Alpha-synuclein conformation-specific epitopes, antibodies thereto, and related methods

Abstract

The present invention provides conformation-specific epitopes of alpha-synuclein, antibodies thereto, and related methods. The present disclosure relates to conformational epitopes of α-synuclein, antibodies thereto, and methods for making and using immunogens and antibodies specific thereto. In particular, antibodies raised against cyclic compounds containing at least three amino acids of EKTKEQ (SEQ ID NO: 1) can selectively recognize misfolded oligomeric α-synuclein and inhibit the propagation and toxicity of α-synuclein. [Selection diagram] Figure 6H

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT application and claims priority from U.S. Provisional Application No. 62 / 742,408, filed October 7, 2018, U.S. Provisional Application No. 62 / 780,599, filed December 17, 2018, U.S. Provisional Application No. 62 / 820,701, filed March 19, 2019, and U.S. Provisional Application No. 62 / 864,060, filed June 20, 2019, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to alpha-synuclein (also referred to as alpha-syn or α-synuclein) epitopes and antibodies thereto, more particularly to conformational alpha-synuclein epitopes that are selectively accessible in disease-associated alpha-synuclein, and related antibody compositions and uses thereof. [Background technology]

[0003] Alpha-synuclein (α-syn or α-synuclein) is a 140-amino acid protein found primarily in the presynaptic terminals of neurons. It is thought to play a functional role in maintaining the supply of synaptic vesicles at presynaptic terminals by clustering synaptic vesicles and in regulating dopamine release [eLife 2013;2:e00592 doi:10.7554 / eLife.00592]. At least three isoforms of synuclein are produced by alternative splicing. The most common form of the protein is the full-length protein of 140 amino acids. Other isoforms are α-syn-126, which lacks residues 41–54 due to the loss of exon 3, and α-syn-112, which lacks residues 103–130 due to the loss of exon 5.

[0004] Monomeric α-synuclein in solution is considered to be an intrinsically disordered protein lacking a single stable 3D structure. The N-terminal residues 1–60 of α-syn are amphipathic and contain four 11-residue repeats containing the consensus sequence KTKEGV (SEQ ID NO: 6). This sequence has a structural α-helical propensity similar to that of the apolipoprotein-binding domain. Residues 61–95 constitute a central hydrophobic region, termed the non-amyloid-β component or NAC region, which is known to be involved in protein aggregation [PNAS December 1, 1993 90(23)11282–11286; doi.org / 10.1073 / pnas.90.23.11282]. Residues 96–140 constitute a highly acidic, proline-rich region with no clear structural propensity.

[0005] α-syn monomers in solution are intrinsically disordered. Membrane-bound monomers have a partial helical structure [Ulmer, TS, Bax, A., Cole, NB, Nussbaum, RL (2005) J Biol Chem 280 9595-9603; Rao, JN, Jao, CC, Hegde, BG, Langen, R., Ulmer, TS (2010) J Am Chem Soc 132 8657-8668]. Membrane-bound α-syn monomers induce membrane curvature [Varkey et al. J Biol Chem v285, no. 42, pp. 32486-32493, (2010) DOI: 10.1074 / jbc.M110.139576]. Alpha-synuclein may exist in stably folded tetramers that resist aggregation [doi:10.1038 / nature10324], or at least in the CNS as a monomer (Fauvet et al., 2012, DOI:10.1074 / jbc.M111.318949).

[0006] Recently, it has been shown that a Parkinson's-like disease develops in mice expressing a mutant α-syn that cannot tetramerize (Nuber et al., 2018).

[0007] In pathological conditions associated with Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy (collectively known as synucleinopathies), α-synuclein aggregates to form insoluble fibrils characteristic of Lewy bodies and Lewy neurites. α-Synuclein is the major structural component of Lewy body fibrils. α-Synuclein pathology is also found in both sporadic and familial cases of Alzheimer's disease [doi:10.1007 / s00401-002-0596-7]. Point mutations in the α-Syn gene, including A53T, A30P, E46K, H50Q, and G51D, are associated with hereditary Parkinson's disease. Overexpression due to genomic duplication and triplication of the SNCA gene, which encodes α-Syn, also appears to cause Parkinson's disease.

[0008] Pathological aggregates of α-synuclein located at presynapses are thought to be responsible for synaptic dysfunction [doi:10.1007 / s00401-010-0711-0]. Therefore, small molecule compounds that inhibit α-synuclein aggregation have been developed as a strategy to treat synucleinopathies [REF DOI:10.1021 / bi0600749].

[0009] Antibodies that specifically recognize phospho-S129 in α-synuclein immunostained Lewy bodies show that S129 is selectively and extensively phosphorylated in synucleinopathy lesions.

[0010] Antibodies have been raised against alpha-synuclein and its related immunogens described.

[0011] US Patent Publication No. US2016 / 0244515A1 describes human anti-alpha-synuclein antibodies.

[0012] US Patent Publication No. US2015 / 0232524A1 discloses compositions comprising one or more immunogens having at least two regions comprising an alpha-synuclein B cell epitope and at least one T helper cell epitope.

[0013] US Patent Publication No. US2014 / 0295465A1 describes the use of anti-alpha-synuclein antibodies to diagnose elevated levels of alpha-synuclein in the brain.

[0014] Oligomeric α-synuclein may be the form of the protein that causes neuronal death [Brown DR 2010, DOI:10.1002 / iub.316]. α-Syn has been detected in the cerebrospinal fluid (CSF) of patients with Parkinson's disease. Oligomers, which are thought to form as prefibrillar intermediates, may be the predominantly toxic component of α-Syn [Karpinar et al. 2009, DOI:10.1038 / emboj.2009.257]. Prefibrillar α-synuclein variants with impaired β structure increase neurotoxicity in Parkinson's disease models [EMBO J. 28, 3256-3268; Outeiro et al., McLean, (2008)]. The formation of toxic oligomeric α-synuclein species can occur intracellularly in living cells [PLoS ONE 3, e1867; Danzer et al., Kostka, (2007)]. Different species of α-synuclein oligomers induce calcium influx and seeding [J. Neurosci. 27, 9220-9232].

[0015] Oligomers lack a defined structure, are conformationally plastic, and exist at concentrations much lower than those of functional monomers or tetramers. The low concentration of misfolded oligomeric α-syn makes this target elusive. Antibodies or drugs targeting healthy α-syn can be harmful to cells.

[0016] Attempts to generate antibodies against oligomeric α-synuclein have been reported. U.S. Patent Publication No. US2016 / 0199522A1 reports the use of preparations of soluble protofibrillar / oligomeric human α-synuclein modified with 4-hydroxy-2-nonenal (HNE) or the α,β-unsaturated alkenal 4-oxo-2-nonenal (ONE) to generate antibodies. However, no evidence of their utility for human samples was provided.

[0017] The survival of neurons with intracellular Lewy bodies suggests that the presence of cytoplasmic α-Syn aggregates is not severely toxic to all cells [Spillantiniet. al (1997) Alpha-synuclein in Lewy bodies. Nature 388, 839-840].

[0018] The fibril structure of full-length human α-synuclein was obtained by solid-state NMR (PDB 2N0A) [doi:10.1038 / nsmb.3194,Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein,Tuttle et al Nature SMB 2016].

[0019] Antibodies that preferentially or selectively bind to misfolded oligomeric α-synuclein over monomeric α-Syn and / or insoluble fibrillar α-Syn are desirable. Summary of the Invention

[0020] Described herein are conformational epitopes of misfolded oligomeric α-synuclein.

[0021] Embodiments include cyclic compounds comprising alpha-synuclein peptides comprising and / or consisting of three or more residues of EKTKEQ (SEQ ID NO: 1), and optionally comprising and / or consisting of residues EKTK (SEQ ID NO: 2) or a portion thereof, residues KTKE (SEQ ID NO: 3) or a portion thereof, or residues TKEQ (SEQ ID NO: 4) or a portion thereof, a portion of which comprises at least three amino acids.

[0022] The α-synuclein peptides incorporated into the cyclic compounds are conformational epitopes that can be used as immunogens, epitopes that are selectively exposed in misfolded oligomeric species of α-synuclein and that are unavailable or less available in, for example, natively folded α-synuclein monomers and / or native tetramers.

[0023] Another embodiment includes antibodies that specifically bind to epitopes in the α-Syn peptides of the cyclic compounds described herein and / or in misfolded oligomeric α-synuclein compared to the corresponding linear compounds and / or native α-Syn and / or insoluble fibrillar α-Syn. The antibodies can be produced using immunogens or compositions comprising the immunogens described herein.

[0024] The epitope is a conformational epitope, e.g., the epitope is selectively presented or accessible on misfolded oligomeric α-Syn. The α-Syn peptide can be three or more residues, optionally four or more residues, five or more residues, or six residues of EKTKEQ (SEQ ID NO: 1), or specifically EKT, KTK, TKE, KEQ, EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), TKEQ (SEQ ID NO: 4), EKTKE (SEQ ID NO: 8), or KTKEQ (SEQ ID NO: 9).

[0025] In embodiments, the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of one or more of the CDRs is the amino acid sequence set forth below: CDR-H1: SEQ ID NO: 61, 67, 73, 79, 91, or 180; CDR-H2: SEQ ID NO: 62, 68, 74, 80, 92, or 181; CDR-H3: SEQ ID NO: 63, 69, 75, 81, 93, or 182; CDR-L1: SEQ ID NO: 64, 70, 76, 94, or 183; CDR-L2: SEQ ID NO: 65, 71, or 77; or CDR-L3: selected from SEQ ID NO: 66, 72, 78, 84, 96, or 184.

[0026] In an embodiment, the CDRs are: In an embodiment, the CDRs are: CDR-H1: SEQ ID NO: 67, CDR-H2: SEQ ID NO: 68, CDR-H3: SEQ ID NO: 69, CDR-L1: SEQ ID NO: 70, CDR-L2: SEQ ID NO: 71, and CDR-L3: SEQ ID NO: 72.

[0027] In embodiments, the CDRs are as follows: CDR-H1: SEQ ID NO: 73, CDR-H2: SEQ ID NO: 74, CDR-H3: SEQ ID NO: 75, CDR-L1: SEQ ID NO: 76, CDR-L2: SEQ ID NO: 77, and CDR-L3: SEQ ID NO: 78.

[0028] In embodiments, the CDRs are as follows: CDR-H1: SEQ ID NO: 79, CDR-H2: SEQ ID NO: 80, CDR-H3: SEQ ID NO: 81, CDR-L1: SEQ ID NO: 76, CDR-L2: SEQ ID NO: 77, and CDR-L3: SEQ ID NO: 84.

[0029] In embodiments, the CDRs are as follows: CDR-H1: SEQ ID NO: 79, CDR-H2: SEQ ID NO: 80, CDR-H3: SEQ ID NO: 81, CDR-L1: SEQ ID NO: 76, CDR-L2: SEQ ID NO: 77, and CDR-L3: SEQ ID NO: 84.

[0030] In embodiments, the CDRs are as follows: CDR-H1: SEQ ID NO: 91, CDR-H2: SEQ ID NO: 92, CDR-H3: SEQ ID NO: 93, CDR-L1: SEQ ID NO: 94, CDR-L2: SEQ ID NO: 71, and CDR-L3: SEQ ID NO: 96.

[0031] In embodiments, the CDRs are as follows: CDR-H1: SEQ ID NO: 180, CDR-H2: SEQ ID NO: 181, CDR-H3: SEQ ID NO: 182, CDR-L1: SEQ ID NO: 183, CDR-L2: SEQ ID NO: 77, and CDR-L3: SEQ ID NO: 184.

[0032] Further aspects include the nucleic acids described herein.

[0033] A further aspect is a vector comprising a nucleic acid described herein.

[0034] Another embodiment includes a recombinant cell that produces the antibody, nucleic acid, or vector described herein. Further embodiments include compositions that include the components described herein (e.g., cyclic compounds, antibodies, nucleic acids, vectors, recombinant cells, etc., and combinations thereof).

[0035] Another embodiment provides an assay for detecting whether a test sample contains misfolded oligomeric α-Syn, comprising: a. contacting a test sample with an antibody or immunoconjugate described herein under conditions permissive to produce antibody:misfolded oligomeric α-Syn polypeptide complexes; b. detecting the presence or absence of any complex; The presence of a detectable complex indicates that the test sample may contain misfolded oligomeric α-Syn polypeptides.

[0036] The detected misfolded oligomeric α-Syn comprises a conformational epitope described herein that is selectively accessible in misfolded oligomeric α-Syn polypeptides, e.g., compared to native α-Syn, e.g., the epitope may be selectively presented or accessible in misfolded oligomeric α-Syn.

[0037] Further aspects include methods of inhibiting misfolded alpha-synuclein toxicity comprising administering to a population of cells or a subject in need thereof an effective amount of an antibody, immunoconjugate, or composition described herein.

[0038] Yet another aspect is a method of treating an α-synucleinopathy comprising administering to a subject in need thereof any of the foregoing antibodies, immunoconjugates or compositions, or combinations described herein, which selectively bind to misfolded oligomeric α-synuclein and / or soluble α-synuclein fibrils (e.g., toxic misfolded species) relative to monomeric, tetrameric (e.g., physiological or native species) and / or insoluble fibrillar α-synuclein species, e.g., as shown herein.

[0039] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0040] Various embodiments of the present disclosure will now be described in connection with the following drawings.

[0041] [Figure 1] Figures 1A-C are graphs depicting predicted epitopes. Figure 1A shows predicted exposure potential as a function of sequence based on solvent-accessible surface area (SASA). The graph in Figure 1A represents epitope predictions resulting from the stress fibril structure PDB 2N0A using the increase in SASA (ΔSASA) as the criterion for epitope selection. The EKTK (SEQ ID NO: 2) (residues 57-60) and TKEQ (SEQ ID NO: 4) (residues 59-62) epitopes appear as predictions for PDB structure 2N0A (Figure 1A). Figure 1B shows epitope predictions resulting from the structure PDB 2N0A using loss of native contacts as the criterion for epitope selection. The EKTK epitope (SEQ ID NO: 2) appears as a prediction using this metric. Figure 1C shows epitope predictions made by several metrics, including increasing SASA (ΔSASA), increasing root mean square fluctuation (RMSF) of atomic positions, which represents increasing epitope dynamics, and decreasing the number of native contacts (ΔContacts). These three metrics predict the epitopes EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4), as well as their subsequences. That is, for one or more chains within the fibril structure, EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4) meet all three criteria, but the adjacent regions do not. [Figure 2A] This figure shows a conformational rendering of a monomer of α-Syn in the context of an unbiased fibril (PDB 2N0A). This structure is obtained from an equilibrium simulation of five chains of α-Syn containing 100 mM NaCl. Residues K58 and K60 are nearly parallel in this structural ensemble. There is a close contact between the Hε3 atom of K60 (weakly positively charged, Q=0.05) and the Nε2 atom of Q62 (negatively charged, Q=-0.64). [Figure 2B]A snapshot of the structure of a monomer of α-Syn in a deflected fibril ensemble is shown. In this ensemble, residues K58 and K60 are no longer parallel, and the contact between K60 and Q62 is no longer present. This suggests that K60 and Q62 may be more accessible for binding in deflected or "stressed" fibrils and oligomeric species of α-Syn compared to undeflected fibrils. [Figure 3] Figure 3A-C shows schematic diagrams of different conformations of α-synuclein. Panel A shows a snapshot of EKTK (SEQ ID NO: 2) in the context of unbiased fibrils (PDB 2N0A). This figure also shows the SASA of this sequence region; since the epitope is largely buried, SASA is minimal. Panel B shows the centroid structure of an ensemble of the cyclic peptide cyclo(CGGGGEKTKGG) (SEQ ID NO: 5). The side chains of the cyclic peptide show increased SASA compared to the side chains of the fibrils. Panel C shows the side chain orientation of both instances of the epitope in the centroid structure of an isolated, native monomer ensemble. The orientations of T59 and K60, compared to K58, are significantly different in the isolated monomer ensemble than in the cyclic peptide ensemble. The conformation of the epitope differs from the majority of conformations in the cyclic peptide ensemble. [Figure 4] Panel A shows a snapshot of TKEQ (SEQ ID NO: 4) in the context of unbiased fibrils (PDB 2N0A). This figure also shows the SASA of this sequence region; since the epitope is largely buried, SASA is minimal. Panel B shows the centroid structure of an ensemble of the cyclic peptide cyclo(CGTKEQGGGG) (SEQ ID NO: 7). The side chains of the cyclic peptide exhibit increased SASA compared to the side chains of the fibrils. Panel C shows the side chain orientation of the epitope in the centroid structure of an isolated, native monomer ensemble. The side chain orientation in the isolated monomer ensemble is significantly different from the orientation of the corresponding side chain in the cyclic peptide ensemble. [Figure 5]Panel A plots the ensemble average solvent-accessible surface area (SASA) of the EKTK (SEQ ID NO: 2) epitope in the fibril ensemble, the stress / deflected fibril ensemble, and the cyclic peptide ensemble cyclo(CGGGGEKTKGG) (SEQ ID NO: 5). Residues show a monotonic increase in surface exposure between undeflected fibrils, deflected fibrils, and the cyclic peptide. Panel B plots the ensemble average solvent-accessible surface area (SASA) of the TKEQ (SEQ ID NO: 4) epitope in the undeflected fibril ensemble, the stress / deflected fibril ensemble, and the cyclic peptide ensemble cyclo(CGTKEQGGGG) (SEQ ID NO: 7). Residues T59, E61, and Q62 show the largest increase in surface exposure between undeflected fibrils and the cyclic peptide. The increase in SASA from undeflected fibrils to deflected fibrils is nearly uniform across the epitope. The SASA increase of the epitopes TKEQ (SEQ ID NO: 4) and EKTK (SEQ ID NO: 2) is also shown in Figure 5 and panels A and C of Figure 1. Panel C plots a histogram of RMSD at the center of gravity of the equilibrium distribution of the cyclic peptide scaffold cylco(CGTKEQGGGG) (SEQ ID NO: 7). Most conformations are very similar to the center of gravity conformation, with the distribution peaking at approximately 1.3 Å. Also shown are the RMSDs corresponding to the conformations of the epitopes for the center of gravity conformation of the monomer ensemble and fibril ensemble. Finally, the RMSDs of the epitopes in the conformations of the PDB structures of α-helical, micelle-bound α-synuclein, 1XQ8, and 2KKW are shown. These conformations differ from most cyclic conformations. [Figure 6]Figures 6A-H are a series of graphs. Figure 6A is a series of graphs showing antibody binding to α-synuclein monomers by SPR. Strong binding is observed with pan-antibody 4D6, low levels of binding with Syn-F1 favoring aggregated α-synuclein, and no binding is observed with the test antibodies. Figure 6B is a series of graphs showing antibody binding to α-synuclein oligomers by SPR. Figure 6C is a series of graphs showing antibody binding to α-synuclein oligomers by sandwich SPR. Figure 6D is a series of graphs showing binding to α-synuclein oligomers. Figure 6E is a series of graphs showing antibody binding responses to soluble LBD brain extracts. Figure 6F is a series of graphs showing antibody binding in HMW and LMW soluble LBD brain fractions. The first dark bar for each condition is HMW (approximately 140-700 kDa), and the second light bar is LMW (approximately 8-70 kDa). Figure 6G is a graph showing the extent of cross-reactive binding of antibodies to small soluble fibrils. Figure 6H is a graph showing a comparison of the binding profiles of test antibodies of the present disclosure with the pan antibody 4D6. [Figure 7] Figures 7A-F are a series of dot blots. Figures 7G and H are a series of graphs. Figure 7G is a graph plotting fold reactivity in Dementia with Lewy Bodies (DLB) brains relative to normal brains. Figure 7H is a graph showing the total amount of alpha-synuclein in brain samples. [Figure 8] 8A-H are graphs and images showing inhibition of α-syn toxicity by various test antibodies. [Figure 9] Figures 9A-B are a series of graphs. Figure 9A shows antibody-selective binding to synthetic α-syn oligomers by SPR, but not to monomers or physiological tetramers. Figure 9B shows antibody-selective binding to α-syn oligomers and sonicated fibrils. [Figure 10]Figures 10A-E show that the α-syn test antibody preferentially stains small aggregates of α-syn on dense Lewy bodies by IHC and immunofluorescence staining, and Figures 10F-K show that the α-syn test antibody does not cause detectable staining of normal brain by IHC. [Figure 11] 1 is a graph showing representative ELISA results for antibody 2E9. [Figure 12] Figures 12A-B are a series of graphs: Figure 12A is a graph showing test antibody binding to DLB soluble brain extract; Figure 12B is a graph showing test antibody binding to soluble DLB extract is epitope specific. [Figure 13] 13A-I are graphs and images showing that the test antibodies reduce the formation of α-synuclein aggregates induced by PFFs. [Figure 14] 14A-H are graphs and images showing that the test antibodies reduce PFF-induced aggregation and phosphorylation of endogenous α-synuclein. [Figure 15] 15A-B are graphs, where FIG. 15A is a graph showing that test antibodies inhibit the in vitro propagation of alpha-synuclein aggregation, and FIG. 15B is a graph showing that cyclic peptides, including alpha-syn peptides containing conformational epitopes, are sufficient to replicate the seeding activity of preformed fibrils and are neutralized by test antibody 2E9. [Figure 16] Figures 16A and B are bar graphs showing the binding responses of test antibodies to brain extracts from patients with multiple system atrophy (MSA). Figure 16C is a bar graph showing the binding responses of test antibodies to a prion-enriched fraction of brain extract. [Figure 17] Figures 17A-C are a series of graphs showing the binding profile of test antibody 12G1. [Figure 18] Figures 18A-C are a series of graphs showing the binding profile of test antibody 9D8. [Figure 19] Figures 19A-C are a series of graphs showing the binding profile of test antibody 10D5. [Figure 20] 1 is a graph showing quantification of misfolded oligomeric α-syn in biological samples. DETAILED DESCRIPTION OF THE INVENTION

[0042] The generation of conformation-specific antibodies has been achieved.

