Human aggregated alpha-synuclein binding molecules

Abstract

Provided herein are human aggregated alpha-synuclein binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human aggregated alpha-synuclein binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) with particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat a disease, such as a synucleinopathy.

Description

[0001] HUMAN AGGREGATED ALPHA- SYNUCLEIN BINDING MOLECULES

[0002] The present application claims priority to U.S. Provisional applications serial numbers 63 / 486,127, filed February 21, 2023, and 63 / 511,933, filed July 5, 2023, both of which are herein incorporated by reference in their entirities.

[0003] This invention was made with government support under GM 136300 and AG059723 awarded by the National Institutes of Health, and 1159943, 1813963, and 1605266 awarded by the National Science Foundation. The government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] Provided herein are human aggregated alpha-synuclein binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human aggregated alpha-synuclein binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) with particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat a disease, such as a synucleinopathy.

[0006] BACKGROUND OF THE INVENTION

[0007] Conformational antibodies are important for studying structure-function relationships for diverse types of proteins, including poorly soluble ones such as membrane1,2and amyloid- forming3-5proteins. In the case of neurodegenerative diseases, conformational antibodies are critically important because of the large number of possible structures that can form for a single polypeptide assembled into diverse types of aggregates ranging from pre-fibrillar soluble oligomers to amyloid fibrils. Immunization6-8and in vitro display technologies9-11, such as phage and yeast surface display, have been widely used to generate antibodies with conformational specificity against A , tau, a-synuclein and other proteins linked to neurodegenerative diseases.

[0008] Despite this progress, the generation of high-quality conformational antibodies with strict conformational specificity for insoluble antigens such as protein aggregates remains largely a trial-and-error process, requires extensive experimentation and, in many cases, results in sub-optimal antibodies. In particular, most previously reported conformational antibodies against protein aggregates lack optimal combinations of three key properties, namely high affinity, strict conformational specificity, and low off-target binding12 13. One key explanation for these sub-optimal combinations of binding properties is that it is challenging to select for all three antibody properties during the discovery process. This commonly results in antibodies generated against protein aggregates that either i) bind linear peptide epitopes and lack conformational specificity, ii) display polyreactivity, and / or iii) possess modest affinity, especially for antibodies generated using in vitro display methods.

[0009] In principle, in vitro directed evolution methods such as yeast surface display should be able to address these challenges because of their ability to select for i) antibody binding at extremely low concentrations of aggregated antigen (high affinity), ii) a lack of antibody binding to non -aggregated antigen (high conformational specificity) and iii) a lack of binding to polyreactivity reagents (low antibody off-target binding). However, in practice, protein aggregates are typically insoluble antigens that are poorly compatible with quantitative cell sorting methods such as fluorescence- activated cell sorting (FACS). Instead, protein aggregates typically must be immobilized on surfaces such as magnetic beads for cell sorting via magnetic-activated cell sorting (MACS). The inability of MACS to perform multiparameter, quantitative cell sorting leads to antibody selections that are strongly influenced both by the antibody expression level (e.g., display levels on the surface of yeast) and the intrinsic antibody affinity, which commonly leads to the enrichment of sub-optimal conformational antibodies and requires extensive secondary screening to isolate rare antibodies with strict conformational specificity, high affinity, and low off-target binding.

[0010] SUMMARY OF THE INVENTION

[0011] Provided herein are human aggregated alpha-synuclein binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human aggregated alpha-synuclein binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) with particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat a disease, such as a synucleinopathy.

[0012] In some embodiments, provided herein are compositions (and kits and systems) comprising: a human aggregated alpha-synuclein binding molecule, or one or more nucleic acid molecules encoding the human aggregated alpha-synuclein binding molecule, wherein the human aggregated alpha-synuclein binding molecule comprises: a) a heavy chain variable region, wherein the heavy chain variable region comprises: i) a CDRH1 amino acid sequence comprising SEQ ID NO: 3, 12, 21, 30, 39, 48, 57, 66, 75, 84, 93, 102, 111, 120, 129, 138, or 147 or any of the preceding with one with one or two conservative amino acid changes, ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 4, 13, 22, 31, 40, 49, 58, 67, 76, 85, 94, 103, 112, 121, 130, 139, or 148, or any of the preceding with one or two conservative amino acid changes, and iii) a CDRH3 amino acid sequence comprising SEQ ID NO: 5, 14, 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, 113, 122, 131, 140, or 149 or any of the preceding with one with one or two conservative amino acid changes, and / or; b) a light chain variable region, wherein the light chain variable region comprises; i) a CDRL1 amino acid sequence comprising SEQ ID NO: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 124, 133, 142, or 151 or any of the preceding with one with one or two conservative amino acid changes, ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 8, 17, 26, 35, 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, or 152, or any of the preceding with one with one or two conservative amino acid changes, and iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 9, 18, 27, 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, or 153, or any of the preceding with one with one or two conservative amino acid changes.

