Ny39 alpha-synuclein antibodies and methods of use thereof

Abstract

The invention features nY39 alpha-synuclein antibodies and antigen binding fragments thereof. The invention also features pharmaceutical compositions and methods of using the nY39 alpha-synuclein antibodies or antigen binding fragments thereof to treat a subject having a synucleinopathy (e.g., Parkinson's disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer's disease with synuclein pathology).

Description

[0001] NY39 ALPHA-SYNUCLEIN ANTIBODIES AND METHODS OF USE THEREOF

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 21 , 2025, is named 51340-012WO3_Sequence_Listing_3_21_25.xml and is 163,424 bytes in size.

[0004] BACKGROUND OF THE INVENTION

[0005] Nitration of proteins has been linked to various disease conditions through inducement of agglomeration of proteins. Nitration is known to play a role in neurodegenerative diseases (e.g., synucleinopathies (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), and Alzheimer’s disease with synuclein pathology).

[0006] For example, Parkinson’s disease is the second most common neurodegenerative disease, affecting one million people in the United States alone. The disease is characterized by a dramatic loss of dopaminergic neurons in the substantia nigra, with up to 80% of these neurons having degenerated by the time of clinical manifestations. Death of these neurons causes tremor, bradykinesia, rigidity, and postural instability. Currently, there is no effective intervention to reverse disease progression, suggesting the need for early biomarkers and prophylactic treatments. Based on the loss of dopamine or dopaminergic dysfunction in PD patients, most of the current therapies mainly rely on the use of dopaminergic pharmacological agents, such as levodopa (L-DOPA), for the treatment of the clinical symptoms of PD. However, as L-DOPA is quickly metabolized, it often leads to poor efficacy and severe peripheral side effects. In particular, while L-DOPA may be effective for symptomatic relief, its efficacy is diminished and it causes medication-related complications, particularly motor fluctuations and dyskinesia, such as L-DOPA-induced dyskinesia, after chronic or long-term use. Despite the intensive efforts in PD research and development, there are clear unmet medical needs for the development of treatment options to slow the trajectory of emergence of PD and / or strengthen treatment options to enhance patients’ quality of life.

[0007] A hallmark of PD pathology is an intracellular accumulation of alpha-synuclein aggregates resulting in Lewy Bodies and Lewy Neurites. It has been hypothesized that these alpha-synuclein aggregates, in some form, are responsible for causing PD. Alpha-synuclein has been shown to be nitrated in the inclusions present in PD, DLB, MSA, and Alzheimer’s disease with synuclein pathology. In particular, nitrated tyrosine 39 (nY39) has been detected in the blood of patients with PD. Several studies have suggested that the insolubility of alpha-synuclein may be a result of nitration since nitrated alpha- synuclein is present in the insoluble (not soluble) fraction of homogenates from PD brains. Alpha- synuclein nitration also induces oligomerization and p-pleated (beta-pleated) sheet formation in a nucleation-dependent manner. Furthermore, nitration of alpha-synuclein results in its removal from lipid vesicles and increases its half-life due to decreased ability of the proteasome to degrade it. Each of these factors contributes to alpha-synuclein accumulation and aggregation and suggests a role of alpha- synuclein nitration in mediating disease pathology and dysfunction in neurodegenerative disease, such as PD. Finding a mechanism to specifically inhibit this nitration event could be highly advantageous for the drug discovery efforts for treatment of neurodegenerative diseases and its symptoms.

[0008] SUMMARY OF THE INVENTION

[0009] The present disclosure relates to methods and compositions for the treatment of subjects having a synucleinopathy (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology).

[0010] In one aspect, the disclosure features an antibody or an antigen-binding fragment thereof that binds to alpha-synuclein nitrated at tyrosine residue 39 (nY39), the antibody or antigen-binding fragment thereof comprising: a complementarity-determining region light chain 1 (CDR-L1 ) comprising the amino acid sequence of QASQNVYKNNYLG (SEQ ID NO: 1 ), RASQNVYKNNY (SEQ ID NO: 2), RASKNVYKNYYLG (SEQ ID NO: 1 13), RASKAVYKNYYLG (SEQ ID NO: 114), RASQNVYKNYYLG (SEQ ID NO: 1 15), or RASKAVYNNYYLG (SEQ ID NO: 116); a complementarity-determining region light chain 2 (CDR-L2) comprising the amino acid sequence of YASTLAS (SEQ ID NO: 3); a complementaritydetermining region light chain 3 (CDR-L3) comprising the amino acid sequence of LGIYDCSSVDCNA (SEQ ID NO: 4) or LGIYDCSSGDCNA (SEQ ID NO: 1 17); a complementarity-determining region heavy chain 1 (CDR-H1 ) comprising the amino acid sequence of GFSLSSYYMS (SEQ ID NO: 5), GFKLSSYYMS (SEQ ID NO: 118), GFSVSSSYMS (SEQ ID NO: 119), or GFKLSSTYMS (SEQ ID NO: 120); a complementarity-determining region heavy chain 2 (CDR-H2) comprising the amino acid sequence of YISAGGYTY (SEQ ID NO: 6); and a complementarity-determining region heavy chain 3 (CDR-H3) comprising the amino acid sequence of LDPSSGDI (SEQ ID NO: 7).

[0011] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of QASQNVYKNNYLG (SEQ ID NO: 1 ). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of RASQNVYKNNY (SEQ ID NO: 2). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of RASKNVYKNYYLG (SEQ ID NO: 1 13). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of RASKAVYKNYYLG (SEQ ID NO: 114). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of RASQNVYKNYYLG (SEQ ID NO: 115). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L1 comprising the amino acid sequence of RASKAVYNNYYLG (SEQ ID NO: 1 16).

[0012] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L3 comprising the amino acid sequence of LGIYDCSSVDCNA (SEQ ID NO: 4). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-L3 comprising the amino acid sequence of LGIYDCSSGDCNA (SEQ ID NO: 1 17).

[0013] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-H1 comprising the amino acid sequence of GFSLSSYYMS (SEQ ID NO: 5). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-H1 comprising the amino acid sequence of GFKLSSYYMS (SEQ ID NO: 118). In some embodiments, the antibody or an antigenbinding fragment thereof comprises a CDR-H1 comprising the amino acid sequence of GFSVSSSYMS (SEQ ID NO: 1 19). In some embodiments, the antibody or an antigen-binding fragment thereof comprises a CDR-H1 comprising the amino acid sequence of GFKLSSTYMS (SEQ ID NO: 120). In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region framework regions (FRs): an FR-L1 comprising the amino acid sequence of DIQMTQSPSAMSASVGDRVTITC (SEQ ID NO: 8), DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26), or QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29); an FR-L2 comprising the amino acid sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9), LGWFQQKPGKAPKRLIY (SEQ ID NO: 27), or LGWFQQKPGKAPKLLIY (SEQ ID NO: 30); an FR-L3 comprising the amino acid sequence of GVSSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 10), GVSSRFKGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 16), or GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ) or FGGGTKVEIK (SEQ ID NO: 28).

[0014] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQMTQSPSAMSASVGDRVTITC (SEQ ID NO: 8); an FR-L2 comprising the amino acid sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9); an FR-L3 comprising the amino acid sequence of GVSSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 10); and an FR-L4 comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ).

[0015] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQMTQSPSAMSASVGDRVTITC (SEQ ID NO: 8); an FR-L2 comprising the amino acid sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9); an FR-L3 comprising the amino acid sequence of GVSSRFKGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 16); and an FR-L4 comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ).

[0016] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKLLIY (SEQ ID NO: 30); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

[0017] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKRLIY (SEQ ID NO: 27); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

[0018] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKLLIY (SEQ ID NO: 30); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

[0019] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKRLIY (SEQ ID NO: 27); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

[0020] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12), EVQLVESGGGLVQPGGPLRLSCAAS (SEQ ID NO: 21 ), or EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13) or WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKTTLYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 14), YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYYCAR (SEQ ID NO: 18), YANWAKGRFTISRDNSKNTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 19), YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 20), YANWAKGRFTISRDNSKTTVDLQMNSPRAEDTAVYFCAR (SEQ ID NO: 22), YADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 25), YADSVKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 31 ), YANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 32), or YANWAKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 33); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0021] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKTTLYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 14); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0022] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYYCAR (SEQ ID NO: 18); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0023] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKNTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 19); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0024] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 20); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0025] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGPLRLSCAAS (SEQ ID NO: 21 ); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKTTVDLQMNSPRAEDTAVYFCAR (SEQ ID NO: 22); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0026] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13); an FR-H3 comprising the amino acid sequence of YADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 25); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0027] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YADSVKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 31 ); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0028] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 32); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0029] In some embodiments, the antibody or an antigen-binding fragment thereof comprises the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 33); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0030] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence with at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity) to any one of SEQ ID NOs: 34-39 or 121 -124. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 34-39 or 121 -124. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 121 . In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 122. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 124.

[0031] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence with at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99%, or 100% sequence identity) to any one of SEQ ID NOs: 40-48 or 125-127. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 40-48 or 125-127. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 41 . In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 45. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 125. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 127.

[0032] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 39 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 121 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 125. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 122 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 125. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 123 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 121 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 124 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 127.

[0033] In some embodiments, the antibody or an antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0034] In some embodiments, the antibody or an antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

[0035] In some embodiments, the antibody or an antigen-binding fragment thereof is a full-length antibody. In some embodiments, the antibody or an antigen-binding fragment thereof is an IgG class antibody. In some embodiments, the IgG class antibody is an IgG 1 subclass antibody. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence with at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any one of SEQ ID NOs: 65-87 or 128-131. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence with at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any one of SEQ ID NOs: 88-1 10 or 132-134. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 65-87 or 128-131 and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 88-110 or 132-134.

[0036] In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 128 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 129 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 130 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 128 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 131 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 134.

[0037] In some embodiments, the antigen-binding fragment thereof is selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

[0038] In a second aspect, the disclosure features a polynucleotide encoding an antibody or antigenbinding fragment thereof disclosed herein.

[0039] In a third aspect, the disclosure features a vector comprising a polynucleotide disclosed herein.

[0040] In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a eukaryotic expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenovirus (Ad), retrovirus, poxvirus, adeno-associated virus, baculovirus, and a herpes simplex virus.

[0041] In a fourth aspect, the disclosure features a host cell comprising a vector disclosed herein.

[0042] In a fifth aspect, the disclosure features a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0043] In a sixth aspect, the disclosure features a kit comprising an agent selected from an antibody or antigen-binding fragment thereof disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0044] In a seventh aspect, the disclosure features a method of treating a subject having a synucleinopathy, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that binds to alpha-synuclein nitrated at tyrosine residue 39 (nY39) disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0045] In some embodiments, the synucleinopathy is Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology.

[0046] In some embodiments, the subject has elevated levels of nY39 alpha-synuclein compared to a subject who does not have the synucleinopathy.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof is administered intravenously.

[0048] In another aspect, the invention features a method of diagnosing a synucleinopathy in a subject, the method comprising: (a) determining the presence or level of an alpha-synuclein protein nitrated at tyrosine residue 39 (nY39 alpha-synuclein) in a cerebrospinal fluid (CSF) sample obtained from the subject; and (b) comparing the level of nY39 alpha-synuclein to a reference level of nY39 alpha- synuclein , wherein an increase in the level of nY39 alpha-synuclein in the CSF sample relative to the reference level of nY39 alpha-synuclein identifies a subject having a synucleinopathy, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

[0049] In some embodiments, a statistically significant increase in the level of nY39 alpha-synuclein in the CSF sample relative to the reference level of nY39 alpha-synuclein identifies a subject having a synucleinopathy. In some embodiments, the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the concentration of nitrated alpha-synuclein protein in the CSF relative to the reference level. In some embodiments, the level of nY39 alpha-synuclein is calculated as the concentration of nY39 alpha-synuclein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample. In some embodiments, the reference level is calculated as the concentration of nY39 alpha-synuclein in a reference sample divided by the concentration of total alpha- synuclein in the reference sample. In some embodiments, the reference sample is a CSF sample from a normal subject that does not have a synucleinopathy.

[0050] In another aspect, the invention features a method of identifying a subject having a synucleinopathy, the method comprising determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein the presence of nY39 alpha-synuclein identifies the subject as having a synucleinopathy, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

[0051] In another aspect, the invention features a method of diagnosing a synucleinopathy in a subject, the method comprising determining the presence or level of nY39 alpha-synuclein in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein the presence of nY39 alpha-synuclein identifies the subject as having a synucleinopathy, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145. In some embodiments, the anti-alpha- synuclein antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

[0052] In some embodiments, the presence or level of nY39 alpha-synuclein is determined by single molecule detection, western blot, ELISA, immunohistochemistry, or mass spectrometry. In some embodiments, the method further comprises determining in the CSF sample the presence or level of alpha-synuclein nitrated at Y125, Y133, Y136, or a combination thereof.

[0053] In some embodiments, comprising administering to the subject a therapeutically effect amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, a neuroprotective agent, and / or an antibody or antigen-binding fragment thereof described herein.

[0054] In another aspect, the invention features a method of treating a subject having a synucleinopathy, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein, wherein the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject has been determined to be increased relative to a reference level of nY39 alpha-synuclein, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145. In some embodiments, the anti-alpha-synuclein antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

[0055] In some embodiments, the method further comprises determining the presence or level in the CSF sample of alpha-synuclein nitrated at Y125, Y133, Y136, or a combination thereof.

[0056] In another aspect, the invention features a method of treating a subject having a synucleinopathy, the method comprising: (a) determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein an increase in the level of nY39 alpha-synuclein relative to a reference level of nY39 alpha-synuclein identifies the subject as having a synucleinopathy; and (b) administering to the subject a therapeutically effective amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, a neuroprotective agent, and / or an antibody or antigen-binding fragment thereof described herein, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145. In some embodiments, the anti-alpha-synuclein antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

[0057] In some embodiments, an increase is a statistically significant increase, wherein the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the level of nY39 alpha-synuclein in the CSF relative to the reference level of nY39 alpha-synuclein. In some embodiments, the level of nY39 alpha-synuclein is calculated as the concentration of nY39 alpha- synuclein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample. In some embodiments, the reference level is calculated as the concentration of nY39 alpha-synuclein in a reference sample divided by the concentration of total alpha-synuclein in the reference sample. In some embodiments, the reference sample is a CSF sample from a normal subject that does not have a synucleinopathy.

[0058] In some embodiments, the synucleinopathy is Parkinson’s disease (PD) or a subtype of the disease thereof, dementia with Lewy bodies (DLB), or multiple-system atrophy (MSA). In some embodiments, the subtype of the disease thereof is a prodromal stage of PD.

[0059] In some embodiments, after administering, the method further comprises determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein a decrease in the level of nY39 alpha-synuclein relative to the level of nY39 alpha-synuclein before administration identifies the subject as having responded to treatment.

[0060] In some embodiments, after administering, the method further comprises determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein an increase in the level of nY39 alpha-synuclein relative to the level of nY39 alpha-synuclein before administration identifies the subject as in need of further treatment.

[0061] In some embodiments, the anti-nY39 alpha-synuclein antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 53 and / or a VH sequence comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-nY39 alpha-synuclein antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

[0062] In another aspect, the invention features a kit for diagnosing a synucleinopathy in a subject, the kit comprising: (a) an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and optionally (b) instructions for use of the anti-nY39 alpha-synuclein antibody to determine the level of nY39 alpha-synuclein a CSF sample from the subject, wherein an increase in the level of one or more of nY39 alpha-synuclein relative to a reference level of nY39 alpha-synuclein indicates that the subject is likely to have a synucleinopathy.

[0063] In some embodiments, the anti-nY39 alpha-synuclein antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 53 and / or a VH sequence comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-nY39 alpha-synuclein antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

[0064] In some embodiments, the synucleinopathy is PD or a subtype of the disease thereof, DLB, or MSA. In some embodiments, the subtype of the disease thereof is a prodromal stage of PD.

[0065] Definitions

[0066] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0067] As used herein, “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms condition, disease, or illness, unless otherwise indicated. As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a disorder. The disorder may be a synucleinopathy (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology). Treatment may be administered to a subject who does not exhibit signs of the disease and / or to a subject who exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0068] As used herein, the terms “subject” and “patient” are interchangeable and refer to a subject (e.g., a mammalian subject, e.g., a human subject) that receives treatment for a disorder as described herein.

[0069] The term “alpha-synuclein” refers to proteins whose amino acid sequence includes or consists of an amino acid sequence of a naturally occurring wild-type alpha-synuclein protein as well as proteins whose amino acid sequence includes or consists of an amino acid sequence of a naturally occurring mutant alpha-synuclein protein. For example, as used herein, the term alpha-synuclein encompasses full- length, unprocessed alpha-synuclein, as well as any form of alpha-synuclein resulting from processing in the cell (e.g., truncations e.g., C-terminal truncated forms of alpha-synuclein and N-terminal truncated forms of alpha-synuclein), as well as any naturally occurring variants of alpha-synuclein (e.g., truncations, splice variants, and allelic variants). Alpha-synuclein is also referred to as synuclein alpha (SNCA). Human alpha-synuclein has NCBI Gene ID NO 6622. Alpha-synuclein is considered an intrinsically disordered protein. Exemplary alpha-synuclein amino acid sequences are provided in SEQ ID NOs: 135- 142. As used herein, alpha-synuclein may refer to the amino acid of SEQ ID NOs: 135-142, any truncated form thereof, or a truncated form of alpha-synuclein including, but not limited to those described in U.S. Patent No. 7306945, incorporated herein by reference in its entirety.

[0070] The nomenclature for describing nitration uses the format “nXB,” where “n” designates the presence of nitration, “X” designates the amino acid found in the wild type variant of the alpha-synuclein protein, and “B” designates the number of the amino acid within the alpha-synuclein peptide chain. For example, a nitrated variant described herein contains a nitration described as nY39, which corresponds to a post-translational nitration at the alpha-synuclein protein at amino acid residue #39, in which a tyrosine is nitrated. The nitrations described herein appear in the context of a wild-type alpha-synuclein protein, for example, corresponding to the amino acid sequence of SEQ ID NOs: 135-142, below, or a truncation thereof:

[0071] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTK EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDP DNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 135);

[0072] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTK EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKEGYQDYEPEA (SEQ ID NO: 136);

[0073] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVVAEKTKEQVTNVGGAVVTGV TAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQ DYEPEA (SEQ ID NO: 137);

[0074] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTK EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKSSLRNECSPEPAASTDQS QNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 138);

[0075] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTK EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDP DNEAYEMPSEPPCCEEAQATWRGHAMCTCSS (SEQ ID NO: 139);

[0076] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTK EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKVWLCTFCVTFISW (SEQ ID NO: 140);

[0077] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTK EQVTNV (SEQ ID NO: 141 ); and MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVVAEKTKEQVTNVGGAVVTGV TAVAQKTVEGAGSIAAATGFVKKDQLGKEGYQDYEPEA (SEQ ID NO: 142).

[0078] Thus, references herein to nitrations made “in an alpha-synuclein protein” indicate that the alpha- synuclein protein containing one or more nitrations is in the context of an alpha-synuclein protein or a variant (e.g. truncation) thereof with the above-referenced sequence (e.g., the reference sequence serves as the backbone for the alpha-synuclein protein with the indicated nitration(s)).

[0079] A “synucleinopathy” is a disorder characterized by misfolding and / or abnormal accumulation of aggregates of alpha-synuclein in the central nervous system (e.g., in neurons or glial cells). Exemplary, non-limiting synucleinopathies include PD, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), pure autonomic failure, incidental Lewy body disease, pantothenate kinase-associated neurodegeneration, Alzheimer's disease (e.g., Alzheimer’s disease with synuclein pathology), Down's Syndrome, Gaucher disease, or the Parkinsonism-dementia complex of Guam.

[0080] The term “nY39 alpha-synuclein antibody” refers to an antibody that is capable of binding alpha- synuclein nitrated at tyrosine 39 with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting alpha-synuclein nitrated at tyrosine 39. In some embodiments, the extent of binding of an anti-nY39 alpha-synuclein antibody to an unrelated, non-nY39 alpha-synuclein protein is less than about 10% of the binding of the antibody to nY39 alpha-synuclein as measured, e.g., by an enzyme- linked immunosorbent assay (ELISA), a biosensor assay, or a surface plasmon resonance (SPR) assay. In some embodiments, the extent of binding of an anti-nY39 alpha-synuclein antibody to an alpha- synuclein protein that is not nitrated at tyrosine 39 is less than about 10% of the binding of the antibody to nY39 alpha-synuclein as measured, e.g., by an ELISA, a biosensor assay, or SPR. In some embodiments, the extent of binding of an anti-nY39 alpha-synuclein antibody to an alpha-synuclein protein that is nitrated at a tyrosine residue other than tyrosine 39 (e.g., alpha-synuclein nitrated at tyrosine 125, 133, and / or 136) is less than about 10% of the binding of the antibody to n Y39 alpha- synuclein as measured, e.g., by an ELISA, a biosensor assay, or SPR. In some embodiments, an nY39 alpha-synuclein antibody has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM. In some embodiments, the binding of an nY39 alpha-synuclein antibody may be evaluated by measuring the binding of the nY39 alpha-synuclein antibody to a peptide fragment of an alpha-synuclein protein containing the nitrated tyrosine 39 (e.g., an nY39 10-mer, 50-mer, or 70-mer alpha-synuclein peptide).