[0043] Antibodies raised against native protein regions tend not to be selective for misfolded proteins, such as denatured oligomeric species, and may therefore bind to native functional protein as well as misfolded protein.

[0044] As described herein, to develop antibodies that may be selective for misfolded oligomeric forms of α-Syn, we sought to identify regions of the α-Syn sequence that may be susceptible to disruption in the context of fibrils and thus exposed on the surface of misfolded protein oligomers that may act as catalytic substrates for misfolding.

[0045] Computational simulations using molecular dynamics with a standardized force field were used, as described in the Examples. Experimentally validated structural models of fibril structures were globally deflected from the reported conformation, which was partially unfolded using molecular dynamics, generating adjacent regions of primary sequence prone to disorder upon external challenge in the abnormal cellular environment.

[0046] We hypothesized that these weakly stable regions may be selectively exposed to misfolded pathogenic species such as denatured oligomers.

[0047] As described in the Examples, we identified conformational epitopes. We designed cyclic compounds containing the identified epitopes to mimic putative selective epitopes by satisfying several criteria, such as higher exposed surface area, loss of contact interactions present in fibrils, and / or conformations that did not readily align by root mean square deviation (RMSD) to isolated monomer ensembles but would align more favorably to biased, partially disordered fibril ensembles. As further shown in the Examples, monoclonal antibodies generated using immunogens containing these cyclic compounds preferentially bound to misfolded, oligomeric α-synuclein and inhibited α-synuclein-induced neurotoxicity.

[0048] I. Definition As used herein, the term "α-Syn," alternately referred to herein as "α-synuclein" or "α-synuclein" or "α-syn," refers to all forms of α-Syn, including wild-type sequence α-Syn and mutant forms, monomeric α-Syn, and their aggregates, such as misfolded oligomeric and soluble fibrillar forms of α-Syn from all species, particularly human α-Syn (i.e., huα-Syn). Human α-Syn is typically a 140 amino acid residue protein, the amino acid sequence (e.g., Uniprot accession number P37840) and nucleotide sequence (e.g., accession number HGNC:11138) of which have been previously characterized.

[0049] As used herein, "wild-type" refers to the primary amino acid sequence of the non-mutant or native protein in humans.

[0050] As used herein, "native alpha-synuclein polypeptide" or "native alpha-Syn" refers to alpha-synuclein monomers, whether associated with the membrane or the cytosol, as well as other multimers found in normal cells, such as tetramers, which may be predicted, for example, when using one of the chains from PDB fibrils (2N0A), as described herein. Native alpha-synuclein polypeptides can be detected, for example, in brains not affected by synucleinopathies, using pan antibodies.

[0051] A model of the native α-Syn tetramer [pnas.org / cgi / doi / 10.1073 / pnas.1113260108] shows that the epitope is stabilized by interactions involving interchain salt bridges between residues in the above epitope, specifically between K60 and E57 and between K34 and E57. Similarly, Q62 is shown within the maximum paramagnetic relaxation effect, indicating that it interacts strongly in the tetramer and isolated monomers. These interactions may result in the isolation of the epitope in the native, native tetramer form, resulting in antibodies targeting the epitope being selective against denatured species (e.g., misfolded, oligomeric α-synuclein).

[0052] As used herein, "structured fibril," "unstressed fibril," or "unbiased fibril" refers to the conformation expected to be observed at thermal equilibrium for α-synuclein fibrils, e.g., PDB 2N0A is a representative example.

[0053] As used herein, "misfolded oligomer," "denatured oligomer," refers to the secondary and tertiary structure of a multisubunit polypeptide or polypeptide aggregate, indicating that the oligomeric polypeptide, or subunits therein, have adopted a conformation (e.g., at one or more positions) that differs from that typically adopted by native monomers and / or tetramers. Misfolding can be caused by mutations in the protein, such as amino acid deletions, substitutions, or additions, but wild-type sequence proteins can also be misfolded in disease, exposing disease-specific or selective epitopes, for example, as a result of altered microenvironmental conditions or oligomerization that may be on or off the pathway to fibril formation (e.g., insoluble fibrils). Thus, as used herein, "misfolded oligomeric α-Syn polypeptide," "misfolded α-Syn," or "misfolded oligomeric α-Syn," when referring to a polypeptide, refers to α-Syn polypeptide oligomers that exhibit a conformation different from nascently folded monomeric α-Syn and / or natively folded tetrameric α-synuclein, including, for example, denatured oligomers, soluble fibrils, protofibrils, and fibril fragments. Soluble fibrils include, for example, a-syn fibril species found in the supernatant of a sample ultracentrifuged at 100,000 x g for 1 hour. Soluble fibrils can be produced by sonicating fibrils to produce fragments. For example, misfolded oligomeric α-Syn may contain partially ordered conformations, including portions of the fibril structure, as well as partially disordered conformations, including polymeric segments of amino acids with alternating conformations that are not found in either monomeric, tetrameric, and / or fibril α-Syn.The misfolded oligomeric α-synuclein provided herein comprises conformational epitopes that are selectively presented or accessible for binding, and the epitope sequence in the misfolded oligomeric α-synuclein may be conformationally distinct from the corresponding sequence in the context of the isolated monomer, as measured, for example, by side chain orientation or root mean square deviation (RMSD). Misfolded α-synuclein may comprise at least one of residues E57, K58, T59, K60, E61, or Q62 in an alternative conformation, rather than being occupied by E57, K58, T59, K60, E61, and / or Q62 in an unmisfolded protein conformation, such as a native monomer and / or tetramer, or in an insoluble fibril, such as those found in Lewy body deposits. Soluble alpha-synuclein fibrils refer to smaller fibrils or fragments, e.g., fibrils that have been sonicated as described in the Examples and are in solution, as well as disease-associated smaller fibrils that are not present in Lewy bodies, which contain insoluble fibrils.

[0054] The term "mutant α-Syn" refers to forms of α-Syn, particularly endogenous forms of α-Syn that arise as a result of genetic mutations that result in, for example, amino acid substitutions, such as those characteristic of familial Parkinson's disease.

[0055] The term "EKTK (SEQ ID NO: 2)" refers to the amino acid sequence: glutamic acid, lysine, threonine, lysine, as set forth in SEQ ID NO: 2. The term "TKEQ (SEQ ID NO: 4)" refers to the amino acid sequence: threonine, lysine, glutamic acid, glutamine, as set forth in SEQ ID NO: 4. Similarly, EKT, KTK, TKE, KEQ, EKTKEQ (SEQ ID NO: 1), EKTKE (SEQ ID NO: 8), KTKE (SEQ ID NO: 3), and KTKEQ (SEQ ID NO: 9) refer to amino acid sequences identified by the single-letter amino acid code. Depending on the context, reference to an amino acid sequence may refer to the sequence of α-Syn or the amino acid sequence of an isolated peptide, e.g., an epitope portion of a cyclic compound. The sequences EKTK (SEQ ID NO: 2) and TKEQ (SEQ ID NO: 4) consist of residues 57-60 and residues 59-62, respectively, of the α-Syn amino acid primary sequence (e.g., Uniprot Accession No. P37840).

[0056] As used herein, the term "epitope in EKTKEQ (SEQ ID NO: 1)" refers to any portion thereof that is specifically bound by an antibody. For example, an antibody may specifically bind to the side chains and / or backbone of a combination of several residues in the epitope, including some of E57, and / or E61, and / or K58, and / or K60, and / or Q62, and / or T59, or specific portions of these residues, or any combination of the foregoing. The epitope may be a conformational epitope.

[0057] As used herein, the term "epitope" refers to a sequence of amino acids in an antigen, wherein the amino acids (or a subset thereof) in the sequence are specifically recognized by an antibody or binding fragment, such as an antibody or binding fragment described herein. An epitope can include one or more antigenic determinants. For example, an antibody generated against an isolated peptide corresponding to a conformational epitope will recognize part or all of the epitope sequence.

[0058] As used herein, the term "epitope selectively presented or accessible on misfolded oligomeric α-Syn" refers to a conformational epitope that is selectively presented or accessible on misfolded oligomeric α-Syn polypeptides, whether in multimeric, oligomeric, or aggregated forms, as present in synucleinopathies such as Parkinson's disease and dementia with Lewy bodies (e.g., diseases associated with misfolded α-Syn), but is not presented or accessible on the molecular surface of the initial monomer peptide or tetrameric forms of α-Syn normally found in vivo.

[0059] As used herein, the term "conformational epitope" refers to a sequence of amino acids or an antigenic determinant thereof that has a specific three-dimensional structure in a species of protein, where at least some aspect of the three-dimensional structure exists or is accessible by antibody binding compared to another species, such as the isolated monomer (native) or other native structure. An antibody that specifically binds to a conformational epitope recognizes the spatial arrangement of one or more of the amino acids of that conformation-specific epitope. For example, a conformational epitope of EKTKEQ (SEQ ID NO: 1) can refer to a conformation of one or more amino acids of EKTKEQ (SEQ ID NO: 1), or a portion thereof, that is selectively recognized by the antibody with at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold or more selectivity compared to the corresponding region in another conformation, optionally α-Syn monomer or insoluble fibril, or an antibody generated using, for example, the corresponding linear peptide or portion thereof.

[0060] Reference herein to a "cyclic peptide" can refer to a fully proteinaceous cyclic compound (e.g., where the linker is 2, 3, 4, 5, 6, 7, or 8 amino acids). It is understood that the properties described for the cyclic peptides determined in the Examples can be incorporated into other compounds (e.g., cyclic compounds) that include non-amino acid linker molecules.

[0061] The term "amino acid" includes all naturally occurring amino acids, as well as modified L-amino acids. The atoms of an amino acid can contain, for example, different isotopes. For example, an amino acid can contain deuterium instead of hydrogen, nitrogen-15 instead of nitrogen-14, and carbon-13 instead of carbon-12, and other similar changes.

[0062] As used herein, the term "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, single-chain, single-domain, humanized, and other chimeric antibodies, as well as binding fragments thereof. Antibodies may be derived from recombinant sources and / or produced in transgenic animals. In embodiments, antibodies comprise a heavy chain variable region, or a heavy chain comprising heavy chain complementarity-determining region 1, heavy chain complementarity-determining region 2, and heavy chain complementarity-determining region 3, and a light chain variable region, or a light chain comprising light chain complementarity-determining region 1, light chain complementarity-determining region 2, and light chain complementarity-determining region 3. Also included are human antibodies, which may be produced using biochemical techniques or isolated from libraries. Humanized or chimeric antibodies may comprise sequences from one or more isotypes or classes. Reference to an antibody or antibodies of the present disclosure refers to, e.g., an antibody or antibodies described herein that are produced using an immunogen described herein and / or that are selective for an epitope described herein, e.g., KTKE (SEQ ID NO: 3), EKTK (SEQ ID NO: 2), or TKEQ (SEQ ID NO: 4), or a portion thereof, e.g., in the context of an epitope, misfolded oligomeric alpha-synuclein, and / or a cyclic compound comprising one of the epitope sequences.

[0063] As used herein, the term "heavy chain complementarity determining region" refers to a region of hypervariability within the heavy chain variable region of an antibody molecule. The heavy chain variable region has three complementarity determining regions, from the amino terminus to the carboxy terminus, called heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3).

[0064] The term "heavy chain variable region" as used herein refers to the variable domain of a heavy chain comprising heavy chain complementarity determining region 1, heavy chain complementarity determining region 2, and heavy chain complementarity determining region 3. One or more amino acids or nucleotides can be modified, e.g., replaced with conservative substitutions, e.g., outside the CDR sequences. The variable region comprises framework region 1 (FR1), followed by CDR1, followed by framework region 2 (FR2), followed by CDR2, followed by framework region 3 (FR3), followed by CDR3, followed by framework region 4 (FR4).

[0065] As used herein, the term "light chain complementarity-determining region" refers to a region of hypervariability within the light chain variable region of an antibody molecule. The light chain variable region has three complementarity-determining regions, designated light chain complementarity-determining region 1, light chain complementarity-determining region 2, and light chain complementarity-determining region 3, from the amino terminus to the carboxy terminus.

[0066] As used herein, the term "light chain variable region" refers to the variable domain of a light chain comprising light chain complementarity determining region 1, light chain complementarity determining region 2, and light chain complementarity determining region 3. The variable region comprises framework region 1 (FR1), followed by CDR1, followed by framework region 2 (FR2), followed by CDR2, followed by framework region 3 (FR3), followed by CDR3, followed by framework region 4 (FR4).

[0067] The phrase "isolated antibody" refers to an antibody produced in vivo or in vitro that has been removed from the source that produces the antibody, e.g., an animal, hybridoma, or other cell line (such as a recombinant cell that produces the antibody). An isolated antibody is optionally "purified," meaning at least 80%, 85%, 90%, 95%, 98%, or 99% pure.

[0068] The term "binding fragment" as used herein refers to a portion or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain and that binds to an antigen or competes with the intact antibody. Exemplary binding fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, nanobodies, minibodies, diabodies, and multimers thereof. Fragments can be obtained by chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges and produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be constructed by recombinant expression techniques.

[0069] When an antibody is said to bind to an epitope such as EKTKEQ (SEQ ID NO: 1) or TKEQ (SEQ ID NO: 4), it means that the antibody specifically binds to a polypeptide or compound that includes the specified residues or a portion thereof, e.g., at least one residue or at least two residues. Such an antibody does not necessarily contact every residue of EKTK (SEQ ID NO: 2) or TKEQ (SEQ ID NO: 4), and not all single amino acid substitutions or deletions within the epitope necessarily affect binding affinity significantly or equally.

[0070] As used herein, the term "detectable label" refers to a moiety, such as a fluorescent protein, that can be attached to or introduced into a peptide sequence, a peptide or compound described herein, and that can directly or indirectly produce a detectable signal. For example, the label can be a radiopaque, positron-emitting radionuclide (e.g., for use in PET imaging), or 3 H, 13 N,14 C. 18 F, 32 P, 35 S, 123 I, 125 I, 131 The detectable label can be a radioisotope such as I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; an imaging agent; or a metal ion. The detectable label can also be indirectly detectable, for example, using a secondary antibody.

[0071] As used herein, the term "higher affinity" refers to the ability of antibody X to bind more strongly (K) to target Y than to target Z. on ), and / or a smaller dissociation constant (K off ), in this context, refers to the degree of antibody binding where antibody X binds weaker and / or with a larger dissociation constant to target Y than to target Z, in this context, antibody X has a lower affinity to target Y than to Z. Similarly, the term "lower affinity" herein refers to the degree of antibody binding where antibody X binds weaker and / or with a larger dissociation constant to target Y than to target Z, in this context, antibody X has a lower affinity to target Y than to Z. The affinity of binding between an antibody and its target antigen is expressed as K A =1 / K D and K D is k on / k off is equal to k on and k off The value may be measured using surface plasmon resonance (eg, measurable using a Biacore system).

[0072] Also, as used herein, the term "immunogenic" refers to a substance that induces the production of antibodies and activates lymphocytes and other reactive immune cells directed against the antigenic portion of the immunogen.

[0073] As used herein, "immunogen" refers to a substance that elicits an immune response and / or elicits the production of antibodies and may include, for example, a cyclic peptide described herein conjugated as a multi-antigenic peptide and / or fused to an immunogenicity enhancer such as keyhole limpet hemocyanin (KLH). In addition to the conjugates described herein, immunogenic peptidomimetics that elicit cross-reactive antibodies against identified epitopes, e.g., EKTKEQ (SEQ ID NO: 1), EKTK (SEQ ID NO: 2), TKEQ (SEQ ID NO: 4), or KTKE (SEQ ID NO: 3), are also included. To function as a useful immunogen, an α-Syn peptide desirably incorporates a minimum of about 3, 4, 5, 6, or 7 α-Syn residues, including E57, K58, T59, K60, E61, and / or Q62.

[0074] For example, the term "inhibiting" as used herein in connection with an antibody of the present disclosure that inhibits alpha-syn phosphorylation means reducing the amount of alpha-syn phosphorylation in the presence of the antibody by at least 10%, at least 20%, or at least 30% compared to the absence of the antibody.

[0075] The term "corresponding linear compound" in relation to a cyclic compound refers to a compound, optionally a peptide, that contains or consists of the same sequence or chemical moieties as the cyclic compound, but in linear (non-cyclized) form.

[0076] As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences, including non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza- and deaza-adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. Nucleic acids can be either double-stranded or single-stranded and represent sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon-optimized or synonymous codon equivalents. As used herein, the term "isolated nucleic acid sequence" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0077] The term "vector" as used herein includes any intermediate vehicle for a nucleic acid molecule that allows, for example, the nucleic acid molecule to be introduced into a prokaryotic and / or eukaryotic cell and / or integrated into the genome, including a plasmid, a phagemid, a bacteriophage, or a viral vector, e.g., a retrovirus-based vector, an adeno-associated virus vector, etc. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, which is capable of replication independently of chromosomal DNA.

[0078] The term "host cell" refers to a cell into which a recombinant DNA expression vector can be introduced to produce a recombinant cell. Host cells can be bacterial cells, such as E. coli, as well as any type of microbial, yeast, fungal, insect, or mammalian host cell. Mammalian host cells can be human cells.

[0079] The term "pharmaceutically acceptable" means the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0080] As used herein, the term "administered" refers to administration of a therapeutically effective amount of a compound or composition of the present disclosure to a cell or subject.

[0081] As used herein, the phrase "effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired result. When administered to a subject, the effective amount may vary depending on factors such as the subject's condition, age, sex, and weight. The administration regimen can be adjusted to provide an optimal therapeutic response.

[0082] As used herein and as is well understood in the art, the terms "treating" or "treatment" refer to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stable disease (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, improvement or palliation of disease symptoms, reduction in disease recurrence, and remission (partial or total), whether detectable or not. "Treating" and "treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. As used herein, "treating" and "treatment" also include prophylactic treatment. For example, a subject with early-stage PD can be treated to prevent progression. Such a subject can be treated with a compound, antibody, immunogen, immunoconjugate, or composition described herein to prevent progression.

[0083] As used herein, "specifically binds" with respect to an antibody means that the antibody binds to its target antigen with higher affinity than a structurally or conformationally different antigen and / or an antigen with a modified or mutated sequence. For example, a multivalent antibody may have a K of at least 5e-5, at least 1e-6, at least 1e-7, at least 1e-8, or at least 1e-9. D It binds to its target with an affinity of at least 1e-7 or greater. An affinity of at least 1e-7 or greater is preferred. An antigen-binding fragment, such as a Fab fragment comprising one variable domain, may bind to its target with an affinity / avidity that is, for example, 10- or 100-fold lower than the multivalent interaction with the intact antibody.

[0084] As used herein, the terms "selective" or "selectively binds" with respect to an antibody that preferentially binds to a form of α-Syn (e.g., misfolded conformations such as misfolded oligomers and small soluble fibrils compared to isolated native monomers or native tetramers and / or insoluble fibrillar α-synuclein) means that the binding protein binds to that form with at least 2-fold, 3-fold, or at least 5-fold, at least 10-fold, at least 100-fold, at least 250-fold, at least 500-fold, or at least 1000-fold or greater affinity. Thus, an antibody that is more selective for a particular conformation (e.g., misfolded oligomers) will preferentially bind to a particular form of α-Syn with at least 3-fold greater affinity compared to another form.

[0085] As used herein, the term "linker" refers to a chemical moiety, preferably low or non-immunogenic, capable of being covalently attached directly or indirectly to the N- and / or C-terminus of an α-Syn peptide, including at least three amino acids of an EKTKEQ (SEQ ID NO: 1), optionally EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4) epitope peptide, attached to the N- and / or C-terminus of the peptide. The linker ends can be joined, for example, to produce a cyclic compound. The linker can contain one or more functionalizable moieties, such as one or more cysteine residues. The linker can be linked to a carrier protein or an immunogenicity enhancer, such as keyhole limpet hemocyanin (KLH), via the functionalizable moiety. Cyclic compounds containing a linker are longer in length than the peptide itself. That is, cyclization of a peptide with a linker (e.g., of three amino acid residues) creates a larger closed circle than a peptide without a linker. Linkers may include, but are not limited to, non-immunogenic moieties such as the amino acids G and A, or repeats of PEG.

[0086] The term "functionalizable moiety," as used herein, refers to a chemical entity having a "functional group," which refers to a group of atoms or a single atom that reacts with another group of atoms or a single atom (a so-called "complementary functional group") to form a chemical interaction between the two groups or atoms. In the case of cysteine, the functional group can be -SH, which can react to form a disulfide bond. Thus, the linker can be, for example, CCC. The reaction with another group of atoms can be a covalent bond or a strong non-covalent bond, as in the case of a biotin-streptavidin bond, which can have a Kd of about 1e-14. A strong non-covalent bond, as used herein, means an interaction with a Kd of at least 1e-9, at least 1e-10, at least 1e-11, at least 1e-12, at least 1e-13, or at least 1e-14.

[0087] Proteins and / or other drugs can be attached to the cyclic compounds to aid in immunogenicity or to act as probes in in vitro studies. For this purpose, any functionalizable moiety that can react (e.g., create a covalent or non-covalent but strong bond) can be used. In a specific embodiment, the functionalizable moiety is a cysteine residue that reacts to form a disulfide bond with an unpaired cysteine on the protein of interest, which can be, for example, an immunogenicity enhancer such as keyhole limpet hemocyanin (KLH) or a carrier protein such as bovine serum albumin (BSA) used in in vitro immunoblotting or immunohistochemical assays.

[0088] As used herein, the term "animal" or "subject" includes all members of the animal kingdom, including mammals, including humans.

[0089] A composition or method that "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" an antibody may contain the antibody alone or in combination with other components.

[0090] In understanding the scope of the present disclosure, the term "consisting of" and its derivatives, as used herein, is intended to be a limiting term that specifies the presence of stated features, elements, components, groups, integers, and / or steps and excludes the presence of other, unstated features, elements, components, groups, integers, and / or steps.