[0013] In some embodiments, provided herein are compositions comprising a human aggregated alpha- sy nuclein binding molecule or one or more nucleic acid molecules encoding said human aggregated alpha- sy nuclein binding molecule, wherein said human aggregated alpha- sy nuclein binding molecule comprises a human aggregated alpha-synuclein antibody, antibody fragment, or nanobody that specifically binds a first epitope of human aggregated alpha-synuclein, wherein said first epitope comprises 33-TKEGVLYVGS-42.

[0014] In certain embodiments, provided herein are methods of treating or preventing a synucleinopathy disease or condition comprising: treating a subject with a human aggregated alpha-synuclein binding molecule, or an expression vector comprising the one or more nucleic acid molecules encoding the human aggregated alpha-synuclein binding molecule, as recited above and herein, and wherein the subject (e.g., human subject) has, or is suspected to develop, a synucleinopathy disease or condition. In particular embodiments, the neurodegenerative disease or condition is selected from the group consisting of: Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and Alzheimer's Disease with Amygdalar Restricted Lewy Bodies (AD / ALB) .

[0015] In certain embodiments, wherein: i) the CDRH1 amino acid sequence comprises SEQ ID NO:12, 21, 30, 39, 48, 57, 66, 75, 84, 93, 102, 111, 120, 129, 138, or 147; ii) the CDRH2 amino acid sequence comprises SEQ ID NO: 13, 22, 31, 40, 49, 58, 67, 76, 85, 94, 103, 1 12, 121, 130, 139, or 148; iii) the CDRH3 amino acid sequence comprises SEQ ID NO:14, 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, 113, 122, 131, 140, or 149; iv) the CDRLl amino acid sequence comprises SEQ ID NO:16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 1 15, 124, 133, 142, or 151; v) the CDRL2 amino acid sequence comprises SEQ ID NO:17, 26, 35, 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, or 152; and vi) the CDRL3 amino acid sequence comprises SEQ ID NO:18, 27, 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, or 153.

[0016] In certain embodiments, the human aggregated alpha-synuclein binding molecule is an antibody, minibody, diabody, scFv, or antibody fragment capable of binding human aggregated alpha-synuclein fibrils. In other embodiments, the antibody fragment is a Fab, F(ab')2 or Fv antibody fragment. In additional embodiments, the antibody or antibody fragment comprises at least an antigen binding portion of: 1) H2.3 antibody, 2) H2.4 antibody, 3) H2.7 antibody; 4) Ll. l antibody; 5) LI.2 antibody; 6) LI.5 antibody; 7) LI.6 antibody; 8) LI.8 antibody; 9) LI.9 antibody; 10) LI.10 antibody; 11) L2.1 Antibody; 12) L2.2 antibody; 13) L2.3 antibody; 14) L2.6 antibody; 15) L2.8 antibody; or 16) L2.9 antibody.

[0017] In some embodiments, the heavy chain and / or light chain variable region comprises a human framework region. In additional embodiments, the human aggregated alpha-synuclein binding molecule further comprises a light chain constant region and a CHI heavy chain constant region. In additional embodiments, the alpha-synuclein binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region. In other embodiments, the light chain constant region is human or a humanized murine, and / or wherein the CHI, CH2, and CH3 heavy chain constant regions are human or are humanized murine.

[0018] In certain embodiments, the human aggregated alpha-synuclein binding molecule comprises an antibody, wherein the light chain constant region of the antibody is selected from: IgG Kappa and IgG Lambda, and wherein the heavy chain constant region of the antibody is selected from: IgGl, IgG2, IgG3, and IgG4. In further embodiments, the human aggregated alpha-synuclein binding molecule comprises an antibody, or antigen binding portion thereof, which is glycosylated or non-glycosylated. In other embodiments, the compositions herein further comprise a physiologically tolerable buffer.

[0019] In further embodiments, the heavy chain variable regions comprises SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74, 83, 92, 101, 110, 119, 128, 137, or 146 or any of the preceding with one or more conservative amino acid changes. In additional embodiments, the light chain variable region comprises SEQ ID NO: 6, 15, 24, 33, 42, 51, 60, 69, 78, 87, 96, 105, 114, 123, 132, 141, or 150, or any of the preceding with one or more conservative amino acid changes.

[0020] In some embodiments, the composition comprises the one or more nucleic acid molecules. In further embodiments, the one or more nucleic acid molecules comprise: i) a first nucleic acid sequence encoding the heavy chain variable region, and ii) a second nucleic acid sequence encoding the light chain variable region. In certain embodiments, the compositions, kits, and systems herein further comprise, or the methods herein employ, an expression vector, and wherein the nucleic acid molecules (e.g., first and / or second nucleic acid sequences) are present in the expression vector.