[0081] The term “antibody” includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab’)2, and Fv). The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (LC) chains and two identical heavy (HC) chains. Each HC chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and e isotypes. Each LC chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The LC chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 6, e, y, and p, respectively. The y and a classes are further divided into subclasses, e.g., humans express the following subclasses: lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2.

[0082] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called complementarity-determining regions (CDRs) both in the light-chain (CDR-L1 , CDR-L2, and CDR- L3) and the heavy chain variable domains (CDR-H1 , CDR-H2, and CDR-H3). The more highly conserved portions of variable domains are called the framework regions (FR), which are those variable-domain residues other than the CDR residues. The variable domains of heavy (FR-H1 , FR-H2, FR-H3, and FR- H4) and light chains (FR-L1 , FR-L2, FR-L3, and FR-L4) each comprise four FR regions, connected by three CDRs.

[0083] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies included in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0084] The terms “full-length antibody” refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Full-length antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0085] “Antibody fragments” include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0086] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 -6855 (1984)).

[0087] "Humanized" forms of non-human antibodies are chimeric antibodies or fragments thereof which contain minimal sequence derived from non-human antibody. For the most part, humanized antibodies are human antibodies (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse or rabbit having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human antibody are replaced by corresponding non-human residues. Further, humanized antibodies may include residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.

[0088] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Suitable native-sequence Fc regions for use in the antibodies of the invention include human lgG1 , lgG2, IgGS, and lgG4. In certain instances, one or more amino acid modifications are introduced into the Fc region of the antibody, thereby generating an Fc region variant (see e.g., US 2012 / 0251531 ). The Fc region variant may comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0089] As used herein, the terms “effective amount,” “therapeutically effective amount,” and “sufficient amount” of an nY39 alpha-synuclein antibody described herein refer to a quantity sufficient to treat a synucleinopathy (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology) as described herein in a subject (e.g., a human). For example, in the context of treating PD, it is an amount of the nY39 alpha-synuclein antibody that reduces the motor and / or cognitive symptoms sufficient to achieve a treatment response as compared to the response obtained without administration of the antibody. The amount of an nY39 alpha- synuclein antibody that reduces a symptom of PD, MSA, DLB, or Alzheimer’s disease with synuclein pathology will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the subtype of the pathology (e.g., prodromal PD), the identity of the subject (e.g., age, sex, and / or weight) or host being treated, and the like, but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, a “therapeutically effective amount” of an nY39 alpha-synuclein antibody of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount an nY39 alpha-synuclein antibody of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response.

[0090] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term present in the art which is identical to any term expressly defined in this disclosure, the term's definition presented in this disclosure will control in all respects. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] FIGS. 1 A-1 E are a series of graphs showing the results of an ELISA assay using rabbit antibodies rbAb1 -rbAb4 with recombinantly produced full-length human nY39 alpha-synuclein (nY39 hu synuclein), chemically nitrated full-length human alpha-synuclein (chemical nitrated hu synuclein), unmodified full-length human alpha-synuclein (unmodified hu a-synuclein), and chemically nitrated bovine serum albumin (BSA). Data are shown as raw MSD units as a function of alpha-synuclein or BSA concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0092] FIGS. 2A-2E are a series of graphs showing the results of an ELISA assay using rabbit antibodies rbAbl -rbAb4 with biotinylated 10-mer alpha-synuclein peptides (Y39, nY39, Y125, nY125, nY133, and nY136). Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0093] FIGS. 3A-3B are a series of graphs showing the results of ELISA assays using rabbit antibodies rbAbl -rbAb3 with biotinylated 50-mer nY39 alpha-synuclein peptides. In FIG. 3A, rbAbl -rbAb3 were used as the capture antibody, with data shown as raw MSD units as a function of alpha-synuclein peptide concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. In FIG. 3B, rbAbl -rbAb3 were used as the detection antibody, with data shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0094] FIGS. 4A-4G are a series of graphs showing the results of ELISA assays using rabbit antibodies rAb1 -rAb3 and mouse chimera antibodies msAb1 -msAb3 with 70-mer alpha-synuclein peptides (amino acids 31 -100 of alpha-synuclein with nitrated tyrosine 39 (nY39_70mer), amino acids 81 -140 of alpha- synuclein with nitrated tyrosine 125 (nY125_70mer), amino acids 81 -140 of alpha-synuclein with nitrated tyrosine 133 (nY133_70mer), and amino acids 81 -140 of alpha-synuclein with nitrated tyrosine 136 (nY136_70mer)). Data are shown as raw MSD units as a function of 70-mer alpha-synuclein peptide concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0095] FIGS. 5A-5E are a series of graphs showing the results of Western blot assays using rabbit antibodies rAb1 -rAb3, a total synuclein antibody, and a general nitration antibody (3-NT) with chemically nitrated alpha-synuclein (human and mouse recombinant protein), recombinantly produced full-length human nY39 alpha-synuclein, unmodified human and mouse alpha-synuclein, BSA, and chemically nitrated BSA.

[0096] FIG. 6 is a graph showing the results of an ELISA target engagement assay using mouse chimera antibody msAbl and human chimera antibody hAb1 with 70-mer nY39 alpha-synuclein peptides. A mouse IgG antibody (Ms IgG) was included as a negative control. Data are shown as raw MSD units as a function of msAbl , hAb1 , or Ms IgG antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0097] FIG. 7A is a graph showing the results of a SIMOA assay assessing the levels of nitrated alpha- synuclein in human cerebrospinal fluid (CSF) samples. FIG. 7B is a graph showing the results of an assay assessing the levels of nY39 synuclein in the insoluble fraction of dopaminergic neurons treated with preformed fibril (PFF) synuclein. FIG. 7C is a graph showing the results of a SIMOA assay assessing the levels of nitrated alpha-synuclein in mouse interstitial fluid (ISF) samples.

[0098] FIGS. 8A-8D are a series of graphs showing the results of an ELISA assay using humAbl - humAb20 with 70-mer peptides containing amino acids 81 -140 of alpha-synuclein with nitrated tyrosine 136 (nY136) or full-length unmodified alpha-synuclein. Data are shown as raw MSD units as a function of alpha-synuclein concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0099] FIG. 9A is a graph showing a sequence alignment between human and cyno alpha-synuclein, with 98.6% sequence similarity. FIG. 9B is a graph showing the results of an ELISA assay using humAb20 with human and cyno alpha-synuclein, either unmodified or chemically nitrated. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0100] FIGS. 10A-10B are a series of graphs showing the results of ELISA assays using affinity matured antibodies humAb24-humAb28 with 50-mer biotinylated nY39 alpha-synuclein peptides. In FIG. 10A, humAb24-humAb28 were used as the capture antibody, with data shown as raw MSD units as a function of alpha-synuclein peptide concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. In FIG. 10B, humAb24-humAb28 were used as the detection antibody, with data shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0101] FIGS. 11 A-1 1 E are a series of graphs showing the results of ELISA assays using affinity matured antibodies humAb24-humAb28 with biotinylated 50-mer alpha synuclein peptides (nY39, Y39, nY125, nY133, and nY136) or biotinylated full length alpha-synuclein biotin (FL a-syn). Data are shown as raw MSD units as a function of alpha-synuclein concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0102] FIG. 12 is a graph showing the results of an ELISA using affinity matured antibodies humAb24- humAb28 or a general nitration antibody (3-NT 06-284, Millipore) with chemically nitrated BSA. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0103] FIG. 13 is a graph showing the results of a target engagement ELISA using affinity matured antibodies humAb24-humAb28 with 70-mer nY39 alpha-synuclein peptides containing amino acids 31 - 100 of alpha-synuclein nitrated at tyrosine 39. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0104] FIG. 14 is a graph showing humAb26 binding to nitrated human or cyno alpha-synuclein. humAb26 as a capture antibody shows selectivity to chemically nitrated human alpha-synuclein full-length protein (EC50 135 ng / ml) and to chemically nitrated cyno alpha-synuclein (EC50 115 ng / ml). humAb26 does not bind to unmodified human or cyno alpha-synuclein protein.

[0105] FIG. 15 is a graph showing humAb26 binding to nitrated human or mouse (ms) alpha-synuclein. humAb26 as a capture antibody shows selectivity to chemically nitrated human alpha-synuclein full-length protein (EC50 152.5 ng / ml) and to chemically nitrated mouse alpha-synuclein (EC50 74.64 ng / ml). humAb26 does not bind to unmodified human or mouse alpha-synuclein protein.

[0106] FIGS. 16A-B are a series of graphs showing humAb26 binding to nitrated mouse (FIG. 16A) or rat (FIG. 16B) alpha-synuclein, either with or without buffer exchange prior to nitration reaction. In FIG. 16A, humAb26 as a capture antibody shows selectivity to chemically nitrated mouse alpha-synuclein full- length protein (EC5033.66 ng / ml, nitrated; EC50 56.84 ng / ml, BE_nitrated). In FIG. 16B, humAb26 as capture antibody shows selectivity to chemically nitrated rat alpha-synuclein (EC50 88.18 ng / ml, nitrated; ECso 50.71 ng / ml, BE_nitrated). humAb26 does not bind to un-nitrated mouse or rat alpha-synuclein protein (NaOH or BE_NaOH).

[0107] FIG. 17 is a sequence alignment of human, cyno, mouse and rat alpha-synuclein.

[0108] FIG. 18 is a graph showing mean plasma PK profile of humAb26 in male CD-1 mice following a single intravenous administration of 10 mg / kg.

[0109] FIG. 19 is a graph showing individual and mean plasma concentration profiles of humAb26 in male cynomolgus monkeys following a single intravenous administration of humAb26 at 2 mg / kg.

[0110] FIGS. 20A-B are a series of graphs showing body weights at baseline (FIG. 20A) or throughout the study duration (FIG. 20B).

[0111] FIGS. 21 A-B are a series of graphs showing substantia nigra IHC results.

[0112] FIGS. 22A-B are a series of graphs showing thalamus IHC results.

[0113] FIGS. 23A-B are a series of graphs showing body weights at baseline (FIG. 23A) and throughout the study duration (FIG. 23B).

[0114] FIGS. 24A-B are a series of graphs showing substantia nigra IHC results.

[0115] FIGS. 25A-B are a series of graphs showing thalamus IHC results.

[0116] DETAILED DESCRIPTION OF THE INVENTION

[0117] Alpha-synuclein has been shown to be nitrated in the inclusions present in Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), and Alzheimer’s disease with synuclein pathology. Several studies have suggested that the insolubility of alpha-synuclein may be a result of nitration since nitrated alpha-synuclein is present in the insoluble (not soluble) fraction of homogenates from PD brains. Alpha-synuclein nitration also induces oligomerization and p-pleated (betapleated) sheet formation in a nucleation-dependent manner. Furthermore, nitration of alpha-synuclein results in its removal from lipid vesicles and increases its half-life due to decreased ability of the proteasome to degrade it. Each of these factors contributes to alpha-synuclein accumulation and aggregation and suggests a role of alpha-synuclein nitration in mediating disease pathology and dysfunction in synucleinopathies, such as PD, DLB, MSA, and Alzheimer’s disease with synuclein pathology. Finding antibodies that target this nitration could be highly advantageous for the treatment of synucleinopathies, such as PD, DLB, MSA, and Alzheimer’s disease with synuclein pathology and symptoms thereof.

[0118] This invention is based, at least in part, on the discovery of nY39 alpha-synuclein antibodies that can be useful for treating subjects having a synucleinopathy (e.g., DLB, MSA, PD, or Alzheimer’s disease with synuclein pathology). Accordingly, the disclosure provides antibodies or antigen-binding fragments thereof that bind specifically to nY39 alpha-synuclein. The disclosure further provides methods of treating subjects (e.g., human patients) having a synucleinopathy (e.g., DLB, MSA, PD, Alzheimer’s disease with synuclein pathology, or a subtype of the disease thereof, e.g., prodromal PD) by administering to the subject an nY39 alpha-synuclein antibody described herein.

[0119] In some instances, the nY39 alpha-synuclein antibodies described herein are diagnostic antibodies. In some embodiments, the diagnostic antibodies can be used in an assay (e.g., a diagnostic assay; e.g., in an enzyme-linked immunosorbent assay (ELISA) or an immunohistochemistry assay), e.g., to determine the presence of nY39 alpha-synuclein. In some embodiments, the diagnostic antibodies described herein can be used to determine the presence of nY39 alpha-synuclein, e.g., in a subject having a synucleinopathy (e.g., DLB, MSA, RD, or Alzheimer’s disease with synuclein pathology).

[0120] I. Anti-nY39 alpha-synuclein antibodies

[0121] In particular, the disclosure features anti-nY39 alpha-synuclein antibodies or antigen-binding fragments described according to any of the CDR, light chain variable region (VL), heavy chain variable region (VH), light chain (LC), and / or heavy chain (HC) sequences provided below. The disclosure specifically contemplates antibodies having any combination of the disclosed CDRs (e.g., a combination of any CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2, and CDR-L3 disclosed herein). The disclosure also specifically contemplates the pairing of a heavy chain including any VH described herein with the pairing of a light chain including any VL disclosed herein. Further, the disclosure specifically contemplates the pairing of a HC described herein with the pairing of a LC disclosed herein. In some embodiments, the anti-nY39 alpha-synuclein antibody or fragment thereof is a therapeutic antibody. In some embodiments, the anti-nY39 alpha-synuclein antibody or fragment thereof is a diagnostic antibody.

[0122] Complementarity-determining region seguences

[0123] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including an amino acid sequence selected from QASQNVYKNNYLG (SEQ ID NO: 1 ), RASQNVYKNNY (SEQ ID NO: 2), RASKNVYKNYYLG (SEQ ID NO: 113), RASKAVYKNYYLG (SEQ ID NO: 114), RASQNVYKNYYLG (SEQ ID NO: 1 15), or RASKAVYNNYYLG (SEQ ID NO: 116).

[0124] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L2 including the amino acid sequence of YASTLAS (SEQ ID NO: 3).

[0125] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L3 including the amino acid sequence of LGIYDCSSVDCNA (SEQ ID NO: 4) or LGIYDCSSGDCNA (SEQ ID NO: 117).

[0126] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-H1 including the amino acid sequence of GFSLSSYYMS (SEQ ID NO: 5), GFKLSSYYMS (SEQ ID NO: 1 18), GFSVSSSYMS (SEQ ID NO: 119), or GFKLSSTYMS (SEQ ID NO: 120).

[0127] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-H2 including the amino acid sequence of YISAGGYTY (SEQ ID NO: 6).

[0128] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-H3 including the amino acid sequence of LDPSSGDI (SEQ ID NO: 7).

[0129] In some embodiments, the antibody or antigen binding fragment thereof includes at least one of: CDR-L1 including the amino acid sequence of SEQ ID NOs: 1 or 2; CDR-L2 including the amino acid sequence of SEQ ID NO: 3; CDR-L3 including the amino acid sequence of SEQ ID NO: 4; CDR-H1 including the amino acid sequence of SEQ ID NO: 5; CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0130] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 ; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 4; a CDR-H1 including the amino acid sequence of SEQ ID NO: 5; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 2; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 4; a CDR-H1 including the amino acid sequence of SEQ ID NO: 5; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0131] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 13; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 1 17; a CDR-H1 including the amino acid sequence of SEQ ID NO: 118; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0132] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 14; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 4; a CDR-H1 including the amino acid sequence of SEQ ID NO: 118; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0133] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 15; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 1 17; a CDR-H1 including the amino acid sequence of SEQ ID NO: 119; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0134] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 13; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 1 17; a CDR-H1 including the amino acid sequence of SEQ ID NO: 120; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0135] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 16; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3; a CDR-L3 including the amino acid sequence of SEQ ID NO: 4; a CDR-H1 including the amino acid sequence of SEQ ID NO: 120; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

[0136] In some embodiments, the antibody or antigen binding fragment thereof includes a CDR-L1 including the amino acid sequence of QASQSVYNNNYLG (SEQ ID NO: 146); a CDR-L2 including the amino acid sequence of YASTLAS (SEQ ID NO: 147); a CDR-L3 including the amino acid sequence of LGIYDCSSVDCNA (SEQ ID NO: 148); a CDR-H1 including the amino acid sequence of GFSLSSYYMS (SEQ ID NO: 143); a CDR-H2 including the amino acid sequence of YISTGGYTY (SEQ ID NO: 144); and a CDR-H3 including the amino acid sequence of LDPSTGDI (SEQ ID NO: 145). In some embodiments the antibody or antibody fragment thereof is a diagnostic antibody, e.g., 8C2, i.e., rbAb3.

[0137] Light chain framework regions

[0138] In some embodiments, the antibody or antigen-binding fragment thereof includes a light chain framework region 1 (FR-L1 ) (e.g., N-terminal to CDR-L1 ) including the sequence DIQMTQSPSAMSASVGDRVTITC (SEQ ID NO: 8), DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26), or QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29).

[0139] In some embodiments, the antibody or antigen-binding fragment thereof includes a light chain framework region 2 (FR-L2) (e.g., between CDR-L1 and CDR-L2) including the sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9), LGWFQQKPGKAPKRLIY (SEQ ID NO: 27), or LGWFQQKPGKAPKLLIY (SEQ ID NO: 30).

[0140] In some embodiments, the antibody or antigen-binding fragment thereof includes a light chain framework region 3 (FR-L3) (e.g., between CDR-L2 and CDR-L3) including the sequence of GVSSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 10), GVSSRFKGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 16), or GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23).

[0141] In some embodiments, the antibody or antigen-binding fragment thereof includes a light chain framework region 4 (FR-L4) (e.g., C-terminal to CDR-L3) including the sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ) or FGGGTKVEIK (SEQ ID NO: 28).

[0142] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of SEQ ID NO: 8; a FR-L2 including the sequence of SEQ ID NO: 9; a FR-L3 including the sequence of SEQ ID NO: 10; and a FR-L4 including the sequence of SEQ ID NO: 11 .

[0143] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of SEQ ID NO: 8; a FR-L2 including the sequence of SEQ ID NO: 9; a FR-L3 including the sequence of SEQ ID NO: 16; and a FR-L4 including the sequence of SEQ ID NO: 11 .

[0144] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of SEQ ID NO: 26; a FR-L2 including the sequence of SEQ ID NO: 30; a FR-L3 including the sequence of SEQ ID NO: 23; and a FR-L4 including the sequence of SEQ ID NO: 28.

[0145] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of SEQ ID NO: 26; a FR-L2 including the sequence of SEQ ID NO: 27; a FR-L3 including the sequence of SEQ ID NO: 23; and a FR-L4 including the sequence of SEQ ID NO: 28.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of SEQ ID NO: 29; a FR-L2 including the sequence of SEQ ID NO: 30; a FR-L3 including the sequence of SEQ ID NO: 23; and a FR-L4 including the sequence of SEQ ID NO: 28.

[0147] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of SEQ ID NO: 29; a FR-L2 including the sequence of SEQ ID NO: 27; a FR-L3 including the sequence of SEQ ID NO: 23; and a FR-L4 including the sequence of SEQ ID NO: 28.

[0148] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-L1 including the sequence of QSLEESGGRLVTPGTPLTLSCTAS (SEQ ID NO: 153); a FR-L2 including the sequence of WVRQAPGKGLEYIG (SEQ ID NO: 154); a FR-L3 including the sequence of YANWAKGRFTISRTSTTVDLKFSSPTTEDTATYFCAR (SEQ ID NO: 155); and a FR-L4 including the sequence of WGPGTLVTVS (SEQ ID NO: 156).

[0149] Heavy chain framework regions

[0150] In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain framework region 1 (FR-H1 ) (e.g., N-terminal to CDR-H1 ) including the sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12), EVQLVESGGGLVQPGGPLRLSCAAS (SEQ ID NO: 21 ), or EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24).

[0151] In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain framework region 2 (FR-H2) (e.g., between CDR-H1 and CDR-H2) including the sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13) or WVRQAPGKGLEYIG (SEQ ID NO: 17).

[0152] In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain framework region 3 (FR-H3) (e.g., between CDR-H2 and CDR-H3) including the sequence of YANWAKGRFTISRDNSKTTLYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 14), YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYYCAR (SEQ ID NO: 18), YANWAKGRFTISRDNSKNTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 19), YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 20), YANWAKGRFTISRDNSKTTVDLQMNSPRAEDTAVYFCAR (SEQ ID NO: 22), YADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 25), YADSVKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 31 ), YANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 32), or YANWAKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 33).