[0091] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also understood that all such numbers and fractions are presumed to be modified by the term "about." Furthermore, "a," "an," and "the" are understood to include plural referents unless the context clearly dictates otherwise. The term "about" means plus or minus 0.1 to 50%, 5 to 50%, or 10 to 40%, preferably 10 to 20%, and more preferably 10% or 15% of the referenced numerical value.

[0092] Furthermore, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments described herein where they are suitable, as understood by those skilled in the art. For example, in the following sections, different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other aspect or aspects, unless expressly indicated to the contrary. In particular, any feature indicated as preferred or advantageous can be combined with any other feature or features indicated as preferred or advantageous.

[0093] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0094] II. Epitopes and Epitope Compounds The inventors have identified sequences of α-Syn protein containing EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4) at amino acids 57-60, 58-61, and 59-62, respectively, which may be conformational epitopes, and therefore, for example, EKTK (SEQ ID NO: 2) and TKEQ (SEQ ID NO: 4), or portions of each, may be selectively accessible to antibody binding in misfolded oligomeric species of α-Syn.

[0095] Based on one or more conformational differences identified between epitopes identified in monomer, fibril, and / or biased α-Syn fibril ensembles, we designed conformationally restricted compounds and immunogens for generating antibodies.

[0096] As shown in the Examples, antibodies produced using the immunogen are useful for detecting or targeting misfolded oligomeric α-Syn.

[0097] As described in the Examples, cyclic compounds such as the cyclic peptides cyclo(CGGGGEKTKGG) (SEQ ID NO: 5), cyclo(CGTKEQGGGG) (SEQ ID NO: 7), cyclo(CGGGEKTKGG) (SEQ ID NO: 10), and cyclo(CGGGGTKEQGG) (SEQ ID NO: 11) were identified to capture conformational differences of corresponding epitopes in misfolded oligomeric species of α-Syn compared to monomeric and / or insoluble fibril species. For example, the RMSD structural alignment of amino acids in the cyclic 10-mer cyclo(CGTKEQGGGG) (SEQ ID NO: 7) was found to be significantly different from the corresponding abundance in the monomeric ensemble. This suggests that cyclic compounds may present conformational epitopes that are conformationally distinct from the sequences presented on nascent monomeric α-Syn and / or insoluble fibrils.

[0098] Thus, the present disclosure identifies conformational epitopes in α-Syn, such as the peptides EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4), or portions thereof, such as EK, which corresponds to amino acid residues 57-58 on α-Syn, and KEQ, which corresponds to amino acids 60-62 on α-Syn. As shown in the Examples, EKTK (SEQ ID NO: 2) and TKEQ (SEQ ID NO: 4) were identified as regions prone to disorder in α-Syn. Residues EKTK (SEQ ID NO: 2) and TKEQ (SEQ ID NO: 4) emerged in predictions using ensemble coordinate methods as described in the Examples.

[0099] Aspects include compounds comprising an α-Syn peptide comprising at least three amino acids of EKTKEQ (SEQ ID NO: 1), optionally EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), and / or a portion of any of the foregoing, such as KEQ. In embodiments, the α-Syn peptide is selected from EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), TKEQ (SEQ ID NO: 4), EKTKE (SEQ ID NO: 8), EKT, KTK, TKE, KEQ, or KTKEQ (SEQ ID NO: 9).

[0100] The alpha-syn peptide may also include an additional 1, 2, or 3 amino acids to alpha-syn either N-terminal and / or C-terminal to EKTKEQ (SEQ ID NO: 1) (or an internal sequence thereof, such as EKT or EKTK (SEQ ID NO: 2) at the 1, 2, or 3 N-terminal amino acid residues), along with the 1, 2, or 3 C-terminal amino acid residues. In some embodiments, the maximum length of the alpha-syn peptide is 9, 8, or 7 amino acids.

[0101] In embodiments, the alpha-Syn peptide comprises or consists of KEQ, TKEQ (SEQ ID NO: 4), KTKEQ (SEQ ID NO: 9), or EKTKEQ (SEQ ID NO: 1).

[0102] In embodiments, the compound further comprises a linker. The linker may comprise one or more functionalizable moieties. The linker may comprise, for example, 1, 2, 3, 4, 5, 6, 7, or 8 amino acids and / or equivalently functional molecules such as polyethylene glycol (PEG) moieties, and / or combinations thereof. In embodiments, the linker amino acids are selected from non-immunogenic or low-immunogenic amino acid residues such as G and A. For example, the linker may be GG, GGG, GAG, G(PEG)G, PEG-PEG (also referred to as PEG2)-GG, etc. One or more functionalizable moieties, e.g., amino acids, having functional groups can be included, for example, to link the compound to a drug or detectable tag, or a carrier such as BSA, or an immunogenicity enhancer such as KLH. The functionalizable moiety may be an amino acid such as cysteine. In embodiments, the linker comprises at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.

[0103] In embodiments, the linker comprises GC-PEG, PEG-GC, GCG, or PEG2-CG. In other embodiments, the linker comprises GCGGGG (SEQ ID NO: 12), GGCGG (SEQ ID NO: 13), GGCGGGG (SEQ ID NO: 14), GGGCGG (SEQ ID NO: 15), or GGGGCGG (SEQ ID NO: 16). Other linkers are provided in Tables 2-4 (presented in constructs containing α-Syn peptide).

[0104] The proteinaceous portion of the compound (or a compound where the linker is also proteinaceous) can be prepared by chemical synthesis using techniques well known in protein chemistry such as solid phase synthesis or synthesis in homogeneous solution.

[0105] The compounds can be linear and can be used, for example, to select antibodies that preferentially bind to the corresponding cyclic compounds. Preferably, the compounds are conformational compounds, such as cyclic compounds. As shown in the examples, this can be achieved using cyclic peptides, including α-Syn peptides.

[0106] Thus, aspects provide cyclic compounds comprising an α-syn peptide comprising at least three amino acids of EKTKEQ (SEQ ID NO: 1), optionally EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), and / or a portion of any of the foregoing, and a linker, wherein the linker is directly or indirectly covalently attached to the α-syn peptide. As shown in the examples, residues of the cyclic peptide are in alternative conformations compared to the corresponding residues of the monomer or fibril ensemble. In embodiments, the cyclic compound comprises an α-syn peptide and a linker as described herein. In embodiments, the cyclic compound comprises EKT, KEQ, EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), and up to six α-Syn residues (e.g., one or two amino acids N- and / or C-terminal to EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4)), and a linker, where the linker is directly or indirectly covalently attached to the N- and C-terminal peptide residues of the α-Syn peptide. The exposure of residues in the cyclic peptide may differ from the corresponding residues in monomer and / or fibrillar ensembles, and in cellular monomeric and insoluble fibrillar α-Syn. For example, in the cyclic compound, at least one of E57, K58, T59, K60, E61, or Q62 has more surface exposure than the conformation occupied in the monomer ensemble.

[0107] Peptides containing EKTK (SEQ ID NO:2), EKT, EKTKE (SEQ ID NO:8), KTKEQ (SEQ ID NO:9), KEQ, or TKEQ (SEQ ID NO:4) contain one, two, or three additional residues found in α-Syn that are N- and / or C-terminal to EKTK (SEQ ID NO:2) or TKEQ (SEQ ID NO:4), and a linker in the cyclized compound is covalently attached to the N- and / or C-termini of the additional α-Syn residues. Similarly, when the α-Syn peptide is EKTK (SEQ ID NO:2), the linker is covalently attached to residues E and K; when the α-Syn peptide is TKEQ (SEQ ID NO:4), the linker is covalently attached to residues T and Q; and when the α-Syn peptide is KTKEQ (SEQ ID NO:9), the linker is covalently attached to residues K and Q.

[0108] In embodiments, the cyclic compound comprises a peptide comprising or consisting of EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), and a linker, wherein the linker is attached to the N-terminus and C-terminus of the peptide.

[0109] In embodiments, the alternate conformation is a more solvent exposed conformation for one or more of residues E57, K58, T59, K60, E61, or Q62.

[0110] In one embodiment, the cyclic compound is a cyclic peptide. In another embodiment, the cyclic peptide comprises or consists of the sequence of any one of SEQ ID NOS: 5, 7, and 10-60. In one embodiment, the cyclic peptide comprises or consists of the sequence CGGGGEKTKGG (SEQ ID NO: 5). In another embodiment, the cyclic peptide comprises or consists of the sequence CGTKEQGGGG (SEQ ID NO: 7). In another embodiment, the cyclic peptide comprises or consists of the sequence CGGGEKTKGG (SEQ ID NO: 10).

[0111] Cyclic peptides and corresponding linear peptides can be referenced, for example, by identifying the position of the linker residue relative to the α-Syn peptide and the functionalizable moiety. For example, CGGGGEKTKGG (SEQ ID NO: 5) can be referred to as a 4,2 construct, CGTKEQGGGG (SEQ ID NO: 7) can be referred to as a 1,4 construct, and CGGGEKTKGG can be referred to as a 3,2 construct.

[0112] Methods for producing cyclized peptides are known in the art and include SS cyclization or amide cyclization (head-to-tail, or backbone cyclization). Methods are further described in the Examples section. For example, a peptide with "C" residues at its N- and C-termini, e.g., CGGEKTKGGC (SEQ ID NO: 17), can be reacted by SS cyclization to produce a cyclic peptide. Cyclic compounds can be synthesized as linear molecules with a linker covalently attached to the N- or C-terminus of an α-Syn peptide, optionally EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), or a peptide containing a related epitope, prior to cyclization. Alternatively, a portion of the linker is covalently attached to the N-terminus and a portion is covalently attached to the C-terminus prior to cyclization. In either case, the linear compound is cyclized, for example, by head-to-tail cyclization (e.g., amide bond cyclization).

[0113] As described in the Examples, cyclic compounds were identified, synthesized, and used to prepare immunogens and evaluated for their relevance to conformational epitopes used to generate antibodies selective for misfolded oligomeric α-Syn. The epitopes EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), described herein, and / or portions thereof, may be potential targets in misfolded transmissible strains of α-Syn, and antibodies recognizing conformational epitopes such as those shown herein are useful for detecting misfolded species and inhibiting such transmissible strains. As described above, cyclic compounds containing α-Syn peptides can be used as immunogens to generate antibodies.

[0114] Thus, another aspect includes immunogens comprising the conformational compounds described herein, optionally cyclic compounds such as cyclic peptides. In embodiments, the immunogen includes an immunogenicity enhancer such as keyhole limpet hemocyanin (KLH) or a carrier such as bovine serum albumin (BSA) or ovalbumin. The immunogenicity enhancer can be attached to the compound directly, such as via an amide bond, or indirectly via a chemical linker. Alternatively, the immunogen can be a multiple antigenic peptide (MAP).

[0115] Immunogens can be produced by conjugating cyclic compounds containing restricted α-Syn epitope peptides to immunogenicity enhancing agents such as KLH or carriers such as BSA, for example, using the methods described in Lateef et al., 2007, incorporated herein by reference. In embodiments, the methods described in Examples 3 and 4 are used.

[0116] III. Antibodies The compounds described herein, particularly cyclic compounds containing any three amino acid residues of the α-syn peptides EKTK (SEQ ID NO:2), KTKE (SEQ ID NO:3), or EKTKEQ (SEQ ID NO:1), such as TKEQ (SEQ ID NO:3), can be used to generate antibodies that selectively bind to compounds containing the α-syn peptide compared to the corresponding linear compounds and / or that also bind to epitopes in the α-syn peptide of misfolded forms of α-syn, including misfolded oligomeric α-syn, compared to monomeric and / or insoluble α-syn fibrils. As shown in the Examples, the cyclic compounds exhibit one or more spatial conformations similar to partially unfolded fibrillar α-syn (biased α-syn), but distinct from unbiased fibrillar α-syn. Furthermore, antibodies generated using the compounds have been shown to be selective for cyclic peptides and to selectively bind misfolded α-syn, such as misfolded oligomeric α-syn, relative to native species, indicating that they preferentially recognize the conformation of these residues in misfolded α-syn. For example, as shown in the Examples, the antibodies generated preferentially bind misfolded oligomeric species relative to monomeric and insoluble fibrillar species.

[0117] Similarly, cyclic compounds comprising, for example, EKT, KTK, TKE, KEQ, EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), TKEQ (SEQ ID NO: 4), EKTKE (SEQ ID NO: 8), or KTKEQ (SEQ ID NO: 9), and / or other related epitope sequences described herein, can be used to generate antibodies that selectively bind, for example, EKT, KTK, TKE, KEQ, EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), TKEQ (SEQ ID NO: 4), EKTKE (SEQ ID NO: 8), or KTKEQ (SEQ ID NO: 9), in the context of misfolded oligomeric α-Syn.

[0118] Thus, aspects include antibodies that bind to an epitope in an α-Syn peptide, the α-Syn peptide comprising or consisting of EKTKEQ (SEQ ID NO: 1), or a related epitope thereof, e.g., a portion thereof comprising at least three or at least four amino acids, in embodiments, an α-Syn peptide selected from EKT, KTK, TKE, KEQ, EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4).

[0119] In embodiments, the epitope is a conformational epitope.

[0120] The α-Syn peptides can be in cyclic compounds and / or misfolded forms of α-Syn, such as misfolded oligomeric α-Syn. In embodiments, the antibodies selectively bind to cyclic compounds containing α-Syn peptides compared to the corresponding linear compounds. In other embodiments, the antibodies selectively bind to α-Syn peptides in misfolded forms of α-Syn, such as oligomeric α-Syn, compared to monomeric or insoluble fibrillar α-Syn.

[0121] In embodiments, the antibody is isolated.

[0122] In embodiments, the antibody does not selectively bind to isolated, native monomeric α-Syn relative to misfolded forms, such as misfolded oligomeric α-Syn. Binding, including selective binding, can be measured, for example, as described herein, using, for example, ELISA or surface plasmon resonance assays.

[0123] Thus, a further aspect is a cyclic compound comprising the α-syn peptide, or an antibody that specifically or selectively binds to a conformational epitope in the α-syn peptide of misfolded oligomeric α-syn, wherein the α-syn peptide or epitope comprises or consists of EKTKEQ (SEQ ID NO: 1), EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), or a portion thereof, such as EKT, KTK, TKE, or KEQ. In some embodiments, the α-syn peptide or epitope is EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4). In one embodiment, the α-syn peptide or epitope is EKTK (SEQ ID NO: 2). In another embodiment, the α-syn peptide or epitope is KTKE (SEQ ID NO: 3). In yet another embodiment, the α-syn peptide or epitope is TKEQ (SEQ ID NO: 4).

[0124] In embodiments, the epitope comprises or consists of at least two consecutive amino acid residues primarily responsible for binding to the antibody, wherein the at least two consecutive amino acids are EK, or KT, or TK, or KE, or EQ embedded within EKTK (SEQ ID NO: 2), or KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), correspondingly.

[0125] In another embodiment, the epitope is a conformational epitope and consists of EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4). In embodiments, the antibody selectively binds to EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4) in a cyclic peptide, optionally cyclo(CGTKEQGGGG) (SEQ ID NO: 7), cyclo(CGGTKEQGG) SEQ ID NO: 48, cyclo(CGGTKEQGGGG) SEQ ID NO: 49, cyclo(CGGGEKTKGG) SEQ ID NO: 10, or cyclo(CGGGGEKTKGG) SEQ ID NO: 5, compared to the corresponding linear compound.

[0126] In embodiments, the antibody selectively binds to an α-Syn peptide or epitope in a cyclic compound, where the α-Syn peptide comprises or consists of EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), optionally in the context of cyclo(CGTKEQGGGG) (SEQ ID NO: 7) or other cyclic peptide sequences set forth in Tables 2-4, compared to the corresponding linear peptide and / or monomeric or insoluble fibrillar α-Syn. For example, in embodiments, the antibody selectively binds to TKEQ (SEQ ID NO: 4) in a cyclic compound, optionally a cyclic peptide such as cyclo(CGTKEQGGGG) (SEQ ID NO: 7), and has greater selectivity (e.g., binding affinity) for TKEQ (SEQ ID NO: 4) in a cyclic conformation compared to the corresponding linear peptide and / or TKEQ (SEQ ID NO: 4) in monomeric or insoluble fibrillar α-Syn, optionally as measured by, for example, ELISA or surface plasmon resonance, using a method described herein, that is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold more selective.

[0127] In embodiments, the antibodies selectively bind to α-Syn peptides or epitopes in misfolded oligomeric α-Syn polypeptides compared to native α-Syn, and in embodiments, the selectivity is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold more selective for misfolded oligomeric α-Syn polypeptides than for monomeric or insoluble fibrillar α-Syn.

[0128] In embodiments, the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of one or more of the CDRs is the amino acid sequence set forth below: CDR-H1: SEQ ID NO: 61, 67, 73, 79, 91, or 180; CDR-H2: SEQ ID NO: 62, 68, 74, 80, 92, or 181; CDR-H3: SEQ ID NO: 63, 69, 75, 81, 93, or 182; CDR-L1: SEQ ID NO: 64, 70, 76, 94, or 183; CDR-L2: SEQ ID NO: 65, 71, or 77; or CDR-L3: selected from SEQ ID NO: 66, 72, 78, 84, 96, or 184.

[0129] In embodiments, the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 63, 69, 75, 81, 93, or 182, and the antibody selectively binds to an α-Syn peptide or epitope described herein in misfolded oligomeric α-Syn compared to native α-Syn.

[0130] In one embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of one or more of the CDRs are selected from the amino acid sequences of SEQ ID NOs: 61 to 66.

[0131] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 61, 62, 63, 64, 65, and 66, respectively.

[0132] In another embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of one or more of the CDRs are selected from the amino acid sequences of SEQ ID NOs: 67 to 72.

[0133] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 67, 68, 69, 70, 71, and 72, respectively.

[0134] In another embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of one or more of the CDRs are selected from the amino acid sequences of SEQ ID NOs: 73 to 78.

[0135] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 73, 74, 75, 76, 77, and 78, respectively.

[0136] In another embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of one or more of the CDRs are selected from the amino acid sequences of SEQ ID NOs: 76-77, 79-81, and 84.

[0137] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 76-77, 79, 80, 81, and 84, respectively.

[0138] In another embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of one or more of the CDRs are selected from the amino acid sequences of SEQ ID NOs: 79-81, 76-77, and 84.

[0139] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 79, 80, 81, 76, 77, and 84, respectively.

[0140] In another embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of one or more of the CDRs are selected from the amino acid sequences of SEQ ID NOs: 71, 91-94, and 96.

[0141] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 91, 92, 93, 94, 71, and 96, respectively.

[0142] In another embodiment, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of one or more of the CDRs is selected from the amino acid sequences of SEQ ID NOs: 180, 181, 182, 183, 77, and 184.

[0143] In certain embodiments, the antibody comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 180, 181, 182, 183, 77, and 184, respectively.

[0144] In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 133, 135, 137, 139, 141, 143, and 190, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 133, 135, 137, 139, 141, 143, and 190, wherein the CDR sequences are maintained.

[0145] In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 133. In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 135. In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 137. In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 139. In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 141. In another embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 143. In a further embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 190, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 190.

[0146] In embodiments, the light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 134, 136, 138, 140, 142, 144, and 191, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 134, 136, 138, 140, 142, 144, and 191, wherein the CDR sequences are maintained.

[0147] In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 134. In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 136. In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 138. In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 140. In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 142. In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 144. In another embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 191, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 191.

[0148] In another embodiment, the heavy and light chain variable regions are the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 133 and SEQ ID NO: 134, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 134. In another embodiment, the heavy and light chain variable regions are the amino acid sequence of SEQ ID NO: 135, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 135 and SEQ ID NO: 136, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 136, respectively. In another embodiment, the heavy and light chain variable regions are the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 137 and SEQ ID NO: 138, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 138. In another embodiment, the heavy and light chain variable regions are the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 139 and SEQ ID NO: 140, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 140. In another embodiment, the heavy chain variable region and the light chain variable region are the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 141 and SEQ ID NO: 142, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 142.In another embodiment, the heavy and light chain variable regions are the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 143 and SEQ ID NO: 144, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 144. In another embodiment, the heavy and light chain variable regions are the amino acid sequence of SEQ ID NO: 190, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 190 and SEQ ID NO: 191, respectively, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 191, respectively.

[0149] Tables 13 and 14 below show the complementarity-determining regions (CDRs) and heavy and light chain nucleic acid and amino acid sequences, respectively, of antibody clones 2E9, 9D8, 12G1, 3C11, 12B12, 10D5, and 11B6, as determined by IgBLAST. The heavy and light chain CDRs in Table 14 are shown in bold. In some embodiments, the CDR sets, heavy and / or light chain variable regions are as described therein. In other embodiments, the antibody or nucleic acid comprises the sequences described therein.

[0150] In embodiments, the antibody is selected from the group consisting of a monoclonal antibody, an immunoglobulin molecule, Fab, Fab', F(ab)2, F(ab')2, Fv, disulfide-linked Fv, scFv, disulfide-linked scFv, single-chain antibody, single-domain antibody, diabody, dimer, minibody, bispecific antibody fragment, chimeric antibody, humanized antibody, and polyclonal antibody.

[0151] In embodiments, the antibody is a monoclonal antibody.

[0152] In embodiments, the antibody is a humanized antibody.

[0153] In embodiments, the antibody is a single chain antibody, optionally a humanized single chain antibody.

[0154] In embodiments, the antibody is a binding fragment, such as a Fab fragment, optionally a humanized Fab fragment.

[0155] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be taken from a subject immunized with an immunogen described herein and fused with myeloma cells by standard somatic cell fusion procedures to immortalize these cells and generate hybridoma cells. Such techniques (e.g., the hybridoma technique first developed by Kohler and Milstein (Nature 256:495-497 (1975)), as well as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67 (1986)), and the screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989)) are well known in the art. Hybridoma cells can be screened immunochemically for the production of antibodies specifically reactive with a desired epitope, and monoclonal antibodies can be isolated.