[0021] In further embodiments, provided herein are methods of detecting human aggregated alpha- sy nuclein a sample comprising: a) contacting a sample with the human aggregated alpha-synuclein binding molecule as described above or herein, wherein the sample (e.g., tissue sample) is suspected of containing human alpha-synuclein fibril, and wherein the human aggregated alpha-synuclein binding molecule forms a complex with the human aggregated alpha-synuclein if present in the sample; and b) detecting the presence or absence of the complex in the sample. In some embodiments, the sample is from a subject that has, or is suspected to develop, a synucleinopathy disease or condition. In particular embodiments, the human aggregated alpha-synuclein binding molecule comprises a detectable label. In other embodiments, the methods further comprise contacting the sample with a conjugate molecule capable of binding to the human aggregated alpha-synuclein binding molecule, wherein the conjugate molecule comprises a detectable label.

[0022] In some embodiments, provided are methods of generating an enriched cell fraction comprising target cells expressing antibodies, or fragments thereof, that bind an aggregated conformation of a target protein comprising: a) contacting a plurality of candidate cells that surface-display an antibody library with an aggregated conformation of a target peptide such that a positive cell sub-population, but not a negative cell sub-population, binds the aggregated conformation of the target protein; b) separating the positive cell sub-population from the negative cell sub-population; c) contacting the positive cell sub-population with a disaggregated conformation of the target peptide such that a positive cell fraction, but not a negative cell fraction, binds the disaggregated conformation of the target protein; d) separating the negative cell fraction from the positive cell fraction; and e) sorting the negative cell fraction against a plurality of QD-AgTP conjugates such than an enriched cell fraction is generated comprising target cells expressing antibodies, or fragments thereof, that bind the aggregated conformation of the target protein, wherein each of said QD-AgTP conjugates comprises a quantum dot attached to, or operably linked (e.g., via an antibody) to an aggregated conformation of said target protein.

[0023] In particular embodiments, the methods further comprise: f) treating the target cells such that target nuclei acid molecules are isolated encoding the antibodies, or fragments thereof, that bind the aggregated conformation of the target protein. In other embodiments, the methods further comprise: g) sequencing at least some of the target DNA to obtain sequencing data comprising the nucleic acid sequences encoding one or more antibodies, or antigen binding fragments thereof, that are specific for the aggregated conformation of the target protein. In additional embodiments the methods further comprise: h) affinity maturing at least one of the target nuclei acid molecules to generate at least one matured target nucleic acid molecule. In additional embodiments, the methods further comprise: i) expressing the at least one matured target nucleic acid molecule in a host cell to generate an antibody, or antibody fragment.

[0024] In additional embodiments, the target protein is selected from: amyloid beta, tau, alpha-synuclein, TAR DNA binding protein 43, a C9orf72 related protein, islet amyloid polypeptide, and transthyretin. In further embodiments, the aggregated conformation of a target peptide in step a) is conjugated to a solid support. In certain embodiments, the solid support comprises magnetic beads.

[0025] In other embodiments, the plurality of candidate cells comprise yeast cells. In additional embodiments, the antibody library comprises at least 1000 . .. 10,000 . . . 100,000 . . . or 1 million ... or 10 million different antibodies or fragments thereof. In certain embodiments, the antibody library comprises at least 100 million or 1 billion different antibodies or fragments thereof. In some embodiments, the sorting comprises flow cytometry, wherein the flow cytometry is optionally fluorescence- activated cell sorting (FACS). In additional embodiments, the antibody library comprises a single-chain antibody library.

[0026] DESCRIPTION OF THE FIGURES

[0027] Figure 1. Overview of approach for selecting and affinity maturing anti-amyloid conformational antibodies. A previously reported human single-chain variable fragment (scFv) library (14) was displayed on the surface of yeast and sorted positively for binding to amyloid fibrils (e.g., a-synuclein) via magnetic-activated cell sorting (MACS) and negatively for a lack of binding to disaggregated polypeptide (e.g., a -synuclein monomer) via fluorescence-activated cell sorting (FACS). The resulting enriched libraries were next positively sorted via FACS for binding to amyloid fibrils immobilized on quantum dot immunoconjugates. Finally, the enriched antibody libraries were sequenced, and soluble antibody candidates were tested for their affinity and conformational specificity for amyloid aggregates using recombinant and biological samples.