[0153] In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain framework region 4 (FR-H4) (e.g., C-terminal to CDR-H3) including the sequence of WGQGTLVTVSS (SEQ ID NO: 15).

[0154] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 12; a FR-H2 including the sequence of SEQ ID NO: 13; a FR-H3 including the sequence of SEQ ID NO: 14; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 12; a FR-H2 including the sequence of SEQ ID NO: 17; a FR-H3 including the sequence of SEQ ID NO: 18; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 12; a FR-H2 including the sequence of SEQ ID NO: 17; a FR-H3 including the sequence of SEQ ID NO: 19; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 12; a FR-H2 including the sequence of SEQ ID NO: 17; a FR-H3 including the sequence of SEQ ID NO: 20; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 21 ; a FR-H2 including the sequence of SEQ ID NO: 17; a FR-H3 including the sequence of SEQ ID NO: 22; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 24; a FR-H2 including the sequence of SEQ ID NO: 13; a FR-H3 including the sequence of SEQ ID NO: 25; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 24; a FR-H2 including the sequence of SEQ ID NO: 17; a FR-H3 including the sequence of SEQ ID NO: 31 ; and a FR-H4 including the sequence of SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 24; a FR-H2 including the sequence of SEQ ID NO: 13; a FR-H3 including the sequence of SEQ ID NO: 32; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of SEQ ID NO: 24; a FR-H2 including the sequence of SEQ ID NO: 17; a FR-H3 including the sequence of SEQ ID NO: 33; and a FR-H4 including the sequence of SEQ ID NO: 15.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof includes a FR-H1 including the sequence of AQVLTQTASPVSAAVGGTVTINC (SEQ ID NO: 149); a FR-H2 including the sequence of WFQQKPGQPPKRLIY (SEQ ID NO: 150); a FR-H3 including the sequence of GVSSRFKGSGSGTQFTLTISDVQCDDAATYYC (SEQ ID NO: 151 ); and a FR-H4 including the sequence of FGGGTEVVV (SEQ ID NO: 152).

[0163] Light chain variable domain sequences

[0164] This disclosure provides an antibody or antigen-binding fragment thereof including a light chain variable domain (VL) including an amino acid sequence with at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity) to an amino acid sequence provided in Table 1 , below (e.g., any one of SEQ ID NOs: 34-39, 121 -124, or 53). CDR regions are indicated in bold.

[0165] Table 1. Exemplary VL sequences of the disclosure

[0166] In some embodiments, the antibody or antigen-binding fragment thereof that includes a light chain variable domain includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 ; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof that includes a light chain variable domain includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 2; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof that includes a light chain variable domain includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 113; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof that includes a light chain variable domain includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 1 14; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof that includes a light chain variable domain includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 115; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof that includes a light chain variable domain includes a CDR- L1 including the amino acid sequence of SEQ ID NO: 116; a CDR-L2 including the amino acid sequence of SEQ ID NO: 3, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 4.

[0167] Heavy chain variable domain sequences

[0168] This disclosure provides an antibody or antigen-binding fragment thereof that includes a heavy chain variable domain (VH) including an amino acid sequence with at least 95% sequence identity (e.g., at least 96%, 97%, 98%, 99% or 100% sequence identity) to an amino acid sequence provided in Table 2, below (e.g., any one of SEQ ID NOs: 40-48, 125-127, or 54). CDR regions are indicated in bold.

[0169] Table 2. Exemplary VH sequences of the disclosure

[0170] In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable domain including a CDR-H1 including the amino acid sequence of SEQ ID NO: 5; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable domain including a CDR-H1 including the amino acid sequence of SEQ ID NO: 1 18; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable domain including a CDR-H1 including the amino acid sequence of SEQ ID NO: 1 19; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable domain including a CDR-H1 including the amino acid sequence of SEQ ID NO: 120; a CDR-H2 including the amino acid sequence of SEQ ID NO: 6, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 7. Light chain sequences

[0171] This disclosure provides an antibody or antigen-binding fragment thereof that includes a light chain (LC) including an amino acid sequence with at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence provided in Table 3, below (e.g., any one of SEQ ID NOs: 65-87, 128-131 , or 157). CDR regions are indicated in bold.

[0172] Table 3. Exemplary light chain sequences of the disclosure

[0173] Heavy chain sequences

[0174] This disclosure provides an antibody or antigen-binding fragment thereof that includes a heavy chain including an amino acid sequence with at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or

[0175] 100% sequence identity) to an amino acid sequence provided in Table 4, below (e.g., any one of SEQ ID NOs: 88-110, 132-134, or 158). CDR regions are indicated in bold.

[0176] Table 4. Exemplary heavy chain sequences of the disclosure

[0177] Antibodies and antigen-binding fragments thereof

[0178] In some embodiments, the antibody is a full-length antibody, e.g., an intact IgG antibody (e.g., an intact IgG 1 antibody) or other antibody class or isotype as defined herein. This disclosure provides an antibody or antigen-binding fragment thereof that includes a light chain including an amino acid sequence with at least 80% sequence identity (e.g., at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 65-87 or 128-131 and a heavy chain including an amino acid sequence with at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 88-1 10 or 132-134.

[0179] In certain instances, one or more amino acid modifications are introduced into the Fc region of the antibody, thereby generating an Fc region variant (see e.g., US 2012 / 0251531 ). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions. In some embodiments, the diagnostic nY39 alpha-synuclein antibody may comprise a rabbit Fc region sequence (e.g., a rabbit

[0180] In some embodiments, the nY39 alpha-synuclein antibody may be a monoclonal antibody, comprising a chimeric, humanized, or human antibody. In some instances, the nY39 alpha-synuclein is an antibody fragment, for example, a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In some embodiments, the diagnostic nY39 alpha-synuclein antibody may be a rabbit monoclonal antibody (e.g., a rabbit IgG Fc region).

[0181] Vectors, host cells, and protein production

[0182] The nY39 alpha-synuclein antibodies and antigen binding fragments thereof described herein can be produced from a host cell. A host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the antibodies and antigen binding fragments thereof described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like). The choice of nucleic acid vectors depends in part on the host cells to be used. Generally, preferred host cells are of either eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin. Nucleic acid vector construction and host cells

[0183] A nucleic acid sequence encoding the amino acid sequence of an nY39 alpha-synuclein antibody or antigen binding fragment thereof described herein may be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. A nucleic acid molecule encoding an nY39 alpha-synuclein antibody or antigen binding fragment thereof described herein may be obtained using standard techniques, e.g., gene synthesis. Alternatively, a nucleic acid molecule encoding an nY39 alpha-synuclein antibody or antigen binding fragment thereof described herein may be mutated to include specific amino acid substitutions using standard techniques in the art, e.g., QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0184] In one embodiment, an isolated nucleic acid encoding an anti-nY39 alpha-synuclein antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided In one such embodiment, a host cell comprises (e.g., has been transformed with): (1 ) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.

[0185] An antibody as described herein can also be produced in various formats, including as a Fab, a Fab', a F(ab')2, a scFv, or a dAB. The antibody fragments can be obtained by a variety of methods, including, digestion of an intact antibody with an enzyme, such as pepsin (to generate (Fab')2fragments) or papain (to generate Fab fragments); or de novo synthesis. Antibody fragments can also be synthesized using recombinant DNA methodology. In some embodiments, an antibody includes F(ab')2fragments that specifically binds nY39 alpha-synuclein. An antibody of the disclosure can also include a human constant region. See, e.g., Fundamental Immunology (Paul ed., 4d ed. 1999); Bird, et al., Science 242:423 (1988); and Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879 (1988).

[0186] A nucleic acid sequence encoding an nY39 alpha-synuclein antibody or antigen binding fragment thereof described herein may be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purpose of the invention. Each vector may include various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding the protein of interest, and a transcription termination sequence.

[0187] In some embodiments, mammalian cells may be used as host cells for the invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. coll cells may also be used as host cells for the invention. Examples of E. coll strains include, but are not limited to, E. coli 294 (ATCC®31 ,446), E. coli A 1776 (ATCC®31 ,537, E. coli BL21 (DE3) (ATCC® BAA- 1025), and E. coli RV308 (ATCC®31 ,608). Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems may be chosen to ensure the correct modification and processing of the polypeptide expressed. The above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein production, host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Methods for expression of therapeutic proteins are known in the art, see, for example, Paulina Baibas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012.

[0188] Protein production, recovery, and purification

[0189] Host cells used to produce the nY39 alpha-synuclein antibodies or antigen binding fragments thereof described herein may be grown in media known in the art and suitable for culturing of the selected host cells. Examples of suitable media tor mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle’s Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10%. The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.

[0190] In some embodiments, depending on the expression vector and the host cells used, the expressed protein may be secreted from the host cells (e.g., mammalian host cells) into the cell culture media. Protein recovery may involve filtering the cell culture media to remove cell debris. The proteins may be further purified. An nY39 alpha-synuclein antibody or antigen binding fragment thereof described herein may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, the protein can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column (e.g., POROS Protein A chromatography) with chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting-out and dialysis procedures.

[0191] In other embodiments, host cells may be disrupted, e.g., by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, cell debris may be removed by centrifugation or filtration. In some instances, a polypeptide can be conjugated to marker sequences, such as a peptide to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His- tag), which binds to nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0192] Alternatively, the nY39 alpha-synuclein antibodies or antigen binding fragments thereof described herein can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administrating a vector (such as a viral vector (e.g., adenovirus (Ad), retrovirus, poxvirus, adeno- associated virus, baculovirus, and a herpes simplex virus) containing a nucleic acid molecule encoding an antibody or antigen binding fragment thereof described herein. The vector, once inside a cell of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.) will promote expression of the antibody or antigen binding fragment thereof, which is then secreted from the cell. If treatment of a disease or disorder is the desired outcome, no further action may be required. If collection of the protein is desired, blood may be collected from the subject and the protein purified from the blood by methods known in the art.

[0193] In some embodiments, a cell-free approach may be used to generate the nY39 alpha-synuclein antibodies or antigen binding fragments thereof described herein. In some embodiments, the cell-free approach comprises using an extract including all the necessary biochemical components for energy production, transcription and translation of a protein, and can be used to support cell-free biochemical protein synthesis by the addition of the specific DNA sequence for the desired protein.

[0194] II. Pharmaceutical Compositions

[0195] Pharmaceutical compositions containing nY39 alpha-synuclein antibodies or antigen binding fragments thereof can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions. In some embodiments, a pharmaceutical composition of the invention includes a nucleic acid molecule encoding an nY39 alpha-synuclein antibody or antigen-binding fragment thereof described herein, or a vector containing such a nucleic acid molecule.

[0196] Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, proteins, hydrophilic polymers, amino acids, and / or carbohydrates. Pharmaceutical compositions of the invention can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a-MEMI). III. Methods of Treatment

[0197] Anti-nY39 alpha-synuclein antibodies or antigen-binding fragments thereof described herein can be used to treat a subject having a synucleinopathy (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology).

[0198] The anti-nY39 alpha-synuclein antibodies described herein can be used to treat, ameliorate, or prevent PD, DLB, MSA, or Alzheimer’s disease with synuclein pathology. Accordingly, the antibodies described herein can be administered to a human having or suspected of having one of the above-listed diseases in an appropriate dosage to ameliorate or treat one of the disease or at least one symptom thereof.

[0199] Synucleinopathies are characterized by deposition of intracellular protein aggregates that are microscopically visible as Lewy bodies and / or Lewy neurites, where the protein alpha-synuclein is the major component. Synucleinopathies frequently have degeneration of the dopaminergic nigrostriatal system, responsible for the core motor deficits in Parkinsonism (rigidity, bradykinesia, resting tremor). Several non-motor signs and symptoms are thought to precede motor symptoms in Parkinson’s disease and other synucleinopathies. Such early signs include, for example, REM sleep behavior disorder and loss of smell and constipation.

[0200] Synucleinopathies include Parkinson’s disease (PD) (including idiopathic and inherited forms of Parkinson’s disease as well as prodromal PD), Diffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies (DLB), multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration, and Shy-Drager Syndrome)), Lewy body variant of Alzheimer’s disease, Alzheimer’s disease with synuclein pathology, combined Alzheimer's and Parkinson disease, and pure autonomic failure.

[0201] In some embodiments, the synucleinopathy is PD. In some embodiments, the PD is a subtype of the disease thereof (e.g., prodromal PD). In some embodiments, the synucleinopathy is DLB. In some embodiments, the synucleinopathy is MSA. In some embodiments, the synucleinopathy is Alzheimer’s disease with synuclein pathology.

[0202] IV. Diagnostic Methods

[0203] Provided herein are methods for diagnosing a synucleinopathy (e.g., DLB, MSA, PD or a subtype of the disease thereof e.g., prodromal PD) in a subject. Any of the methods may be based on the protein presence or level of a nitrated alpha-synuclein biomarker provided herein, for example, an increased expression of nY39. Any of the methods may further include administering to the subject a cognitionenhancing agent, an antidepressant agent, a dopamine promoter (e.g., agonist), an anti-tremor agent, a neuroprotective agent, or an anti-synuclein antibody (e.g., anti-nY39 alpha-synuclein antibody) or antigenbinding fragment thereof described above.

[0204] The invention features a method of diagnosing a synucleinopathy (e.g., DLB, MSA, PD or a subtype of the disease thereof e.g., prodromal PD) in a subject, the method including determining the presence or level of at least one nitrated alpha-synuclein protein (e.g., nY39) in an alpha-synuclein protein in a CSF sample obtained from the subject; and comparing the level of the at least one nitrated alpha-synuclein protein to a reference level of the at least one nitrated alpha-synuclein protein, wherein an increase in the level of the at least one nitrated alpha-synuclein protein in the sample relative to the reference level of the at least one nitrated alpha-synuclein protein identifies a subject having a synucleinopathy (e.g., DLB, MSA, PD or a subtype of the disease thereof e.g., prodromal PD).

[0205] The invention further provides a method of identifying a subject having a synucleinopathy (e.g., DLB, MSA, PD or a subtype of the disease thereof e.g., prodromal PD), the method including determining the presence or level of the nitrated alpha-synuclein protein (e.g., nY39) in an alpha-synuclein protein in a sample obtained from the subject; comparing the level of the nitrated alpha-synuclein protein to a reference level of the nitrated alpha-synuclein protein; wherein an increase in the level of the nitrated alpha-synuclein protein in the sample relative to the reference level of the at least one nitrated alpha- synuclein protein identifies a subject having a synucleinopathy (e.g., DLB, MSA, PD or a subtype of the disease thereof e.g., prodromal PD). In some instances, the method includes the step of distinguishing which synucleinopathy the subject has (e.g., PD vs AD).

[0206] In any of the preceding methods, the presence or level of alpha-synuclein nY39 in an alpha- synuclein protein can be determined.

[0207] The presence and / or level / amount of alpha-synuclein nY39 in a CSF sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, a single molecule array (Simoa™) assay, single molecule detection, immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, biochemical enzymatic activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR) and other amplification type detection methods, such as, for example, SISBA, TMA and the like, RNA-seq, microarray analysis, gene expression profiling, and / or serial analysis of gene expression (“SAGE”), as well as any one of the wide variety of assays that can be performed by protein and / or fluid array analysis. In some embodiments, the presence and / or level / amount of alpha-synuclein nY39 in a CSF sample is analyzed with a Simoa™ assay.

[0208] In any of the preceding methods, the presence and / or level / amount of alpha-synuclein nY39 can be measured by determining protein levels of the biomarker. In certain embodiments, the method includes contacting the biological sample (e.g., CSF) with antibodies that specifically bind to a biomarker (e.g., anti-alpha-synuclein antibodies) under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibodies and biomarker. Such method may be an in vitro method. Any method of measuring protein levels known in the art or provided herein may be used. For example, in some embodiments, a protein level of a biomarker is determined using a method selected from the group consisting of a Simoa™ assay, single molecule detection, low cytometry (e.g., fluorescence-activated cell sorting (FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC. In some embodiments, the protein level of the biomarker can be determined in a CSF sample. In some embodiments, the diagnostic methods described herein (e.g., for determining protein levels of the biomarker (e.g., alpha-synuclein n Y39)), is performed using the anti-alpha-synuclein antibody 8C2 (i.e., rbAb3) as described herein (e.g., comprising a VL sequence comprising the amino acid sequence of SEQ ID NO: 53 and a VH sequence comprising the amino acid sequence of SEQ ID NO: 54). In some instances, the anti-alpha-synuclein antibody is an anti-nY39 alpha-synuclein antibody. In some instances, anti-nY39 alpha-synuclein antibody includes a CDR-L1 including the amino acid sequence of SEQ ID NO: 146; a CDR-L2 including the amino acid sequence of SEQ ID NO: 147; a CDR- L3 including the amino acid sequence of SEQ ID NO: 148; a CDR-H1 including the amino acid sequence of SEQ ID NO: 143; a CDR-H2 including the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 including the amino acid sequence of SEQ ID NO: 145. In some embodiments, the anti-alpha-synuclein antibody is an anti-nY39 alpha-synuclein antibody comprising a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

[0209] In some embodiments of any of the methods, the biomarker is nitrated alpha-synuclein (e.g., nY39). In some embodiments, the biomarker is nY39. In some embodiments, presence of nY39 is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control CSF sample or a CSF sample from non-PD patient, a CSF sample from a non-MSA patient, or a CSF sample from a non-DLB patient).

[0210] In some instances, the level of the protein in the sample is an average (e.g., mean expression or median expression) of the detected nitrated alpha-synuclein variants, the reference level of the nitrated alpha-synuclein variants is an average (e.g., mean expression or median expression) of the nitrated alpha-synuclein variants of the reference, and the average of the nitrated alpha-synuclein variants of the sample is compared to the average of the nitrated alpha-synuclein variants of the reference.

[0211] In certain embodiments, the presence and / or levels / amount of a biomarker in a first sample is increased or elevated as compared to presence / absence and / or levels / amount in a second sample. In certain embodiments, the second sample is a reference sample, reference fluid (e.g., CSF), control sample, or control fluid (e.g., CSF). Additional disclosures for determining the presence / absence and / or levels / amount of a nitrated alpha-synuclein variant are described herein. For example, in some embodiments, the level of the nitrated alpha-synuclein protein is calculated as the concentration of the nitrated alpha-synuclein protein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample. In some embodiments, the reference level is calculated as the concentration of the nitrated alpha-synuclein protein in a reference sample divided by the concentration of total alpha- synuclein in the reference sample.

[0212] In certain embodiments, a reference sample, reference fluid, control sample, or control fluid is a single sample or combined multiple samples from the same subject or individual that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample, reference fluid, control sample, or control fluid is obtained at an earlier time point from the same subject or individual than when the test sample is obtained. Such reference samples, reference fluids, control samples, or control fluids may be useful if the reference sample is obtained prior to prodromal PD and the test sample is later obtained when the subject has been diagnosed with prodromal PD.

[0213] In certain embodiments, a reference sample, reference fluid, control sample, or control fluid is a combined multiple samples from one or more healthy individuals who are not the patient. In certain embodiments, a reference sample, reference fluid, control sample, or control fluid is a combined multiple samples from one or more individuals with a disease (e.g., PD) who are not the subject or individual. In certain embodiments, a reference sample, reference fluid, control sample, or control fluid is pooled protein samples from normal samples from one or more individuals who are not the patient. In certain embodiments, reference samples, reference fluids, control samples, or control fluids are pooled protein samples from CSF samples from one or more individuals with a disease (e.g., RD) who are not the patient.

[0214] In some embodiments, any of the diagnostic methods described herein further includes determining the presence or level in a sample (e.g., a CSF sample) from the subject of alpha-synuclein nitrated at Y125, Y133, Y136, or a combination thereof.

[0215] In some embodiments of any of the methods, elevated or increased expression refers to an overall increase of about any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., nitrated alpha-synuclein protein, e.g., nY39 alpha-synuclein, alone or in combination with one or more of nY125, nY133, or nY136 alpha-synuclein), detected by standard art-known methods such as those described herein, as compared to reference sample, reference fluid, control sample, or control fluid. In certain embodiments, the elevated expression refers to the increase in level / amount of a biomarker in the sample wherein the increase is at least about any of 1 .5x, 1 .75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 25x, 50x, 75x, or 100x the level / amount of the respective biomarker in reference sample, reference fluid, control sample, or control fluid. In some embodiments, elevated expression refers to an overall increase of greater than about 1 .5 fold, about 1 .75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25 fold as compared to reference sample, reference fluid, control sample, or control fluid. In some embodiments, the increase is a statistically significant increase (e.g., an increase with a p value of less than or equal to 0.05 (e.g., less than or equal to 0.04, 0.03, 0.02, or 0.01 ) relative to a reference level. In some embodiments, the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the concentration of the at least one nitrated alpha-synuclein protein in the CSF relative to the reference level.

