain sample, a brain section, a tissue section, a tissue sample, a urine sample, a saliva sample or a tear sample.

[0139] E26. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to embodiment 24, wherein said sample is a CSF sample, a blood sample, a serum sample, a plasma sample or a brain section, and wherein the aggregated / oligomeric alpha-synuclein content has been amplified prior to detection.

[0140] E27. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E25, wherein said assay is an immune-based assay.

[0141] E28. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E25, wherein said assay is a high sensitivity immunoassay.

[0142] E29. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E25, wherein said assay is an ELISA based assay.

[0143] E30. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E25, wherein said assay employs electro-chemiluminescent (ECL) detection.

[0144] E31. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 which has high specificity for alpha-synuclein fibrils.

[0145] E32. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 which has high specificity and affinity for alpha-synuclein fibrils.E33. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use as a capture or detection antibody in an immunoassay.

[0146] E34. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use as a capture antibody in an immunoassay.

[0147] E35. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8 for use as a detection antibody in an immunoassay.

[0148] E36. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E30, wherein the assay is an immunoassay selected from: a sandwich immunoassay or a bead-based multiplex assay.

[0149] E37. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E30, wherein the assay comprises quantification of the amount of alpha-synuclein fibrils in the sample.

[0150] E38. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E30, wherein the assay is performed continuously on samples from the same patient on a regular basis, such as every 1 month, every 2 months, every 3 month, every 4 months, every 5 month, every 6 month or yearly.

[0151] E39. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E30, wherein the antibodies are employed as either a capture or a detection antibody.

[0152] E40. The monoclonal antibody or a fragment thereof according to any of embodiment E1-E18 for use according to any of embodiment E24-E30, wherein the antibody inhibits PFF-induced accumulation of pSer129 alpha-synuclein in human samples.E41. the monoclonal antibody or a fragment thereof according to any of embodiment El-E18 for use according to any of embodiment E24-E30, wherein the assay is an in vitro assay.

[0153] E42. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein said antibody binds an epitope on human alpha-synuclein which is conformationally exposed on fibrils but not on monomers.

[0154] E43. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein the antibody retains full fibril-binding capacity following preincubation with alpha-synuclein monomers at a concentration of 1000 nM for 15 minutes.

[0155] E44. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein the antibody retains full fibril-binding capacity following preincubation with alpha-synuclein monomers at 15-fold molar excess of monomer for 15 minutes.

[0156] E45. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein the antibody retains full fibril-binding capacity following preincubation with alpha-synuclein monomers at 150-fold molar excess of monomer for 15 minutes.

[0157] E46. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein the antibody retains full fibril-binding capacity following preincubation with alpha-synuclein monomers at 500-fold molar excess of monomer for 15 minutes.

[0158] E47. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein the antibody retains full fibril-binding capacity following preincubation with alpha-synuclein monomers at 1000-fold molar excess of monomer for 15 minutes.E48. The monoclonal antibody or a fragment thereof according to any of embodiment El -El 8, wherein the antibody retains full fibril-binding capacity following preincubation with alpha-synuclein monomers at 1500-fold molar excess of monomer for 15 minutes.

[0159] E49. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of any of the antibodies of embodiment E1-E18.

[0160] E50. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of HLu12_14.

[0161] E51. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of HLu10_16.

[0162] E52. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of HLu13_11.

[0163] E53. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of HLu14_21.

[0164] E54. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of HLu15_15.

[0165] E55. An alpha-synuclein monoclonal antibody or a fragment thereof with >90%, >95%, or >98%, sequence similarity to the variable regions of HLu11_51.

[0166] E56. A method of diagnosing or following synucleinopathy progression in a patient comprising the steps of:

[0167] a) Obtaining a sample from said patient

[0168] b) Applying the sample on an immunoassay for measuring aggregated / oligomeric alpha-synuclein in said sample,c) wherein the assay of step b) employs an antibody of any of embodiments El -El 8 as a capture or a detection antibody; and

[0169] d) Optionally, comparing the results to a control.

[0170] E57. The method of embodiment E56, wherein the sample in step b) is selected form CSF, serum, plasma, blood, a detergent-insoluble fraction extracted from a brain sample or a tissue section.