[0028] Figure 2. Affinity and conformational specificity analysis of a-synuclein antibodies. (A) a-synuclein antibodies (IgG 1 s) were incubated with Dynabeads coated with a-synuclein aggregates and their binding was detected with fluorescently labeled secondary antibodies via flow cytometry. (B) a-synuclein antibodies at a fixed concentration (10 nM) were preincubated with disaggregated a-synuclein monomer (0-1000 nM) followed by incubation with Dynabeads coated with a-synuclein aggregates. Antibodies bound to fibril-coated beads were detected by flow cytometry. The percentage of antibody binding to a-synuclein fibrils was calculated as the binding signal in the presence of a-synuclein monomer pre-incubation (0.1-1000 nM) relative to the control without pre-incubation. The data are averages of two independent repeats, and the error bars are standard deviations. (C) a-synuclein antibodies (IgG Is) at 5 nM were incubated with Dynabeads coated with a-synuclein aggregates and their binding was detected with fluorescently labeled secondary antibodies via flow cytometry. Background is binding of a-synuclein antibodies (IgG Is) at 5 nM to Dynabeads followed by detection with fluorescently labeled secondary antibodies.

[0029] Figure 3. a-synuclein immunofluorescence analysis of human brain tissues. Fixed human brain tissues from Parkinson’ s disease (PD) or Diffuse Lewy body disease (DLBD) patients were co-stained with H2.7 (conformational a-synuclein antibody) and EP1536Y (phospho-antibody specific for pSynl29) or LB509 (pan-synuclein) antibody. H2.7 was detected with anti-human Fc Alexa Fluor 647, LB509 was detected with anti-mouse Alexa Fluor 488, and pSynl29 was detected with anti-rabbit Alexa Fluor 488. Merged staining results with DAPI are shown. The scale bars within the images are approximately 50 pm wide, and the scale bars within the insets are approximately 20 pm wide.

[0030] Figure 4. Biophysical characterization of a-synuclein antibodies. (A) Non-specific binding of a-synuclein antibodies to biotinylated soluble membrane proteins from CHO cells. The antibodies were immobilized on Protein A-Dynabeads, and binding of the soluble membrane proteins was detected via flow cytometry. The binding signals were normalized between the signals for emibetuzumab (high non-specific binding) and elotuzumab (low nonspecific binding). (B) Antibody melting temperatures (first unfolding transition) were analyzed by differential scanning fluorimetry. (C) Self-association of antibodies was analyzed by charge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS). The dashed line represents a CS-SINS score of 0.35, and values <0.35 indicate low levels of selfassociation23. a-synuclein antibodies were analyzed in comparison to omalizumab (high selfassociation) and denosozmab (low self-association), and all data is normalized to a previously published scale. In (A-C), the data are averages of 2-3 independent repeats, and the error bars are standard deviations.

[0031] Figure 5. Summary of library sorting to enrich for a-synuclein conformational antibodies. In the initial discovery stage (top row), the human scFv library was enriched four times against aggregated alpha-synuclein fibrils via MACS (rounds 1 -4; percentage of cells collected is reported), depleted once against aggregated alpha-synuclein monomer to remove non-conformational antibodies via FACS (round 5), and enriched one additional time against a-synuclein fibrils immobilized on QD immunoconjugates via FACS (round 6). A lead clone was then identified (aS2. 1), and an scFab library was prepared via soft randomization of HCDR2. This library (bottom row) was then enriched against a-synuclein fibrils using MACS (round 1) and QD-fibril conjugates using FACS (round 2). A negative selection against a polyspecificity reagent (soluble membrane proteins) was then performed to deplete non-specific antibodies from the library (round 3). Next, a positive selection was performed by FACS using QD-fibril conjugates (round 4), a negative selection was performed by FACS against -synuclein monomer (round 5), and a final positive selection was performed by FACS using QD-fibril conjugates. The terms for a-synuclein in the figure refer to fibrils (-syn agg.) and disaggregated -synuclein (-synuclein monomer). The percentages in the green boxes represent the percentage of cells collected in MACS selections, and the percentages in the cytogram gates represent the percentage of cells collected in the FACS selections. Blue numbers represent the % of cells collected for the library and wild-type samples that were evaluated at the same time, which can be directly compared.

[0032] Figure 6a shows the amino acid sequence for antibody aS2.1 (SEQ ID NO:1), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:2), including CDRH1 (SEQ ID NO:3), CDRH2 (SEQ ID NO:4), and CDRH3 (SEQ ID NO:5), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:6), including CDRL1 (SEQ ID NO:7), CDRL2 (SEQ ID NO:8), and CDRL3 (SEQ ID NO:9).

[0033] Figure 6b shows the amino acid sequence for antibody H2.3 (SEQ ID NOTO), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:11), including CDRH1 (SEQ ID NO:12), CDRH2 (SEQ ID NO:13), and CDRH3 (SEQ ID NO: 14), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO: 15), including CDRL1 (SEQ ID NO: 16), CDRL2 (SEQ ID NO: 17), and CDRL3 (SEQ ID NO: 18).

[0034] Figure 6c shows the amino acid sequence for antibody H2.4 (SEQ ID NO: 19), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:20), including CDRH1 (SEQ ID NO:21), CDRH2 (SEQ ID NO:22), and CDRH3 (SEQ ID NO:23), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:24), including CDRL1 (SEQ ID NO:25), CDRL2 (SEQ ID NO:26), and CDRL3 (SEQ ID NO:27).