[0156] Specific antibodies or antibody fragments reactive with specific antigens or molecules can also be generated by screening expression libraries encoding immunoglobulin genes or portions thereof expressed in bacteria bearing cell surface components. For example, complete Fab fragments, VH regions, and FV regions can be expressed in bacteria using phage expression libraries (see, e.g., Ward et al., Nature 41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); and McCafferty et al., Nature 348:552-554 (1990)).

[0157] Humanization of antibodies from non-human species has been well described in the literature. See, for example, EP-B10239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997, which are incorporated herein by reference in their entirety). Humanized antibodies are also readily available commercially (e.g., Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).

[0158] Humanized versions of rodent antibodies are easily produced by CDR grafting (Riechmann et al. Nature, 332:323-327, 1988). In this approach, the six CDR loops that make up the antigen-binding site of a rodent monoclonal antibody are linked to corresponding human framework regions. Because amino acids in the framework regions can affect antigen recognition, CDR grafting often produces antibodies with reduced affinity (Foote & Winter. J Mol Biol, 224:487-499, 1992). To maintain antibody affinity, it is often necessary to replace specific framework residues by site-directed mutagenesis or other recombinant techniques, which can be aided by computer modeling of the antigen-binding site (Co et al. J Immunol, 152:2968-2976, 1994).

[0159] Humanized forms of antibodies are optionally obtained by resurfacing (Pedersen et al. J Mol Biol, 235:959-973, 1994). In this approach, only the surface residues of a rodent antibody are humanized.

[0160] Human antibodies specific to a particular antigen can be identified by phage display strategies (Jespers et al., Bio / Technology, 12:899-903, 1994). In one approach, the heavy chain of a rodent antibody directed against a particular antigen is cloned and paired with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage are selected by binding to the antigen. The selected human light chains are then paired with a repertoire of human heavy chains for display on the phage, and the phage are again selected by binding to the antigen. This results in human antibody Fab fragments specific to the particular antigen. In another approach, a library of phage is produced, each member of which displays different human antibody fragments (Fab or Fv) on its outer surface (Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047). Phage-displayed antibodies with the desired specificity are selected by affinity enrichment against a specific antigen. Human Fab or Fv fragments identified from either approach can be recloned for expression as human antibodies in mammalian cells.

[0161] Human antibodies are optionally obtained from transgenic animals (U.S. Patent Nos. 6,150,584, 6,114,598, and 5,770,429). In this approach, the heavy chain joining region (JH) gene of chimeric or germline mutant mice is deleted. The human germline immunoglobulin gene array is then transferred into such mutant mice. The resulting transgenic mice are capable of producing a full repertoire of human antibodies upon antigen challenge.

[0162] Humanized or human antibodies can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, or IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized or human antibodies can contain sequences from more than one isotype or class. Furthermore, these antibodies are typically produced as antigen-binding fragments and single-domain antibody fragments, such as Fab, Fab', F(ab'), Fd, and Fv, or as single-chain antibodies in which heavy and light chains are linked by a linker. Human or humanized antibodies can also exist in monomeric or polymeric form. Humanized antibodies optionally contain one non-human chain and one humanized chain (i.e., one humanized heavy or light chain).

[0163] Furthermore, antibodies specific to the epitopes described herein can be easily isolated by screening antibody phage display libraries. For example, antibody phage libraries can be optionally screened using the disease-specific epitopes of the present disclosure to identify antibody fragments specific to the disease-specific epitopes. The identified antibody fragments can be optionally used to produce various recombinant antibodies that are useful in different embodiments of the present disclosure. Antibody phage display libraries are commercially available, for example, through Xoma (Berkeley, California). Methods for screening antibody phage libraries are well known in the art.

[0164] Another aspect is an immunoconjugate comprising an antibody disclosed herein and a moiety such as a detectable label or particle.

[0165] The detectable label can be, for example, a polypeptide fused to the antibody (e.g., a fusion moiety), such as a fluorescent protein, or an optionally cleavable purification tag, such as a FLAG tag, a histidine tag, etc. For example, the detectable label can be streptavidin, or a fluorescent dye (e.g., Cy3, Cy4, Cy5).

[0166] The detectable label can also be a positron-emitting radionuclide. Other detectable labels are described elsewhere herein.

[0167] The particle can be, for example, a magnetic particle, such as a magnetic bead, to which an antibody is conjugated, a gold particle, a resin, or an agarose.One or more different antibodies described herein can be conjugated to the particle.Antibodies can be conjugated to the particle by covalent bonding, for example, via amine, carboxyl, or maleimide functional groups.

[0168] A further aspect is a nucleic acid encoding the amino acid residues of a compound or immunogen described herein.

[0169] A further embodiment is a nucleic acid encoding an antibody or immunoconjugate described herein, such as a single-chain, single-domain, and / or humanized antibody, optionally contained in a vector. The nucleic acid sequence can be determined, for example, by sequencing the immunoglobulin gene transcripts expressed by the hybridoma using cDNA generated therefrom using standard RT-PCR, e.g., using standard dye terminator capillary sequencing.

[0170] The nucleic acid can also encode any portion thereof, such as a CDR, for example, comprising at least 10 nucleotides. The nucleic acid can also be a primer for amplifying one or more of the sequences described herein.

[0171] The nucleic acid may include other components, such as a signal sequence.

[0172] In embodiments, the nucleic acid encoding the antibody comprises a nucleic acid sequence encoding a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of one or more of the CDRs being as set forth below: CDR-H1: SEQ ID NO: 61, 67, 73, 79, 91, or 180; CDR-H2: SEQ ID NO: 62, 68, 74, 80, 92, or 181; CDR-H3: SEQ ID NO: 63, 69, 75, 81, 93, or 182; CDR-L1: SEQ ID NO: 64, 70, 76, 94, or 183; CDR-L2: SEQ ID NO: 65, 71, or 77; or CDR-L3: selected from SEQ ID NO: 66, 72, 78, 84, 96, or 184.

[0173] In embodiments, the nucleic acid encoding the antibody comprises a nucleic acid sequence encoding a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequence of CDR-H3 is selected from SEQ ID NOs: 63, 69, 75, 81, 93, and 182.

[0174] In embodiments, the nucleic acid encoding the antibody comprises a nucleic acid sequence encoding a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, the amino acid sequence of one or more of the CDRs being selected from the nucleic acid sequences set forth below: CDR-H1: SEQ ID NO: 97, 103, 109, 115, 127, or 185; CDR-H2: SEQ ID NO: 98, 104, 110, 116, 128, or 186; or CDR-H3: encoded by SEQ ID NO: 99, 105, 111, 117, 123, 129, or 187.

[0175] For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 97 to 99, respectively. For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 103 to 105, respectively. For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 109 to 111, respectively. For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 115 to 117, respectively. For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 115 to 116 and 123, respectively. For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can each be encoded by a nucleic acid sequence selected from SEQ ID NOs: 127 to 129. For example, the complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 can each be encoded by the nucleic acid sequences of SEQ ID NOs: 185 to 187.

[0176] In another embodiment, the nucleic acid encoding the antibody comprises a nucleic acid encoding a light chain variable region, the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of one or more of the CDRs is selected from the group consisting of the nucleic acid sequences set forth below: CDR-L1: SEQ ID NO: 100, 106, 112, 115, 130, or 188; CDR-L2: SEQ ID NO: 101, 107, or 113; or CDR-L3: encoded by SEQ ID NO: 102, 108, 114, 120, 132, or 189.

[0177] For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 100 to 102, respectively. For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 106 to 108, respectively. For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 112 to 114, respectively. For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 112 to 113, and 120, respectively. For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 112 to 113, and 120, respectively. For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 107, 130, and 132, respectively. For example, the complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 can be encoded by the nucleic acid sequences of SEQ ID NOs: 188, 113, and 189, respectively.

[0178] In embodiments, the nucleic acid encodes an antibody and comprises: i) a first nucleic acid molecule encoding a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3; and ii) a second nucleic acid molecule encoding a light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of one or more of the CDRs being selected from the nucleic acid sequences set forth below: CDR-H1: SEQ ID NO: 97, 103, 109, 115, 127, or 185; CDR-H2: SEQ ID NO: 98, 104, 110, 116, 128, or 186; CDR-H3: SEQ ID NO: 99, 105, 111, 117, 123, 129, or 187; CDR-L1: SEQ ID NO: 100, 106, 112, 130, or 188; CDR-L2: SEQ ID NO: 101, 107, or 113; or CDR-L3: encoded by SEQ ID NO: 102, 108, 114, 120, 132, or 189.

[0179] The first and second nucleic acid molecules can be fused together as an expression cassette or can be included in a vector as separate expression cassettes.

[0180] The nucleic acid can comprise a set of CDRs described herein.

[0181] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 97, 98, 99, 100, 101, and 102, respectively.

[0182] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 103, 104, 105, 106, 107, and 108, respectively.

[0183] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 109, 110, 111, 112, 113, and 114, respectively.

[0184] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 115, 116, 117, 112, 113, and 120, respectively.

[0185] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 115, 116, 123, 112, 113, and 120, respectively.

[0186] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 127, 128, 129, 130, 107, and 132, respectively.

[0187] In certain embodiments, CDRs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L1, CDR-L2, and CDR-L3 are encoded by SEQ ID NOs: 185-188, 113, and 189, respectively.

[0188] In another embodiment, the heavy chain variable region of the antibody is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs: 145, 147, 149, 151, 153, 155, and 192; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to any of the foregoing, wherein the amino acid sequences of the CDR regions are maintained; or a sequence encoding any one of SEQ ID NOs: 133, 135, 137, 139, 141, 143, and 190, or encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 133, 135, 137, 139, 141, 143, and 190, wherein the CDR amino acid sequences are maintained.

[0189] In further embodiments, the light chain variable region of the antibody is encoded by a nucleic acid sequence comprising any one of SEQ ID NOs: 146, 148, 150, 152, 154, 156, and 193; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to any of the foregoing, wherein the amino acid sequences of the CDR regions are maintained; or a sequence encoding any one of SEQ ID NOs: 134, 136, 138, 140, 142, 144, and 191, or encoding an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 134, 136, 138, 140, 142, 144, and 191, wherein the CDR sequences are maintained.

[0190] In embodiments, the nucleic acid is an isolated nucleic acid.

[0191] The vector can be any vector, including vectors suitable for producing antibodies or expressing the peptide sequences described herein.

[0192] The nucleic acid molecule can be incorporated into an appropriate expression vector, ensuring protein expression, using known methods. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The vector must be compatible with the host cell used. The expression vector is suitable for transforming a host cell, which means that the expression vector contains a nucleic acid molecule encoding an antibody described herein. For example, nucleic acid molecules encoding the heavy chain variable region and the light chain variable region can be inserted into separate vectors. For example, nucleic acid molecules encoding the heavy chain variable region and the light chain variable region can be inserted into the same expression vector.

[0193] In embodiments, the vector comprises one or more of the nucleic acid sequences encoding any of the amino acid sequences described herein, or comprises any of the nucleic acids described herein, e.g., one or more of SEQ ID NOs: 97-117, 120, 123, 127-130, 132, 145-156, and 185-189.

[0194] In some embodiments, the vector is suitable for expressing single-chain antibodies, for example, by gene therapy. The vector can be adapted for specific expression in neural tissue, for example, by using a neural-specific promoter. In some embodiments, the vector contains an IRES, allowing the expression of light chain variable regions and heavy chain variable regions. Such vectors can be used to deliver antibodies in vivo.

[0195] Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes.

[0196] Examples of such regulatory sequences include transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosomal binding sequences including translation initiation signals, etc. Furthermore, depending on the host cell selected and the vector used, other sequences such as replication origins, additional DNA restriction sites, enhancers, and sequences that confer inducibility of transcription can be incorporated into the expression vector.

[0197] In embodiments, the regulatory sequences direct or increase expression in neural tissues and / or cells.

[0198] In embodiments, the vector is a viral vector.

[0199] The recombinant expression vectors may also contain a marker gene that facilitates selection of host cells that have been transformed, infected or transfected with the vector to express the antibodies or epitopic peptides described herein.

[0200] Recombinant expression vectors can also contain expression cassettes encoding fusion moieties (i.e., "fusion proteins") that provide increased expression or stability of the recombinant peptide, increase the solubility of the recombinant peptide, and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, the tags and labels described herein. Additionally, a proteolytic cleavage site can be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Exemplary fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the recombinant protein.

[0201] For example, systems for transferring genes into neurons and neural tissues both in vitro and in vivo include vectors based on viruses, particularly herpes simplex viruses, adenoviruses, adeno-associated viruses (AAVs), and retroviruses, including lentiviruses. Alternative approaches to gene delivery include the use of naked plasmid DNA and liposome-DNA complexes. Another approach is the use of AAV plasmids, in which DNA is polycationically condensed and lipid-entrapped, and introduced into the brain via intracerebral gene delivery (Leone et al., U.S. Application No. 2002 / 076394).

[0202] In embodiments, the vector includes a nucleic acid sequence containing a signal sequence (enabling intracellular antibody expression). Any signal peptide suitable for expression of secretable chain precursors can be used that allows proper externalization, including folding and disulfide formation, to produce the desired antibody as a secreted, dimerized, and processed protein. For example, the signal sequence can be selected from any one of SEQ ID NOs: 157-164, 169-177, and 179.

[0203] In embodiments, the vector comprises a nucleic acid sequence, optionally one in Table 15, from which the signal sequence has been deleted.

[0204] In another aspect, cells expressing the antibodies described herein are also provided.

[0205] In embodiments, the cells are fusion cells such as hybridomas.

[0206] In embodiments, the cell is a recombinant cell. In embodiments, the cell is a mammalian cell, optionally a CHO cell.

[0207] Recombinant cells can be generated using any cell suitable for producing polypeptides, for example, suitable for producing antibodies and / or binding fragments thereof.

[0208] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, or mammalian cells.

[0209] More specifically, bacterial host cells suitable for generating recombinant antibody-producing cells include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to those skilled in the art. Suitable bacterial expression vectors preferably contain a promoter functional in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the β-lactamase (penicillinase) and lactose promoter systems, the trp promoter, and the tac promoter. Representative selectable markers include various antibiotic resistance markers, such as kanamycin or ampicillin resistance genes. Suitable expression vectors include, but are not limited to, bacteriophages such as lambda derivatives or plasmids such as pBR322, pUC plasmids pUC18, pUC19, pUC118, pUC119, and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).

[0210] Suitable yeast and fungal host cells include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, various species of Pichia or Kluyveromyces, and Aspergillus. Examples of vectors for expression in yeast S. cerivisiae include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for transformation of yeast and fungi are well known to those skilled in the art.

[0211] Suitable mammalian cells include, among others, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells, and any derivatives of these lines.

[0212] In embodiments, the mammalian cells used to produce the recombinant antibody are selected from CHO, HEK293 cells, or FreeStyle™ 293-F cells (Life Technologies). The FreeStyle 293-F cell line is derived from the 293 cell line and can be used in the FreeStyle™ MAX 293 Expression System, the FreeStyle™ 293 Expression System, or other expression systems.

[0213] Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, adenovirus 2, cytomegalovirus, and simian virus 40) and other transcriptional and translational control sequences.

[0214] Suitable insect cells include cells and cell lines from the species Bombyx or Spodotera. Baculovirus vectors available for expression of proteins in cultured insect cells (SF9 cells) include the pAc series and pVL series.

[0215] Recombinant expression vectors can also contain genes encoding fusion moieties (i.e., "fusion proteins") that provide increased expression or stability of the recombinant peptide, increase the solubility of the recombinant peptide, and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, the tags and labels described herein. Additionally, a proteolytic cleavage site can be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Exemplary fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the recombinant protein.

[0216] In embodiments, expression of the antibody or binding fragment thereof is under the control of an inducible promoter. Examples of inducible non-fusion expression vectors include pTrc (ThermoFisher Scientific) and pET 11d.

[0217] The recombinant expression vector may also contain a marker gene that facilitates the selection of host cells transformed or transfected with the recombinant molecule of the present invention. Examples of selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, such as the Fc portion of immunoglobulins, preferably IgG. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein, such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein that confers antibiotic resistance, such as neomycin-resistant transformants, selection can be performed with G418. Cells that have incorporated the selectable marker gene will survive, while other cells will die. This allows for visualization and assay of the expression of the recombinant expression vector of the present invention, particularly to determine the effects of mutations on expression and phenotype. It will be understood that the selectable marker can be introduced into a vector separate from the nucleic acid of interest. Other selectable markers include fluorescent proteins such as GFP, which may be co-transfected with the nucleic acid of interest.

[0218] IV. Composition A further embodiment is a composition comprising the compounds, immunogens, immunoconjugates, antibodies, nucleic acids, vectors, and / or cells described herein. The composition may comprise two or more, three or more, or any combination of the components described herein. For example, the composition may comprise two or more antibodies, two or more immunoconjugates, two or more immunogens, etc.

[0219] In an embodiment, the composition comprises a diluent.

[0220] Suitable diluents for polypeptides, including antibodies and / or cells, include, but are not limited to, saline, pH buffers, and glycerol solutions, or other solutions suitable for freezing polypeptides and / or cells. Suitable diluents for nucleic acids include, but are not limited to, water, saline, and ethanol.

[0221] In embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. In embodiments, the composition is a pharmaceutical composition, e.g., for a method described herein, e.g., for treating a subject having a synucleinopathy or in need of inhibiting misfolded alpha-synuclein toxicity.

[0222] One or more antibodies can be administered in combination or with other treatments for the conditions or diseases described herein.

[0223] The compositions described herein can be prepared by methods known per se for preparing pharmaceutically acceptable compositions, which can optionally be administered to a subject as a vaccine, such that an effective amount of the active substance is combined in admixture with a pharmaceutically acceptable vehicle.

[0224] In embodiments comprising a compound or immunogen described herein, the composition comprises an adjuvant.

[0225] For example, adjuvants that can be used include intrinsic adjuvants (such as lipopolysaccharides), which are typically components of killed or attenuated bacteria used in vaccines. Extrinsic adjuvants are typically immunomodulators formulated to noncovalently bind to antigens and enhance the host's immune response. Aluminum hydroxide, aluminum sulfate, and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants. A wide range of extrinsic adjuvants can elicit a strong immune response to immunogens. These include saponins such as Stimulons (QS21, Aquila, Worcester, Mass.), or particles derived therefrom, such as ISCOMs complexed to membrane protein antigens (immunostimulating complexes) and ISCOMATRIX; mineral oil, killed mycobacteria, and pluronic polymers containing mineral oil; Freund's complete adjuvant; Lipid A and Bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS) ,oh and liposomes.

[0226] In one embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is aluminum phosphate. Oil-in-water emulsions include squalene, peanut oil, MF59 (WO 90 / 14387), SAF (Syntex Laboratories, Palo Alto, Calif.), and Ribi™ (Ribi Immunochem, Hamilton, Mont.). Oil-in-water emulsions can be used with immunostimulants such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), -acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) ceramide (TM)), or other bacterial cell wall components.

[0227] The adjuvant can be administered with the immunogen as a single composition, or the adjuvant can be administered before, simultaneously with, and / or after administration of the immunogen.

[0228] In embodiments, the composition comprises an antibody described herein. In another embodiment, the composition comprises an antibody described herein and a diluent. In embodiments, the composition is a sterile composition.

[0229] Pharmaceutical compositions include, but are not limited to, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats, and solutes that render the compositions substantially compatible with the tissues or blood of the intended recipient. Other components that may be present in such compositions include, for example, water, surfactants (such as Tween), alcohols, polyols, glycerin, and vegetable oils. Extemporaneous infusion solutions and suspensions can be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. Compositions can be supplied, for example, but not limited to, as lyophilized powders that are reconstituted with sterile water, saline, or other pharmaceutically acceptable diluents before administration to a patient.

[0230] The pharmaceutical composition may contain a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and non-toxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleylphosphotidylethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound together with a suitable amount of carrier to provide a form for direct administration to a patient.

[0231] The term "compound as used herein" can refer to, for example, a peptide, an immunogen, an antibody, an immunoconjugate, and the like.

[0232] Another embodiment includes an antibody complex comprising an antibody described herein and α-syn (e.g., misfolded α-syn oligomers or soluble fibrils). The complex can be in solution.

[0233] V. Kit Further embodiments relate to kits comprising i) an antibody or immunoconjugate, ii) a nucleic acid or vector, iii) a peptide, cyclic compound, or immunogen, iv) a composition, and / or v) a recombinant cell as described herein, contained in a vial, e.g., a sterile vial or other housing, and optionally a reference agent and / or instructions for its use.

[0234] The kit can include an antibody described herein and a particle such as a bead, a plate such as a multi-well plate for immunoassays, or other matrix, which can be conjugated to or provided separately from one or more coupling reagents.

[0235] The kit can include one or more reagents, such as a test sample preparation or dilution solution, a complexation solution or a washing solution for washing away unbound antibody, one or more detection or coupling reagents, etc.

[0236] The reagent can be a reagent described herein, for example, a reagent described in the Examples.

[0237] The kit can be, for example, for use in a method or methods described herein.

[0238] The compounds, immunogens, antibodies, immunoconjugates, nucleic acids, vectors, cell compositions, and kits described herein can be used to inhibit misfolded alpha-syn toxicity, treat synucleinopathies, or in other methods or assays described herein. They can also be used in the manufacture of medicaments for inhibiting misfolded alpha-syn toxicity, treating synucleinopathies, or in other methods or assays described herein.

[0239] VI. Methods and Assays Included are methods for producing the compounds, immunogens, nucleic acids, vectors, antibodies, and immunoconjugates described herein.

[0240] In particular, methods are provided for generating antibodies selective for conformational epitopes of EKTKEQ (SEQ ID NO: 1), EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), TKEQ (SEQ ID NO: 4), or related epitopes.

[0241] In one embodiment, the method comprises administering to a subject a cyclic compound or immunogen described herein, or a composition comprising the cyclic compound or immunogen, isolating antibodies and / or cells specific for the administered cyclic compound or immunogen, and optionally isolating one or more antibodies that selectively bind to misfolded oligomeric α-Syn polypeptides.