[0216] V. Routes, Dosage, and Administration

[0217] Pharmaceutical compositions that include the anti-nY39 alpha-synuclein antibodies or antigen binding fragments thereof of the invention as the therapeutic agents may be by a variety of routes, such as intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, topical, intratracheal, intraperitoneal, intraarterial, intravascular, intrathecal, intracerebroventricular, intraparenchymal, inhalation, stereotactic, perfusion, lavage, and oral administration. In some embodiments, the anti-nY39 alpha-synuclein antibodies or antigen binding fragments thereof may be administered intravenously. The pharmaceutical composition may also be formulated for, or administered via, oral, ocular, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, e.g., ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).

[0218] The most suitable route and dosage for administration in any given case will depend on the particular composition administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the disease being treated, the patient’s diet, and the patient’s excretion rate. A pharmaceutical composition of the invention may include a dosage of an anti-nY39 alpha-synuclein antibody or an antigen binding fragment thereof of the invention ranging from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg) and, in a more specific embodiment, about 0.1 to about 30 mg / kg and, in a more specific embodiment, about 0.3 to about 30 mg / kg. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.

[0219] The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Pharmaceutical compositions that include an anti-nY39 alpha-synuclein antibody or an antigen binding fragment thereof of the invention may be administered to a subject in need thereof, for example, one or more times (e.g., 1 -10 times or more) daily, weekly, biweekly, monthly, bimonthly, quarterly, biannually, annually, or as medically necessary. In some embodiments, pharmaceutical compositions that include an anti-nY39 alpha-synuclein antibody or an antigen binding fragment thereof of the invention may be administered to a subject in need thereof weekly, biweekly, monthly, bimonthly, or quarterly. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may decrease as the medical condition improves or increase as the health of the patient declines.

[0220] VI. Kits

[0221] The compositions described herein can be provided in a kit for use in treating a synucleinopathy (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology). Compositions may include an anti-nY39 alpha-synuclein antibody or antigen binding fragment thereof described herein, and may be provided in unit dosage form, optionally in a pharmaceutically acceptable excipient (e.g., saline), in an amount sufficient to treat a synucleinopathy (e.g., Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology). The kit can further include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

[0222] EXAMPLES

[0223] Example 1. Design of exemplary nY39 alpha-synuclein antibodies

[0224] Parental rabbit antibodies and mouse chimera antibodies that bind specifically to nY39 alpha- synuclein were prepared. The sequences of the VL and VH domains of the rabbit antibodies are provided in Table 5. The sequences of the light chain (LC) and heavy chain (HC) of the mouse chimera antibodies are provided in Table 6. CDR regions are indicated in bold. Table 5. VH and VL domain amino acid sequences of exemplary rabbit antibodies

[0225] Table 6. LC and HC amino acid sequences of exemplary mouse chimera antibodies rbAbl and corresponding mouse chimera antibody msAbl were selected for further investigation.

[0226] Human chimera antibodies, hAb1 and hAb2, were produced, with LC and HC amino acid sequences provided in Table 7. CDR sequences are indicated in bold.

[0227] Table 7. LC and HC amino acid sequences of exemplary human chimera antibodies rbAbl and corresponding mouse chimera antibody msAbl were then humanized (producing humanized antibodies humAb1 -humAb23), with sequences of the VL and VH domains provided in Table 8 and sequences of the light chains and heavy chains provided in Table 9. CDR regions are indicated in bold.

[0228] Table 8. VL and VH domain amino acid sequences of exemplary humanized antibodies Table 9. LC and HC amino acid sequences of exemplary humanized antibodies humAb20 was then affinity matured (producing antibodies humAb24-humAb28), with sequences of the VL and VH domains provided in Table 10 and sequences of the light chains and heavy chains provided in Table 11 . CDR regions are indicated in bold. Table 10. VL and VH domain amino acid sequences of exemplary affinity matured antibodies

[0229] Table 11. LC and HC amino acid sequences of exemplary affinity matured antibodies

[0230] Example 2. ELISA binding studies of rabbit antibodies rbAb1-rbAb4 with full-length alpha- synuclein

[0231] To assess the specificity of rabbit antibodies rbAb1-rbAb4 for nY39 alpha-synuclein, an ELISA assay was performed with full-length human nY39 alpha-synuclein (prepared recombinantly with targeted nitration site), chemically nitrated full-length human alpha-synuclein, unmodified full-length human alpha- synuclein, and chemically nitrated bovine serum albumin (BSA). Chemically nitrated proteins were prepared by treating alpha synuclein (rPeptide, S-1001 -2) or BSA at 0.1 mg / ml with 500uM peroxynitrite solution (Nitrase) for 10 minutes in PBS. Rabbit antibodies rbAbl -rbAb4 were coated at 1 ug / ml in PBS onto MSD 96-well standard sector plates overnight. After blocking for 1 hour in 1 X casein, alpha-synuclein or BSA was added in titration and incubated for 1 hour at room temperature on a shaker. Plates were washed and a total synuclein detection antibody (610787, BD Transduction Labs conjugated to sulfotag) was added and plates were incubated for 1 hour at room temperature on a shaker. Next, plates were washed, 150 pL of read buffer was added, and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0232] Results are shown in FIGS. 1 A-1 E. Data are shown as raw MSD units as a function of alpha- synuclein or BSA concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0233] Rabbit antibodies rbAbl -rbAb4 showed binding to chemically nitrated full length alpha-synuclein and full-length nY39 alpha-synuclein but not to unmodified full-length alpha-synuclein or chemically nitrated bovine serum albumin by ELISA (FIGS. 1A-1 D). rbAb1 -rbAb3 showed a similar degree of binding to nY39 alpha-synuclein; rbAb4 showed a lower level of binding (FIG. 1 E).

[0234] Example 3. ELISA binding studies of rabbit antibodies rbAb1-rbAb4 with biotinylated 10-mer alpha-synuclein peptides

[0235] To assess the specificity of rabbit antibodies rbAb1-rbAb4 for nY39 alpha-synuclein, an ELISA assay was performed with biotinylated 10-mer alpha-synuclein peptides (Y39, nY39, Y125, nY125, nY133, and nY136).

[0236] The biotinylated 10-mer alpha-synuclein peptides were coated on streptavidin MSD plates for 1 hour in 0.1 X casein / PBS. Plates were washed and rabbit antibodies rAb1 -rAb4 were added in titration in 0.1 X casein / PBS for 1 hour. Plates were washed and antibodies were detected with anti-rabbit sulfotag, with incubation for 1 hour at room temperature on a shaker. Next, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0237] Results are shown in FIGS. 2A-2E. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism.

[0238] Rabbit antibodies rbAb1 -rbAb4 showed binding to nY39 but did not bind Y39, Y125, nY125, nY133, or nY136 by ELISA (FIGS. 2A-2D). rbAb1 -rbAb3 show similar degree of binding to nY39; rbAb4 showed a lower level of binding (FIG. 2E).

[0239] Example 4. ELISA binding studies of rabbit antibodies rbAb1-rbAb3 with biotinylated 50-mer nY39 alpha-synuclein peptides

[0240] To assess the binding affinity of rabbit antibodies rbAb1 -rbAb3 for nY39 alpha-synuclein, ELISA assays were performed with biotinylated 50-mer nY39 alpha-synuclein peptides (amino acids 14-64 with tyrosine 39 nitrated), either with rAb1 -rAb3 as the capture antibody or as the detection antibody.

[0241] For ELISA assays with rbAb1 -rbAb3 as the capture antibody, antibodies were coated at 1 ug / mL in PBS onto MSD 96-well standard sector plates overnight. After blocking for 1 hour in 1 X casein, biotinylated 50-mer nY39 alpha-synuclein peptides were added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and peptide binding was detected with streptavidin sulfotag and incubated for 1 hour at room temperature on a shaker. After washing, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0242] Results are shown in FIG. 3A. Data are shown as raw MSD units as a function of alpha-synuclein peptide concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. EC50 values are summarized in Table 12. The binding affinity of rbAbl -rbAb3 for biotinylated 50-mer nY39 peptides was ~2 nM by ELISA (using antibody as capture).

[0243] Table 12. EC50 of antibodies for biotinylated 50-mer nY39 peptides

[0244] For ELISA assays using rbAbl -rbAb3 as the detection antibody, biotinylated 50-mer nY39 alpha- synuclein peptides were coated at 1 ug / ml on streptavidin MSD plates for 1 hour in 0.1 X casein / PBS. Plates were washed and antibodies were added in titration in 0.1X casein / PBS for 1 hour. Plates were then washed and antibodies were detected with anti-rabbit sulfotag, with incubation for 1 hour at room temperature on a shaker. After washing, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SO 120MM plate reader.

[0245] Results are shown in FIG. 3B. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. EC50 values are summarized in Table 13. The binding affinity of rbAb1 -rbAb3 for 50-mer nY39 peptides was -46-73 pM by ELISA (using antibody as detection, EC50 values may include avidity component).

[0246] Table 13. EC50 of antibodies for biotinylated 50-mer nY39 peptides.

[0247] Example 5. ELISA binding studies of rabbit antibodies rbAb1-rbAb3 and mouse chimera antibodies msAb1-msAb3 with 70-mer alpha-synuclein peptides

[0248] To assess the specificity and binding affinity of rabbit antibodies rAb1 -rAb3 and mouse chimera antibodies msAb1 -msAb3 for nY39 alpha-synuclein, an ELISA assay was performed with 70-mer alpha- synuclein peptides (amino acids 31 -100 of alpha-synuclein with nitrated tyrosine 39 (nY39_70mer), amino acids 81 -140 of alpha-synuclein with nitrated tyrosine 125 (nY125_70mer), amino acids 81 -140 of alpha- synuclein with nitrated tyrosine 133 (nY133_70mer), and amino acids 81 -140 of alpha-synuclein with nitrated tyrosine 136 (nY136_70mer)).

[0249] Rabbit antibodies rAb1 -rAb3 and mouse chimera antibodies msAb1 -msAb3 were coated at 1 ug / ml in PBS onto MSD 96-well standard sector plates overnight. After blocking for 1 hour in 1 X casein, 70-mer alpha-synuclein peptides were added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and a total synuclein detection antibody (610787, conjugated to sulfotag) was added and incubated for 1 hour at room temperature, on a shaker. After washing, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0250] Results are shown in FIGS. 4A-4G. Data are shown as raw MSD units as a function of 70-mer alpha-synuclein peptide concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. EC50 values are summarized in Table 14. The rabbit and mouse chimera antibodies showed similar binding specificity and affinity to 70-mer nY39 peptides (FIG. 4G) but do not bind to 70-mer nY125, nY133, or nY136 peptides by ELISA (FIGS. 4A-4F).

[0251] Table 14. EC50 of antibodies for 70-mer nY39 peptides Example 6. Biosensor binding studies of rabbit antibodies rbAbl and rbAb3 and mouse chimera antibodies msAb1-msAb3 with 50-mer nY39 alpha-synuclein peptides

[0252] To assess the binding affinity of rabbit antibodies rAb1 and rAb3 and mouse chimera antibodies msAb1 -msAb3 for nY39 alpha-synuclein, a biosensor binding assay was performed using 50-mer nY39 alpha-synuclein peptides.

[0253] For rabbit antibodies, binding experiments were performed on Octet HTX at 25 °C. The biotinylated anti-Rabbit Fc (Jackson 11 1 -066-046) (3 pg / mL) was loaded onto SAX sensors. The loaded sensor then were blocked with 100 uM dPEG-biotin acid for 10 minutes. Then, the loaded sensors were used to capture rbAbl and rbAb3. The antibody loaded sensors were dipped into a serial dilutions of analyte 50-mer nY39 alpha-synuclein peptides (1 :3 dilution, 8 points from top concentration, 3 uM as top concentration). The lowest and three highest concentrations were excluded from analysis to improve fitting. Reference sample well (buffer) was used for data analysis. Kinetic constants were calculated using a monovalent (1 :1 ) binding model. Results are shown in Table 15.

[0254] Table 15. Binding affinity by standard format Octet analysis of rabbit antibodies

[0255] For mouse chimera antibodies, binding experiments were performed on Octet HTX at 25 °C. The anti-mouse IgG capture (AMC) was blocked with 100 uM dPEG-biotin acid for 10 minutes. msAb1 -msAb3 were loaded onto blocked AMC sensors. The antibody loaded sensors were dipped into a serial dilutions of analyte 50-mer nY39 alpha-synuclein peptides (1 :3 dilution, 8 points from top concentration, 3 uM as top concentration). The lowest and three highest concentrations were excluded from analysis to improve fitting. Reference sample well (buffer) was used for data analysis. Kinetic constants were calculated using a monovalent (1 :1 ) binding model. Results are shown in Table 16.

[0256] Table 16. Binding affinity by standard format Octet analysis of mouse chimera antibodies

[0257] Binding affinity by standard format Octet analysis for rabbit antibodies and mouse chimera antibodies were similar (~2-5 nM).

[0258] Example 7. Biosensor binding studies of rbAbl with biotinylated 50-mer nY39 alpha-synuclein peptides

[0259] To assess the binding affinity of rabbit antibody rAb1 for nY39 alpha-synuclein, a biosensor binding assay was performed using 50-mer nY39 alpha-synuclein peptides. Binding experiments were performed on Octet HTX at 30 °C. The biotinylated 50-mer nY39 alpha-synuclein peptide (nY39) (10 ug / mL) was loaded onto SAX sensors. The loaded sensors then were blocked with 100 uM dPEG-biotin acid for 10 minutes. The peptide loaded sensors were dipped into a serial dilutions of antibodies (1 :3 dilution, 7 points from top concentration, 300 nM top concentration). Reference sample well (buffer) was used for data analysis. Kinetic constants were calculated using a monovalent (1 :1 ) binding model (inverted format with peptide on the chip and antibody flowed over, may include avidity component). Results are shown in Table 17.

[0260] Table 17. Binding affinity using inverted format Octet analysis

[0261] Example 8. Western blot binding studies of rabbit antibodies rbAb1-rbAb3 with full-length alpha- synuclein

[0262] To assess the specificity of rabbit antibodies rAb1 -rAb3 for nY39 alpha-synuclein, an ELISA assay was performed with chemically nitrated alpha-synuclein (human and mouse recombinant protein), recombinantly produced full-length human nY39 alpha-synuclein, unmodified human and mouse alpha- synuclein, BSA, and chemically nitrated BSA. A total synuclein antibody (610787) and a general nitration antibody (3-NT 06-284, Millipore) were included as controls.

[0263] Alpha-synuclein and BSA were run on BOLT 4-12% bis-tris gels, transferred to nitrocellulose by i Blot dry transfer, then blocked with Licor blocking buffer and probed with rbAbl -rbAb3, 610787, and 3-NT antibodies. Bands were detected with Licor secondary antibodies and imaged on an Odyssey CLx.

[0264] Results are shown in FIGS. 5A-5E. Western blot analysis demonstrates specificity of rbAbl - rbAb3 to chemically nitrated alpha-synuclein, alpha-synuclein nitrated at tyrosine 39 and chemically nitrated mouse alpha-synuclein.

[0265] Example 9. Target engagement assays using 70-mer nY39 alpha-synuclein peptides

[0266] To assess the binding affinity of mouse chimera antibody msAbl and human chimera antibody hAb1 for nY39 alpha-synuclein, an ELISA target engagement assay was performed with 70-mer nY39 alpha-synuclein peptides containing amino acids 31 -100 of alpha-synuclein nitrated at tyrosine 39. A mouse IgG antibody (Ms IgG) was included as a negative control.

[0267] 70-mer nY39 peptides (10 ng / ml) were preincubated with msAbl , hAb1 , or Ms IgG in titration. The samples were run in the nitrated synuclein ELISA assay (msAb3 antibody capture with 610787 detection). MSD 96-well standard sector plates were coated with msAb3 at 1 ug / ml in PBS overnight. After blocking for 1 hour in 1 X casein, the mixture of 70-mer peptides with test antibodies in titration was added and plates were incubated for 1 hour at room temperature, on a shaker. Plates were washed and a total synuclein detection antibody 610787 conjugated to sulfotag was added and incubated for 1 hour, at room temperature on a shaker. After washing, 150 uL read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0268] Results are shown in FIG. 6. Data are shown as raw MSD units as a function of msAbl , hAb1 , or Ms IgG antibody concentration with a 4 parameter logistic curve fit performed in Graph Pad Prism. EC50 values are summarized in Table 18. Target engagement assays demonstrated effective competition of binding to target with msAbl and hAb1 .

[0269] Table 18. EC50 of antibodies for 70-mer nY39 peptides

[0270] Example 10. Nitrated synuclein levels in cerebrospinal fluid

[0271] To assess the levels of nitrated alpha-synuclein in human cerebrospinal fluid (CSF) samples, samples of human CSF from healthy controls (HC) or from Parkinson’s disease (PD) patients were tested in a SIMOA assay for levels of total synuclein and nY39 synuclein. N>50 samples of each were tested. Nitrated synuclein levels are presented as % of total synuclein.

[0272] To assess the levels of nitrated alpha-synuclein in the insoluble fraction of dopaminergic neurons treated with preformed fibril (PFF) synuclein, monomer synuclein (a-syn) or growth media (GM), iCell dopaminergic neurons (Fujifilm-CDI) were plated per manufacturer directions and grown for 14 days. Cells were then treated with human alpha synuclein PFF (Stress Marq, SPR-322, material sonicated before use) or human alpha synuclein monomer (rPeptide S-1001 -2) at 5 ug / ml for 3 days. Treatments were washed out and growth media added for another 3 days. Cells were then lysed in 1% triton / TBS with HALT protease / phosphatase inhibitor for 15 min on wet ice. Lysed cells were transferred to microfuge tubes and spun down at 18,000xg for 15 minutes. Supernatant was removed and the pellet solubilized in 1% SDS / TBS with HALT (insoluble fraction). The insoluble fraction was then subjected to MSD ELISA assay to measure nY39 synuclein. Interpolated values were normalized to total protein in the assay.

[0273] To assess the levels of nitrated alpha-synuclein in mouse interstitial fluid (ISF) samples, ISF samples were collected from M83 human alpha synuclein transgenic mice treated with a single striatal injection of human alpha synuclein preformed fibrils (Stress Marq, SPR-322, material sonicated before use), M83 sham treated animals or wild-type mice sham treated. The samples were tested in a SIMOA assay for levels of total synuclein and nY39 synuclein.

[0274] Results are shown in FIGS. 7A-7C. Nitrated synuclein levels are elevated in PD CSF, a mouse model of PD, and in a cell culture model of PD, suggesting it may play an important role in the disease.

[0275] Example 11. Surface plasmon resonance binding studies of human chimera and humanized antibodies with biotinylated 50-mer nY39 alpha-synuclein peptide

[0276] To assess the binding affinity of human chimera antibodies hAb1 and hAb2, and humanized antibodies humAb1 -humAb20 for nY39 alpha-synuclein, a surface plasmon resonance assay was performed with biotinylated 50-mer nY39 alpha-synuclein peptide. The affinity of the antibodies was individually determined using a Surface Plasmon Resonance (SPR) biosensor, Biacore (Cytiva). The antibodies were captured onto the Protein A sensor chip as ligand and antigen flowed over the sensor chip surface as analyte. The dissociation (kd) and association (ka) rate constants were obtained using Evaluation Software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd over ka.

[0277] Results from single point SPR experiments are shown in Table 19. Single point SPR data demonstrates that many of the humanized antibodies have high affinity for nY39.

[0278] Table 19. Surface plasmon resonance single point binding data of human chimera and humanized antibodies with biotinylated 50-mer nY39 alpha-synuclein peptides

[0279] Results from dose-response SPR experiments are shown in Table 20. Dose-response SPR binding data for human chimera and humanized antibodies demonstrated nM binding affinity.

[0280] Table 20. Surface plasmon resonance dose-response binding data of human chimera and humanized anti-nY39 alpha-synuclein antibodies with biotinylated 50-mer nY39 alpha-synuclein peptides

[0281] Example 12. ELISA binding studies of human chimera and humanized msAbl antibodies with biotinylated 50-mer nY39 alpha-synuclein peptides

[0282] To assess the binding affinity of human chimera and humanized antibodies for nY39 alpha- synuclein, an ELISA assay was performed with biotinylated 50-mer nY39 alpha-synuclein peptides.