[0171] E58. The method of embodiment E56-E57, wherein the control is a sample from a healthy control or a previous sample obtained from the same patient.

[0172] E59. The method of embodiment E56-E58, wherein the control is a matched sample form a healthy control or a matched previous sample obtained from the same patient.

[0173] E60. A method of measuring aggregated alpha-synuclein in a blood sample, serum sample, plasma sample, CSF sample, a detergent-insoluble fraction extracted from a brain sample or a tissue section comprising, performing an assay to quantify the level of aggregated alpha-synuclein present in the sample, wherein the assay comprises the use of a capture antibody and a detection antibody which both specifically bind to aggregated alpha-synuclein.

[0174] E61. The method of embodiment E60, wherein the assay uses a Single Molecule Counting (SMC) technology platform.

[0175] E62. The method of embodiment E60, wherein the assay is a Meso Scale Discovery (MSD) assays.

[0176] E63. The method of embodiment E60, wherein the assay is a high sensitivity immunoassay.

[0177] E64. The method of embodiment E60, wherein the assay is a sandwich immunoassay.E65. The method of embodiment E60, wherein the assay is a multiplex assay.

[0178] E66. The method of embodiment E60, wherein the assay is a bead-based multiplex assay.

[0179] E67. The method of embodiment E60, wherein the assay is an immunohistochemical staining.

[0180] E68. The method of any of embodiments E60-E67, which employs any of the monoclonal antibodies or fragments thereof of embodiments E1-E18.Examples

[0181] Example 1 - immunisation protocols for antibodies of the invention

[0182] Protocol 1 (llamas):

[0183] Preformed fibrils (PFFs) of alpha-synuclein were produced according to published procedures (Polinski, N. K. et al. Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents. J Parkinsons Dis 8, 303-322 (2018). https: / / doi.org:10.3233 / JPD-171248).

[0184] Wild-type human alpha-synuclein monomer was produced recombinantly in HEK293 cells. These reagents were utilized in the immunization process to generate antibodies against alpha-synuclein fibrils in llamas.

[0185] A total of four llamas were immunized to generate VHH immune libraries.

[0186] Immunization was done with fibrils mixed 1: 1 with Incomplete Freund’s adjuvant (IF A) and with subsequent weekly booster injections (up to 6 weeks) according to following schedule:

[0187] 1. Initial Immunization:

[0188] • Time: Week 0

[0189] • Dosage: 100 pg human a-synuclein fibrils with (IF A) in a 1:1 ratio.

[0190] • Sample collected: Collect 20 ml of blood for pre-immune serum.

[0191] 2. First Booster:

[0192] • Time: Week 1

[0193] • Dosage: 100 pg with IF A in a 1: 1 ratio.

[0194] 3. Second Booster:

[0195] • Time: Week 2

[0196] • Dosage: 50 pg with IFA in a 1:1 ratio.

[0197] 4. Third Booster:

[0198] • Time: Week 3• Dosage: 50 pg with IFA in a 1: 1 ratio.

[0199] 5. Fourth Booster:

[0200] • Time: Week 4

[0201] • Dosage: 50 pg with IFA in a 1: 1 ratio.

[0202] 6. Fifth Booster:

[0203] • Time: Week 5

[0204] • Dosage: 50 pg with IFA in a 1: 1 ratio.

[0205] 7. Sixth Booster:

[0206] • Time: Week 6

[0207] • Dosage: 50 pg with IFA in a 1: 1 ratio.

[0208] • Sample collected: Collected 400 ml of blood with anticoagulant for RNA preparation. The supernatant of Ficoll treatment was used as post-immune serum.

[0209] The pre-immune and post-immune sera were analyzed using ELISA to determine the antibody titers and binding specificity to monomeric alpha-synuclein and fibrils to assess the immune response and the effectiveness of the immunization protocol.