[0035] Figure 6d shows the amino acid sequence for antibody H2.7 (SEQ ID NO:28), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:29), including CDRH1 (SEQ ID NO:30), CDRH2 (SEQ ID NO:31), and CDRH3 (SEQ ID NO: 32), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:33), including CDRL1 (SEQ ID NO:34), CDRL2 (SEQ ID NO:35), and CDRL3 (SEQ ID NO:36).

[0036] Figure 6e shows the amino acid sequence for antibody Ll.l (SEQ ID NO:37), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO: 38), including CDRH1 (SEQ ID NO:39), CDRH2 (SEQ ID NO:40), and CDRH3 (SEQ ID NO:41), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:42), including CDRL1 (SEQ ID NO:43), CDRL2 (SEQ ID NO:44), and CDRL3 (SEQ ID NO:45).

[0037] Figure 6f shows the amino acid sequence for antibody LI.2 (SEQ ID NO:46), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:47), including CDRH1 (SEQ ID NO:48), CDRH2 (SEQ ID NO:49), and CDRH3 (SEQ ID NO: 50), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:51), including CDRL1 (SEQ ID NO:52), CDRL2 (SEQ ID NO:53), and CDRL3 (SEQ ID NO:54).

[0038] Figure 6g shows the amino acid sequence for antibody LI.5 (SEQ ID NO:55), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:56), including CDRH1 (SEQ ID NO:57), CDRH2 (SEQ ID NO:58), and CDRH3 (SEQ ID NO: 59), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:60), including CDRL1 (SEQ ID NO:61), CDRL2 (SEQ ID NO:62), and CDRL3 (SEQ ID NO:63).

[0039] Figure 6h shows the amino acid sequence for antibody LI.6 (SEQ ID NO:64), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:65), including CDRH1 (SEQ ID NO:66), CDRH2 (SEQ ID NO:67), and CDRH3 (SEQ ID NO: 68), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:69), including CDRL1 (SEQ ID NQ:70), CDRL2 (SEQ ID NO:7I), and CDRL3 (SEQ ID NO:72).

[0040] Figure 6i shows the amino acid sequence for antibody LI.8 (SEQ ID NO:73), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:74), including CDRH1 (SEQ ID NO:75), CDRH2 (SEQ ID NO:76), and CDRH3 (SEQ ID NO:77), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:78), including CDRL1 (SEQ ID NO:79), CDRL2 (SEQ ID NO:80), and CDRL3 (SEQ ID N0:81).

[0041] Figure 6j shows the amino acid sequence for antibody LI.9 (SEQ ID NO:82), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:83), including CDRH1 (SEQ ID NO:84), CDRH2 (SEQ ID NO:85), and CDRH3 (SEQ ID NO: 86), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:87), including CDRL1 (SEQ ID NO:88), CDRL2 (SEQ ID NO:89), and CDRL3 (SEQ ID NO:90).

[0042] Figure 6k shows the amino acid sequence for antibody LI.10 (SEQ ID NO:91), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:92), including CDRH1 (SEQ ID NO:93), CDRH2 (SEQ ID NO:94), and CDRH3 (SEQ ID NO:95), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:96), including CDRL1 (SEQ ID NO:97), CDRL2 (SEQ ID NO:98), and CDRL3 (SEQ ID NO:99).

[0043] Figure 61 shows the amino acid sequence for antibody L2.1 (SEQ ID NO: 100), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:101), including CDRH1 (SEQ ID NO: 102), CDRH2 (SEQ ID NO:103), and CDRH3 (SEQ ID NO: 104), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO:105), including CDRL1 (SEQ ID NO:106), CDRL2 (SEQ ID NO:107), and CDRL3 (SEQ ID NO: 108).

[0044] Figure 6m shows the amino acid sequence for antibody L2.2 (SEQ ID NO: 109), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:110), including CDRH1 (SEQ ID NO:111), CDRH2 (SEQ ID NO:112), and CDRH3 (SEQ ID NO: 113), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO: 114), including CDRL1 (SEQ ID NO:115), CDRL2 (SEQ ID NO: 116), and CDRL3 (SEQ ID NO: 117). Figure 6n shows the amino acid sequence for antibody L2.3 (SEQ ID NO: 118), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO: 119), including CDRH1 (SEQ ID NQ: 120), CDRH2 (SEQ ID NO:121), and CDRH3 (SEQ ID NO: 122), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO: 123), including CDRL1 (SEQ ID NO:124), CDRL2 (SEQ ID NO:125), and CDRL3 (SEQ ID NO: 126).

[0045] Figure 6o shows the amino acid sequence for antibody L2.6 (SEQ ID NO: 127), which includes the: 1 ) amino acid sequence of the heavy chain variable region (SEQ ID NO: 128), including CDRH1 (SEQ ID NO:129), CDRH2 (SEQ ID NO:130), and CDRH3 (SEQ ID NO: 131), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO: 132), including CDRL1 (SEQ ID NO:133), CDRL2 (SEQ ID NO: 134), and CDRL3 (SEQ ID NO:135).