[0283] 96-well ELISA microtiter plates were coated with 100 nM antibody in PBS overnight at 4°C (nonrelated isotype antibody was the negative control, 100 pL / well). Plates were washed three times and blocked with 4% MPBS at 37°C for 1 hour. Next, 1 ug / mL biotinylated 50-mer nY39 alpha-synuclein peptides in PBS was added. After rinsing plates three times with wash buffer, SA-HRP was added to the plates for 45 minutes at room temperature. The plates were washed an additional 6 times and the substrate solution was added to the wells for the developing reaction. The absorption was measured at 450 nm using a spectrometer. Results are shown in Table 21 , demonstrating nM binding affinity to nY39 by ELISA. Table 21. Binding affinity to biotinylated 50-mer nY39 alpha-synuclein peptides

[0284] Example 13. ELISA binding studies of human chimera and humanized antibodies with 70-mer nY136 alpha-synuclein peptides To assess the specificity of human chimera antibody hAb2 and humanized antibodies humAbl - humAb20 for nY39 alpha-synuclein, an ELISA assay was performed with 70-mer peptides containing amino acids 81 -140 of alpha-synuclein with nitrated tyrosine 136 (nY136) or full-length unmodified alpha- synuclein (a-syn). Antibodies specific to c-terminal synuclein nitration (NSYN12, EMD Millipore Cat No. 36-011 ) and antibodies that bind amino acids 118-123 of human alpha synuclein (MJFR1 , Abeam Cat. No. ab138501 ) were included as positive controls.

[0285] Antibodies were coated at 1 ug / mL in PBS onto MSD 96-well standard sector plates overnight. After blocking for 1 hour in 1 X casein, 70-mer nY136 alpha synuclein peptides or full-length unmodified alpha-synuclein were added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and a total synuclein detection antibody (610787, conjugated to sulfotag) was added and incubated for 1 hour, at room temperature on a shaker. After washing, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0286] Results are shown in FIGS. 8A-8D. Data are shown as raw MSD units as a function of alpha- synuclein concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. hAb2 and humAb1 -humAb20 do not bind to 70-mer nY136 alpha synuclein peptides or full-length unmodified alpha- synuclein.

[0287] Example 1 . Target engagement assays using 70-mer nY39 alpha-synuclein peptides

[0288] To evaluate the binding affinity of the human chimera and humanized antibodies, engagement assay determinations were performed. Plates containing 1 ug / mL rbAb3 in PBS at a volume of 100 uL / well were incubated at 4 °C overnight. Plates were washed two times and blocked with 4% dry milk in 1 X PBS (MPBS) at 37°C for 1 hour. 70-mer nY39 alpha-synuclein peptides at 100 ng / mL were added to the plates and incubate for 2 hours at room temperature. The plates were washed three times, then 1 ug / mL 610787 was added to the wells for the developing reaction and incubated at room temperature for 1 hour. The plates were washed an additional 6 times, then the anti-mouse HRP antibody was added to the plates for 45 minutes at room temperature. The plates were then washed an additional 6-times, and the substrate solution was added to the wells for the developing reaction. The absorption was measured at 450 nm using a spectrometer.

[0289] Results are shown in Table 22. Target engagement assay using 70-mer nY39 peptides demonstrated effective competition with many human chimera and humanized antibodies.

[0290] Table 22. Binding affinity to 70-mer nY136 alpha-synuclein peptides

[0291] Example 15. ELISA binding studies of humAb20 with human and cyno nitrated alpha-synuclein

[0292] To compare the binding of humAb20 with human and cyno nitrated alpha-synuclein, ELISA binding studies were performed. Antibodies were coated at 1 ug / ml in PBS onto MSD 96 well standard sector plate overnight. After blocking for 1 hour in 1 X casein, proteins (chemically nitrated full length human alpha synuclein, unmodified full length human alpha synuclein, chemically nitrated full length cynomolgus monkey synuclein and unmodified full length cynomolgus monkey synuclein) were added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and a total anti- alpha-synuclein detection antibody (MJFR1 , Abeam, Cat# ab138501 ) was added and incubated for 1 hour. Plates were then washed and an anti-rabbit sulfotag was added and incubated for 1 hour at room temperature, on a shaker. After final wash, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader. Raw MSD units were graphed as a function of protein concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. A sequence alignment between human and cyno alpha-synuclein showed 98.6% sequence similarity FIG. 9A).

[0293] Results are shown in FIG. 9B. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. Humanized humAb20 demonstrates similar cross-reactivity for human and cyno nitrated alpha-synuclein protein.

[0294] Example 16. Surface plasmon resonance binding studies of affinity matured antibodies with biotinylated 50-mer nY39 alpha-synuclein peptide

[0295] To assess the binding affinity of affinity matured antibodies hAb24-hAb28 for nY39 alpha- synuclein, a surface plasmon resonance assay was performed with biotinylated 50-mer nY39 alpha- synuclein peptide.

[0296] The affinity of the antibodies was individually determined using a Surface Plasmon Resonance (SPR) biosensor, Biacore 8K (Cytiva). The antibodies were captured onto the Protein A sensor chip as ligand and antigen (biotinylated 50-mer nY39 alpha-synuclein peptide) flowed over the sensor chip surface as analyte. The dissociation (kd) and association (ka) rate constants were obtained using Biacore 8K Evaluation Software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd over ka.

[0297] Results are shown in Table 23. Binding affinities by SPR analysis for affinity matured antibodies humAb24-humAb28 were in a comparable range (~2-3 nM).

[0298] Table 23. Surface plasmon resonance binding data of affinity matured antibodies with biotinylated 50-mer nY39 alpha-synuclein peptides

[0299] Example 17. ELISA binding studies of affinity matured antibodies with biotinylated 50-mer nY39 alpha-synuclein peptides

[0300] To assess the binding affinity of affinity matured antibodies humAb24-humAb28 for nY39 alpha- synuclein, ELISA assays were performed with biotinylated 50-mer nY39 alpha-synuclein peptides (amino acids 14-64 with tyrosine 39 nitrated), either with humAb24-humAb28 as the capture antibody or as the detection antibody.

[0301] For ELISA assays with humAb24-humAb28 as the capture antibody, antibodies were coated at 1 ug / mL in PBS onto MSD 96-well standard sector plates overnight. After blocking for 1 hour in 1 X casein, biotinylated 50-mer nY39 alpha-synuclein peptides were added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and peptide binding was detected with streptavidin sulfotag and incubated for 1 hour at room temperature on a shaker. After washing, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0302] Results are shown in FIG. 10A. Data are shown as raw MSD units as a function of alpha- synuclein peptide concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. EC50 values are summarized in Table 24. The binding affinity of the affinity matured antibodies humAb24- humAb28 as capture antibodies ranged from 4-7 nM.

[0303] Table 24. EC50 of antibodies for biotinylated 50-mer nY39 alpha-synuclein peptides

[0304] For ELISA assays using humAb24-humAb28 as the detection antibody, biotinylated 50-mer nY39 alpha-synuclein peptides were coated at 1 ug / ml in 0.1X casein / PBS onto MSD 96 well streptavidin plates for 1 hour at room temperature, on a shaker. Antibodies were then added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and anti-human sulfotag was added and incubated for 1 hour at room temperature on a shaker. After washing, 150 uL of read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0305] Results are shown in FIG. 10B. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. EC50 values are summarized in Table 25 (using antibody as detection, EC50 values may include avidity component). The binding affinity of affinity matured antibodies humAb24-humAb28 as detection were about 0.01 1 -0.013 nM or 10-13 pM.

[0306] Table 25. EC50 of antibodies for biotinylated 50-mer nY39 alpha-synuclein peptides

[0307] Example 18. ELISA binding studies of affinity matured antibodies with biotinylated 50-mer alpha- synuclein peptides and full-length alpha-synuclein

[0308] To assess the specificity of affinity matured antibodies humAb24-humAb28 for nY39 alpha- synuclein, an ELISA assay was performed with biotinylated 50-mer alpha-synuclein peptides (Y39, nY39, nY125, nY133, and nY136) and biotinylated full-length alpha-synuclein.

[0309] Antibodies were coated at 1 ug / ml in PBS onto MSD 96well standard sector plates overnight. After blocking for 1 hour in 1 X casein, nY39-50-mer biotin, Y39-50-mer biotin, nY125 50-mer biotin, nY133 50-mer biotin, nY136 50-mer biotin, and full length alpha-synuclein biotin (FL a-syn; Anaspec AS- 55581 ) were added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and streptavidin sulfotag was added and incubated for 1 hour at room temperature, on a shaker. After washing, 150 uL read buffer was added and plates were read on an MSD MESO QuickPlex SQ 120MM plate reader.

[0310] Results are shown in FIGS. 11 A-1 1 E. Data are shown as raw MSD units as a function of alpha- synuclein concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. The affinity matured antibodies humAb24-humAb28 showed specific binding to nY39-50-mer peptides, but not to the Y39-50-mer peptide, the C-terminal synuclein nitration sites (nY125, nY133 or nY136-50-mer peptides), or full length unmodified synuclein.

[0311] Example 19. ELISA binding studies of affinity matured antibodies with chemically nitrated BSA

[0312] To assess the specificity of affinity matured antibodies humAb24-humAb28 for nY39 alpha- synuclein, an ELISA assay was performed with chemically nitrated BSA. A general nitration antibody (3- NT 06-284, Millipore) was included as a control.

[0313] Chemically nitrated BSA was coated at 1 ug / ml in PBS onto MSD 96 well standard sector plates for 2 hours at room temperature. Antibodies were then added in titration and incubated for 1 hour at room temperature, on a shaker. Plates were washed and anti-human sulfotag was added and incubated for 1 hour at room temperature, on a shaker. After washing, 150 uL of read buffer was added and plates were read on a MSD MESO QuickPlex SQ 120MM plate reader. Results are shown in FIG. 12. Data are shown as raw MSD units as a function of antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. None of the affinity matured antibodies humAb24-humAb28 showed reactivity with nitrated BSA.

[0314] Example 20. Target engagement assays using 70-mer nY39 alpha-synuclein peptides

[0315] To assess the binding affinity of affinity matured antibodies humAb24-humAb28 for nY39 alpha- synuclein, an ELISA target engagement assay was performed with 70-mer nY39 alpha-synuclein peptides containing amino acids 31 -100 of alpha-synuclein nitrated at tyrosine 39.

[0316] 10 ng / mL 70-mer nY39 alpha-synuclein peptides was preincubated with test antibodies in titration. The samples were then run in the nitrated synuclein ELISA assay (msAb3 antibody capture with 610787 detection). MSD 96 well standard sector plates were coated with msAb3 at 1 ug / mL in PBS overnight. After blocking for 1 hour in 1 X casein, the mixture of 70-mer nY39 peptides with test antibodies in titration was added and plates were incubated for 1 hour at room temperature, on a shaker. Plates were washed and a total synuclein detection antibody (610787 conjugated to sulfotag) was added and incubated for 1 hour at room temperature, on a shaker. After washing, 150 uL of read buffer was added and plates were read on MSD MESO QuickPlex SQ 120MM plate reader.

[0317] Results are shown in FIG. 13. Data are shown as raw MSD units as a function of humAb24- humAb28 antibody concentration with a 4 parameter logistic curve fit performed in GraphPad Prism. IC50 values are summarized in Table 26. Target engagement assays demonstrated effective competition of binding to target with humAb24-humAb28 with EC50 of about 0.3-0.4 nM.

[0318] Table 26. IC50 of antibodies for 50-mer nY39 peptides.

[0319] Example 21. Treatment of a synucleinopathy in human subjects

[0320] Using the compositions and methods of the disclosure, a subject (e.g., a human subject), may be treated for a synucleinopathy (e.g., DLB, MSA, PD, or Alzheimer’s disease with synuclein pathology). To treat the subject, a physician of skill in the art can administer to the subject a composition containing an nY39 alpha-synuclein antibody or an antigen binding fragment thereof (e.g., any one of humAbl - humAb28 (e.g., any one of humAb24-humAb28)). The composition containing the nY39 alpha-synuclein antibody or an antigen binding fragment thereof may be administered to the subject, for example, intravenously. The nY39 alpha-synuclein antibody or an antigen binding fragment thereof may be administered in a therapeutically effective amount, such as from 0.01 to 500 mg / kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the nY39 alpha-synuclein antibody or an antigen binding fragment thereof is administered bimonthly, once a month, once every two weeks, or at least once a week or more (e.g., 1 , 2, 3, 4, 5, 6, or 7 times a week or more).

[0321] Following administration of the composition to a patient, a practitioner of skill in the art can monitor the patient’s improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient’s motor and / or cognitive symptoms. A finding that the patient does not develop or exhibits improved motor and / or cognitive symptoms following administration of the composition compared to an untreated subject or compared to measurements from the patient prior to treatment indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

[0322] Example 22. Detection of nitrated alpha-synuclein nY39 using the anti-synuclein antibody 8C2 (i.e., rbAb3)

[0323] Alpha-synuclein nitrated at tyrosine 39 was measured using clone 8C2 (Nitrase) as a capture antibody. Total a-syn levels were assayed using a-syn antibodies as capture (either antibody #610787, BD Transduction Labs or #10842-1 , Proteintech) with detection by another total synuclein antibody (either #ab138501 , MJFR1 , Abeam or 6H7, Elan / lmago Pharmaceuticals). ELISAs specific for nitrated a-syn (e.g., nY39) and total a-syn (including unmodified, nitrated and phosphorylated full length a-syn) were developed using a sandwich ELISA either with HRP / TMB absorbance detection or with electrochemiluminescence detection on the MSD platform (Mesoscale Diagnostics). Alpha-synuclein could be detected in either samples obtained from whole blood lysis or in samples obtained from lysis of brain tissue. For whole blood lysis: 1 mL of 1 X RBC lysis buffer (50 mM Tris, pH 7.5, 20 mM NaCI, 1 % Triton 100X, protease inhibitor cocktail, Sigma #P2714) was added to 0.1 mL of blood and incubated for 10-15 minutes at room temperature until the solution was clear. Then, the samples were heated to 95 °C for 10 minutes, followed by centrifugation at 5000 x g for 5 minutes at 4 °C to pellet the cell membranes. Total protein in the supernatants was quantified by BCA assay (Pierce #23227), and the samples were stored at -80 °C until analysis by MSD ELISA for a-syn and nitrated a-syn. For lysis of brain tissue: Tissue and 10x volume (uL) per weight (mg) of 50 mM Tris pH 7.5, 150 mM NaCI, and 1 X protease inhibitor (Sigma #P2714) were added to Omni Bead Ruptor Elite tubes containing beads and homogenized according to the manufacturer’s directions. The samples were centrifuged at 10,000 x g for 20 minutes at 4 °C, and the supernatant was transferred to a fresh tube on ice. The amount of tissue was quantified for total protein using a BCA assay, and the lysate was stored at -80 °C until use in an MSD ELISA for synuclein and nitrated synuclein.

[0324] Example 23. Binding of humAb26 to chemically nitrated human, cynomolgus monkey, mouse and rat alpha-synuclein Summary

[0325] The purpose of this study is to confirm comparable binding of humAb26 to chemically nitrated human, cynomolgus monkey, mouse, and rat alpha-synuclein. humAb26 was characterized as a capture antibody in Meso Scale Discovery (MSD) ELISA setting for binding to unmodified or chemically nitrated human, cynomolgus monkey, mouse, and rat recombinant alpha-synuclein. humAb26 showed binding only to chemically nitrated forms of synuclein and not unmodified synuclein. The binding affinity was similar for human, cynomolgus monkey, mouse and rat nitrated alpha-synuclein. Materials and Methods

[0326] Test articles and study design

[0327] Test articles are summarized in Table 27. The test system was Meso Scale Discovery (MSD) Plate Reader, MESO QuickPlex SQ 120. Additional reagents are summarized in Table 28.

[0328] Table 27. Test articles.

[0329] Table 28. Additional reagents. Chemical nitration of recombinant alpha-synuclein

[0330] The human, cynomolgus monkey, mouse and rat alpha-synuclein were chemically nitrated by incubating the protein at 100pg / ml concentration with 0.5 mM peroxynitrite solution. Chemically nitrated proteins were then aliquoted and stored at -80 °C for long term storage. Aliquots were used for further experiments. As a control for the peroxynitrite-mediated nitration reaction, proteins were treated with 0.03mM sodium hydroxide (NaOH).

[0331] Testing humAb26 as a Capture Antibody in MSD ELISA Assay

[0332] For the antibody capture MSD assays, MSD MULTI-ARRAY® 96, SECTOR Plates were coated with 1 pg / ml humAb26 overnight at 4eC, 30 pl / well, and plates were spun at 10Oxg briefly to spread solution on well. Plates were then stored at 4°C overnight without shaking. The next day, 10X casein was diluted to 1 X concentration in 1 X PBS to block the plates. Total volume per well was 150 pl. Plates were sealed and kept shaking at 750 rpm at room temperature for 1 -2 hours. Plates were then washed 3 times with TBS containing 0.05% Tween-20 (TBST) with BioTek EL406 Washer Dispenser and were tapped to remove residual TBST. Test antigens, i.e. nitrated or unmodified full-length human, cynomolgus monkey, mouse and rat alpha-synuclein proteins were serially diluted in 0.1 X Casein in 1 X PBS and added to the plates. Antigen concentrations ranged from 0.24 to 1000 ng / ml, and total volume added per well was 25 pl. Plates were sealed and kept shaking at 750 rpm at room temperature for 1 -2 hours. Plates were then washed 3 times with TBST as before and were tapped to remove residual TBST. BD610787 antibody, either sulfotag conjugated (per MSD protocol for SULFO-TAG NHS-ester conjugation) or biotin conjugated (followed by streptavidin-sulfotag reporter) was diluted to 1 pg / ml in 0.1 X Casein in 1 X PBS and was added to the plates. Total volume per well was 25 pl. Plates were sealed (protected from light) and kept shaking at 750 rpm at room temperature for 1 -2 hours. Plates were then washed 3 times with TBST as before and were tapped to remove residual TBST. MSD GOLD Read Buffer B was then added to the plates. Total volume per well was 150 pl. Plates were read with MSD Plate Reader, MESO QuickPlex SO 120, that utilizes electrochemiluminescence technology for detections.

[0333] Data analysis

[0334] MSD data was plotted in GraphPad Prism 10. Sigmoidal, 4PL, least squares fit was performed to generate a nonlinear fit to the data. ECso calculations were done in GraphPad Prism 10.

[0335] Results humAb26 as capture antibody binds to nitrated human, cyno, mouse and rat alpha-synuclein

[0336] As shown in FIG. 14, as a capture antibody, humAb26 showed selectivity to chemically nitrated human and cyno alpha-synuclein protein. There was no binding to the unmodified human or cyno alpha- synuclein.

[0337] As shown in FIG. 15, as a capture antibody, humAb26 showed selectivity to chemically nitrated human and mouse (ms) alpha-synuclein protein (FIG. 15). There was no binding to the unmodified human or mouse alpha-synuclein. As shown in FIGS. 16A-B, as a capture antibody, humAb26 showed selectivity to chemically nitrated mouse and rat alpha-synuclein protein (FIGS. 16A-B). There was no binding to the unmodified mouse and rat alpha-synuclein protein. In this experiment, some of the synuclein protein was buffer exchanged (BE) prior to the chemical nitration reaction. This did not appear to change the signal detectable by humAb26.

[0338] The calculated EC50 values for the nitrated proteins are within 2-3-fold of each other, indicating a similar degree of humAb26 binding affinity (Table 29). In all cases, the unmodified synuclein was not detected with humAb26.

[0339] Table 29. Summary of calculated EC50 values for humAb26 binding to human, cyno, mouse and rat alpha-synuclein

[0340] The sequence alignment shown here demonstrates that the sequence in the region of tyrosine 39 (red box), the nitration site that humAb26 antibody binds to, is identical from aa1 -52 (FIG. 17). At position 53, both the mouse and rat sequences feature a T instead of A. However, that does not appear to affect the humAb26 binding.

[0341] Conclusion humAb26 demonstrates specificity to chemically nitrated human, cyno, mouse, and rat alpha- synuclein as a capture antibody. There is no capture of unmodified human, cyno, mouse, or rat alpha- synuclein. The binding affinities for the capture of human, cyno, mouse, and rat alpha-synuclein are comparable, as would be expected from the identical sequences of synuclein in the region of tyrosine 39.

[0342] Example 24. Preclinical pharmacokinetic evaluation of humAb26 in CD-1 mouse plasma and brain following a single intravenous bolus dose

[0343] Summary

[0344] The purpose of this study is to determine the pharmacokinetic parameters of humAb26 in plasma and brain following an intravenous bolus to male CD-1 mice. 8-week old male CD-1 mice (N=3 / timepoint) were administered humAb26 by single intravenous bolus administration at 10 mg / kg. Plasma samples were collected at 0.5, 3, 6, 24, 72, and 168 hours post-dose, and brains were collected at 24, 72, and 168 hours post-dose. Concentrations of humAb26 in plasma and brain homogenate samples were determined by Meso Scale Discovery (MSD) immunoassay method.

[0345] For the IV administration of humAb26 at 10 mg / kg in male CD-1 mice, humAb26 had a mean brain concentration of 0.334, 0.216, and 0.189 pg / g at 24, 72, and 168 hours post-dose, respectively. The mean brain to plasma ratio was ~0.4%, which is typical for mAbs. All animals tolerated humAb26 well during the entire course of the study. No adverse effect was observed during the in-life phase of the study. Materials and Methods

[0346] Test articles and study design

[0347] Test articles are provided in Table 30.