[0210] The RNA was extracted from the blood to prepare cDNA and obtain the sequences of the encoded VHHs. Furthermore, these were cloned into vectors for phage display to construct a phage library of potential fibril binders. Selection of binders were done in multiple rounds of phage display selections using alpha-synuclein fibrils as the target antigen. Pre-depletion steps were performed using plates coated with monomeric alpha-synuclein to remove monomeric binders. Positive selection was then done on phages in blocking buffer to prevent non-specific interaction, these were incubated with alpha-synuclein fibrils to allow specific binders to attach. Extensive washing steps were performed to remove unbound and weakly bound phages. The bound phages were eluted, enriched, and taken to the next round of selection. After several rounds of selection, phages displaying high affinity and specificity for alpha-synuclein fibrils were enriched. Master plates were generated for further screening and characterization of individual clones.Following selection, 28 phages displaying the selected VHH sequences were used to infect a bacterial host strain for expression (E. coli). Periplasmic extracts (P. E.) were produced from the bacteria expressing the VHH antibodies as VHH- human Fc fusions. The periplasmic extracts were subjected to binding ELISA to assess the binding affinity and specificity of the VHH antibodies to a-synuclein fibrils.

[0211] The ELISA protocol involved coating plates with alpha-synuclein fibrils, blocking, incubating with P. E., washing, and detecting bound VHH antibodies using a secondary antibody against human Fc.

[0212] The dissociation rate (off-rate) of the VHH antibodies was measured to determine the stability of the antibody-antigen interaction. Based on this, 10 clones were sequenced to determine the nucleotide sequences of the VHH regions. Sequence cluster analysis was done to identify unique VHH antibodies and to assess their diversity and potential liabilities. Furthermore, the selected clones were expressed and purified. One clone was selected for its high affinity and specificity to alpha-synuclein fibrils. The selected VHH clone was cloned into an expression vector fused with a human Fc domain (huFc) to create a full-length IgGl antibody. The antibody, HLu 12_14 (VHH Fc Fusion antibody format) demonstrated high specificity and affinity for alpha-synuclein fibrils, with minimal binding to monomeric alpha-synuclein.

[0213] Protocol 2 (mice):

[0214] Different fibrillar assemblies of alpha-synuclein were produced according to published protocols, e.g. preformed fibrils (PFFs) (as for protocol 1), p91 (Makky, A., Bousset, L., Polesel-Maris, J. & Melki, R. Nanomechanical properties of distinct fibrillar polymorphs of the protein alpha-synuclein. Sci Rep 6, 37970 (2016). https: / / doi.org: 10.1038 / srep37970) and ribbons (Bousset, L. et al. Structural and functional characterization of two alpha-synuclein strains. Nat Commun 4, 2575 (2013). https: / / doi.org: 10.1038 / ncomms3575).Wild-type human alpha-synuclein monomer was produced recombinantly in HEK293 cells. Furthermore, aggregated alpha-synuclein was isolated from brain tissue obtained from cases of multiple system atrophy according to published protocol (Tarutani, A., Arai, T., Murayama, S., Hisanaga, S. I. & Hasegawa, M. Potent prion-like behaviors of pathogenic alpha-synuclein and evaluation of inactivation methods. Acta Neuropathol Commun 6, 29 (2018). https: / / doi.org:10.1186 / s40478-018-0532-2), sample material termed MSA.

[0215] Mice transgenic with human antibody genes (Alloy mice) were used for immunization and screening. Three groups (6 or 8 mice each) were made based on the three alpha-synuclein immunogens: PFF, p91 and ribbons.

[0216] Two adjuvants, Ribi and Alum, were used to further divide each group. For the PFF immunogen, 4 animals were immunized with Ribi as adjuvant, while 2 were immunized with Alum as adjuvant. For the p91 immunogen, 4 animals were immunized with Ribi as adjuvant, while 2 were immunized with Alum as adjuvant. For the ribbon immunogen, 4 animals were immunized with Ribi as adjuvant, and 4 were immunized with Alum as adjuvant. The mice immunized with PFF or p91 and with alum as adjuvant would further receive an additional booster injection. The immunized mice were sacrificed and isolated plasma cell placed in microwells for secretion of antibodies to generate a plate microarray (‘device’) for screening. For plasma cell screening, the animals would be divided into four groups. Screening was done to assess binding to monomer, PFF (Group 1), p91 (Group 2), PFF+p91 (Group 3), ribbons (Group 4) and MSA material on each group.