[0046] Figure 6p shows the amino acid sequence for antibody L2.8 (SEQ ID NO: 136), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO:137), including CDRH1 (SEQ ID NO:138), CDRH2 (SEQ ID NO: 139), and CDRH3 (SEQ ID NO: 140), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO: 141), including CDRL1 (SEQ ID NO:142), CDRL2 (SEQ ID NO:143), and CDRL3 (SEQ ID NO: 144).

[0047] Figure 6q shows the amino acid sequence for antibody L2.9 (SEQ ID NO: 145), which includes the: 1) amino acid sequence of the heavy chain variable region (SEQ ID NO: 146), including CDRH1 (SEQ ID NO: 147), CDRH2 (SEQ ID NO:148), and CDRH3 (SEQ ID NO: 149), and 2) amino acid sequence of the kappa (light) chain variable region (SEQ ID NO: 150), including CDRL1 (SEQ ID NO: 151), CDRL2 (SEQ ID NO: 152), and CDRL3 (SEQ ID NO: 153).

[0048] Figure 6r shows the amino acid sequence for antibody Cinpanemab HC (SEQ ID NO: 154) and Cinpanemab LC (SEQ ID NO: 155).

[0049] Figure 7. Characterization of aggregated alpha-synuclein antibodies using SDS-PAGE and SEC. (A) SDS-PAGE analysis of antibodies before (-) and after (+) being heated and reduced with -mercaptoethanol. (B) SEC analysis of the percentage of antibody monomer following Protein A purification.

[0050] Figure 8. Immunodot blot analysis of brain samples from wild-type and a-synuclein transgenic (A53T) mice. (A) Immunodot blotting analysis of wild-type and transgenic (A53T) a-synuclein mouse brains. Short (left) and long (right) time exposures are shown. The loading control (LC) was Ponceau stain. The experiments were performed three times and a representative example is shown.

[0051] Figure 9 shows a schematic representation of an exemplary IgG molecule with the various regions and sections labeled. The CDRs and framework regions (FR) of one of the two variable region light chains, and one of the two variable region heavy chains, are also labeled.

[0052] Figure 10. Immunohistochemical staining of human brain tissues. Free floating and fixed brain tissue from (A) Alzheimer’s disease (AD) plus neocortical Lewy pathology samples in which both tau and a-synuclein aggregates were present and (B) control samples in which aggregates of both proteins were largely absent was stained with conformational and control antibodies (n=2). (A) Staining of AD tissue with ATA1.459.3 and zagotenemab shows similar staining of tau aggregates (distrophic neutrites and neutrophil threads) by both antibodies. Staining of the same tissue with H2.7 and cinpanemab shows similar staining of a-synuclein aggregates (Lewy bodies) by both antibodies. Images display the same region of consecutive sections from the frontal cortex from the same tissue in which both tau and a- synuclein aggregates are present. However, staining of a-synuclein aggregates by either of the conformational tau antibodies or staining of tau aggregates by either of the a-synuclein conformational antibodies was not observed. Control tau (Tau-5) and a-synuclein (LB509) antibodies were also included for representative staining of these regions by non- conformational antibodies. (B) Both ATA1.459.3 and H2.7 showed no observable staining when tested using control tissue. In (A) and (B), ATA 1.459.3 was examined as an scFv-Fc fusion with a human IgGl Fc. H2.7, cinpanemab, and zagotenemab were analyzed in human IgGl format. Tau-5 and LB 509 are IgGs with mouse Fc.

[0053] Figure 11. Analysis of a-synuclein antibody conformational specificity. (A-C) For ELISA measurements, (A) H2.7, (B) cinpanemab, and (C) LB509 were used as capture antibodies for recombinant a-synuclein monomer or fibrils. Captured monomer and fibrils were detected using a monoclonal rabbit anti-a-synuclein primary antibody and a goat antirabbit HRP-conjugated secondary antibody. (D) The binding of H2.7 to recombinant a- synuclein monomer and fibrils was also examined using western blotting, and it was observed to bind a-synuclein monomer and fibrils under denaturing conditions. Three repeats were performed (n=3), and a representative image is shown. (E) The western blotting data was quantified compared to a total a-synuclein antibody. The lines represent the average binding signal. For (A-C), the data are averages, and error bars are the standard deviations for three independent experiments (n=3). Figure 12. Peptide scanning analysis of H2.7 epitope. (A) The epitope of H2.7 was first probed (n=l) using a membrane containing 15-mer overlapping peptides for human a- synuclein, which are shifted in an interval of three amino acids. The epitope was identified in the N-terminal region using this first array. (B) A focused array containing overlapping a- synuclein 15-mer peptides from this N-terminal region, which differ by one amino acid, was then probed (n=l). This refined scanning identified a more focused epitope containing residues 33-TKEGVLYVGS-42 of a-synuclein. Residues within the identified epitope are shown in green for peptides in which H2.7 binding was observed.