[0242] As described above, the antibodies described herein comprise a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDRs are as follows: CDR-H1: SEQ ID NO: 61, 67, 73, 79, 91, or 180; CDR-H2: SEQ ID NO: 62, 68, 74, 80, 92, or 181; CDR-H3: SEQ ID NO: 63, 69, 75, 81, 93, or 182; CDR-L1: SEQ ID NO: 64, 70, 76, 94, or 183; CDR-L2: SEQ ID NO: 65, 71, or 77; or CDR-L3: selected from SEQ ID NO: 66, 72, 78, 84, 96, or 184.

[0243] The antibody can comprise any of the CDR sets or variable regions described herein.

[0244] In another embodiment, the antibodies produced are isolated and purified.

[0245] In another embodiment, the isolated and purified antibodies are affinity matured, which can be performed as described for the initial selection using, for example, a phage library containing variants of the CDR sequences, with antigen adsorbed to plastic plates.

[0246] Those skilled in the art will appreciate that several methods can be used to generate antibodies with specific binding affinity for misfolded oligomeric α-synuclein. One method that can be used is phage display.

[0247] Further embodiments provide assays for detecting whether a test sample contains, for example, misfolded oligomeric α-Syn, wherein EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), or a related conformational epitope, comprises at least one of residues E57, K58, T59, K60, E61, or Q62 and is in an alternative conformation than that occupied by E57, K58, T59, K60, E61, and / or Q62 in the non-misfolded protein conformation (e.g., native monomer and tetramer) or in insoluble fibrils.

[0248] In embodiments, the assay comprises: a. contacting a test sample with an antibody described herein under conditions permissive to produce antibody:misfolded oligomeric α-Syn polypeptide complexes; b. detecting the presence of any complexes; The presence of a detectable complex indicates that the test sample may contain misfolded α-Syn polypeptide.

[0249] In another embodiment, the assay comprises: a. contacting the subject's test sample with an antibody or immune complex described herein under conditions that allow antibody-antigen complexes to form; b. quantitating the amount of antibody-antigen complexes in the test sample; and c. Comparing the amount of antibody-antigen complex in the test sample with a control. For example, a comparison with a control can indicate whether a test sample contains misfolded α-Syn, such as misfolded oligomeric α-Syn.

[0250] In embodiments, the test sample comprises brain tissue or an extract thereof, saliva, and / or CSF. In embodiments, the test sample is obtained from a human subject.

[0251] The control can be a range or cutoff value derived from a control population known to have or not have the disease. The assay can also include a negative or positive control (such as recombinant misfolded oligomeric α-syn or a cyclic peptide).

[0252] For example, a negative control sample can be included in the assay to provide the background value of the assay.The results obtained from the test sample can be compared with the values obtained from a control population (e.g., control).The control can be, for example, a range or cut-off value determined from age-matched control subjects who are known not to have or have the synucleinopathy of the subject from whom the test sample is obtained.In methods that involve monitoring the progression of disease, it can also be compared with one or more previous levels.

[0253] In some embodiments, the test sample is from a subject that contains a genetic mutation in the alpha-synuclein gene.

[0254] In another embodiment, the test sample is derived from a subject suffering from or suspected of suffering from a synucleinopathy, optionally Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy.

[0255] Many methods can be used to determine whether misfolded oligomeric α-Syn polypeptides are present in a test sample using the antibodies described herein, including flow cytometry, dot blot, Western blot, ELISA, and immunoprecipitation followed by SDS-PAGE immunocytochemistry, and immunoassays on other detection platforms (e.g., SIMOA, MSD, etc.).

[0256] Detection of misfolded oligomeric α-Syn in test samples using several methods could be used to diagnose and track treatment for synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). For example, assays capable of detecting levels in the 1-1000 pg / mL sample range could be useful. For example, the "Single Molecule Counting" (SMC™) assay from EMD Millipore is ultrasensitive and suitable for detecting low-abundance biomarkers such as misfolded oligomeric α-Syn.

[0257] In some embodiments, immunoconjugates containing particles such as magnetic beads coated with an α-Syn antibody or an antibody described herein are used to capture α-Syn from a test sample. Because the antibodies described herein selectively bind to toxic oligomeric species compared to monomeric species, this method limits interference from physiologically abundant monomers. The measurement step can include detecting the captured misfolded oligomeric α-Syn with a pan α-Syn antibody containing a label, such as one of the detectable labels described elsewhere, followed by elution of the bound detector antibody for quantification of the signal from the label. The determined signal is proportional to the amount of misfolded oligomeric α-Syn polypeptide in the sample, which can be calculated from a standard curve. Comparison with a range or cutoff value obtained in a normal control sample can be used for diagnostic purposes, and longitudinal measurements can be used to evaluate the effectiveness of treatment. The feasibility of this approach has been established using soluble MSA brain extracts (Example 19).

[0258] As described in the Examples, surface plasmon resonance can be used to assess conformation-specific binding.

[0259] Further embodiments include methods of inducing an immune response in a subject, comprising administering to the subject a compound, immunogen, and / or composition comprising a compound described herein, and optionally isolating cells and / or antibodies that specifically bind to the administered compound or immunogen. Antibodies can be tested using one or more of the assays described in the Examples.

[0260] The Examples also demonstrate that antibodies of the present disclosure were able to inhibit the toxicity of misfolded α-syn oligomers in a Parkinson's disease rat dopaminergic neuron assay. Furthermore, the present disclosure also demonstrates that antibodies of the present disclosure can prevent α-synuclein aggregation and phosphorylation induced by exposure to small soluble fibrils (sonicated synthetic preformed fibrils (PFFs)) in a hippocampal neuron culture model of Parkinson's disease. For example, as shown in Example 11, antibodies of the present disclosure were able to reduce the amount of internalized synthetic α-syn and the recruitment of endogenous α-syn to pathological phosphorylated forms induced by exposure to PFFs.

[0261] Recently, it was also shown that mice expressing the familial PD E46K mutation and two homologous E→K mutations in the adjacent KTKEGV sequence disrupted the tetramer and increased the number of monomers (Nuber 2018). The inability to form physiological tetramers in these mice resulted in a Parkinson's disease-like disorder.

[0262] Accordingly, another aspect is a method of inhibiting misfolded α-syn toxicity and / or propagation comprising administering to a cell population and / or a subject in need thereof an effective amount of an antibody, immunoconjugate, or composition comprising an antibody or immunoconjugate described herein, wherein the antibody selectively binds to misfolded oligomeric α-syn but not to physiological tetramers, e.g., as determined using the assays described herein, or inhibits misfolded oligomeric α-syn toxicity as assessed in a dopaminergic neurotoxicity assay, e.g., the model assay described in the Examples.

[0263] Also provided is the use of an effective amount of an antibody, immunoconjugate, or composition comprising an antibody or immunoconjugate described herein for inhibiting misfolded α-syn toxicity in a cell population or a subject in need thereof, wherein the antibody selectively binds to misfolded oligomeric α-syn and / or an epitope sequence in the context of an immunogen described herein.

[0264] In embodiments, the method or use is for inhibiting intercellular transmission of misfolded alpha-syn and / or for reducing the amount of endogenous aggregated phosphorylated alpha-syn.

[0265] In embodiments, the cell population is a neuronal cell population. In embodiments, the neuronal cell population is an in vitro model of Parkinson's disease.

[0266] In embodiments, the subject is a human.

[0267] In embodiments, the human has, is suspected of having, or is likely to develop Parkinson's disease (PD), Lewy body disease (LBD, also known as dementia with Lewy bodies or DLB), or multiple system atrophy (MSA) (collectively known as synucleinopathies). For example, a person who carries a mutation associated with familial PD is considered likely to develop PD.

[0268] Also provided is a method of treating an alpha-synucleinopathy, comprising administering to a subject in need thereof an effective amount of an antibody or immunoconjugate of the present disclosure described herein, or a composition comprising the antibody or immunoconjugate.

[0269] In embodiments, the α-synucleinopathy is PD, LBD, or multiple system atrophy (MSA). α-synuclein is also associated with Alzheimer's disease (AD). Accordingly, a further aspect is a method of treating a subject in need thereof, comprising administering to the subject an effective amount of an antibody or immunoconjugate of the present disclosure described herein, or a composition comprising the antibody or immunoconjugate, optionally in combination with another AD treatment. The other AD treatment may be, for example, an antibody described in any of WO / 2017 / 079833, WO / 2017 / 079834, WO / 2017 / 079831, WO / 2017 / 079832, and WO / 2017 / 079835, each of which was filed on September 11, 2016, and is incorporated herein by reference.

[0270] The antibodies can be included in the compositions described herein, for example, in combination with a pharmaceutically acceptable carrier, diluent, and / or excipient, for example, formulated into vesicles to improve delivery. Combinations of antibodies (e.g., two or more antibodies) and / or immunoconjugates can also be used.

[0271] The compositions, antibodies, immunogens, and immunoconjugates described herein can be administered, for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal, intraorbital, ophthalmic, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.

[0272] In certain embodiments, the composition is administered systemically.

[0273] Other embodiments contemplate the co-administration of the compositions, antibodies, and immunoconjugates described herein with biologically active molecules known to facilitate transport across the blood-brain barrier.

[0274] Also contemplated in certain embodiments are methods for administering the compositions, antibodies, and immunoconjugates described herein across the blood-brain barrier, such as those aimed at temporarily increasing the permeability of the blood-brain barrier, as described in U.S. Pat. No. 7,012,061, entitled "Method for increasing the permeability of the blood-brain barrier," which is incorporated herein by reference.

[0275] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are set forth for illustrative purposes only and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms are employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0276] The following non-limiting examples are illustrative of the present disclosure. [Example]

[0277] Example 1 We identified epitopes selectively or preferentially displayed on misfolded α-synuclein using molecular dynamics-based simulations, which impose a global coordinate bias on a protein (or peptide aggregate) to force it to misfold and then predict the most likely unfolded regions of the partially unstructured protein (or peptide aggregate). Biased simulations were performed and the change in solvent-accessible surface area (SASA) corresponding to each residue was measured (relative to the change in the initial fibril structure of the protein under consideration). SASA represents the surface area accessible to HO. A positive change in SASA (relative to the change in the initial structure of the protein under consideration) can be considered indicative of unfolding in the region of the relevant residue index. In addition to SASA, we used two other methods to identify candidate epitopes. These are the loss of fibril contacts defined by non-hydrogen atoms within a cutoff length, and the root mean square fluctuation (RMSF), which measures the degree of deviation with respect to the mean of the structural ensemble, where an increase in the RMSF of some amino acids indicates an increase in the dynamics of those amino acids.

[0278] This method was applied to α-Syn fibrils (PDB entry 2N0A).

[0279] The structure of the 10-strand α-Syn fibril has been determined and is listed in the Protein Data Bank as PDB entry 2N0A. The PDB 2N0A structure, or portions thereof, can be computationally equilibrated to obtain an equilibrated ensemble, which is used to measure all fibril conformations of epitopes within the α-Syn fibril structure and is referred to herein indefinitely as a "structured fibril" or "unbiased fibril structure of α-Syn," "fibril ensemble of α-Syn," "equilibrium fibril ensemble of α-Syn," or "α-Syn fibril structural ensemble."

[0280] A monomer ensemble can be obtained by, for example, taking one of the chains from the PDB fibril (2N0A) as the starting structure. Then, a pivoting algorithm is used to induce large conformational changes in the configuration, generating 1500 different unfolded structures to be used as initial configurations. For each of these 1500 structures, a 3 ns equilibration simulation is performed, and 1 ns snapshot configurations are collected and added to the monomer ensemble (three equilibration snapshots for each initial condition). The final result is a monomer ensemble of 4500 configurations.

[0281] The collective coordinate method described in WO / 2017 / 079836, as well as K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, O. Guvench, P. Lopes, I. Vorobyov, and A.D. Mackerell, "The CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields," Journal of Computational Chemistry, 31(4):671-690, 2010, and P. Bjelkmar, P. Larsson, M.A. Cuendet, B. Hess, and E. Lindahl, "Implementation of the CHARMM force field in GROMACS: analysis of protein stability effects from correlation maps, virtual interaction sites, and water" Simulations of this initial structure were performed using the CHARMM force field parameters described in models. J. Chem. Theo. Comp., 6:459-466, 2010 (both of which are incorporated herein by reference) with TIP3P water as the solvent.

[0282] I. Epitope Prediction The epitopes EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4) appear as predicted epitopes from PDB structure 2N0A using, for example, a cluster coordinate approach as shown in Figures 1A and 1B. The EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4) epitopes appear as predictions of PDB structure 2N0A when considering either increased SASA, loss of fibril contacts, or increased RMSF (Figure 1, panel C). Cyclic compounds containing the epitopes EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4) in an amino acid scaffold (e.g., including a linker) were evaluated for their suitability for presenting the epitopes described in Example 2 and used for further analysis.

[0283] For the plots of Figures 1-5 discussed herein, data are obtained from equilibrium simulations in explicit solvent (TIP3P) using the Charmm36m force field, as previously described.

[0284] Figure 2 shows the conformation of an α-Syn monomer in the context of an unbiased fibril. This structure is a center-of-mass conformation obtained from an equilibrium simulation of five α-Syn chains containing 100 mM NaCl. Residues K58 and K60 are nearly parallel in this structural ensemble. There is a close contact between the Hε3 atom of K60 (weakly positively charged, Q = 0.05) and the Nε2 atom of Q62 (negatively charged, Q = -0.64). Panel B shows a snapshot of the structure of an α-Syn monomer in the biased ensemble. In this ensemble, residues K58 and K60 are no longer parallel, and the contact between K60 and Q62 no longer exists.

[0285] II. Solvent-epitope exposure Figure 3, panels A-C, show epitope snapshots showing the solvent-accessible surface area (SASA) of each residue at the center of gravity of the unstressed fibril ensemble, the identified cyclic peptide, and the equilibrium ensemble of isolated (native) monomers of sequence EKTK (SEQ ID NO: 2). Figure 4, panels A-C, show snapshots showing the solvent-accessible surface area (SASA) of each residue in the unstressed fibril ensemble, the equilibrium ensemble of the cyclic peptide, and the isolated monomer ensemble of sequence TKEQ (SEQ ID NO: 4). Figures 1A and 1C show that the SASA of residues in EKTKEQ (SEQ ID NO: 1), including EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4), in the deflected fibril ensemble increases compared to the undeflected fibrils. Figures 5A and B show that the SASA of the cyclic peptides is increased over that of the unbiased or biased ensembles (except for K60 in TKEQ (SEQ ID NO: 4)), indicating that more surface area is exposed and therefore accessible for antibody binding. For TKEQ (SEQ ID NO: 4), the increased exposure is most pronounced at residues E61 and Q62, which show the largest increase in SASA over the unbiased ensemble. For E61, this difference between the cyclic and unbiased fibrils is 101 Å. 2 However, in the case of Q62, this difference is 67 Å. 2 In the case of T59, the difference between the circular fibrils of TKEQ (SEQ ID NO: 4) and the unbiased fibrils is 80 Å. 2 However, in the case of EKTK (SEQ ID NO: 2), the difference is 65 Å. 2 is.

[0286] III. Ensembles of cyclic peptide conformational clusters distinct from ensembles of either linear or fibrillar conformations. Figure 5C plots a histogram of RMSD at the center of gravity of the cyclic peptide equilibrium distribution for the cyclic peptide scaffold cyclo(CGTKEQGGGG) (SEQ ID NO: 7). Most conformations are very similar to the center of gravity conformation, with the distribution peaking at approximately 1.3 Å. Also shown are the RMSDs corresponding to the epitope conformations for the center of gravity conformation in the native monomer ensemble and fibril ensemble. Finally, the RMSDs for the epitope in the conformations of the PDB structures of α-helix, micelle-bound α-synuclein, 1XQ8, and 2KKW are shown. This figure demonstrates that these conformations are distinct from most cyclic conformations. The dissimilarity between the epitope conformation in the cyclic peptide ensemble and its conformation in either the fibril ensemble or the isolated native monomer ensemble can be quantified using the Jensen-Shannon distance. This distance provides the effective separation between any two pairs of ensembles, which can be recast as the effective separation between two Gaussian ensembles. The cyclic peptide conformation cyclo(CGGGGEKTKGG) (SEQ ID NO:5) of the epitope EKTK (SEQ ID NO:2) is distinct from that of the fibrils, and the effective distance between the two Gaussian distributions representing these ensembles is 7.8 standard deviations. Many cyclic peptide conformations of the epitope are also distinct from that of the isolated, native monomer ensemble, and the effective distance between the two Gaussian distributions representing these ensembles is 3.1 standard deviations.

[0287] Similarly, the cyclic peptide conformation cyclo(CGTKEQGGGG) (SEQ ID NO:7) of the epitope TKEQ (SEQ ID NO:4) differs from that of the fibrils, with an effective distance of 7.8 standard deviations between the two Gaussian distributions representing these ensembles. Most cyclic peptide conformations of the epitope also differ from that of the isolated, native monomer ensemble, with an effective distance of 5.2 standard deviations between the two Gaussian distributions representing these ensembles.

[0288] The conformations of α-helical membrane-bound α-synuclein (PDB IDs 1XQ8 and 2KKW) also differ from most conformations of cyclic peptide ensembles, as seen, for example, in Figure 5C for the TKEQ (SEQ ID NO: 4) epitope. The degree of similarity was quantified by calculating the embedding depth of the epitopes in these PDB structures into the cyclic peptide ensembles. A shallower embedding depth indicates fewer ensembles outside the PDB structure, and therefore a greater proportion of the cyclic conformations differ from those in the PDB structure. For EKTK (SEQ ID NO: 2), the embedding depth of the epitope in 2KKW is 20%, and the embedding depth of the epitope in 1QX8 is 38%. For TKEQ (SEQ ID NO: 4), the embedding depth of the epitope in 2KKW is 16%, and the embedding depth of the epitope in 1QX8 is 22%. These values indicate that many of the cyclic peptide ensembles are conformationally distinct from the conformation of the epitope in α-helical membrane-bound α-synuclein, and therefore antibodies raised against the cyclic peptides are unlikely to bind to this epitope in its native form.

[0289] Example 2 Scaffolds that could be used to present the identified epitopes in a cyclic conformation were evaluated. Table 2 below shows several cyclic epitope scaffolds of EKTK (SEQ ID NO: 2), obtained by flanking the epitope with a variable number of glycine amino acids N- and C-terminal to the epitope. Suitability was assessed by measuring the Jenson-Shannon distance between the ensemble of cyclic peptides and the equilibrium ensemble of α-synuclein monomers, β-synuclein monomers, and γ-synuclein monomers, as well as the equilibrium ensemble of stressed (i.e., deflected) fibrils. While similarity to the stressed / deflected fibrils is desired, dissimilarity to the monomer ensemble is also desired to avoid interference with in vivo function. Cyclic peptide scaffolds predicted to be suitable based on these criteria are shown in Table 2. [Table 1]

[0290] A similar analysis was performed for the epitope KTKE (SEQ ID NO: 3). Suitable scaffolds are provided in Table 3. [Table 2]

[0291] A similar analysis was performed for the epitope TKEQ (SEQ ID NO: 4). Suitable scaffolds are provided in Table 4. [Table 3]

[0292] Example 3 Construction of cyclic compounds containing conformationally restricted epitopes Compounds containing conformationally restricted epitope sequences can be prepared by making a linear peptide that includes or consists of EKTKEQ (SEQ ID NO: 1), EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), or a portion thereof, such as KEQ, and a linker sequence, which can be cyclized to make a cyclic compound, such as cyclo(CGTKEQGGGG) (SEQ ID NO: 7) or cyclo(CGGTKEQGGGG) (SEQ ID NO: 49). For example, a cyclic compound can be made by cyclizing the linear peptide from head to tail.

[0293] For example, a peptide corresponding to an epitope such as EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), or TKEQ (SEQ ID NO: 4), or a portion thereof such as KEQ, can be synthesized with or conjugated to a linker, preferably containing 1, 2, 3, or 4 amino acids and / or PEG units at the C-terminus and / or N-terminus of the epitope sequence. When the linker is composed of an amino acid sequence, it can be synthesized using known methods such as Fmoc-based solid-phase peptide synthesis, alone or in combination with other methods. PEG molecules can be attached to amine groups at the N-terminus using, for example, the coupling chemistry described in Hamley 2014 [Biomacromolecules, 2014, 15(5), pp 1543-1559, DOI:10.1021 / bm500246w] and Roberts et al 2012 [Advanced Drug Delivery Reviews, Volume 64, Supplement, December 2012, Pages 116-127, M.J. Roberts, M.D. Bentley, J.M. Harris doi.org / 10.1016 / j.addr.2012.09.025] (each of which is incorporated herein by reference). The compounds can be cyclized by 1) the amino and carboxy termini of the peptide plus linker to form a peptide bond (e.g., cyclizing the backbone), 2) the amino or carboxy termini of the peptide plus linker bearing a side chain, or 3) covalently linking two side chains of the peptide plus linker.

[0294] The bonds in the cyclic compounds may all be regular peptide bonds (homodetic cyclic peptides) or may include other types of bonds such as ester, ether, amide, or disulfide bonds (heterodetic cyclic peptides).

[0295] Peptides can be cyclized by oxidation of thiol- or mercaptan-containing residues at the N- or C-terminus, or internally in peptides containing, for example, cysteine and homocysteine. For example, two adjacent cysteine residues in a peptide can be oxidized to form a disulfide bond. Oxidizing reagents that can be used include, for example, oxygen (air), dimethyl sulfoxide, oxidized glutathione, cystine, copper (II) chloride, potassium ferricyanide, trifluorothallium (III) acetate, or other oxidizing reagents that may be known to those skilled in the art and can be used in a manner known to those skilled in the art.

[0296] Methods and compositions related to cyclic peptide synthesis are described in U.S. Patent Publication No. 2009 / 0215172. U.S. Patent Publication No. 2010 / 0240865, U.S. Patent Publication No. 2010 / 0137559, and U.S. Patent No. 7,569,541 describe various methods of cyclization. Other examples are described in PCT Publication No. WO 01 / 92466 and Andreu et al., 1994. Methods in Molecular Biology 35:91-169.