[0217] Group 1 were formed by PFF immunogen and Ribi adjuvant (4 mice total). Group 2 were formed by p91 immunogen and ribi as adjuvant (4 mice total). Group 3 were formed by p91 or PFF immunogen with Alum as adjuvant (4 mice total). Group 4 were formed by ribbon immunogen and ribi or alum as adjuvant (8 mice total).Furthermore, sequences were obtained from the plasma cells by next generation sequencing (NGS) used for sub-clustering of candidates. From group 1, 14 candidates were selected for expression, purification and further characterization based on screening and subclustering (based on NGS sequencing). From group 2 and 3, 24 and 30 candidates were selected as these antibodies demonstrated high specificity and affinity for alpha-synuclein fibrils, with minimal binding to monomeric alpha-synuclein. From group 4, no candidates were selected. The best performing antibodies from this campaign were HLu 10_16, HLu 13_11, HLu 14_21, HLu 15_15 and HLu 11_51 which were all fully human IgGl formats.

[0218] Example 2- Antibody expression and purification

[0219] Cloning in expression vectors: Sequences encoding heavy and light chains for each antibody were cloned into separate pTT5 vectors between EcoRI and HindIII cloning sites.

[0220] Expression and purification of antibodies: Antibodies were produced by transient transfection of pTT5 vectors (National Research Council (1:1 mass ratio of each plasmid encoding paired heavy chain and light chain) into ExpiCHO cells according to ThermoFishers Max titer protocol. Transfected cells were fed according to the manufacturers protocol, monitored, and harvested when viability approached 75%. The expression level of antibodies was measured on a CEDEX Bio Analyser (Roche) using the IgG Bio test kit. Cell supernatant was filtered over 0.2 pm filters. Capture purification of the expressed antibodies from the media was done by flowing media over a Mab Select Sure resin equilibrated in PBS on a high-performance liquid chromatography (HPLC) system (Äkta Pure, Cytiva). Captured antibody was eluted using 0.1 sodium acetate pH 3.5 and collected in fractions. The fractions were pooled and added 10 % 0.1 M piperazine for neutralization. The pool was the further dialyzed against 20 mM piperazine pH 6.0 overnight and centrifuged at 4500 g for 10 min before the supernatant was further purified by anion exchange. A 5 mL Q Sepharose (GE Healthcare) column was equilibrated in 20 mM piperazine pH 6.0. The dialyzed andcentrifuged pool was then applied and the flow through collected. The flow through was dialyzed overnight into PBS and concentrated to storage concentration before sterile filtration (0.22 um) and freezing at -80 °C. No precipitation observed during this step. Purity and mono-dispersity was assessed by analytical size-exclusion chromatography (SEC) on an ultra-performance liquid chromatography (UPLC) system (Acquity, Waters).

[0221] Example 3 - Further characterization of binding properties of the antibodies of the invention

[0222] Antibodies specific for fibrillar alpha-synuclein species were selected on binding to PFF prepared from recombinant alpha-synuclein and counter screened on alpha-synuclein monomer. For this purpose, an ELISA was established using the Meso Scale Discovery (MSD) platform. An MSD plate was coated with 5 nM alpha-synuclein in the form of either PFF or monomer. The coating concentration was selected to identify antibodies with high avidity due to bivalent interaction with alpha-synuclein. This way, antibodies displaying specific binding to PFFs, but not to densely coated monomeric alpha-synuclein, could be selected. Detection of binding antibody was done using a sulfo-tagged anti¬ human Fc antibody. To verify the selectivity extended beyond PFFs, isolation of insoluble material from brain from cases of PD and MSA, as well as unaffected controls, was done. This material was coated onto MSD plates and used for ELISA as described for PFFs. The insoluble material was prepared according to published protocol (Tarutani, A., Arai, T., Murayama, S., Hisanaga, S. I. & Hasegawa, M. Potent prion-like behaviors of pathogenic alpha-synuclein and evaluation of inactivation methods. Acta Neuropathol Commun 6, 29 (2018). https: / / doi.org: 10.1186 / s40478-018-0532-2). Exemplary data on specific binding of HLu10_16 to PFFs, MSA and PD insoluble brain material, but not to monomeric alpha-synuclein and control material, is provided in figures 1A and IB.