[0054] Figure 13. a-synuclein antibody western blotting analysis of human brain lysates. The binding of (A) H2.7 and (B) cinpanemab to a-synuclein in human lysates from Lewy Body Dementia (LBD) and age-matched controls was examined via western blotting (n=2). Binding was detected using a goat anti-human HRP conjugated secondary antibody. Binding to a-synuclein was observed for both H2.7 and cinpanemab in both LBD and control tissues.

[0055] Figure 14. Immunoblotting analysis of tau and a-synuclein antibody sequence specificity. The binding of ATA1.459.3, zagotenemab, H2.7, and cinpanemab to various conformers of tau and a-synuclein was examined via immunoblotting. Binding of ATA 1.459.3 and zagotenemab was observed to human 2N4R tau fibrils as well as human 0N3R fibrils. Binding of H2.7 and cinpanemab was observed to human a-synuclein oligomers as well as human and mouse a-synuclein fibrils. The blots were run under nondenaturing conditions, and the loading control was detected via silver stain. Immunoblotting was repeated twice (n=2), and a representative image is shown.

[0056] Figure 15. Immunoblotting analysis of tau and a-synuclein antibody specificity. The binding of ATA1.459.3, zagotenemab, H2.7, and cinpanemab to tau and a-synuclein monomer, oligomers, and fibrils and Ap oligomers was examined via immunoblotting under non-denaturing conditions (n= 1). Binding of ATA1.459.3 was observed for tau monomer, oligomers, and fibrils, and binding of zagotenemab was observed for both tau monomer and fibrils. Tau loading was confirmed with Tau-5 staining. Binding of H2.7 and cinpanemab was observed for a-synuclein monomer, oligomers, and fibrils, a-synuclein loading was confirmed with LB509 staining. A oligomer loading was confirmed with 6E10 staining.

[0057] DEFINITIONS

[0058] To facilitate an understanding of the invention, a number of terms are defined below.

[0059] The term "antibody," as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each variable region (VH or VL) contains 3 CDRs, designated CDR1, CDR2 and CDR3 (see, Figure 9). Each variable region also contains 4 framework sub-regions, designated FR1, FR2, FR3 and FR4 (see, Figure 9), which may be human framework sub-regions.

[0060] As used herein, the term "antibody fragment or portion" refers to a portion of an intact antibody. Examples of antibody fragments or portions include, but are not limited to, linear antibodies, single-chain antibody molecules, Fv, Fab and F(ab') fragments, and multispecific antibodies formed from antibody fragments. The antibody fragments preferably retain at least part of the heavy and / or light chain variable region.

[0061] As used herein, the terms "complementarity determining region" and "CDR" refer to the regions that are primarily responsible for antigen-binding. There are three CDRs in a light chain variable region (CDRL1, CDRL2, and CDRL3), and three CDRs in a heavy chain variable region (CDRH1, CDRH2, and CDRH3).

[0062] As used herein, the term "framework" refers to the residues of the variable region other than the CDR residues. There are four separate framework sub-regions that make up the framework: FR1, FR2, FR3, and FR4 (see, Figure 9). In order to indicate if the framework sub-region is in the light or heavy chain variable region, an "L" or "H" may be added to the sub-region abbreviation (e.g., "FRL1" indicates framework sub-region 1 of the light chain variable region). It is noted that, in certain embodiments, the human aggregated alpha- sy nuclein binding molecules of the present invention may have less than a complete framework (e.g. the human aggregated alpha- sy nuclein binding molecule may have a portion of a framework that only contains one or more of the four sub-regions).

[0063] As used herein, the term "fully human framework" means a framework with an amino acid sequence found naturally in humans. Examples of fully human frameworks, include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al., (1970) J. Exp. Med. 132, 211-250, both of which are herein incorporated by reference). In certain embodiments, the human aggregated alpha- synuclein binding molecules herein have a fully human framework.

[0064] As used herein, the terms "subject" and "patient" refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.

[0065] As used herein, the term "codon" or "triplet" refers to a group of three adjacent nucleotides which specify one of the naturally occurring amino acids found in polypeptides. The term also includes codons which do not specify any amino acid. It is also noted that, due to the degeneracy of the genetic code, there are many codons that code for the same amino acid. As such, many of the bases of the nucleic acid sequences of the present invention can be changed without changing the actual amino acid sequence that is encoded. The present disclosure is intended to encompass all such nucleic acid sequences.