[0297] The linker can contain one or more cysteine residues adjacent to and / or inserted in the linker. The peptide can be structured into a cyclic conformation by creating a disulfide bond between non-natural cysteine residues added to the N- and C-termini of the peptide.

[0298] The cyclic peptide can be linked to a carrier, optionally a BSA moiety, or an immunogenicity enhancing agent such as KLH.

[0299] Example 4 The following linear and cyclic peptides were prepared: [Table 4]

[0300] Peptide synthesis was performed by CPC Scientific Inc. (Sunnyvale, CA, USA). Peptides were synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on 2-chlorotrityl chloride resin followed by cleavage from the resin. Peptide sequences were confirmed by electrospray MS, and purity was assessed by HPLC to confirm at least 95% purity. Cyclization was performed via a head-to-tail (CG) amide bond. The uncyclized linear CGHHQKG peptide was also produced by CPC Scientific.

[0301] Construction of immunogens The cyclic compounds in Table 1 were then conjugated to KLH (for immunization) or BSA (for screening) via maleimide-based coupling (CPC Scientific Inc, Sunnyvale Calif.).

[0302] Example 5 Antibody generation and selection The coupled peptide was used to generate mouse monoclonal antibodies according to protocols approved by the Canadian Council on Animal Care (Immunoprecise Antibodies LTD, Victoria, BC, Canada).

[0303] immunity Briefly, 50-day-old female BALB / c mice (Charles River Laboratories, Quebec) were immunized. A series of subcutaneous injections of an aqueous solution containing antigen but no adjuvant was administered over a 19-day period. Mice were immunized with 100 μg of cyclic peptide-KLH per injection of a 0.5 mg / mL solution in sterile saline. On day 19, all mice were euthanized, and lymphocytes were harvested for generation of hybridoma cell lines.

[0304] Fusion / hybridoma development Lymphocytes were isolated and fused with murine SP2 / 0 myeloma cells in the presence of polyethylene glycol (PEG 1500). Fused cells were cultured using HAT selection. This method combines hybridoma selection and cloning into a single step using semi-solid methylcellulose-based HAT selection medium. Single-cell-derived hybridomas were expanded to form monoclonal colonies on semi-solid medium. Ten days after the fusion event, the resulting hybridoma clones were transferred to 96-well tissue culture plates and grown in HT-containing medium until they reached mid-logarithmic growth phase (5 days).

[0305] Hybridoma analysis (screening) Tissue culture supernatants from hybridomas were tested by indirect ELISA during screening of antigens (cyclic peptide-BSA and linear peptide-BSA) and probed for both IgG and IgM antibodies using a goat anti-IgG / IgM (H&L)-HRP secondary and developed with TMB substrate.

[0306] Positive cultures were retested on an irrelevant antigen (human transferrin) during antigen screening to confirm secretion. Clones were isotyped by antibody capture ELISA to determine whether they were IgG or IgM isotypes and tested by indirect ELISA on other cyclic peptide-BSA conjugates containing the same epitope to assess cross-reactivity.

[0307] Isotyping Hybridoma antibodies were isotyped using an antibody capture experiment. Capture plates were coated overnight at 4°C with 100 μL / well of 1:10,000 goat anti-mouse IgG / IgM (H&L) antibody in carbonate coating buffer (pH 9.6). Primary antibodies (hybridoma supernatants) were added at 100 μg / mL. Secondary antibodies were added at 1:5,000. Goat anti-mouse IgGγ-HRP or 1:10,000 goat anti-mouse IgMμ-HRP was added at 100 μL / well in PBS-Tween for 1 hour at 37°C with shaking. All washing steps were performed for 30 minutes using PBS-Tween. Substrate TMB was added at 50 μL / well, and color was developed in the dark and stopped with an equal volume of 1 M HCl.

[0308] result TKEQ (SEQ ID NO: 4) construct Mice immunized with cyclo(CGTKEQGGGG)-KLH (SEQ ID NO: 7), cyclo(CGGTKEQGGGG)-KLH (SEQ ID NO: 49), and cyclo(CGGTKEQGG)-KLH (SEQ ID NO: 48) produced clones selective for the cyclic peptide (free or bound to BSA) compared to the linear construct. Clones were tested at least three times for each clone for reactivity to cyclic peptide-BSA, with similar results. [Table 5-1] [Table 5-2] [Table 6] [Table 7-1] [Table 7-2]

[0309] The hybridoma antibodies were also tested for their ability to selectively bind to cyclic peptides containing different linkers as shown in Tables 2-4. [Table 8-1] [Table 8-2]

[0310] The results are shown in Figure 11, where hybridoma antibody 2E9 selectively binds to the cyclic peptide compared to the linear or unrelated peptides HT, BSA, and x-reactive self (a potentially cross-reactive human protein from in silico analysis). Also shown is a lack of reactivity to α-syn, β-syn, and γ-syn monomers. [Table 9] [Table 10-1] [Table 10-2]

[0311] Example 6 EKTK epitope (SEQ ID NO: 2) Similarly, the binding selectivity of hybridoma antibodies raised against cyclo(CGGGEKTKGG) (SEQ ID NO: 10) to free and BSA-bound cyclic peptides compared to linear or unrelated peptides HT, BSA, and human complement factor H (HCFH) is shown in Table 11. Reactivity to cyclic and linear peptides of CGGGGEKTKGG (SEQ ID NO: 5) is also shown. [Table 11]

[0312] Additionally, the binding selectivity of hybridoma antibodies raised against cyclo(CGGGGEKTKGG) (SEQ ID NO: 5) to free and BSA-bound cyclic peptides compared to linear or unrelated peptides HT, BSA, and HCFH is shown in Table 12. Reactivity to cyclic and linear peptides of CGGGEKTKGG (SEQ ID NO: 10) is also shown. [Table 12]

[0313] Mouse anti-α-synuclein hybridomas were cross-tested for reactivity to β-synuclein and γ-synuclein by indirect ELISA.

[0314] ELISA plates were coated with 0.1 micrograms / well of β-synuclein or γ-synuclein antigen at 100 microL / well in carbonate coating buffer (pH 9.6) overnight at 4°C. Plates were blocked with 3% skim milk powder in PBS at room temperature for 1 hour. Hybridoma antibody (100 microL / well) was added, and the plates were incubated at 37°C for 1 hour with shaking. Secondary antibody (goat anti-mouse IgGy-HRP) was added at 1:5000 in PBS-Tween at 100 microL / well with shaking for 1 hour at 37°C. All washing steps were performed for 30 minutes using PBS-Tween. TMB substrate was added at 50 microL / well, developed in the dark, and stopped with an equal volume of 1M HCl.

[0315] result Hybridomas generated using cyclo(CGTKEQGGGG) (SEQ ID NO: 7) were tested. Both β-synuclein and γ-synuclein counts were similar to background.

[0316] Similarly, hybridomas raised against cyclo(CGGTKEQGG) (SEQ ID NO: 48) and cyclo(CGGTKEQGGGG) (SEQ ID NO: 49) were tested. Both β-synuclein and γ-synuclein counts were similar to background.

[0317] Ten hybridomas raised against cyclo(CGGGEKTKGG) (SEQ ID NO: 10) were tested. Both β-synuclein and γ-synuclein counts were similar to background.

[0318] Similarly, hybridomas raised against cyclo(CGGGGEKTKGG) (SEQ ID NO: 5) were tested. Both β-synuclein and γ-synuclein counts were similar to background.

[0319] Example 7 Characterization of anti-misfolded α-Syn antibodies Antibodies were tested for their ability to bind to native monomeric and misfolded oligomeric α-Syn polypeptides using surface plasmon resonance.

[0320] Surface plasmon resonance analysis of biological samples. Homogenization: Human neural tissue samples were weighed and then immersed in fresh, ice-cold TBS (supplemented with 5 mM EGTA, 5 mM EDTA (both from Sigma) and an EDTA-free protease inhibitor cocktail from Roche Diagnostics, Laval QC, Canada) to a final tissue concentration of 20% (w / v). Tissues were homogenized in this buffer using a mechanical probe homogenizer (3 × 30-second pulses with a 30-second pause between, all performed on ice). The TBS-homogenized samples were then ultracentrifuged (70,000 × g for 90 minutes). The supernatant was collected, aliquoted, and stored at -80°C. Protein concentrations of TBS homogenates were determined using a BCA protein assay (Pierce Biotechnology Inc., Rockford, IL, USA).

[0321] Surface plasmon resonance analysis: Neural tissue samples from PD and LBD patients were analyzed. Test antibodies, a positive control antibody (4D6), and an IgG isotype control were immobilized at high density (approximately 10,000 RU) on the flow cell of a sensor chip (approximately 9500-13,000 RU). Diluted samples were injected sequentially over the surface for approximately 300-900 seconds, followed by a 150-second dissociation period in buffer to regenerate the surface. In some experiments, 4D6 (BioLegend) was used to detect captured material. Binding responses were double-referenced by subtracting the IgG reference surface binding and normalized to assay buffer to compare different sample groups.

[0322] Test antibodies included clones: 2E9, 3B5, 2D2, 8B12, 9A7. Control antibodies used were pan-αSyn antibody (4D6) (Biolegend) and a mouse IgG1 isotype control.

[0323] Analytes included SynAging α-Syn oligomers, unfractionated Lewy body disease (LBD) (also known as dementia with Lewy bodies (DLB)) soluble brain extracts, high molecular weight (HMW) and low molecular weight (LMW) LBD and PD fractions.

[0324] The unfractionated soluble brain extract was diluted 1:4 and the HMW and LMW fractions were diluted to 100 ug / mL total protein.

[0325] Analytes were injected over the immobilized antibodies at 10 uL / min for 15 min. Antibody 4D6 was injected over the captured analytes at 10 uL / min for 5 min.

[0326] result 4D6 (pan α-syn antibody) showed strong binding to SynAging α-syn oligomers. Test clone 2E9 shows strong binding to SynAging α-syn oligomers. Test clones 2D2 and 8B12 show weaker binding (Figure 6D). The left panel shows direct binding of captured α-synuclein. The right panel shows α-synuclein captured by antibody along the x-axis and detected with pan antibody 4D6.

[0327] As shown in the right panel of Figure 6E, the test antibodies bound α-syn directly in unfractionated, soluble LBD brain extracts. Subsequent detection with the pan α-syn antibody 4D6 confirmed the presence of α-syn in the material captured by the test antibodies (left panel). Test clone 2E9, and to a lesser extent clones 2D2 and 8B12, bound α-syn in both the unfractionated (Figure 6E) and LMW and HMW fractions of brain extracts (Figure 6F).

[0328] Test clones were also evaluated for binding of soluble fibrils. In separate SPR runs, antibodies were immobilized and soluble, sonicated fibril preparations were injected. As shown in Figure 6G, most of the test antibodies showed some cross-reactivity with small, soluble fibrils.

[0329] Figure 6H compares the binding profiles of test clone 2E9 and the pan α-syn 4D6 antibody. Clone 2E9 does not bind to monomers, but shows strong binding to soluble oligomers and cross-reactivity with small soluble fibrils.

[0330] Example 8 Binding to misfolded α-synuclein oligomers Additional SPR experiments were performed on an IBIS 96X SPR biosensor spotted with Wastach protein. Test mAbs, along with control mAbs Syn-F1 and 4D6, were amine-coupled to the 200M biosensor surface using standard NHS / EDC activation. Controls Syn-F1 and 4D6 were immobilized at four positions each. Test mAbs were immobilized at two positions each. Oligomeric α-synuclein was purchased from SynAging (Nancy, France).

[0331] Alpha-synuclein monomer purchased from rPeptide (Georgia, USA) was tested in a 3-fold concentration series up to 500 μM. Syn-F1 shows weak binding to alpha-synuclein monomer. 4D6 (a commercially available pan mAb) showed a higher level of binding to alpha-synuclein (see Figure 6A). None of the test antibodies showed binding to alpha-synuclein monomer (Figure 6A).

[0332] SynAging α-synuclein oligomers were tested in a 3-fold titration up to 6 μM. Synaging oligomers bound well to Syn-F1 and 4D6 surfaces. They also bound well to clone 2E9 (Figure 6B).

[0333] Test mAb 2E9 and control 4D6 were also tested at a 1 / 10 dilution on surfaces with captured oligomers. 2E9 was injected first, followed by 4D6 (see arrows). 2E9 (raised against cyclo(CGTKEQGGGG) (SEQ ID NO: 7)) binds to surfaces with SynAging oligomers on them but not to surfaces without them. 4D6 also binds to surfaces with SynAging oligomers present but not to surfaces without them. See Figure 6. Binding was also detected with clones 3B5 (raised against cyclo(CGTKEQGGGG) (SEQ ID NO: 7)), 8B12, 9A7 (raised against cyclo(CGGTKEQGG) (SEQ ID NO: 48)), and 2D2 (raised against cyclo(CGGTKEQGGGG) (SEQ ID NO: 49)).

[0334] Figure 6C shows that other clones also specifically bound to SynAging oligomers.

[0335] In a separate SPR run using a MASS2 instrument (Sierra Biosensors), antibodies were directly immobilized on a sensor chip via amine coupling, and α-syn analytes were injected over the chip to measure binding responses. All clones tested, particularly 1C7, 8D6, 8B12, 9D8, 12B12, 2D2, and 2E9, selectively bound α-syn oligomers with little or no binding to monomers or physiological tetramers (Figure 9). Control 4D6 reacted with all species of α-syn. Control 1 (Prasinezumab, human IgG, PRX002 / RG7935, Creative Biolabs) behaved similarly to a pan α-syn antibody, binding to all species. Control 2 (BAN0805, mAb49 / G, mouse IgG, α-syn antibody, Creative Biolabs) bound α-syn oligomers with some reactivity to monomers.

[0336] The results for test antibody 2E9 are shown in Figure 9B in comparison with other α-syn-directed antibodies. Control antibodies 1 and 3 (NI-202.12F4 and PRX002 from Creative Biolabs) behave similarly to pan α-syn antibodies, binding to all species. Control antibody 2 (mAb 49 / G) binds primarily to α-syn oligomers and sonicated fibrils.

[0337] Example 9 Several antibodies were also tested in dot blot assays using Lewy body dementia (LBD) and control brain homogenates. LBD frontal cortex high-speed pellet Triton X extract (NDBB220_HP-TX) and control brain ("72_HP-TX") were tested using Syn-F1 aggregate / fibril-preferring antibody (0.5 μg / mL), 4D6 pan α-Syn antibody (BioLegend, 1 μg / mL), and test antibodies 2E9, 12B12, 3C11, and 2D6 (4 μg / mL). Loading was confirmed by staining for β-actin (abm, 1 μg / mL). Nitrocellulose membranes were dotted with 10 μg of control brain or 10 μg of LBD brain. 10 μg of β-actin was dotted as a loading reference.

[0338] As shown in Figure 7A, Syn-F1 preferentially bound to the LBD compared to control brain. Also shown in Figure 7A, 4D6 strongly bound to both LBD and control brain. Strong binding to the LBD was demonstrated by the test antibodies 2E9, 12B12, 3C11, and 2D6 (Figures 7B-E). A β-actin control confirmed that the amount of protein in each dot was comparable (Figure 7F).

[0339] The relative amount of staining in the LBD compared to control brain extracts was assessed for antibodies Syn-F1, 4D6, 2E9, 12B12, and 3C11. As shown in Figure 7G, the test antibodies preferentially bound to the LBD by 15-26 fold, which was several fold higher than that seen with Syn-F1 or 4D6. The total amount of α-Syn detected by the pan α-syn antibody 4D6 in control and LBD brains is shown in Figure 7H.

[0340] Example 10 Neuroprotective effects of antibodies against α-Syn toxicity in a rat primary dopaminergic neuron model of Parkinson's disease The neuroprotective effects of several antibodies were also tested in rat primary dopaminergic neurons damaged by exposure to α-syn oligomers using an in vitro Parkinson's disease model.

[0341] method Rat dopaminergic neurons were cultured as described by Schinelli et al., 1988. Briefly, pregnant female rats (Wistar rats, Janvier) were killed by cervical dislocation on day 15 of gestation, and fetuses were removed from the uterus. The embryonic midbrain was removed and placed in ice-cold Leibovitz 15 (L15, PanBiotech, Ref. P04-27055, Batch: 4511117) medium containing 2% penicillin-streptomycin (PS: PanBiotech, Ref. P06-07100, Batch: 7050218) and 1% bovine serum albumin (BSA, PanBiotech, Ref. P06-1391100, Batch: H170807). The midbrain was dissociated by trypsinization. The cells were then mechanically dissociated by passing them three times through a 10 mL pipette. Cells were resuspended in a synthetic culture medium consisting of B27 2% (Invitrogen, ref: 17504, batch: 1950376), L-glutamine (2 mM, PanBiotech, ref: P04-80100, batch: 8440517), and Neurobasal (Invitrogen, ref: 11570556, batch: 1944312) supplemented with 2% PS, 10 ng / mL brain-derived neurotrophic factor (BDNF) (PeproTech, ref: 450-02, batch: 081761), and 1 ng / mL glial cell line-derived neurotrophic factor (GDNF) (PanBiotech, ref: CB-1116001, batch: H170806). Viable cells were counted using a Neubauer cytometer using the trypan blue exclusion test. Cells were seeded at a density of 40,000 cells / well in 96-well plates (precoated with poly-D-lysine; Greiner, Ref: 655940, Batch: E170938V) and cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere.

[0342] Half of the medium was replaced with fresh medium every 2 days. In these conditions, after 5 days of culture, astrocytes were present in the culture and released growth factors that allowed neuronal differentiation.

[0343] Preparation of α-synuclein and cell culture injury Briefly, α-syn peptide (rPeptide, ref: S1001-1, batch: 080817AS) was reconstituted in synthetic culture medium at 4 μM and incubated at +37°C for 3 days in the dark with slow shaking to generate oligomers. Control medium was prepared under the same conditions. A second α-syn oligomer preparation from SynAging was also tested. After 6 days of culture, the test antibody and α-syn toxin (oligomer) were preincubated at room temperature for 30 minutes before the mixture was added to the neuronal cultures. The culture medium was removed and the α-syn oligomer preparation was added. The test compound was left in place during α-syn intoxication. The following conditions were tested: Control (vehicle) / vehicle for 4 days α-synuclein oligomer / vehicle 4-day injury α-synuclein oligomers (0.5 μM) + test antibody at two concentrations (0.05 μM and 0.25 μM) α-synuclein oligomer (0.5 μM) + BDNF 50 ng / mL Only test antibodies were evaluated at the highest concentration (0.25 μM).

[0344] To assess dopaminergic neuron survival, one culture (six wells per condition) was performed.

[0345] Total number of TH-positive neurons After 4 days of intoxication with or without test compound, cells were fixed with 4% paraformaldehyde (Sigma, ref. 6148, batch: SZBE2390V) for 20 minutes at room temperature. Control cells were fixed in the same manner. Cells were then permeabilized and nonspecifically blocked with a solution of 0.1% saponin (Sigma, ref. S7900, batch: BCBJ8417V) and 1% fetal calf serum (FCS) in phosphate-buffered saline (PBS, PanBiotech; ref. P04-36500, batch: 2300518) for 15 minutes at room temperature. Cells were then incubated with a monoclonal anti-tyrosine hydroxylase antibody raised in chicken (TH, Abcam; ref. ab76442, batch: GR3190915) in PBS containing 1% FCS and 0.1% saponin for 2 hours at room temperature. TH antibody stained dopaminergic neurons.

[0346] The antibody was exposed for 1 h at room temperature with Alexa Fluor 488 goat anti-chicken IgG (Molecular Probes, ref: 13417227, batch: SC2359411A) in PBS containing 1% FCS and 0.1% saponin. Cell nuclei were labeled with a fluorescent marker (Hoechst solution, Sigma; ref: B1155, batch: 046M4048V) in the same solution.

[0347] For each condition, 20 photographs were taken per well using an InCell Analyzer™ 2200 (GE Healthcare) at 20x magnification. Images of each culture well were taken under the same conditions. Analysis of the cell bodies of TH-positive neurons was performed using Developer software (GE healthcare). A total of six data points were provided for each experimental condition.

[0348] statistics Data were expressed as mean + / - standard error of the mean (six data per culture, per condition). Global analysis of the data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p<0.05.

[0349] result As shown in Figure 8A, SynAging α-synuclein oligomer preparations induced a significant decrease in dopaminergic neuron viability (p<0.001, ***, 58.43% of control). Similar experiments showed a similar decrease in neuronal viability (e.g., 56.07% of control). BDNF at 50 ng / mL rescued neurons from cell death (p<0.001, ***, 97.19% of control). In similar experiments, BDNF rescued neurons from cell death at a similar level (e.g., 99.42% of control).

[0350] Test antibody 1A12 (raised against cyclo(CGGTKEQGGGG) (SEQ ID NO: 49)) at 250 nM shows a statistically significant effect on dopaminergic neuron survival (p<0.05, *, 83.15% of control).

[0351] Test antibodies 3C11 at 50 nM and 12B12 at 50 nM are able to rescue dopaminergic neurons from oligomer-induced cell death in a statistically significant manner (**, p<0.01, 89.60% and *, 79.77% of control, respectively; Figure 8A).

[0352] Test antibodies 2D6 at 250 nM and 11B6 at 250 nM (both raised against cyclo(CGGGGEKTKGG) (SEQ ID NO: 5)) are able to rescue dopaminergic neurons from oligomer-induced cell death in a statistically significant manner (**, p<0.01, 83.80%, and *, p<0.05, 81.01% of control, respectively).

[0353] Antibodies 1A12, 3C11, 12B12, 2D6, and 11B6 were able to rescue dopaminergic neurons from oligomer-induced cell death in a statistically significant manner. Antibody 2E9 approached statistical significance.