[0223] Example 4 - Oligomer assay for aggregated alpha-synuclein

[0224] Quantitation of aggregated alpha-synuclein has been developed by screening optimal antibody pairs and conditions using Single Molecule Counting (SMC) technologyplatform. The antibody pair HLu 13 11 and HLu 14 21 was found to deliver most sensitive detection.

[0225] The reagents to run the assay includes the detection antibody (HLu 14 21) labeled with 647-fluorophore (Ab-Fluo) and capture antibody (HLu 13 11) biotin-labeled and combined with magnetic particles (MP) coating (MP -Ab). Equipment needed: Magnetic particle separator and fluorescence reader.

[0226] The protocol for the assay is:

[0227] 1. Preparation of Reagents

[0228] Dilute the Detection Antibody (Ab-Fluo) to the required concentration. Prepare the Capture Antibody (MP -Ab) by biotin labeling and coating onto Magnetic Particles. Optimal concentrations to use in the assay are 6.25 ug ab / mg beads of the MP -Ab capture and 5 ul / mg of the Ab-Fluo for the detection.

[0229] 2. Sample Incubation

[0230] Add the sample containing the analyte to a tube or well. Add the MP- Ab to the sample and incubate for 1 hour at room temperature with gentle shaking to allow the analyte to bind to the MP- Ab.

[0231] 3. Washing

[0232] Place the tube or well on a Magnetic Particle Separator to collect the MP-Ab bound analyte. Remove the supernatant carefully without disturbing the Magnetic Particles. Wash the Magnetic Particles three times with wash buffer to remove unbound substances.

[0233] 4. Detection Antibody Binding

[0234] Add the Detection Antibody (Ab-Fluo) to the tube or well containing the washed Magnetic Particles. Incubate for 1 hour at room temperature with gentle shaking to allow the Ab-Fluo to bind to the analyte.

[0235] 5. Final WashingPlace the tube or well on the Magnetic Particle Separator to collect the MP-Ab bound analyte with the Detection Antibody. Remove the supernatant carefully without disturbing the Magnetic Particles. Wash the Magnetic Particles three times with wash buffer to remove unbound Detection Antibody.

[0236] 6. Measurement

[0237] Resuspend the Magnetic Particles in an appropriate buffer for fluorescence measurement. Transfer the suspension to a fluorescence reader and measure the fluorescence signal at the appropriate wavelength for the 647-fluorophore.

[0238] 7. Data Analysis

[0239] Analyze the fluorescence data to quantify the amount of analyte in the sample based on a standard curve. Calculate the recovery rates and coefficient of variation (CV%) for quality control.

[0240] This protocol outlines the steps required to perform the SMC assay, including preparation, incubation, washing, and measurement procedures.

[0241] A standard curve based on alpha-synuclein converted to pre-formed fibrils (PFFs) according to published protocol (Polinski, N. K. et al. Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents. J Parkinsons Dis 8, 303-322 (2018). https: / / doi.org:10.3233 / JPD-171248) indicated a lower limit of quantification (LLOQ) of 1.37 pg / mL of aggregated alpha-synuclein.

[0242] Example 5 - Inhibition of PFF driven seeded aggregation in presence and absence of monomeric alpha-synuclein