[0348] Table 30. Test articles

[0349] Three groups of three male CD-1 mice were administered humAb26 by single intravenous bolus (retro-orbital) administration at 10 mg / kg. The vehicle was 10 mM HAC / NaAC pH 5.5. The detailed dosing and sampling regimens are described in Table 31 .

[0350] Table 31 . Study design, p = plasma; b = brain

[0351] Intravenous (IV) Dose

[0352] Animals were dosed at 5 mL / kg with humAb26 prepared in a vehicle consisting of 10 mM HAC / NaAC pH 5.5.

[0353] Animal studies

[0354] Nine 7-week old CD-1 mice were allowed a week to acclimate prior to study initiation. The room was on a 12 hour light / dark cycle.

[0355] Animals were weighed on the day of dosing to calculate the actual dose volume required for administration. All animals received a single IV bolus dose via a retro-orbital injection.

[0356] Blood Collection and Plasma Preparation

[0357] Interim blood collection was performed at 0.5, 3, or 6 hours post-dose via submandibular bleeds, with 50-100 pL of blood collected from each animal. For terminal collections, blood was collected via cardiac puncture under anesthesia (2.5% Avertin in 0.9% saline, i.p. injection) with 150-300 pL of blood collected from each animal. The actual time for each sample collection was recorded. Acceptable deviations on sampling time were + 10% of the nominal timepoint.

[0358] All blood samples were collected into tubes containing K2EDTA and placed on wet ice. Samples were centrifuged (5000 x g for 10 min at 4°C) within 1 hours of collection. The plasma layer (-25-150 pL) was pipetted into a fresh labeled tube and frozen at -80°C until analyzed.

[0359] Brain Dissection and Homogenate Preparation

[0360] At the end of study (24, 72, or 168 hours post-dose), mice were sacrificed and perfused with 50 mL of PBS prior to dissecting the brains which were then placed into individually labeled tubes and stored at -80°C.

[0361] For preparation of brain homogenate, a slice of brain sample was cut from the middle of the brain, weighed, and then transferred to a tube containing ceramic beads. Based on the brain weight, 5- fold (w / v) RIPA buffer (with 1X HALT protease inhibitor) was added to the tube for a final 6-fold dilution. Samples were homogenized on a Bead Rupter (OMNI International) at RT for 20 sec, centrifuged at 18,000 x g for 20 min at 4°C, and supernatants transferred to a fresh labeled tube. Samples were stored at -80°C until analyzed.

[0362] Reagents and Materials

[0363] Reagents and materials are provided in Table 32.

[0364] Table 32. Reagents and Materials

[0365] Instrumentation and Parameters

[0366] Instrumentation and parameters are provided in Table 33.

[0367] Table 33. Instrumentation and parameters.

[0368] Bioanalytical Analysis

[0369] Assay procedure

[0370] The free concentrations of humAb26 in plasma were determined using an MSD immunoassay method. MSD streptavidin coated plates (pre-blocked) were coated in each well with 30 pL of 0.5 pg / mL biotinylated nY39 alpha-synuclein 50-mer peptide and incubated on a shaker (750 rpm) at room temperature for 1 -2 h. Plates were washed with three cycles of 0.05% Tween in TBS prior to adding the samples.

[0371] An 8-point calibration curve was prepared using a reference standard of humAb26 that was titrated 4x for each calibrator level. The curve range was 0 ng / mL to 50 ng / mL, with a limit of quantitation (LOQ) of 24 pg / mL. The curve was analyzed in triplicate by transferring 25 pL / well for each calibrator level.

[0372] Study plasma samples were diluted 10,000-, 20,000-, and 40,000-fold and brain homogenates were diluted 5-.10-, 20- and 40-fold into assay buffer to minimize potential matrix effects while achieving detection within the curve range. Diluted samples were transferred at 25 pL / well and the plate was incubated on a shaker (750 rpm) at RT for 1 -2 h.

[0373] A 1 pg / mL anti-human sulfotag antibody in 0.1 X casein / PBS buffer was added to each well and incubated on a shaker (750 rpm) at RT for 1 -2 h. The plate was then washed with 3 cycles of 0.05% Tween in TBS.

[0374] For detection on the MSD instrument, 100 pL of Read buffer B is added to each well and detection initiated on the instrument within 30 minutes after addition.

[0375] Data Analysis and Results Criteria

[0376] The data output from the MSD were reviewed against the acceptance criteria for precision and accuracy using a sigmoidal, 4PL non-linear regression fit in GraphPad Prism. For the calibration curve, back-calculated concentrations of triplicate wells for each calibrator should be within ± 25% of the nominal value. If a calibrator level did not meet the acceptance criteria, that level was removed from the curve and a new regression is performed. The regression fit must have an r-squared value of >0.95. A minimum of 6 calibrators must achieve the acceptance criteria, with at least one point per level.

[0377] For unknown samples, concentrations must be detected within the assay curve range and are reported using the average of the multiple replicate analysis wells. If a dilution was performed prior to analysis, a dilution factor is applied to the calculated concentration for final reporting of unknown sample concentrations.

[0378] Bioanalytical Reagents, Materials and Instrumentation

[0379] Bioanalytical reagents, materials and instrumentation are provided in Table 34. Bioanalytical instrumentation and parameters are provided in Table 35. Table 34. Bioanalytical Reagents and Materials

[0380] Table 35. Bioanalytical Instrumentation and Parameters

[0381] Pharmacokinetic Data Analysis

[0382] The reported plasma concentrations of humAb26 were subjected to a non-compartmental pharmacokinetic analysis using the Phoenix WinNonlin software (version 8.4, Certara). The linear / log trapezoidal rule was applied in obtaining the PK parameters.

[0383] Individual plasma concentration values were averaged per timepoint and BLQ results were excluded from the PK parameter calculation. The nominal dose levels and nominal sampling times were used in the calculation of all pharmacokinetic parameters.

[0384] Results

[0385] Clinical Observations

[0386] During the entire course of the study, humAb26 was well tolerated in all animals. No adverse effects were observed during the in-life phase of the study.

[0387] Plasma and Brain Levels of humAb26

[0388] Results for plasma and brain levels of humAb26 are summarized in Tables 36 and 37 and in FIG. 18.

[0389] Table 36. Individual and Mean Plasma Concentrations (pg / mL) of humAb26 in Male CD-1 Mice Following a Single Intravenous Administration of 10 mg / kg Table 37. Individual and Mean Brain Concentrations (pg / g) of humAb26 in Male CD-1 Mice Following a Single Intravenous Administration of 10 mg / kg

[0390] Pharmacokinetic Parameters of humAb26 in Plasma

[0391] For the IV administration of humAb26 at 10 mg / kg in male CD-1 mice, a plasma clearance (CL) of 0.0014 mL / min / kg, half-life (T1 / 2) of 187 h, volume of distribution (Vd) of 0.1 16 L / kg, systemic exposure (AUCo-iast) value of 10526957 ng-h / mL were determined (Table 38). humAb26 had a mean brain concentration of 0.334, 0.216, and 0.189 pg / g at 24, 72, and 168 hours post-dose, respectively. The mean brain to plasma ratio is ~0.4%, which is typical for mAbs.

[0392] Table 38. Mean Pharmacokinetic Parameters of humAb26 in Male CD-1 Mice Following a Single Intravenous Administration of 10 mg / kg

[0393] Conclusion

[0394] This study determined the plasma pharmacokinetic parameters and brain exposure for humAb26 following a 10 mg / kg intravenous bolus dose to male CD-1 mice. All animals tolerated humAb26 well during the entire course of the study. No adverse effects were observed during the in-life phase of the study.

[0395] Example 25. Preclinical pharmacokinetic evaluation of humAb26 in cynomolgus monkey plasma following a single intravenous bolus dose

[0396] Summary

[0397] The purpose of this study is to determine the pharmacokinetics of humAb26 in plasma, following intravenous bolus to male cynomolgus monkeys. A group of three non-naive male cynomolgus monkeys were assigned into this study. Animals were administered humAb26 by single intravenous bolus administration at 2 mg / kg. Plasma samples were collected at pre-dose (0), 3, 6, 24, 48, 96, 168, and 336 hours post-dose. Concentrations of humAb26 in plasma samples were determined by enzyme-linked immunosorbent assay (ELISA) method.

[0398] For the IV administration of humAb26 at 2 mg / kg in male cynomolgus monkeys, humAb26 showed a plasma clearance (Cl) of 0.00328+0.00152 mUmin / kg, half-life (T1 / 2) at 243+138 h. The volume of distribution at steady state (Vdss) was 0.0555±0.0155 L / kg, the area under the plasma concentration-time curve from time zero to the last quantifiable concentration (AUCo-iast) value was 7026164±681937 ng-h / mL.

[0399] All animals tolerated humAb26 well at the dosing levels during the entire course of the study. No adverse effect was observed during the in-life phase of the study.

[0400] Materials and Methods

[0401] Test articles and study design

[0402] Test articles are provided in Table 39.

[0403] Table 39. Test articles

[0404] A group of three non-naive male cynomolgus monkeys were assigned into this study. Animals were administered humAb26 by single intravenous bolus administration at 2 mg / kg. The vehicle was 10 mM HAC / NaAC pH5.5. The detailed dosing and sampling regimens are described in Tables 40 and 41 .

[0405] Table 40. Study design. Note: no overnight fast.

[0406] Table 41. Study design. Anticoagulant: K2-EDTA. Intravenous (IV) Dose

[0407] Animals were dosed with humAb26 prepared in a vehicle consisting of 10 mM HAC / NaAC pH 5.5.

[0408] Animal Studies

[0409] Three non-naive male cynomolgus monkeys were used in this study. The room(s) were controlled and monitored for relative humidity (targeted range 40% to 70%) and temperature (targeted range 18°C to 26°C) with no less than 10 air changes / hour. The room (s) were on a 12-hour light / dark cycle except when interruptions were necessitated by study activities.

[0410] Dose Administration

[0411] Animals were weighed prior to dose administration on the day of dosing to calculate the actual dose volume. The body weights were in the range from 3.43 to 3.67 kg for males on the dosing day. All animals received a single intravenous bolus administration of humAb26 via the peripheral vein.

[0412] Clinical Observation

[0413] Cage-side observations for the general health condition and appearance of the animal were performed before and after dosing and at each time point of sample collection. Unusual observations were recorded throughout the duration of the study.

[0414] Blood Collection and Plasma Preparation

[0415] Approximately 0.8 mL blood was collected at each time point via peripheral vessel from each study animal. The actual time for each sample collection was recorded. The acceptable deviations on sampling time were +1 minute for the time points pre-dose through 1 -hour post-dose, and ±5% of the nominal time for time points after 1 hour post-dose.

[0416] All blood samples were transferred into commercial tubes containing K2-EDTA and placed on wet ice. Plasma samples were centrifuged (3200xg for 10 minutes at 2 to 8°C) within one hour of collection. The plasma samples about 0.3 mL were divided to approximate 0.15 mLx2 aliquots (one for BA, and the other one for back up) were transferred into labeled polypropylene micro-centrifuge tubes and stored frozen in a freezer set to maintain -60°C or lower until bioanalysis.

[0417] Bioanalytical Analysis

[0418] The concentrations of humAb26 in plasma were determined by using an ELISA method.

[0419] Pharmacokinetics Data Analysis

[0420] The plasma concentrations of humAb26 in study animals were subjected to a non-compartmental pharmacokinetic analysis by using the Phoenix WinNonlin software (version 8.3.5, Certara). The linear / log trapezoidal rule was applied in obtaining the PK parameters.

[0421] Individual plasma concentration values that were below the lower limit of quantitation (LLOQ) were excluded from the PK parameter calculation. The nominal dose levels and nominal sampling times were used in the calculation of all pharmacokinetic parameters. Results

[0422] Clinical Observations

[0423] All animals tolerated humAb26 well at the dosing levels during the entire course of the study. No adverse effect was observed during the in-life phase of the study.

[0424] Pharmacokinetics of humAb26 in Animals

[0425] Following a single IV bolus administration of humAb26 at 2 mg / kg in male animals, the individual and mean plasma concentrations of humAb26 are shown in Table 42, and are illustrated in FIG. 19.

[0426] Table 42. Individual and mean plasma concentrations (ng / ml) of humAb26 in male cynomolgus monkeys following a single intravenous administration at 2 mg / kg. BQL= below the quantifiable limit. ND= not determined as more than half (>50%) of the individual values are not quantifiable. C#

[0427] =animal number.

[0428] Following a single IV bolus administration of humAb26 at 2 mg / kg in male animals, the individual and mean plasma pharmacokinetic parameters of humAb26 are shown in Table 43. For the IV administration of humAb26 at 2 mg / kg in male cynomolgus monkeys, humAb26 showed a plasma clearance (Cl) of 0.00328+0.00152 mL / min / kg, half-life (T1 / 2) at 243±138 h. The volume of distribution at steady state (Vdss) was 0.0555+0.0155 L / kg, systemic exposure (AUCo-iast) value was 7026164±681937 ng-h / mL.

[0429] Table 43. Individual and Mean Plasma Pharmacokinetic Parameters of humAb26 in Male Cynomolgus Monkeys Following a Single Intravenous Administration at 2 mg / kg

[0430] Example 26. Preclinical efficacy evaluation of recombinant human alpha synuclein preformed fibril-inoculated A53T M83 transgenic mice following 7 weeks of treatment with msAbl Summary

[0431] The purpose of this study is to evaluate the efficacy of msAbl in reducing alpha-synuclein aggregation and spread in the brain of hPFF-inoculated A53T M83 mouse model of Parkinson’s Disease following 7 weeks of treatment. Eight to nine week-old M83 Tg mice of mixed sex (homozygous, JAX Strain #004479) were administered msAbl , 9E4 (mouse parental to prasinezumab), and lgG1 control antibodies. Weekly dosing of 100 mg / kg msAbl for 7 weeks was well tolerated by Tg M83 mice, with no test article-related effects on general health or body weights. Mean steady-state plasma concentrations were 2042 pg / mL and 518 pg / mL for msAbl and 9E4, respectively. Two days after the last dose, the mean brain concentrations were 5.65 pg / g and 1 .33 pg / g for msAbl and 9E4, respectively. This represents 0.26-0.28% penetrance of systemically administered antibody into the CNS. As compared to an lgG1 control antibody, msAbl was associated with dose-dependent reductions in phospho-alpha- synuclein aggregates in the substantia nigra (88% reduction, p<0.0001 ) and outperformed 9E4 at an equivalent dose (31 % reduction, not significant). For the thalamus region, msAbl demonstrated a significant reduction in phospho-alpha-synuclein aggregates (up to 80% reduction, p<0.0001 ) and outperformed 9E4 at an equivalent dose (38% reduction, p<0.05).

[0432] Materials and Methods

[0433] Test articles and study design

[0434] Test articles are provided in Table 44. Table 44. Test articles

[0435] The study design is summarized in Table 45.

[0436] Table 45. Study design, i.p. = intraperitoneal injection; QW = once weekly administration; DPI = days post hPFF inoculation

[0437] Formulation Animals were dosed at 18.47 mL / kg with msAbl , 19.64 ml / kg 9E4, or 20 ml / kg mouse IgG 1 control. Test articles were stored at -80 °C until needed. Each week, an aliquot of each test article was thawed for use within 1 -2 weeks and stored at 4 °C. Test articles were administered once per week via intraperitoneal (i.p.) dosing starting 2 hours before inoculations and continuing until the week of tissue collection (Study Week 7). Animal studies

[0438] Intraperitoneal (i.p. dose)

[0439] Eight- to nine-week old homozygous Tg M83 mice (mixed sex - JAX colony No. 004479) were bred and genotyped. Each animal received a unique identification number and was examined and weighed prior to study initiation to ensure adequate health and suitability. Throughout the course of the study, a 12 / 12 hr I ight / dark cycle was maintained. All enrolled mice were checked twice daily for survival and any deaths during study period were recorded. Homozygous Tg M83 mice were divided into Cohorts 1 and 2 based on the dates they were born. One group of 4 mice was used for alpha-synuclein monomer- treated animals to serve as an inoculation control group.

[0440] Preformed Fibril (hPFF) Inoculations

[0441] Recombinant human alpha-synuclein preformed fibrils (hPFF, catalog no. SPR-322) and recombinant human alpha-synuclein protein monomer (catalog no. SPR-321 ) were purchased from StressMarq. The hPFFs and monomers were stored at -80 °C until needed. Immediately before use, a frozen 2 mg / mL hPFF aliquot was thawed at room temperature. The hPFFs were pulse sonicated at room temperature with 20% power output, 1 second on and 1 second off for 10 cycles and repeated twice by utilizing a Fisher Scientific FB120 sonicator. Following sonication, the fibril aliquot was mixed by gently tapping the tube and visually inspecting to ensure that fibril suspension was no longer opaque, but instead clear (a small amount of visible light scattering small fragments was considered acceptable). Immediately prior to stereotaxic injection, the aliquot of sonicated hPFFs was gently tapped to ensure adequate mixing and an aliquot was removed for intracerebral administration. Sonicated hPFFs were maintained at room temperature and were utilized for injections on the same day as they were thawed and sonicated. hPFF suspensions were only used after a single thawing and did not undergo multiple freeze thaw cycles. Alpha-synuclein protein monomer was used as control and no sonication was performed.

[0442] Mice were anesthetized with isoflurane and stereotaxically injected into the right hemisphere with recombinant human alpha-synuclein fibrils or monomer control (4 pg per hemisphere; 2 pg / pL) (unilateral injection). Animals received 4 mg / kg of Meloxicam-SR subcutaneously immediately prior to surgery. Using a high-speed air-turbine drill with a burr tip size of <0.7 mm in diameter, one small hole was drilled into the right side of the skull (coordinates: +1 mm relative to Bregma, -1 .5 mm from midline). A single needle was inserted (coordinates: +1 mm relative to Bregma, -1 .5 mm from midline) into the right forebrain and was used to target the inoculum (2 pL of a 2 mg / mL solution) to the dorsal neostriatum (-2.5 mm beneath the dura). Injections were performed using a 33-gauge stainless steel cannula at an infusion rate of 300 nL / min. Following the injection, the needle was kept in place for 3 min post-injection at the target site and then slowly withdrawn. Animals were monitored regularly following recovery from surgery.

[0443] Dose Administration

[0444] Each animal enrolled in the study received an i.p. dose of test article once per week for a total of seven doses. Test article administration began the day of hPFF inoculation and continued until the week of terminal tissue collection. Biological Sample Collections

[0445] For each treatment group, interim blood sampling was performed on 5 animals at 1 hr pre-dose (Study Week 5). For all animals that survived until the end of study, terminal blood collections at 2 days post last dose were performed via cardiac puncture (Study Week 7).

[0446] Whole blood samples (100-400 pL) were processed for plasma by centrifugation for 10 min at 5,000 x g, plasma layer (-50-200 pL) transferred to prelabeled tubes, and stored at -80 °C until analyzed.

[0447] For terminal brain collections, mice were transcardially perfused with approximately 50 mL of 1X PBS (pH 7.4). Following perfusion, whole brains were dissected, olfactory bulbs removed and placed into dedicated tubes for storage at -80 °C, and remaining brain fixed by immersion into 4% paraformaldehyde in 1 X PBS for 48 hr at 4 °C. After fixation step, brains were transferred into 1X PBS with 0.02% sodium azide and stored at 4 °C until immunohistochemistry staining and image analysis.

[0448] Brain Tissue Histology and Analysis

[0449] Tissue Embedding

[0450] A total of 45 mouse brains were treated with 20% glycerol and 2% dimethylsulfoxide to prevent freeze artifacts. Twenty-five brains at a time were arranged on a block and embedded in gelatin matrix and allowed to cure in a formaldehyde solution. The blocks were then rapidly frozen by immersion in 2- methylbutane chilled with crushed dry ice.

[0451] Tissue Sectioning

[0452] Tissue blocks were sectioned with a microtome in the coronal orientation at a thickness of 30 pm. The entire block (through whole brain volume) was sectioned and collected onto slides and placed into a solution of Antigen Preserve solution until staining.

[0453] Tissue Staining

[0454] For immunohistochemistry (IHC), 2 sets of every sixth section (180 pm interval) were stained using a primary antibody for phosphoserine at amino acid position 129 (pSer129) of alpha-synuclein. Following rinses, a biotinylated secondary antibody was applied to the sections, treated with a chromogen, dehydrated in alcohol / xylene rinses, and then air dried. The mounted slides were counterstained with Neutral Red counterstain and coverslipped.

[0455] Slide Scanning

[0456] IHC stained sections were scanned using an Olympus VS200 Research Slide Scanner at 20x magnification (0.273 pm / pixel resolution). Images were uploaded to a repository on Concentriq (Proscia).