[0354] Results using additional clones are shown in Figure 8B. Figures 8C-G are exemplary immunohistochemistry images showing dopaminergic neurons (stained with TH) and nuclei (stained with Hoechst solution), as described in the methods. Figure 8C is a control, untreated neuron, showing dopaminergic neuronal processes. Figure 8D is a cell treated with α-synuclein oligomers. No neuronal processes are detectable. Neuronal loss is prevented by the application of antibodies. Figure 8E is a cell treated with the 2E9 antibody and α-synuclein oligomers. Figure 8F is a cell treated with the 12G1 antibody and α-synuclein oligomers, and Figure 8G is a cell treated with the 12B12 antibody and α-synuclein oligomers, all demonstrating protection of dopaminergic neurons from oligomer toxicity.

[0355] In repeated studies, the α-synuclein oligomer preparation was shown to induce a significant decrease in dopaminergic neuron survival (p<0.01, ***, 62.98-64.50% of control). Furthermore, 50 ng / mL of BDNF was able to rescue neurons from preparation-induced cell death (p<0.001, ***, 96.31-103.76% of control).

[0356] The test antibodies 9D8 at 250 nM and 12G1 at 250 nM are able to rescue dopaminergic neuronal death in a statistically significant manner (**, p<0.01, 96.47% and *, p<0.05, 90.59% of control, respectively).

[0357] The test antibody 12B12 (250 nM and 50 nM) and the antibody 10D5 at 250 nM are able to rescue dopaminergic neuronal death in a statistically significant manner (**, p<0.01, 92.63% and *, p<0.05, 91.24% and *, p<0.05, 88.94% of the control, respectively).

[0358] The test antibody 8B12 (250 nM and 50 nM) is able to rescue dopaminergic neuronal death in a statistically significant manner (**, p<0.01, 101.44% and *, p<0.05, 99.52% of control, respectively).

[0359] The test antibody 7F6 at 250 nM is able to rescue dopaminergic neuron death in a statistically significant manner (**, p<0.01, respectively 98.39% of control).

[0360] Example 11 Effect of antibodies against α-synuclein aggregates using preformed fibrils (PFFs) in a hippocampal neuron culture model of Parkinson's disease. Sonicated synthetic preformed fibrils (PFFs, small soluble fibrils) have been shown to recruit endogenous α-syn and induce LB / LN pathology in vitro and in vivo, implicating the propagation and cell-to-cell communication of pathological α-syn as a mechanism for the gradual spread of LB / LN ( Costanzo and Zurzolo, 2013 , Guo and Lee, 2014 ).

[0361] Intercellular diffusion of misfolded disease proteins can involve their release and subsequent internalization. Immunotherapy can treat neurodegenerative diseases by neutralizing them in the extracellular space (Prusiner, 2012; Jucker and Walker, 2013).

[0362] The effect of test antibodies on the internalization of synthetic α-syn and the recruitment of endogenous α-syn to pathological phosphorylated forms was also examined using PFFs in hippocampal neuron cultures.

[0363] method Rat hippocampal neurons were cultured as described by Harrison (1990). Pregnant females (Wistar, Janvier) were sacrificed by cervical dislocation on day 17 of gestation. The hippocampus was rapidly and aseptically dissected from each brain in ice-cold Leibovitz medium (L15, Panbiotech, Ref. P04-27055, Batch: 4511117), then the meninges were removed and minced. The hippocampal tissue was then digested with trypsin (Trypsin EDTA 1X; PanBiotech, Ref. P10-023100, Batch: 8970318) at 37°C for 20 min. The reaction was stopped by the addition of DMEM (Panbiotech, Ref P04-03600, Batch: 5181217) containing DNAase I Grade II (0.1 mg / mL Panbiotech, ref: P60-37780100, Batch: H170706) and 10% fetal calf serum (FCS, Invitrogen, ref: 10270-098, Batch 42G2068K). Cells were mechanically dissociated by passing them three times through a 10 mL pipette. Cells were then centrifuged at 515 x g for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in synthetic culture medium consisting of Neurobasal (Nb, Invitrogen, ref. 21103049, batch 1979084) supplemented with 2% B27 (Invitrogen, ref. 17504-044, batch: 1969926), 2 mM L-glutamine (PanBiotech, ref. P04-80100, batch: 8440517), 2% PS solution, and 10 ng / mL BDNF (Peprotech, ref. 450-02, batch: 021861). Viable cells were counted using a Neubauer cytometer using the trypan blue exclusion test. Under these conditions, after 3 days of culture, hippocampal neuron cultures contained less than 5% astrocytes.

[0364] Cells were seeded at a density of 20,000 cells / well in 96-well plates (wells precoated with poly-D-lysine (Greiner)) and cultured at 37°C in a humidified air (95%) / CO2 (5%) atmosphere. Half of the medium was replaced with fresh medium every 2 days. Cultures were used after 7 days of culture.

[0365] Preparation of α-synuclein and cell culture injury Human α-syn peptide (Proteos) was prepared as described by Volpicelli-Daley et al. (2014). Human α-syn peptide was thawed and centrifuged at 12,000 g for 10 minutes at 4°C to pellet any aggregated material. The supernatant was used to generate PFF. The concentration was adjusted to 5 mg / mL, and 500 μL was shaken at 37°C and 1000 RPM for 7 days. At this step, the PFF was aliquoted and stored at -80°C until use.

[0366] The α-syn PFF preparation was used for primary hippocampal neurons after 7 days of culture.

[0367] PFFs were diluted to 0.1 mg / mL in sterile PBS, and the suspension was sonicated with 60 pulses of 0.5 seconds at 10% power. The sonicated PFF solution was then diluted to 1 μg / mL in hippocampal neuron medium and added to the neuronal cell cultures.

[0368] Test antibodies and α-syn toxin (sonicated PFF) were pre-incubated together for 30 min at room temperature before adding the mixture to the neuronal cultures.

[0369] The cells were simultaneously incubated with the test compounds. The following conditions were performed: Control (vehicle) / vehicle 14 days α-synuclein PFF (1 μg / mL, 14 days) α-synuclein PFF (1 μg / mL, 14 days) + test antibody (0.25 μM and 0.05 μM) The medium was changed once a week without adding fresh fibrils. One culture and six wells were performed per condition.

[0370] Assessment of α-synuclein aggregates Fourteen days after intoxication, cells were fixed for 15 min at room temperature with a solution of 4% paraformaldehyde (Sigma, ref. 6148, batch: SZBE2390V) / 4% sucrose (Sigma, ref. S7903-250G, batch: BCBV9208) / 1% Triton X-100 (Sigma). Cells were then permeabilized and blocked for 15 min at room temperature with a solution of 3% bovine serum albumin (BSA, Dutcher, ref. P06-1391100, batch: H160810) and 0.1% Triton X-100 in phosphate-buffered saline (PBS, PanBiotech, ref. P04-36500, batch: 6760918).

[0371] For quantification of human α-synuclein, cells were Chicken primary antibody against microtubule-associated protein 2 (MAP2, Abcam, ref: ab5392, batch: GR3209140-2) The sections were incubated overnight at 4°C in blocking buffer (PBS, 3% BSA) containing 1 / 500 rabbit primary antibody anti-α-synuclein (Thermofisher, ref 701085, batch 1920377-3).

[0372] For quantification of endogenous pathological α-synuclein, cells were Chicken primary antibody against microtubule-associated protein 2 (MAP2, Abcam, ref: ab5392, batch: GR3209140-2) The sections were incubated overnight at 4°C in blocking buffer containing 1 / 500 rabbit primary antibody anti-phosphorylated Ser129 α-synuclein (abcam, ref ab51253, batch: GR3232346-1).

[0373] These antibodies were exposed to Alexa Fluor 633 goat anti-rabbit IgG (Molecular Probes, ref: A21070, batch: 1700326) and Alexa Fluor 568 goat anti-chicken (Molecular Probes, ref: A110041, batch: 1776042) in PBS 3% BSA for 1 h at room temperature. Cell nuclei were labeled with a fluorescent marker (Hoechst solution, SIGMA, ref: B1155, batch: 046M4048V) in the same solution.

[0374] For each condition, multiple photographs were taken per well using an InCell Analyzer™ 2200 (GE Healthcare) at 20x magnification. Analysis of MAP2-positive neurons and α-synuclein aggregates was performed using Developer software (GE healthcare). All values were expressed as mean ± SEM. Statistical analysis was performed across conditions.

[0375] statistics Data are expressed as mean ± SEM (six data per culture, per condition). Global analysis of data was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. The significance level was set at p<0.05.

[0376] result The effects of the test antibodies on human α-synuclein aggregates in hippocampal neurons damaged by PFF preparations are shown in Figures 13-14.

[0377] As shown in FIG. 13A, the PFF preparation induces a large and significant increase in human α-synuclein aggregates (p<0.001, ***, 20376% of control).

[0378] Antibodies 2E9 (***, p<0.001, 12168% of control at 0.25 μM; *, p<0.05 15387% of control at 0.05 μM), 9D8 (***, p<0.001, 9181% of control at 0.25 μM), and 12G1 (***, p<0.001, 11694% of control at 0.25 μM and 13156% of control at 0.05 μM) are able to reduce human α-synuclein aggregates in a statistically significant manner.

[0379] As shown in Figure 13B, PFF preparations induce a large and significant increase in human α-synuclein aggregates (p<0.001, ***, 53941% of control). Antibodies 12B12 (*, p<0.05, 40717% of control at 0.25 μM and 41007% of control at 0.05 μM) and 1A12 (**, p<0.01, 38641% of control at 0.25 μM and ***, p<0.001, 30064% of control at 0.05 μM) are able to reduce human α-syn aggregates in a statistically significant manner.

[0380] As shown in FIG. 13C, the PFF preparation induces a large and significant increase in human α-synuclein aggregates (p<0.001, ***, 41528% of control).

[0381] Antibodies 3C11 (*, p<0.05, 28829% of control at 0.05 μM) and 11B6 (***, p<0.001, 23690% of control at 0.25 μM and **, p<0.01, 24897% of control at 0.05 μM) are able to reduce human α-syn aggregates in a statistically significant manner.

[0382] As shown in Figure 13I, the effects of 10D5 and 1C7 antibodies on the internalization of preformed α-Syn fibrils (PFFs) were tested according to the protocol described in this example. Both antibodies significantly reduced PFF uptake and aggregation induction. For mean + SEM, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and # indicates PFFs only.

[0383] Exemplary images are shown in Figures 13D-H. Figure 13D shows control cells stained for the neuronal marker MAP2, revealing the long neuronal processes and cell bodies of neurons. Nuclei are stained as described in the Methods. Figure 13E shows cells treated with α-synuclein PFFs. The α-synuclein PFFs are visible as bright punctate staining, indicating aggregates. Figures 13F-H show cells where α-synuclein PFFs were first incubated with the test antibody. There are visibly fewer aggregates.

[0384] The effect of test antibodies on the recruitment of phosphorylated endogenous rat α-synuclein aggregates in hippocampal neurons exposed to human PFF α-synuclein preparations is shown in Figures 14A and 14B.

[0385] As observed in FIG. 14A, the PFF preparation induces a large and significant increase in endogenous phosphorylated α-synuclein aggregates (p<0.001, ***, 215.26% of control).

[0386] Antibody 2E9 at 0.05 μM (**, p<0.01, 131.13% of control) and 12G1 at 0.25 μM and 0.05 μM (*, p<0.05, 144.81% and 147.77% of control, respectively) are able to reduce endogenous phosphorylated α-syn aggregates in a statistically significant manner.

[0387] As observed in FIG. 14B, the PFF preparation induces a large and significant increase in endogenous phosphorylated α-synuclein aggregates (p<0.001, ***, 225.18% of control).

[0388] Antibody 12B12 at 0.25 μM and 0.05 μM can statistically reduce endogenous phosphorylated α-syn aggregates (*, p<0.05, 140.44% and 144.88% of control, respectively).

[0389] Antibodies 3C11 at 0.05 μM (*, p<0.05, 152.89% of control) and 11B6 at 0.25 μM (*, p<0.05, 156.71% of control, respectively) are able to statistically reduce endogenous phosphorylated α-syn aggregates.

[0390] As shown in Figure 14H, the effects of 10D5 and 1C7 antibodies on the recruitment of endogenous α-Syn to its pathological phosphorylated form were tested according to the protocol described in this example. Both antibodies significantly reduced PFF uptake and aggregation induction. For mean + SEM, ** indicates p<0.01, *** indicates p<0.001, and # indicates PFFs only.

[0391] Exemplary images are shown in Figures 14C-G. Figure 14C shows control cells. Figure 14D shows PFF-treated cells, showing extensive phosphorylated α-syn aggregate staining. Examples of phosphorylated aggregates within neurons are identified by arrows. Figures 14E-G show that preincubation of PFFs with test antibodies dramatically reduces endogenous phosphorylated α-syn aggregate staining.

[0392] The tested antibodies 2E9, 12G1, 12B12, 3C11, and 11B6 are able to reduce endogenous phosphorylated alpha-syn aggregates in a statistically significant manner.

[0393] Example 12 Immunohistochemistry (IHC) staining and immunofluorescence of LBD and normal brains Frozen sections from the frontal cortex of patients with Lewy body dementia (LBD) were exposed to test antibodies (2E9, 12B12, or 3C11) or control antibodies at a concentration of 4 μg / mL. Similarly, frozen sections from the frontal cortex of normal individuals were exposed to test antibodies (12B12, 12G1, 3C11, 2E9, 11B6, and 9D8) at a concentration of 10 μg / mL. Bound antibodies were detected by adding horseradish peroxidase-conjugated sheep anti-mouse IgG (ECL, 1:1000 dilution) or rabbit anti-human IgG (Abcam, 1:5000 dilution). Diaminobenzidine (DAB) chromogen reagent, HRP enzyme substrate (Vector Laboratories), was then added to the sections, resulting in a brown color. To visualize cells and cell nuclei, the sections were counterstained with hematoxylin (blue-purple staining). For immunofluorescence, detection of bound antibodies was performed using Alexa fluor 568-conjugated goat anti-mouse IgG (Invitrogen) at a working concentration of 1:1000 with DAPI counterstaining.

[0394] result IHC staining demonstrates that α-syn antibodies according to the present disclosure preferentially bind to small aggregates over dense Lewy bodies (insoluble fibril deposits), as shown in Figures 10A (2E9), 10B (12B12), and 10C (3C11). Figure 10D shows pan α-syn 4D6 antibody staining of Lewy bodies, and Figure 10E shows a mouse IgG1 control. While arrows in Figures 10A, B, and C point to staining of small aggregates by the test antibody, the arrow in Figure 10D identifies Lewy bodies stained by the pan α-syn antibody. The test antibody demonstrates higher selectivity for small disease-promoting aggregates over Lewy bodies. Consistent with the results seen in IHC, immunofluorescence staining of the test antibody was also performed (Figure 10A, top left panel).

[0395] IHC staining also shows that none of the tested antibodies 12B12, 12G1, 3C11, 2E9, 11B6, and 9D8 produced detectable staining of normal brain (100x magnification), as shown in Figure 10F (12B12), Figure 10G (12G1), Figure 10H (3C11), Figure 10I (2E9), Figure 10J (11B6), and Figure 10K (9D8).

[0396] Example 13 Specificity - Ligand Blocking The binding specificity of the test antibodies to α-syn in soluble DLB brain extracts was also tested in a ligand blocking assay. As shown in Figure 12A, the test antibodies bound to DLB brain extracts. As shown in Figure 12B, the binding of the test antibodies to DLB extracts was epitope-specific, i.e., inhibited by exposure to a peptide containing the epitope sequence used to generate the antibody. Because the Pan α-syn 4D6, Control 1 (human IgG, prasinezumab, PRX002 / RG7935, Creative Labs), and Control 2 (mouse IgG, BAN0805, mAb49 / G, Creative Labs) antibodies recognize different epitopes, their binding to DLB extracts was not blocked by the test antibody peptide containing the epitope sequence.

[0397] Example 14 Prion-like propagation of aggregated α-synuclein (α-Syn) underlies the progression of Parkinson's disease (PD), dementia with Lewy bodies (LBD), and multiple system atrophy (MSA). α-Syn oligomers and small soluble fibrils have been implicated in α-Syn neurotoxicity and propagation, respectively (Fusco 2017 Science, Choi 2018 Cell Reports). We have identified epitopes that enable targeting of these pathogenic species while sparing normal α-Syn monomers and physiological tetramers (Nuber 2018 Neuron).

[0398] Methods: Population coordinates (described in WO / 2017 / 079836 and Peng et al., 2018) were used to identify conformational epitopes predicted to be exposed on a-Syn oligomers and, to a lesser extent, fibril fragments / protofibrils, but not on large fibrils, physiological tetramers, or α-Syn monomers. Using a cyclic peptide scaffold replicating the conformational epitope, mouse monoclonal antibodies were generated, which were then screened for selectivity of binding and bioactivity in vitro.

[0399] Results: Using surface plasmon resonance (SPR), we identified antibody candidates that showed selective binding to synthetic α-Syn oligomers and soluble sonicated fibrils, with little or no binding to monomers or physiological tetramers. Recognition of native α-Syn aggregates in LBD brain extracts was confirmed by SPR and dot blot. Immunohistochemistry confirmed minimal binding to Lewy bodies. In vitro, the antibodies protected primary rodent neurons from α-Syn oligomer toxicity and inhibited mechanisms involved in α-Syn propagation, namely, uptake of sonicated preformed fibrils and induction of phosphorylated α-Syn aggregates.

[0400] Conclusions: "Tuning" epitopes by population coordinates allows the generation of selective antibodies with protective activity against pathogenic α-Syn.

[0401] Example 15 Antibody sequencing The variable regions of the heavy and light chain immunoglobulin genes of murine hybridoma clones 2E9, 9D8, 12G1, 3C11, 12B12, 10D5, and 11B6 were identified and sequenced.

[0402] method Total RNA was isolated from hybridoma cells and reverse transcribed into cDNA using either an isotype-specific antisense primer or a universal primer. Heavy and light chain antibody fragments were amplified by rapid amplification of cDNA ends (RACE). The amplified antibody fragments were separately cloned into standard cloning vectors. Colony PCR was performed to screen for clones with the correct insert size. For each hybridoma cell line, five clones were selected and sequenced for both the heavy and light chains. Sequence alignment was performed using the five clones to confidently determine the heavy and light chain sequences of each monoclonal antibody.

[0403] Amino acid and nucleic acid sequence analysis Tables 13 and 14 below show the nucleic acid and amino acid sequences of the heavy and light chains and complementarity determining regions (CDRs) of each of the antibody clones 2E9, 9D8, 12G1, 3C11, 12B12, 10D5, and 11B6, respectively, as determined by IgBLAST. The CDRs of the heavy and light chains in Table 14 are shown in bold. [Table 13-1] [Table 13-2] [Table 14-1] [Table 14-2] [Table 14-3]

[0404] Table 15 below shows signal sequences that can optionally be linked amino-terminally to antibody chains. [Table 15]

[0405] Evaluating CDR and antibody sequencing consensus The VH and VL sequences were confirmed by sequencing five cloning vectors containing amplified antibody fragments of the variable IgG1 heavy chain and variable kappa light chain. For antibodies 2E9, 9D8, 12G1, 3C11, 12B12, 10D5, and 11B6, 100% alignment was obtained across the five sequencing traces, ensuring the reported framework and CDR1, CDR2, and CDR3 regions of both the heavy and light chains. No alternative nucleotides and / or amino acids were identified for the reported heavy and light chain sequences.

[0406] Example 16 Relative binding of α-Syn species Purified antibodies 2E9, 12B12, and 12G1, as well as three α-syn comparator antibodies purchased from Creative Biolabs, were immobilized on a sensor chip surface for SPR analysis. Approximately 5000 RU of each antibody was immobilized. Serial dilutions of α-syn monomer, tetramer, or sonicated preformed fibrils (soluble fibrils) were injected over the antibodies. Binding interactions were measured, and the relative binding of soluble fibril:monomer and tetramer forms was calculated.

[0407] The ratio of antibody binding to soluble fibrils relative to native monomer and tetramer for the tested antibodies is shown below. [Table 16]

[0408] Unlike the three comparator antibodies, the test antibody showed over 10-fold greater binding to soluble fibrillar α-syn compared to either the monomeric or tetrameric species.

[0409] Example 17 In vitro propagation of α-synuclein aggregation Alpha-synuclein monomers (100 uM) were incubated with 10 nM soluble human preformed fibrils (huPFFs), which acted as seeds to initiate aggregation.

[0410] As shown in Figure 15A, monomers incubated alone did not aggregate under the conditions tested. Aggregation triggers the formation of β-sheets that are bound by thioflavin-T (25 μM), resulting in a fluorescent signal proportional to the amount of aggregation. 2E9 antibody added at 0.1 nM (1:100 molar ratio of 2E9:huPFF) inhibited the propagation of aggregation.

[0411] Seeding by cyclic peptides and inhibition by 2E9 Sonicated preformed fibrils of α-Syn (PFF) are known to act as seeds to trigger aggregation. The ability of the cyclic peptide (CGTKEQGGGG) (SEQ ID NO: 7) alone to replicate the seeding activity of PFF was tested using the thioflavin-T assay described in this example. Aggregation triggers the formation of β-sheets that are bound by thioflavin-T (25 μM), producing a fluorescent signal proportional to the amount of aggregation.

[0412] α-Syn monomer (100 μM) was incubated with 100 nM of a BSA-conjugated cyclic peptide (SEQ ID NO: 7, the cyclic peptide used to generate antibody 2E9) as a seed, or the corresponding BSA-conjugated linear peptide as a control. As shown in Figure 15B, monomers incubated alone or in the presence of the linear peptide did not aggregate. In contrast, the cyclic peptide possessed a conformation that could act as a seed and induce the progressive aggregation of α-Syn over time. 2E9 antibody added at 0.1 nM inhibited the propagation of aggregation (Figure 15B).