[0243] Cortical primary neurons prepared from E16 embryos from the F28 mouse overexpressing wildtype human alpha-synuclein under the endogenous mouse alpha-synuclein promoter, were used to assess the conformational selectivity of antibody HLu10_16 in a cellular seeding model. HLu10_16 was pre-incubated with 1000 nM recombinant monomeric human alpha-synuclein for 15 minutes at room temperature inorder to challenge binding to monomer-accessible epitopes. The antibody / monomer mixture was then added to 5 days in vitro (DIV) old primary neurons and incubated for 1 hour. Subsequently, recombinant alpha-synuclein pre-formed fibrils (PFFs) were added at a final concentration of 33.3 nM (0.5 pg / mL). Cells were maintained under standard culture conditions for 7 days to allow development of seeded alpha-synuclein pathology. At DIV12 cells were fixed and labelled for phosphorylated alpha-synuclein (pS129) as a marker for aggregated alpha-synuclein and Hoechst. Seeding was measured as pS129 fluorescence / cell by Cellomics Array Scan VTI HCS Reader and normalized to PFF seed control (w / o mAb). The experiment was performed once with three technical replicates per condition. HLu10_16 retained its ability to inhibit PFF-induced accumulation of pSer129 alpha-synuclein in cortical primary neurons despite pre-incubation with a molar excess of monomeric alpha-synuclein and exposure to monomer during the initial incubation period. The presence of excess monomeric alpha-synuclein did not substantially diminish the functional neutralization of fibril-induced seeding. These results demonstrate that HLu10_16 selectively recognizes aggregated / fibrillated alpha-synuclein species over monomeric alpha-synuclein and exhibits functional selectivity for pathological alpha-synuclein conformations in a cellular model of seeded aggregation.

[0244] Example 6 - Immunodepletion of aggregated alpha-synuclein species from brain samples derived from MSA and PD patients

[0245] Human brain homogenates were prepared from putamen tissue obtained from subjects diagnosed with Parkinson’s disease (PD) and Multiple System Atrophy (MSA), as well as from corresponding brain regions of neurologically unaffected control subjects. Tissue samples were homogenized under conditions designed to preserve both soluble and aggregated protein species.

[0246] For immunoprecipitation experiments, HLu10_16 and HLu12_14 were immobilized on magnetic beads and incubated with the respective brain homogenates under conditions that permitted antigen-antibody complex formation. Following incubation, immune complexes were magnetically separated and washed to remove non-specific binding. Bound proteins were then eluted for downstream analysis. An isotype-matched humanIgG1 antibody directed against hen egg lysozyme (HEL) served as a negative control, and a total alpha-synuclein antibody was used as a positive control.

[0247] To quantify the depletion of aggregated alpha-synuclein by HLu10_16 and HLu12_14, post-immunoprecipitation supernatants (immunodepleted fractions) were analyzed using a Homogeneous Time-Resolved Fluorescence (HTRF)-based Cisbio assay specific for aggregated alpha-synuclein. Treatment with HLu10_16 or HLu12_14 resulted in a measurable reduction of aggregated alpha-synuclein signal in PD and MSA homogenates down to baseline levels, comparable to unaffected controls. In contrast, the isotype-matched IgG1 control antibody exhibited minimal or no binding to alpha-synuclein and did not substantially reduce alpha-synuclein levels in the homogenates.

[0248] These results demonstrate that HLu10_16 and HLu12_14 are capable of binding aggregated forms of human alpha-synuclein and efficiently immunoprecipitate pathological alpha-synuclein present in brain tissue from subjects with PD and MSA. Western blot analysis was performed on the immunoprecipitated material using both total and pS129 alpha-synuclein antibodies (Figure 4B). Phosphorylated alpha-synuclein (pS129), which serves as a pathology marker, was detected in HLu12_14 and HLu10_16, as well as in positive control immunoprecipitates from PD and MSA, but not from healthy controls (CTRL).

[0249] Notably, full-length alpha-synuclein, along with its truncated forms, was enriched from CTRL, PD, and MSA lysates. Densitometry analysis was conducted to quantify the levels of pS129 and full-length alpha-synuclein. These results show that HLu10_16 and HLu12_14, unlike the positive control alpha-synuclein antibody, enriched primarily the phosphorylated (pathological) alpha-synuclein, exhibiting relatively lower binding to total alpha-synuclein across MSA, PD, and CTRL samples.Example 7 Detection of pathological alpha-synuclein in brains from MSA patients by immunohistochemistry

[0250] Paraffin-embedded tissue sections of human pons from patients suffering from MSA were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. After cooling and washing, endogenous peroxidase activity was blocked with hydrogen peroxide, followed by incubation with a protein blocking solution to reduce nonspecific binding. The sections were incubated with a primary antibody specific for the target antigen. For human or humanized primary antibodies applied to human tissue, the antibody was pre-treated with blocking reagents (human-on-human approach) prior to application. After washing, an enzyme-conjugated secondary or polymer-based detection reagent (e.g., HRP -linked) was applied. Signal was visualized using DAB. The sections were counterstained, dehydrated, cleared, and mounted. Data was analyzed using QuPath v.0.5.1 with squares placed at similar position covering pons. Positive pixels were defined as >0.35 in DAB intensity.