[0457] Signal Quantification

[0458] For pSerl 29 object quantification in substantia nigra reticulata, pSerl 29 images were uploaded to a remote image analysis platform (Aiforia) for neural network algorithm development. The algorithm was trained to recognize total tissue area, substantia nigra reticulata regions (SNr), and pSerl 29-positive Lewy bodies and neurites within SNr. The finished algorithm was then applied bilaterally to 3 selected levels of SNr for each animal (anatomically matched across all animals). Data were exported from Aiforia and organized into a Microsoft Excel spreadsheet.

[0459] For pSerl 29 object quantification in thalamus, pSerl 29 images were uploaded to Aiforia for neural network algorithm development. The algorithm was trained to recognize total tissue area and pSerl 29-positive Lewy bodies and neurites within the thalamus region. The finished algorithm was then applied bilaterally to 3 selected levels of thalamus for each animal (levels manually traced and anatomically matched across all animals). Data were exported from Aiforia and organized into a Microsoft Excel spreadsheet.

[0460] Bioanalysis

[0461] Brain Homogenate Preparation

[0462] At necropsy, the olfactory bulbs were subdissected from the brain and placed into 1 .5 mL Eppendorf tubes with flip-caps, snap-frozen on dry ice, and stored at -80 °C. Weight of each olfactory bulb was collected and recorded. For preparation of brain homogenate, the olfactory bulb was transferred to a tube containing ceramic beads. Based on the brain weight, 10-fold (w / v) homogenization buffer (10 mM Tris-HCI pH 7.5, 150 mM NaCI, 1 % Triton X-100, 2 mM EDTA with 1 X HALT protease inhibitor) was added to the tube for a final 11 -fold dilution. Samples were homogenized on a Bead Rupter (OMNI International) at RT for 20 sec, centrifuged at 18,000 x g for 20 min, and supernatants transferred to a fresh, labeled tube. Samples were stored at -80 °C until analyzed.

[0463] Assay Procedure

[0464] The free concentrations of msAbl and 9E4 in plasma were determined using an MSD immunoassay method. MSD streptavidin coated plates (preblocked) were coated in each well with 30 pL of 0.5 pg / mL biotinylated nY39 alpha-synuclein 50-mer peptide (for msAbl ) or 0.5 pg / mL biotinylated Y136 alpha-synuclein 50-mer peptide (for 9E4) and incubated on a shaker (750 rpm) at RT for 1 -2 hr. Plates were washed with three cycles of 0.05% Tween in TBS prior to adding the samples.

[0465] An 8-point calibration curve was prepared using a reference standard of either msAbl or 9E4 that was titrated 4X for each calibrator level. The curve range was 0 ng / mL to 100 ng / mL, with a limit of quantitation (LOQ) of 24 pg / mL. The curve was analyzed in duplicate by transferring 25 pL / well for each calibrator level.

[0466] Unknown plasma samples were diluted 40,000- or 80,000-fold and brain homogenates were diluted 10- or 20-fold into assay buffer to minimize potential matrix effects while achieving detection within the curve range. Diluted samples were transferred at 25 pL / well and the plate was incubated on a shaker (750 rpm) at RT for 1 -2 hr.

[0467] 1 pg / mL anti-mouse sulfotag antibody in 0.1 X casein / PBS buffer was added to each well and incubated on a shaker (750 rpm) at RT for 1 -2 hr. The plate was then washed with 3 cycles of 0.05% Tween in TBS.

[0468] For detection on the MSD instrument, 100 pL of Read buffer B is added to each well and detection initiated on the instrument within 30 minutes after addition. Data Analysis and Results Criteria

[0469] The data output from the MSD were reviewed against the acceptance criteria for precision and accuracy using a sigmoidal, 4PL non-linear regression fit in GraphPad Prism. For the calibration curve, back-calculated concentrations of duplicate wells for each calibrator were required to be within + 25% of the nominal value. If a calibrator level did not meet the acceptance criteria, that level was removed from the curve and a new regression was performed. The regression fit was required to have an r-squared value of >0.95. A minimum of 6 calibrators must achieve the acceptance criteria, with at least one point per level. For unknown samples, concentrations must be detected within the assay curve range and were reported using the average of the multiple replicate analysis wells. If a dilution was performed prior to analysis, a dilution factor was applied to the calculated concentration for final reporting of unknown sample concentrations. Bioanalytical reagents and materials

[0470] Bioanalytical reagents and materials are provided in Table 46.

[0471] Table 46. List of Bioanalytical Reagents and Materials

[0472] Bioanalytical Instrumentation and Parameters

[0473] Bioanalytical instrumentation and parameters are provided in Table 47.

[0474] Table 47. Bioanalytical Instrumentation and Parameters

[0475] Results

[0476] Clinical Observations

[0477] In the lgG1 control group, one Tg M83 mouse was found dead 48 hours after alpha-synuclein inoculation surgery, which was considered to be due to surgery-induced damage and complications. The 55 remaining Tg M83 mice survived to the terminal time point and maintained a relatively healthy status, with no animals experiencing severe health issues over the course of the study. After dosing onset on day of inoculation, each animal received the test articles once per week until the end of the study (total of 7 doses). Body weights were recorded each week (prior to dosing) and there were no differences in body weights among the 3 different treatment groups at termination (2 way ANOVA with Tukey’s multiple comparisons test, p > 0.56, N=17 for each treatment group, N=4 for monomer group) (FIGS. 20A-B). msAbl had no effect on either body weight or survival.

[0478] Immunohistochemistry ofpSer129

[0479] Quantitative counting of alpha-synuclein aggregates in Lewy Bodies and Lewy Neurites was performed on the pSer129 IHC brain sections using an unsupervised algorithm. The algorithm was applied to the substantia nigra (FIGS. 21 A-B) and thalamus (FIGS. 22A-B) regions of the brain to assess pathology and spread beyond the striatum inoculation site. Both the ipsilateral (same hemisphere as inoculation site) and the contralateral (opposite hemisphere from inoculation site) sides of the brain were assessed.

[0480] Aggregate counts reported by the algorithm for each region per hemisphere were normalized to the area sampled, averaged within a treatment group, and plotted using GraphPad Prism. Statistical analysis using an ordinary one-way ANOVA or Welch’s T-test was performed to determine significance of each test article treatment (Prism). * p < 0.05, ** p < 0.01 , *“ p < 0.001 , **** p < 0.0001 .

[0481] Plasma and Brain Levels of msAb 1 and 9E4

[0482] Tg mice treated with 100 mg / kg msAbl had on average 2042 pg / mL free plasma concentrations (Table 48). For 100 mg / kg 9E4, mice had 518 pg / mL mean free plasma concentrations. The average free msAbl exposure in brain was 5.65 pg / g while the average free 9E4 exposure was 1 .33 pg / g in brain. This translates to a 0.26-0.28% ratio of brain :plasma levels for both antibodies. These ratios are consistent with expectations for antibodies accessing the brain via peripheral administration.

[0483] Table 48. Results for Free Plasma and Brain Concentrations of msAbl and 9E4

[0484] Conclusion

[0485] In this model, human synuclein became overexpressed and was prone to aggregation and spread throughout the mouse brain over time. The PFF-inoculation into the right striatum was performed to serve as a seed to rapidly induce the synuclein pathology and spread, forming aggregates in multiple brain regions over a shorter duration than would naturally occur in this Tg model. The high expression synuclein pathology and spread closely mimics the Parkinson’s Disease progression phenotype observed in humans. Thus, this model is ideal for evaluating the potential efficacy against specifically targeting the nitrated subspecies of alpha synuclein that is hypothesized to be present in the disease state and is the driver of pathology. Relevant brain regions were selected based on known pathology in human Parkinson’s Disease progression (motor and non-motor deficits), including the substantia nigra and thalamus regions.

[0486] Mice were inoculated with hPFF in the right striatum (4 pg per hemisphere; 2 pg / pL) during Study Week 1 . Test articles were dosed starting 2 hours before inoculation and were administered to each animal via i.p. injection once per week for 7 weeks (Study Weeks 1 through 7). Interim blood samples were collected via submandibular bleed at 1 timepoint throughout the study (Study Week 5) and terminal tissue collections took place 2 days after the final dose during Study Week 7.

[0487] In the isotype control group, one mouse of the 18 Tg M83 animals was found dead 48 hours after hPFF inoculation which was deemed to be due to surgery-induced damage and complications. The remaining 17 Tg M83 mice survived to the terminal time point (study week 7) and no deaths were observed in the msAbl (n=17) and 9E4 (n=17) antibody treatment groups. Weekly dosing of 100 mg / kg msAbl for 7 weeks was well tolerated by Tg M83 mice, with no test article-related effects on general health or body weights. Mean steady-state plasma msAbl concentrations were 2042 pg / mL and, for 9E4, the mean steady-state plasma concentrations were 518 pg / mL. Two days after the last dose, the mean brain msAbl concentrations were 5.65 pg / g while the mean brain 9E4 concentrations were 1 .33 pg / g (representing -0.26-0.28% penetrance into CNS). In the substantia nigra region, when compared to an irrelevant lgG1 control mAb, msAbl was associated with significant reductions in pSer129 aggregates (up to 88% reduction, p<0.0001 ) and outperformed 9E4 (31% reduction, not significant) at an equivalent dose. For the thalamus region, msAbl demonstrated a significant reduction in pSerl 29 aggregates (up to 80% reduction, p<0.0001 ) and outperformed 9E4 at an equivalent dose (38% reduction, p<0.05).

[0488] Example 27. Preclinical efficacy evaluation of preformed fibril-inoculated A53T M83 transgenic mice following 12 weeks of treatment

[0489] Summary

[0490] The purpose of this Example is to evaluate the efficacy of msAbl in reducing alpha-synuclein aggregation and spread in the brain of PFF-inoculated A53T M83 mouse model of Parkinson’s Disease following 12 weeks of treatment. Eight to ten week-old M83 Tg mice of mixed sex (homozygous, JAX Strain #004479) were administered msAbl , 9E4 (mouse parental to prasinezumab), and lgG1 control antibodies. Weekly dosing of 30 or 100 mg / kg msAbl for 12 weeks was well tolerated by Tg M83 mice, with no test article-related effects on general health or body weights. Mean steady-state plasma msAbl concentrations were -775 and 1750 ug / mL, for 30 mg / kg and 100 mg / kg respectively. Two days after the last dose, the mean brain msAbl concentrations were -1 .35 and 2.85 ug / g for 30 mg / kg and 100 mg / kg groups, respectively (representing -0.1 % penetrance in CNS). As compared to an lgG1 control mAb, msAbl was associated with dose-dependent reductions in phospho-alpha-synuclein aggregates in the substantia nigra (33% reduction, p=0.0442) and outperformed 9E4 at an equivalent dose (14% reduction, not significant).

[0491] Materials and Methods

[0492] Test articles and study design

[0493] Test articles are summarized in Table 49. Study design is summarized in Table 50.

[0494] Table 49. Test articles

[0495] Table 50. Study design, i.p. = intraperitoneal injection; QW = once weekly administration; DPI = days post PFF inoculation

[0496] Intraperitoneal (i.p.) Dose

[0497] Animals were dosed at 10 or 20 mUkg with msAbl , 9E4, or IgG 1 Control prepared in 10 mM histidine or PBS.

[0498] Animal studies

[0499] A total of 109 eight- to ten-week old M83 Tg mice of mixed sex (homozygous, JAX Strain #004479) were used. Mice were assigned unique identifier numbers and examined for health status clearance. Throughout the course of the study, a 12 / 12 hr I ig ht / dark cycle was maintained.

[0500] Preformed Fibril (PFF) Inoculations

[0501] Human alpha-synuclein PFF were purchased from StressMarq (Cat No. SPR-322) at a 2 mg / mL concentration. Mice were anesthetized with isoflurane and a unilateral injection of 4 ug PFF into the right striatum was performed using stereotactic guidance. Using the same inoculation procedure, a small cohort of wild-type C57BI6 mice (N=4) were also injected with 4 ug PFF.

[0502] A subset of M83 Tg mice (N=7) were inoculated with human alpha-synuclein monomer. Both the wild-type PFF mice (Group 6) and the monomer M83 Tg mice (Group 7) were used as model controls and were carried out to end of study (12 weeks) without any test article treatments. Dose Administration

[0503] Each animal enrolled in the study received an i.p. dose of test article once per week for a total of eleven doses. Test article administration began 7 days after PFF inoculation and continued until the week of terminal tissue collection.

[0504] Biological Sample Collections

[0505] For each treatment group, interim blood sampling was performed on 4 animals at 24 hr post dose No. 1 , 1 hr pre dose No. 2, and 24 hr post dose No. 6. For all animals that survived until the end of study, terminal blood collections at 2 days post dose No. 11 were performed via cardiac puncture.

[0506] Whole blood samples (100-400 pL) were processed for plasma by centrifugation for 10 min at 5,000 x g, plasma layer (-50-200 pL) transferred to prelabeled tubes, and stored at -80°C until shipped for bioanalysis.

[0507] For terminal brain collections, mice were transcardially perfused with approximately 50 ml_ of 1X PBS (pH 7.4). Following perfusion, whole brains were dissected, olfactory bulbs removed and placed into dedicated tubes for storage at -80°C, and remaining brain fixed by immersion into 4% paraformaldehyde in 1 X PBS for 48 hours at 4°C. After fixation step, brains were transferred into 1 X PBS with 0.02% sodium azide and stored at 4°C until immunohistochemistry staining and image analysis.

[0508] Brain Tissue Histology and Analysis

[0509] Tissue Embedding

[0510] A total of 75 mouse brains were treated with 20% glycerol and 2% dimethylsulfoxide to prevent freeze artifacts. The 25 brains at a time were arranged on a block and embedded in gelatin matrix and allowed to cure in a formaldehyde solution. The blocks were then rapidly frozen by immersion in 2- methylbutane chilled with crushed dry ice.

[0511] Tissue Sectioning

[0512] Tissue blocks were sectioned with a microtome in the coronal orientation at a thickness of 30 pm. The entire block (through whole brain volume) was sectioned and collected onto slides and placed into a solution of Antigen Preserve solution until staining.

[0513] Tissue Staining

[0514] For immunohistochemistry (IHC), 2 sets of every sixth section (180 pm interval) were stained using a primary antibody for phosphoserine at amino acid position 129 (pSer129) of alpha-synuclein. Following rinses, a biotinylated secondary antibody was applied to the sections, treated with a chromogen, dehydrated in alcohol / xylene rinses, and coverslipped.

[0515] Slide Scanning

[0516] IHC stained sections were scanned using an Olympus VS200 Research Slide Scanner at 20x magnification (0.273 pm / pixel resolution). Images were uploaded to a repository on Concentriq (Proscia). Signal Quantification

[0517] For pSerl 29 object quantification in substantia nigra reticulata, pSerl 29 images were uploaded to a remote image analysis platform (Aiforia) for neural network algorithm development. The algorithm was trained to recognize total tissue area, substantia nigra reticulata regions (SNr), and pSerl 29-positive synuclein aggregates within SNr. The finished algorithm was then applied bilaterally to 3 selected levels of SNr for each animal (anatomically matched across all animals). Data were exported from Aiforia and organized into a Microsoft Excel spreadsheet.

[0518] For pSerl 29 object quantification in thalamus, pSerl 29 images were uploaded to Aiforia for neural network algorithm development. The algorithm was trained to recognize total tissue area and pSerl 29-positive synuclein aggregates within thalamus. The finished algorithm was then applied bilaterally to 3 selected levels of thalamus for each animal (levels manually traced and anatomically matched across all animals). Data were exported from Aiforia and organized into a Microsoft Excel spreadsheet.

[0519] Bioanalysis

[0520] Brain Homogenate Preparation

[0521] For preparation of brain homogenate, a slice of brain sample was cut from the middle of the brain, weighed, and then transferred to a tube containing ceramic beads. Based on the brain weight, 5- fold (w / v) RIPA buffer (with 1X HALT protease inhibitor) was added to the tube for a final 6-fold dilution. Samples were homogenized on a Bead Rupter (OMNI International) at RT for 20 seconds, centrifuged at 18,000 x g for 20 min, and supernatants transferred to a fresh labeled tube. Samples were stored at - 80°C until analyzed.

[0522] Assay Procedure

[0523] The free concentrations of msAbl and 9E4 in plasma were determined using an MSD immunoassay method. MSD streptavidin coated plates (preblocked) were coated in each well with 30 pL of 0.5 pg / mL biotinylated nY39 alpha-synuclein 50-mer peptide (for msAbl ) or 0.5 pg / mL biotinylated Y136 alpha-synuclein 50-mer peptide (for 9E4) and incubated on a shaker (750 rpm) at RT for 1 -2 hr. Plates were washed with three cycles of 0.05% Tween in TBS prior to adding the samples.

[0524] An 8-point calibration curve was prepared using a reference standard of either msAbl or 9E4 that was titrated 4x for each calibrator level. The curve range was 0 ng / mL to 100 ng / mL, with a limit of quantitation (LLOQ) of 24 pg / mL. The curve was analyzed in duplicate by transferring 25 pL / well for each calibrator level.

[0525] Unknown plasma samples were diluted 100-, 10,000-, 80,000-, or 160,000-fold and brain homogenates were diluted 10-, 40-, or 160-fold into assay buffer to minimize potential matrix effects while achieving detection within the curve range. Diluted samples were transferred at 25 pL / well and the plate was incubated on a shaker (750 rpm) at RT for 1 -2 hr.

[0526] A 1 pg / mL anti-mouse sulfotag antibody in 0.1 X casein / PBS buffer was added to each well and incubated on a shaker (750 rpm) at RT for 1 -2 hr. The plate was then washed with 3 cycles of 0.05% Tween in TBS. For detection on the MSD instrument, 100 pL of Read buffer B was added to each well and detection initiated on the instrument within 30 minutes after addition.

[0527] Data Analysis and Results Criteria

[0528] The data output from the MSD were reviewed against the acceptance criteria for precision and accuracy using a sigmoidal, 4PL non-linear regression fit in GraphPad Prism. For the calibration curve, back-calculated concentrations of triplicate wells for each calibrator should be within ± 25% of the nominal value. If a calibrator level did not meet the acceptance criteria, that level was removed from the curve and a new regression is performed. The regression fit must have an r-squared value of >0.95. A minimum of 6 calibrators must achieve the acceptance criteria, with at least one point per level.

[0529] For unknown samples, concentrations must be detected within the assay curve range and reported using the average of the multiple replicate analysis wells. If a dilution was performed prior to analysis, a dilution factor was applied to the calculated concentration for final reporting of unknown sample concentrations.

[0530] Bioanalytical Reagents, Materials, and Instrumentation

[0531] Bioanalytical reagents and materials are provided in Table 51 . Bioanalytical instrumentation and parameters are provided in Table 52.

[0532] Table 51. Bioanalytical Reagents and Materials Table 52. Bioanalytical Instrumentation and Parameters

[0533] Results

[0534] Clinical Observations

[0535] A total of 109 homozygous A53T M83 Tg mice were provided for the study. One animal was found dead on arrival and one animal was euthanized on the day of arrival due to an injury. One female animal was found housed with males upon arrival and was not enrolled. The remaining animals were evenly enrolled into study groups (N=18-21 mice) based on body weight prior to surgery.

[0536] Some animals from each treatment group experienced health issues and died or were euthanized over the course of the study. Of the 106 mice that were inoculated with PFF, 20 animals required early euthanasia prior to dosing initiation due to health issues following surgery.

[0537] After dosing onset, each animal received the test articles once per week until the end of the study. Twenty-three animals required early euthanasia after receiving at least one dose of a test article but prior to the scheduled tissue collection on Study Week 12. Of these animals, 6 were from Group 1 (TA10), 7 were from Group 2 (TA20), 2 were from Group 3 (TA30), 2 were from Group 4 (TA40), and 6 were from Group 5 (TA50). The remaining 63 animals survived until the terminal tissue collection during Week 12. msAbl had no effect on either body weight or survival (FIGS. 23A-B). Body weights were taken once per week starting at baseline (W1 ) and continued until tissue collection (W12). Animals underwent surgery at W1 . Any deaths were considered to be model related or procedural deaths related to the surgery and were not considered to be treatment-related.

[0538] Immunohistochemistry ofpSer129

[0539] Quantitative counting of alpha-synuclein aggregates was performed on the pSer129 IHC brain sections using an unsupervised algorithm. The algorithm was applied to the substantia nigra (FIGS. 24A- B) and thalamus (FIGS. 25A-B) regions of the brain to assess pathology and spread beyond the striatum inoculation site. Both the ipsilateral (same hemisphere as inoculation site) and the contralateral (opposite hemisphere from inoculation site) sides of the brain were assessed.

[0540] Aggregate counts reported by the algorithm for each region per hemisphere were normalized to the area sampled, averaged within a treatment group, and plotted using GraphPad Prism.

[0541] Statistical analysis using an ordinary one-way ANOVA or Welch’s T-test was performed to determine significance of each test article treatment (Prism). * p < 0.05, ** p < 0.01 .