[0413] Example 18 Antibody binding to brain extracts from patients with multiple system atrophy (MSA) Antibodies were directly immobilized on the sensor chip via amine coupling. A pan α-synuclein antibody (4D6) was used as a positive control, and mouse IgG1 (mIgG1) was used as a negative isotype control. Soluble brain extract (200 μg / mL) from the cerebellum of a 50-year-old female MSA patient was infused onto the immobilized antibody for 8 minutes, followed by a 5-minute dissociation period. The binding response (in terms of response units, RU) 30 seconds into the dissociation phase is shown in Figure 16A. As can be seen, the test antibodies 2E9, 12G1, 11B6, 12B12, 3C11, 9D8, and 10D5 exhibited binding responses that exceeded the background binding response obtained with mIgG1, and the binding responses were greater than those seen with the control pan α-syn (4D6) antibody.

[0414] SPR analysis of binding to unfractionated human soluble MSA brain extract SPR analysis of unfractionated human soluble MSA brain extract from the cerebellum of a 50-year-old woman with multiple system atrophy (MSA) was performed as described in this example. Test antibodies, a positive control antibody (pan α-Syn 4D6), a Creative Biolabs comparator anti-α-Syn antibody (mAb49 / G, NI-202.12F4, PRX002), and a mouse IgG isotype control (mIgG1) were immobilized at high density (approximately 12,000-20,000 RU) on the flow cell of a sensor chip. Brain soluble extract diluted to 200 μg / mL was injected over the surface at 10 μL / min for approximately 8 minutes, followed by a 5-minute dissociation period (quadruplicate assay). As shown in Figure 16B, all test antibodies and the comparator exhibited binding responses exceeding the background binding response obtained with mIgG1. The binding responses of the test antibodies were also greater than those seen with the control pan α-syn (4D6) antibody: the NI-202.12F4 antibody binds to N-terminal residues 1-10, and the PRX002 antibody binds to residues 118-126.

[0415] SPR analysis of binding to a "prion-enriched" fraction from human MSA brain extracts Brain samples from the cerebellum of a 50-year-old woman with MSA were homogenized as described above (Example 7). The homogenate was then processed as described by Aoyagi et al. (Science Translational Medicine, eaat8462, 2019) to isolate a "prion-enriched fraction" containing self-propagating species of α-Syn. Briefly, 2% sarkosyl and 0.5% benzonase were added to the homogenate and incubated with shaking at 37°C for 2 hours. Phosphotungstic acid (PTA) was then added to a final concentration of 2%, and the mixture was incubated with shaking at 37°C overnight. The material was then centrifuged at 16,100 x g for 30 minutes, and the resulting pellet was resuspended in 2% sarkosyl and 2% PTA and incubated with shaking at 37°C for 1 hour (to wash away residual sarkosyl-soluble proteins). The mixture was then centrifuged at 16,100 x g for 30 minutes, and the final pellet was resuspended in PBS for SPR analysis.

[0416] Test antibodies, a positive control antibody (pan α-Syn 4D6), and a mouse IgG isotype control (mIgG1) were immobilized at high density (approximately 18,000-24,000 RU) on the flow cell of a sensor chip. Resuspended pellet material (estimated protein concentration 250-375 μg / mL) was injected over the surface at 10 μL / min for approximately 8 minutes, followed by a dissociation period of approximately 200 seconds (in duplicate). Binding responses were double-referenced by subtracting the IgG reference surface binding and normalized to assay buffer. Results are shown in Figure 16C. All test antibodies exhibited binding responses above the background binding response obtained with mIgG1. The binding responses of the test antibodies were comparable to or greater than those seen with the control pan α-syn (4D6) antibody, which is expected to bind both prions and any residual contaminating α-Syn species remaining in the prion-enriched preparation.

[0417] Example 19 Measurement of misfolded α-syn oligomers in biological samples The 12G1 antibody was used on the EMD Millipore SMC™ platform. To generate a standard curve, magnetic particles were coated with 12.5 μg / mL of 12G1 antibody and exposed to various concentrations of α-syn oligomers ranging from 0 to 156 pg / mL. The captured α-Syn was then detected using a labeled pan α-Syn antibody (4D6) at a concentration of 1,500 ng / mL. The standard curve shows the signal (response units) from the eluted detection antibody at different α-Syn concentrations. The 12G1-coated magnetic particles were then exposed to MSA brain extract (1,277 μg / mL total protein) following the same protocol. The resulting signal was used to derive the amount of α-Syn oligomers present in the sample using the standard curve. The amount was estimated to be approximately 113 pg / mL (Figure 20).

[0418] Example 20 Selectivity analysis using Millipore's "Single Molecule Counting" (SMC™) platform The binding of test antibodies 12G1 (Figures 17A-C), 9D8 (Figures 18A-C), and 10D5 (Figures 19A-C) to α-Syn monomers, oligomers, and sonicated fibrils was evaluated on the Millipore SMC™ platform to determine the lower limit of quantitation (LLoQ) and relative selectivity of the antibodies for these species. Briefly, magnetic particles were coated with test antibody at 12.5 μg / mL (12G1, 10D5) or 25 μg / mL (9D8). The coated particles were exposed to a wide range of α-Syn concentrations, up to 1400 ng / mL for monomers, 10,000 pg / mL for oligomers, and 1,000 pg / mL for soluble sonicated fibrils. Captured α-Syn was then detected using a labeled pan α-Syn antibody (4D6) at a concentration of 1,500 ng / mL. The binding curves show the signal (response units) from the eluted detection antibody for different α-Syn concentrations. The LLoQ is defined as the interpolated value where the signal is 2.5 times background.

[0419] All three antibodies tested demonstrated much higher reactivity with α-Syn oligomers and fibrils (pathogenic species of α-Syn) compared to monomers. Based on LLoQ values, the fold selectivity for oligomers versus monomers ranged from 9,300-35,000-fold, and the fold selectivity for soluble fibrils versus monomers ranged from 11,200-175,000-fold. Specifically, the 12G1 fold selectivity for oligomers versus monomers was 35,000-fold and for fibrils versus monomers was 175,0000-fold (Figure 17A-C). The 9D8 fold selectivity for oligomers versus monomers was 9,300-fold and for fibrils versus monomers was 93,300-fold (Figure 18A-C). The 10D5-fold selectivity for oligomers versus monomers was 9,300-fold and for fibrils was 11,200-fold (Figure 19A-C).

[0420] Example 21 SPR affinity measurement For SPR analysis of antibody binding parameters for various α-Syn species (monomer, physiological tetramer, oligomer, and soluble fibrils), antibodies were immobilized at approximately 2,000–4,500 RU on the flow cell of a sensor chip. α-Syn analytes diluted two-fold from 1,000–0.5 nM (12-point dilution series) were injected sequentially over the surface for approximately 4 min, followed by dissociation with buffer for approximately 5 min to regenerate the surface. Binding parameters were calculated using steady-state (monomer, physiological tetramer) or kinetic (oligomer, sonicated fibrils) curve fitting and a Langmuir 1:1 binding model. The results are summarized in Table 17.

[0421] Compared with other commercially available α-Syn antibodies (Creative Biolabs, clones PRX002 and NI-202.12F4), the tested antibodies (2E9, 12G1, 12B12) showed higher selectivity for pathogenic α-Syn species, negligible binding to monomers and physiological tetramers, but strong affinity for oligomers and sonicated fibrils. [Table 17]

[0422] While the present application has been described with reference to what are presently considered to be preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0423] All publications, patents, and patent applications are incorporated herein 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. Specifically, the sequences associated with each accession number provided herein, including, for example, the accession number and / or biomarker sequences (e.g., proteins and / or nucleic acids) provided in the tables or elsewhere, are incorporated by reference in their entirety.

[0424] The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole. References Aoyagi et al. (2019).Aβ and tau prion-like activities decline with longevity in the Alzheimer's disease human brain.Sci.Transl.Med.,eaat8462. Schinelli,S.,Zuddas,A.,Kopin,I.J.,Barker,J.L.,& DI Porzio,U.(1988).1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolism and 1-methyl-4- phenylpyridinium uptake in dissociated cell cultures from the embryonic mesencephalon.J Neurochem.,50,1900-1907. Costanzo,M.,and Zurzolo,C.(2013).The cell biology of prion-like spread of protein aggregates: mechanisms and implication in neurodegeneration.Biochem.J.452,1-17. Guo,J.L.,and Lee,V.M.(2014).Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases.Nat.Med.20,130-138. Harrison NL.(1990)On the presynaptic action of baclofen at inhibitory synapses between cultured rat hippocampalneurones.J Physiol.1990 Mar;422:433-46. Jucker M and Walker LC.(2013)Self-propagation of pathogenic protein aggregates in neurodegenerative diseases.Nature.Sep 5;501(7465):45-51. Prusiner,S.B.(2012).Cell biology.A unifying role for prions in neurodegenerative diseases.Science 336,1511-1513. Volpicelli-Daley L.,Luk K.,Lee V.(2014)Addition of exogenous α-Synuclein Pre-formed fibrils to Primary Neuronal Cultures to seed recruitment of endogenous α-Synuclein to Lewy body and Lewy Neurite-like aggregates.Nat Protoc.9(9):2135-2146. Nuber,Silke,Molly Rajsombath,Georgia Minakaki...Barbara Caldarone,Ulf Dettmer,and Dennis J.Selkoe.Abrogating Native α-Synuclein Tetramers in Mice Causes a LDOPA-Responsive Motor Syndrome Closely Resembling Parkinson’s Disease.NEURON October 10,2018. Peng 2018 Journal Physical Chemistry B.Prediction of Misfolding-Specific Epitopes in SOD1 Using Collective Coordinates Xubiao Peng,Neil R.Cashman,and Steven S.Plotkin,The Journal of Physical Chemistry B 2018 122(49),11662-11676.

Claims

1. A cyclic compound comprising an α-Syn peptide consisting of at least four consecutive residues of EKTKEQ (SEQ ID NO: 1), and a linker, wherein the linker is covalently attached to the N-terminal and C-terminal residues of the peptide, and the cyclic compound consists of the amino acid sequence of any one of SEQ ID NOs: 5, 7, 10, 11, or 18-60.

2. 2. The cyclic compound of claim 1, wherein the α-Syn peptide is selected from EKTK (SEQ ID NO: 2), KTKE (SEQ ID NO: 3), and TKEQ (SEQ ID NO: 4).

3. The cyclic compound of claim 1 or 2, wherein the α-Syn peptide is KTKE (SEQ ID NO: 3).

4. The cyclic compound of claim 1 or 2, wherein the α-Syn peptide is selected from EKTK (SEQ ID NO: 2) and TKEQ (SEQ ID NO: 4).

5. The cyclic compound of any one of claims 1, 2, or 4, wherein the α-Syn peptide is EKTK (SEQ ID NO: 2).

6. The cyclic compound of any one of claims 1, 2, or 4, wherein the α-Syn peptide is TKEQ (SEQ ID NO: 4).

7. The cyclic compound of claim 1 , wherein the cyclic compound is selected from compounds consisting of the amino acid sequence of any one of SEQ ID NOs: 5, 7, 10, 48, or 49.

8. 2. The cyclic compound of claim 1, wherein the cyclic compound is selected from cyclo(CGTKEQGGG) (SEQ ID NO: 57), cyclo(CGGTKEQGGG) (SEQ ID NO: 47), cyclo(CGGTKEQGG) (SEQ ID NO: 48), cyclo(CGGGEKTKGG) (SEQ ID NO: 10), and cyclo(CGGGGEKTKGG) (SEQ ID NO: 5).

9. An immunogen comprising the cyclic compound according to any one of claims 1 to 8.

10. 10. The immunogen of claim 9, wherein the cyclic compound is conjugated to a carrier protein or immunogenicity enhancing agent and / or is a multiple antigenic peptide (MAP).

11. The immunogen of claim 10, wherein the carrier protein is bovine serum albumin (BSA) or the immunogenicity enhancer is keyhole limpet hemocyanin (KLH).

12. An antibody that binds to the α-Syn peptide in the cyclic compound of any one of claims 1 to 8.

13. The antibody of claim 12, wherein the antibody binds to misfolded oligomeric α-Syn polypeptides.

14. An antibody described in any one of claims 12 or 13, wherein the antibody selectively binds to a cyclic compound described in any one of claims 1 to 8 compared to a corresponding linear compound and / or native α-Syn polypeptide.

15. The antibody of claim 13, wherein the antibody is at least 2-fold, 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold more selective for the cyclic compound compared to the corresponding linear compound and / or native α-Syn polypeptide.

16. The antibody of any one of claims 13 to 15, wherein the antibody selectively binds to misfolded, oligomeric α-Syn polypeptides compared to native α-Syn polypeptides.

17. 17. The antibody of claim 16, wherein the antibody is at least 2-fold, 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold more selective for misfolded oligomeric alpha-Syn polypeptides compared to native alpha-Syn polypeptides.

18. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of any one of claims 13 to 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 61 to 66, respectively.

19. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of any one of claims 13 to 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 67 to 72, respectively.

20. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of any one of claims 13 to 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 73 to 78, respectively.

21. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of any one of claims 13 to 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 79 to 81, 76, 77, and 84, respectively.

22. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of claim 16 or 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 79, 80, 81, 76, 77, and 84, respectively.

23. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of any one of claims 13 to 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 91 to 94, 71, and 96, respectively.

24. the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3; The antibody of any one of claims 13 to 17, wherein the complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 have the amino acid sequences of SEQ ID NOs: 180, 181, 182, 183, 77, and 184, respectively.

25. 19. The antibody of claim 18, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 133, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 134, and the CDR sequences are maintained.

26. 20. The antibody of claim 19, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 135, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 136, and the CDR sequences are maintained.

27. 21. The antibody of claim 20, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 137, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 138, and the CDR sequences are maintained.

28. 22. The antibody of claim 21, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 139, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 140, and the CDR sequences are maintained.

29. 23. The antibody of claim 22, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 141, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 142, and the CDR sequences are maintained.

30. 24. The antibody of claim 23, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 143, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 144, and the CDR sequences are maintained.

31. 25. The antibody of claim 24, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 190 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 190, and the CDR sequences are maintained; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 191 or an amino acid sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 191, and the CDR sequences are maintained.

32. 32. The antibody of any one of claims 13 to 31, wherein the antibody is selected from the group consisting of a monoclonal antibody, an immunoglobulin molecule, Fab, Fab', F(ab)2, F(ab')2, Fv, disulfide-linked Fv, scFv, disulfide-linked scFv, single-chain antibody, single-domain antibody, diabody, dimer, minibody, bispecific antibody fragment, chimeric antibody, humanized antibody, and polyclonal antibody.

33. An immunoconjugate comprising an antibody according to any one of claims 13 to 32 and a detectable label or particle, optionally a moiety such as a magnetic particle.

34. A nucleic acid comprising a nucleic acid sequence encoding the amino acid residues of the cyclic compound or immunogen of any one of claims 1 to 11, or encoding the antibody of any one of claims 13 to 32 or the immune complex of claim 33.

35. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 97-102 encoding complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

36. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 103-108, which encodes complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

37. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 109-114, which encodes complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

38. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 115-117, 112, 113, and 120, which encode complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

39. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 115, 116, 123, 112, 113, and 120, which encode complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

40. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 127-130, 107, and 132, which encode complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

41. 35. The nucleic acid of claim 34, wherein the nucleic acid comprises a nucleic acid sequence having SEQ ID NOs: 185-188, 113, and 189, which encode complementarity determining regions CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively.

42. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 145; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 145, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 133; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 133, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 146; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 146, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 134; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 134, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 36. The nucleic acid of claim 35.

43. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 147; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 147, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 135; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 135, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 148; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 148, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 136; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 136, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 37. The nucleic acid of claim 36.

44. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 149; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 149, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 137; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 137, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 150; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 150, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 138; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 138, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 38. The nucleic acid of claim 37.

45. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 151; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 151, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 139; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 139, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 152; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 152, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 140; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 140, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 39. The nucleic acid of claim 38.

46. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 153; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 153, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 141; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 141, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 154; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 154, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 142; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 142, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 40. The nucleic acid of claim 39.

47. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 155; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 155, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 143; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 143, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 156; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 156, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 144; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 144, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 41. The nucleic acid of claim 40.

48. the heavy chain variable region a nucleic acid sequence comprising SEQ ID NO: 192; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 192, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 190; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 190, wherein the amino acid sequence of the CDR sequences is maintained; is coded by the light chain variable region a nucleic acid sequence comprising SEQ ID NO: 193; a sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 193, wherein the amino acid sequence of the CDR regions is maintained; A sequence encoding SEQ ID NO: 191; or a sequence encoding an amino acid sequence having at least 80%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 191, wherein the amino acid sequence of the CDR sequences is maintained; is coded by 42. The nucleic acid of claim 41.

49. A vector comprising the nucleic acid of any one of claims 34 to 48.

50. 50. The vector of claim 49, further comprising a signal sequence selected from any of SEQ ID NOs: 157-164, 169-177, and 179.

51. A recombinant cell expressing the antibody of any one of claims 13 to 32.

52. 52. The recombinant cell of claim 51, wherein the recombinant cell is a mammalian cell, optionally a hybridoma cell or a CHO cell.

53. 53. A composition comprising a cyclic compound, immunogen, antibody, immunoconjugate, nucleic acid, vector, or recombinant cell according to any one of claims 1 to 52, optionally comprising one or more, optionally two or more, or three or more of the cyclic compound, immunogen, antibody, immunoconjugate, nucleic acid, vector, or recombinant cell according to any one of claims 1 to 52.

54. 54. A kit comprising a cyclic compound, immunogen, antibody, immunoconjugate, nucleic acid, vector, recombinant cell, or composition of any one of claims 1 to 53, optionally together with one or more reagents, particles, or plates.

55. 33. A method of making the antibody of any one of claims 13 to 32, comprising administering to a non-human subject a cyclic compound or immunogen of any one of claims 1 to 11, or a composition comprising said cyclic compound or immunogen, isolating antibodies and / or cells expressing antibodies specific for said administered cyclic compound or immunogen, and optionally selecting and / or isolating one or more antibodies that selectively bind to misfolded oligomeric alpha-Syn polypeptides.

56. 1. An assay for determining whether a test sample contains a misfolded α-Syn polypeptide, the method comprising: contacting the test sample with the antibody or immune complex comprising said antibody of any one of claims 13 to 32, optionally the immune complex of claim 33, under conditions that allow the formation of an antibody:misfolded α-Syn polypeptide complex; b. detecting and / or quantifying the presence of any antibody:misfolded α-Syn polypeptide complex; The presence of a detectable complex indicates that the test sample may contain a misfolded α-Syn polypeptide.

57. 57. The assay of claim 56, wherein the amount of antibody:misfolded α-Syn polypeptide complex is quantified and / or compared to a control.

58. 58. The assay of claim 56 or 57, wherein the test sample comprises blood and / or serum and / or plasma and / or brain tissue extract and / or CSF.

59. 59. The assay of any one of claims 56 to 58, wherein the test sample is a human sample.

60. 60. The assay of any one of claims 56 to 59, wherein detecting the complex comprises contacting the complex with a pan α-Syn antibody.

61. 61. The assay of any one of claims 56 to 60, wherein the assay further comprises detecting and / or quantifying the presence of antibody:misfolded alpha-Syn polypeptide complexes in a subsequent test sample and optionally comparing with the test sample.

62. 62. The assay of any one of claims 56 to 61, wherein the subject is undergoing treatment for an alpha-synucleinopathy.

63. 63. The assay of any one of claims 56 to 62, wherein the subject is not undergoing treatment for an alpha-synucleinopathy.

64. 64. The assay of any one of claims 56 to 63, wherein the antibody contacted with the test sample is conjugated to a particle, optionally a magnetic bead.

65. 65. The assay of any one of claims 56-64, wherein said detecting or quantifying said complex comprises contacting said complex with a labeled pan alpha-syn antibody.

66. 66. An assay according to any one of claims 56 to 65 for use in a method for diagnosing whether a subject has an alpha-synucleinopathy, comprising: a. detecting the amount of misfolded alpha-synuclein in a test sample of said subject; b. comparing the amount of misfolded alpha-synuclein to a control, wherein the control is a cut-off or range found in a population of control samples; If the amount of misfolded alpha-synuclein is higher than the level or range found in a normal control sample or within the range found in a control sample from a subject with the synucleinopathy, then the subject is likely to have an alpha-synucleinopathy.

67. 67. The assay of claim 66, wherein the alpha-synucleinopathy is selected from Parkinson's disease (PD), Lewy body disease (LBD), or multiple system atrophy.

68. 34. A pharmaceutical composition for inhibiting misfolded α-syn toxicity in a cell population or a subject, comprising an effective amount of the antibody or immunoconjugate of any one of claims 12 to 33, or a composition comprising said antibody or immunoconjugate, wherein said antibody selectively binds to misfolded oligomeric α-Syn compared to monomeric, native tetrameric, and / or insoluble fibrillar α-synuclein species in said cell population or said subject.

69. 69. The pharmaceutical composition of claim 68, wherein the composition comprises one or more antibodies and / or immunoconjugates according to any one of claims 12 to 33.

70. 70. The pharmaceutical composition of claim 68 or 69 for treating an α-synucleinopathy, comprising an effective amount of the antibody of any one of claims 14 to 32, the immunoconjugate of claim 33, or the composition of claim 53, wherein the antibody selectively binds to misfolded oligomeric α-synuclein compared to monomeric, native tetrameric, and / or insoluble fibrillar α-synuclein species in a subject.

71. 71. The pharmaceutical composition of claim 70, wherein the α-synucleinopathy is selected from Parkinson's disease (PD), Lewy body disease (LBD), or multiple system atrophy.

72. The cyclic compound of claim 3 , wherein the cyclic compound is selected from compounds consisting of the amino acid sequence of any one of SEQ ID NOs: 33, 35, and 36.

73. 72. A pharmaceutical composition according to claims 68 to 71, comprising the immunogen of any one of claims 9 to 11 and an adjuvant selected from aluminium phosphate, aluminium hydroxide, aluminium sulphate, saponin, particles produced from saponin, pluronic polymers containing mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products, liposomes, or oil-in-water emulsions.

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