[0251] Collectively these examples indicate that the antibodies of the invention can be used in diagnosing patients suffering from alpha-synucleinopathy as well as to detect and monitor aggregated / oligomeric alpha-synuclein levels in samples obtained from patients suffering from alpha-synucleinopathy. Further, the data indicate that the invention provides antibodies with high sensitivity which are suitable for use in diagnostic assays for diagnosing alpha-synucleinopathies and for use in assays for detecting and / or monitoring of aggregated / oligomeric alpha-synuclein in patients suffering from alpha-synucleinopathies.

Claims

1. Claims1. An alpha-synuclein monoclonal antibody or a fragment thereof comprising:a. a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:19; b. a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:20; c. a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:21; d. a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:22; e. a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:23; and f. a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:24.

2. The monoclonal antibody or a fragment thereof according to claim 1, wherein said antibody further comprises a heavy chain variable domain consisting of SEQ ID NO: 16 and a light chain variable domain consisting of SEQ ID NO: 18.

3. The monoclonal antibody of any of claims 1-2, wherein said antibody is an IgGl antibody.

4. The monoclonal antibody or fragment thereof of any of claims 1-3, wherein said antibody has high specificity for alpha-synuclein fibrils.

5. The monoclonal antibody or fragment thereof of any of claims 1-4, wherein said antibody has high specificity and affinity for alpha-synuclein fibrils.

6. The monoclonal antibody or fragment thereof of any of claims 1-5, wherein the antibody is capable of inhibiting pre-formed-fibril -induced accumulation of pSer129 alpha-synuclein in human samples.

7. The monoclonal antibody or fragment thereof of any of claims 1-5, wherein the antibody retains full fibril-binding capacity following preincubation with alpha- synuclein monomers at 15-fold molar excess of monomer for 15 minutes.

8. The monoclonal antibody or fragment thereof of any of claims 1-5, wherein the antibody retains full fibril-binding capacity following preincubation with alpha- synuclein monomers at 1500-fold molar excess of monomer for 15 minutes.

9. The monoclonal antibody or fragment thereof of any of claims 1-8, wherein said antibody is used as a capture or detection antibody in an immunoassay.

10. The monoclonal antibody or fragment thereof of any of claims 1-8 for use in measuring alpha-synuclein aggregates / fibrils in a human sample.

11. The monoclonal antibody or fragment thereof of any of claims 1-8 for use in diagnosing a patient suspected of suffering from an alpha-synucleinopathy, such as PD or MSA.

12. The monoclonal antibody or fragment thereof of any of claims 1-8 for use in an assay to quantify or visualize the amount of alpha-synuclein fibrils in a sample obtained from a patient suffering from an alpha-synucleinopathy, such as PD or MSA.

13. The monoclonal antibody or fragment thereof of any of claims 1-8 for use according to any of claims 9-12, wherein said sample is a CSF sample, a blood sample, a serum sample, a plasma sample, a detergent-insoluble fraction extracted from a brain sample or a tissue-section.

14. The monoclonal antibody or fragment thereof of any of claims 1-8 for use according to any of claims 9-12, wherein the assay is an in vitro immunoassay.

15. A method of diagnosing or following synucleinopathy progression, such as PD or MSA progression, in a human comprising the steps of:a) Obtaining a sample from said humanb) Applying the sample on an immunoassay for measuring aggregated / oligomeric alpha-synuclein in said sample,c) wherein the assay of step b) employs the monoclonal antibody or fragment thereof of any of claims 1-8 as a capture or a detection antibody; andd) Optionally, comparing the amount of aggregated / oligomeric alpha-synuclein in said sample to a control.

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