[0542] Plasma and Brain Levels of msAb 1 and 9E4

[0543] Tg mice treated with 30 mg / kg msAbl had on average 776 pg / mL plasma concentrations while the 100 mg / kg msAbl treated mice had on average 1749 pg / mL plasma concentrations (Tables 53 and 56). For 9E4, mice had 193 pg / mL and 481 pg / mL mean plasma concentrations for the 30 mg / kg and 100 mg / kg groups, respectively (Tables 53 and 57).

[0544] On average, msAbl was present in the brain at 1 .35 pg / g in the 30 mg / kg group and 2.86 pg / g in the 100 mg / kg group (Tables 54 and 56). This translates to a 0.1% ratio of brainplasma for free msAbl levels (Tables 55 and 56).

[0545] On average, 9E4 was present in brain at 0.79 pg / g in the 30 mg / kg group and 1 .69 pg / g in the 100 mg / kg group (Tables 54 and 56). This translates to a 0.4% ratio of brain plasma for free 9E4 levels (Tables 55 and 56). These ratios are consistent with expectations for antibodies accessing the brain via peripheral administration.

[0546] Table 53. Individual Free Plasma Concentrations of msAbl and 9E4

[0547] Table 54. Individual Free Brain Concentrations of msAbl and 9E4 Table 55. Individual Brain to Plasma Ratios of msAbl and 9E4

[0548] Table 56. Mean Free Plasma and Brain Concentrations of msAbl and 9E4 Conclusion

[0549] In this model, human synuclein became overexpressed and was prone to aggregation and spread throughout the mouse brain over time. The PFF-inoculation into the right striatum was performed to serve as a seed to rapidly induce the synuclein pathology and spread, forming aggregates in multiple brain regions over a shorter duration than would naturally occur in this Tg model. The high expression synuclein pathology and spread closely mimics the Parkinson’s Disease progression phenotype observed in humans. Thus, this model is ideal for evaluating the potential efficacy against specifically targeting the nitrated subspecies of alpha synuclein that is hypothesized to be present in the disease state and is the driver of pathology. Relevant brain regions were selected based on known pathology in human Parkinson’s Disease progression (motor and non-motor deficits), including the substantia nigra and thalamus regions.

[0550] Weekly dosing of 30 or 100 mg / kg msAbl for 12 weeks was well tolerated by Tg M83 mice, with no test article-related effects on general health or body weights. Mean steady-state plasma msAbl concentrations over time were ~775 and 1750 pg / mL, for 30 mg / kg and 100 mg / kg treatment groups, respectively. Two days after the last dose, the mean brain msAbl concentrations were ~1 .35 and 2.85 pg / g for 30 mg / kg and 100 mg / kg groups, respectively (representing -0.1% penetrance in CNS). In the substantia nigra region, when compared to an irrelevant lgG1 control mAb, msAbl was associated with the dose-dependent reductions in phospho-alpha-synuclein aggregates (33% reduction, p=0.0442) and outperformed 9E4 (14% reduction, not significant) at an equivalent dose. For the thalamus region, msAbl demonstrated a dose-dependent reduction in phospho-alpha-synuclein aggregates and outperformed 9E4 at an equivalent dose.

[0551] OTHER EMBODIMENTS

[0552] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0553] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0554] Other embodiments are within the following claims.

Claims

CLAIMS1 . An antibody or an antigen-binding fragment thereof that binds to alpha-synuclein nitrated at tyrosine residue 39 (nY39), the antibody or antigen-binding fragment thereof comprising: a complementarity-determining region light chain 1 (CDR-L1 ) comprising the amino acid sequence of QASQNVYKNNYLG (SEQ ID NO: 1 ), RASQNVYKNNY (SEQ ID NO: 2), RASKNVYKNYYLG (SEQ ID NO: 1 13), RASKAVYKNYYLG (SEQ ID NO: 114), RASQNVYKNYYLG (SEQ ID NO: 1 15), or RASKAVYNNYYLG (SEQ ID NO: 116); a complementarity-determining region light chain 2 (CDR-L2) comprising the amino acid sequence of YASTLAS (SEQ ID NO: 3); a complementarity-determining region light chain 3 (CDR-L3) comprising the amino acid sequence of LGIYDCSSVDCNA (SEQ ID NO: 4) or LGIYDCSSGDCNA (SEQ ID NO: 117); a complementarity-determining region heavy chain 1 (CDR-H1 ) comprising the amino acid sequence of GFSLSSYYMS (SEQ ID NO: 5), GFKLSSYYMS (SEQ ID NO: 1 18), GFSVSSSYMS (SEQ ID NO: 1 19), or GFKLSSTYMS (SEQ ID NO: 120); a complementarity-determining region heavy chain 2 (CDR-H2) comprising the amino acid sequence of YISAGGYTY (SEQ ID NO: 6); and a complementarity-determining region heavy chain 3 (CDR-H3) comprising the amino acid sequence of LDPSSGDI (SEQ ID NO: 7).

2. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L1 comprising the amino acid sequence of QASQNVYKNNYLG (SEQ ID NO: 1 ).

3. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L1 comprising the amino acid sequence of RASQNVYKNNY (SEQ ID NO: 2).

4. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L1 comprising the amino acid sequence of RASKNVYKNYYLG (SEQ ID NO: 113).

5. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L1 comprising the amino acid sequence of RASKAVYKNYYLG (SEQ ID NO: 114).

6. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L1 comprising the amino acid sequence of RASQNVYKNYYLG (SEQ ID NO: 1 15).

7. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L1 comprising the amino acid sequence of RASKAVYNNYYLG (SEQ ID NO: 116).

8. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L3 comprising the amino acid sequence of LGIYDCSSVDCNA (SEQ ID NO: 4).

9. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-L3 comprising the amino acid sequence of LGIYDCSSGDCNA (SEQ ID NO: 117).

10. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-H1 comprising the amino acid sequence of GFSLSSYYMS (SEQ ID NO: 5).11 . The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-H1 comprising the amino acid sequence of GFKLSSYYMS (SEQ ID NO: 118).

12. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-H1 comprising the amino acid sequence of GFSVSSSYMS (SEQ ID NO: 119).

13. The antibody or an antigen-binding fragment thereof of claim 1 , comprising a CDR-H1 comprising the amino acid sequence of GFKLSSTYMS (SEQ ID NO: 120).

14. The antibody or antigen binding fragment thereof of any one of claims 1 -13, comprising the following light chain variable region framework regions (FRs): an FR-L1 comprising the amino acid sequence of DIQMTQSPSAMSASVGDRVTITC (SEQ ID NO: 8), DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26), or QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29); an FR-L2 comprising the amino acid sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9), LGWFQQKPGKAPKRLIY (SEQ ID NO: 27), or LGWFQQKPGKAPKLLIY (SEQ ID NO: 30); an FR-L3 comprising the amino acid sequence of GVSSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 10), GVSSRFKGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 16), or GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ) or FGGGTKVEIK (SEQ ID NO: 28).

15. The antibody or antigen binding fragment thereof of any one of claims 1 -14, comprising the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQMTQSPSAMSASVGDRVTITC (SEQ ID NO: 8); an FR-L2 comprising the amino acid sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9); an FR-L3 comprising the amino acid sequence of GVSSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 10); and an FR-L4 comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ).

16. The antibody or antigen binding fragment thereof of any one of claims 1 -14, comprising the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQMTQSPSAMSASVGDRVTITC (SEQ IDNO: 8);an FR-L2 comprising the amino acid sequence of WFQQKPGKVPKRLIY (SEQ ID NO: 9); an FR-L3 comprising the amino acid sequence of GVSSRFKGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 16); and an FR-L4 comprising the amino acid sequence of FGGGTKLEIK (SEQ ID NO: 1 1 ).

17. The antibody or antigen binding fragment thereof of any one of claims 1 -14, comprising the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKLLIY (SEQ ID NO: 30); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

18. The antibody or antigen binding fragment thereof of any one of claims 1 -14, comprising the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 26); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKRLIY (SEQ ID NO: 27); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

19. The antibody or antigen binding fragment thereof of any one of claims 1 -14, comprising the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKLLIY (SEQ ID NO: 30); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).

20. The antibody or antigen binding fragment thereof of any one of claims 1 -14, comprising the following light chain variable region FRs: an FR-L1 comprising the amino acid sequence of QQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 29); an FR-L2 comprising the amino acid sequence of LGWFQQKPGKAPKRLIY (SEQ ID NO: 27); an FR-L3 comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23); and an FR-L4 comprising the amino acid sequence of FGGGTKVEIK (SEQ ID NO: 28).21 . The antibody or antigen binding fragment thereof of any one of claims 1 -20, comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12), EVQLVESGGGLVQPGGPLRLSCAAS (SEQ ID NO: 21 ), or EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13) or WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence ofYANWAKGRFTISRDNSKTTLYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 14),YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYYCAR (SEQ ID NO: 18), YANWAKGRFTISRDNSKNTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 19), YANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 20), YANWAKGRFTISRDNSKTTVDLQMNSPRAEDTAVYFCAR (SEQ ID NO: 22), YADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 25), YADSVKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 31 ), YANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 32), or YANWAKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 33); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

22. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13); an FR-H3 comprising the amino acid sequence ofYANWAKGRFTISRDNSKTTLYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 14); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

23. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence ofYANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYYCAR (SEQ ID NO: 18); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

24. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17);an FR-H3 comprising the amino acid sequence ofYANWAKGRFTISRDNSKNTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 19); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

25. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 12); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence ofYANWAKGRFTISRDNSKTTVDLQMNSLRAEDTAVYFCAR (SEQ ID NO: 20); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

26. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGPLRLSCAAS (SEQ ID NO: 21 ); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence ofYANWAKGRFTISRDNSKTTVDLQMNSPRAEDTAVYFCAR (SEQ ID NO: 22); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

27. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13); an FR-H3 comprising the amino acid sequence ofYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 25); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

28. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence ofYADSVKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 31 ); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

29. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEWIG (SEQ ID NO: 13); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 32); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).

30. The antibody or antigen binding fragment thereof of any one of claims 1 -21 , comprising the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 24); an FR-H2 comprising the amino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 17); an FR-H3 comprising the amino acid sequence of YANWAKGRFTISRDNSKNTVYLQINSLRAEDTAVYYCAR (SEQ ID NO: 33); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 15).31 . The antibody or antigen binding fragment thereof of claim 1 , comprising a light chain variable domain comprising an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 34-39 or 121 -124.

32. The antibody or antigen-binding fragment thereof of claim 31 , comprising a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 34-39 or 121 -124.

33. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 34.

34. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 35.

35. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 36.

36. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 37.

37. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 38.

38. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 39.

39. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 121.

40. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 122.41 . The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 123.

42. The antibody or antigen-binding fragment thereof of claim 32, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 124.

43. The antibody or antigen binding fragment thereof of any one of claims 1 or 31 -42, comprising a heavy chain variable domain comprising an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 40-48 or 125-127.

44. The antibody or antigen-binding fragment thereof of claim 43, comprising a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 40-48 or 125-127.

45. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 40.

46. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 41 .

47. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 42.

48. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 43.

49. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 44.

50. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 45.51 . The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 46.

52. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 47.

53. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 48.

54. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 125.

55. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 126.

56. The antibody or antigen-binding fragment thereof of claim 44, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 127.

57. The antibody or antigen-binding fragment thereof of claim 1 , comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 39 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 48.

58. The antibody or antigen-binding fragment thereof of claim 1 , comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 121 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 125.

59. The antibody or antigen-binding fragment thereof of claim 1 , comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 122 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 125.

60. The antibody or antigen-binding fragment thereof of claim 1 , comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 123 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 126.61 . The antibody or antigen-binding fragment thereof of claim 1 , comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 121 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 127.

62. The antibody or antigen-binding fragment thereof of claim 1 , comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 124 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 127.

63. The antibody or antigen-binding fragment thereof of any one of claims 1 -62, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

64. The antibody or antigen-binding fragment thereof of any one of claim 1 -63, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

65. The antibody or antigen-binding fragment thereof of any one of claim 1 -64, wherein the antibody is a full-length antibody.

66. The antibody or antigen-binding fragment thereof of any one of claim 1 -65, wherein the antibody is an IgG class antibody.

67. The antibody or antigen-binding fragment thereof of any one of claim 1 -66, wherein the IgG class antibody is an IgG 1 subclass antibody.

68. The antibody or antigen-binding fragment thereof of claim 1 or 63-67, wherein the antibody comprises a light chain comprising an amino acid sequence with at least 80% sequence identity to any one of SEQ ID NOs: 65-87 or 128-131 .

69. The antibody or antigen-binding fragment thereof of claim 68, wherein the antibody comprises a heavy chain comprising an amino acid sequence with at least 80% sequence identity to any one of SEQ ID NOs: 88-1 10 or 132-134.

70. The antibody or antigen-binding fragment thereof of claim 1 or 63-69, wherein the antibody comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 65-87 or 128-131 and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 88-1 10 or 132- 134.71 . The antibody or antigen-binding fragment thereof of claim 70, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 84 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 107.

72. The antibody or antigen-binding fragment thereof of claim 70, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 128 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 132.

73. The antibody or antigen-binding fragment thereof of claim 70, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 129 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 132.

74. The antibody or antigen-binding fragment thereof of claim 70, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 130 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 133.

75. The antibody or antigen-binding fragment thereof of claim 70, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 128 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 134.

76. The antibody or antigen-binding fragment thereof of claim 70, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 131 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 134.

77. The antibody or antigen-binding fragment thereof of any one of claims 1 -64, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

78. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 -77.

79. A vector comprising the polynucleotide of claim 78.

80. The vector of claim 79, wherein the vector is an expression vector.81 . The vector of claim 80, wherein the expression vector is a eukaryotic expression vector.

82. The vector of claim 81 , wherein the vector is a viral vector.

83. The vector of claim 82, wherein the viral vector is selected from the group consisting of adenovirus (Ad), retrovirus, poxvirus, adeno-associated virus, baculovirus, and a herpes simplex virus.

84. A host cell comprising the vector of any one of claims 79-83.

85. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 -77, the polynucleotide of claim 78, the vector of any one of claims 79-83, or the host cell of claim 84, and a pharmaceutically acceptable carrier or excipient.

86. A kit comprising an agent selected from the antibody or antigen-binding fragment thereof of any one of claims 1 -77, the polynucleotide of claim 78, the vector of any one of claims 79-83, the host cell of claim 84, or the pharmaceutical composition of claim 85.

87. A method of treating a subject having a synucleinopathy, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1 -77, the polynucleotide of claim 78, the vector of any one of claims 79-83, the host cell of claim 84, or the pharmaceutical composition of claim 85.

88. The method of claim 87, wherein the synucleinopathy is Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), Multiple-System Atrophy (MSA), or Alzheimer’s disease with synuclein pathology.

89. The method of claim 87 or 88, wherein the subject has elevated levels of nY39 alpha- synuclein compared to a subject who does not have the synucleinopathy.

90. The method of any one of claims 87-89, wherein the antibody or antigen-binding fragment thereof is administered intravenously.91 . A method of diagnosing a synucleinopathy in a subject, the method comprising:(a) determining the presence or level of an alpha-synuclein protein nitrated at tyrosine residue 39 (nY39 alpha-synuclein) in a cerebrospinal fluid (CSF) sample obtained from the subject; and(b) comparing the level of nY39 alpha-synuclein to a reference level of nY39 alpha- synuclein, wherein an increase in the level of nY39 alpha-synuclein in the CSF sample relative to the reference level of nY39 alpha-synuclein identifies a subject having a synucleinopathy, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha- synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

92. The method of claim 91 , wherein a statistically significant increase in the level of nY39 alpha- synuclein in the CSF sample relative to the reference level of nY39 alpha-synuclein identifies a subject having a synucleinopathy.

93. The method of claim 91 or 92, wherein the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the concentration of the at least one nitrated alpha-synuclein protein in the CSF relative to the reference level.

94. The method of any one of claims 91 -93, wherein the level of nY39 alpha-synuclein is calculated as the concentration of nY39 alpha-synuclein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample.

95. The method of any one of claims 91 -94, wherein the reference level is calculated as the concentration of nY39 alpha-synuclein in a reference sample divided by the concentration of total alpha- synuclein in the reference sample.

96. The method of claim 95, wherein the reference sample is a CSF sample from a normal subject that does not have a synucleinopathy.

97. A method of identifying a subject having a synucleinopathy, the method comprising determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein the presence of nY39 alpha-synuclein identifies the subject as having a synucleinopathy, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha- synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

98. A method of diagnosing a synucleinopathy in a subject, the method comprising determining the presence or level of nY39 alpha-synuclein in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein the presence of nY39 alpha-synuclein identifies the subject as having a synucleinopathy, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha- synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

99. The method of any one of claims 91 -98, wherein the presence or level of nY39 alpha- synuclein is determined by single molecule detection, western blot, ELISA, immunohistochemistry, or mass spectrometry.

100. The method of any one of claims 91 -99, wherein the method further comprises determining in the CSF sample the presence or level of alpha-synuclein nitrated at Y125, Y133, Y136, or a combination thereof.101 . The method of any one of claims 91 -100, further comprising administering to the subject a therapeutically effect amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, a neuroprotective agent, and / or the antibody or antigen-binding fragment thereof of any one of claims 1 -77.

102. A method of treating a subject having a synucleinopathy, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 -77, wherein the level of nY39 alpha-synuclein in a CSF sample obtained from the subject has been determined to be increased relative to a reference level of nY39 alpha- synuclein,wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha- synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

103. The method of claim 102, wherein the method further comprises determining the presence or level in the CSF sample of alpha-synuclein nitrated at Y125, Y133, Y136, or a combination thereof.

104. A method of treating a subject having a synucleinopathy, the method comprising:(a) determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein an increase in the level of nY39 alpha-synuclein relative to a reference level of nY39 alpha-synuclein identifies the subject as having a synucleinopathy; and(b) administering to the subject a therapeutically effective amount of a cognitionenhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, a neuroprotective agent, and / or the antibody or antigen-binding fragment thereof of any one of claims 1 -77, wherein the presence or level of nY39 alpha-synuclein is determined using an anti-nY39 alpha- synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145.

105. The method of any one of claims 102-104, wherein an increase is a statistically significant increase, wherein the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the level of nY39 alpha-synuclein in the CSF relative to the reference level of nY39 alpha-synuclein.

106. The method of any one of claims 102-105, wherein the level of nY39 alpha-synuclein is calculated as the concentration of nY39 alpha-synuclein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample.

107. The method of any one of claims 102-106, wherein the reference level is calculated as the concentration of nY39 alpha-synuclein in a reference sample divided by the concentration of total alpha- synuclein in the reference sample.

108. The method of claim 107, wherein the reference sample is a CSF sample from a normal subject that does not have a synucleinopathy.

109. The method of any one of claims 91 -108, wherein the synucleinopathy is Parkinson’s disease (PD) or a subtype of the disease thereof, dementia with Lewy bodies (DLB), or multiple-system atrophy (MSA).

110. The method of claim 109, wherein the subtype of the disease thereof is a prodromal stage of PD.11 1 . The method of any one of claims 102-110, wherein, after administering, the method further comprises determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein a decrease in the level of nY39 alpha-synuclein relative to the level of nY39 alpha- synuclein before administration identifies the subject as having responded to treatment.

112. The method of any one of claims 102-110, wherein, after administering, the method further comprises determining the presence or level of nY39 alpha-synuclein in a CSF sample obtained from the subject, wherein an increase in the level of nY39 alpha-synuclein relative to the level of nY39 alpha- synuclein before administration identifies the subject as in need of further treatment.

113. The method of any one of claims 91 -1 12, wherein the anti-nY39 alpha-synuclein antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 53 and / or a VH sequence comprising the amino acid sequence of SEQ ID NO: 54.

114. The method of any one of claims 91 -1 13, wherein the anti-nY39 alpha-synuclein antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 157 and / or a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 158.

115. A kit for diagnosing a synucleinopathy in a subject, the kit comprising:(a) an anti-nY39 alpha-synuclein antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 146; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 147; a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 148; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 144; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 145; and optionally(b) instructions for use of the anti-nY39 alpha-synuclein antibody to determine the level of nY39 alpha-synuclein a CSF sample from the subject, wherein an increase in the level of one or more of nY39 alpha-synuclein relative to a reference level of nY39 alpha-synuclein indicates that the subject is likely to have a synucleinopathy.

116. The kit of claim 1 15, wherein the anti-nY39 alpha-synuclein antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 53 and / or a VH sequence comprising the amino acid sequence of SEQ ID NO: 54.

117. The kit of claim 1 15 or 116, wherein the anti-nY39 alpha-synuclein antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 53 and / or a VH sequence comprising the amino acid sequence of SEQ ID NO: 54.

118. The kit of any one of claims 115-117, wherein the synucleinopathy is PD or a subtype of the disease thereof, DLB, or MSA.

119. The kit of claim 1 18, wherein the subtype of the disease thereof is a prodromal stage of PD.

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