[0066] As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a polypeptide," "polynucleotide having a nucleotide sequence encoding a polypeptide," and "nucleic acid sequence encoding a peptide" means a nucleic acid sequence comprising the coding region of a particular polypeptide. The coding region may be present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0067] Also, as used herein, there is no size limit or size distinction between the terms "oligonucleotide" and "polynucleotide. " Both terms simply refer to molecules composed of nucleotides. Likewise, there is no size distinction between the terms "peptide" and "polypeptide." Both terms simply refer to molecules composed of amino acid residues.

[0068] As used herein, the term "the complement of" a given sequence is used in reference to the sequence that is completely complementary to the sequence over its entire length. For example, the sequence 5'-A-G-T-A-3' is "the complement" of the sequence 3'-T-C-A-T-5'.

[0069] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" or "isolated nucleic acid sequence encoding an aggregated alpha-synuclein binding molecule" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated (e.g. host cell proteins).

[0070] As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, aggregated alpha-synuclein binding molecules (e.g., antibodies or antibody fragments) may be purified by removal of contaminating nonimmunoglobulin proteins; they are also purified by the removal of immunoglobulins that do not bind to the same antigen. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind the particular antigen results in an increase in the percentage of antigen specific immunoglobulins in the sample. In another example, recombinant antigen-specific polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percentage of recombinant antigen-specific polypeptides is thereby increased in the sample.

[0071] As used herein, the term "Fc region" refers to a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region (e.g., with increased or decreased effector functions).

[0072] DESCRIPTION OF THE INVENTION

[0073] Provided herein are human aggregated alpha-synuclein binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human aggregated alpha-synuclein binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) with particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat a disease, such as a synucleinopathy.

[0074] Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or current in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against insoluble (amyloid fibril) antigens using a single, off-the-shelf human library (14). Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against aggregated alpha- synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for aggregated alpha-synuclein (cinpanemab). Our antibody generation method enables isolation of high-quality conformational antibodies specific for amyloid fibrils using a single human antibody library. This approach allows for conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

[0075] In certain embodiments, the human aggregated alpha-synuclein binding molecules comprise one or more of the antibodies, variable regions, or CDRs shown in SEQ ID NOS: 1- 153 and / or variable regions or CDRs with one or more conservative or non-conservative amino acid changes in these SEQ ID NOS:1-153. Changes to the amino acid sequences of the CDRs or variable regions or antibodies (see Figure 6) may be generated by changing the nucleic acid sequence encoding the amino acid sequence. A nucleic acid sequence encoding a variant of a given CDR or variable region may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid encoding the CDR or variable region.

[0076] Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

[0077] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting CDR (see, e.g., Vallette et. al., (1989) Nucleic Acids Res. 17: 723-733, hereby incorporated by reference). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., (1985) Gene 34: 315-323, hereby incorporated by reference. The starting material is the plasmid (or other vector) comprising the starting CDR or variant region DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There should be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double- stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

[0078] Alternatively, or additionally, the desired amino acid sequence encoding a CDR variant, or variable region variant, can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically. Conservative modifications in the amino acid sequences of the CDRs or variable region may also be made. Naturally occurring residues are divided into classes based on common side-chain properties:

[0079] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0080] (2) neutral hydrophilic: cys, ser, thr;

[0081] (3) acidic: asp, glu;

[0082] (4) basic: asn, gin, his, lys, arg;

[0083] (5) residues that influence chain orientation: gly, pro; and

[0084] (6) aromatic: trp, tyr, phe.

[0085] Conservative substitutions will entail exchanging a member of one of these classes for another member of the same class in a particular variable region or CDR, such as in SEQ ID NOS: 1-153.

[0086] The CDRs of the present invention may be employed with any type of suitable framework. In some embodiments, the CDRs are used with fully human frameworks, or framework sub-regions. For example, the NCBI web site contains the sequences for known human framework regions. Examples of human VH sequences include, but are not limited to, VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81, which are provided in Matsuda et al., (1998) J. Exp. Med. 188:1973-1975, that includes the complete nucleotide sequence of the human immunoglobulin chain variable region locus, herein incorporated by reference. Examples of human VK sequences include, but are not limited to, Al, A10, Al l, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, LI , LIO, LI 1 , L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4 / 18a, L5, L6, L8, L9, 01, Oi l, 012, 014, 018, 02, 04, and 08, which are provided in Kawasaki et al., (2001) Eur. J. Immunol. 31:1017-1028; Schable and Zachau, (1993) Biol. Chem. Hoppe Seyler 374: 1001-1022; and Brensing- Kuppers et al., (1997) Gene 191 :173-181, all of which are herein incorporated by reference. Examples of human VL sequences include, but are not limited to, V1-11, V1-13, V1-16, VI- 17, Vl-18, Vl-19, Vl-2, Vl-20, Vl-22, Vl-3, Vl-4, Vl-5, Vl-7, Vl-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6, which are provided in Kawasaki et al., (1997) Genome Res. 7:250-261, herein incorporated by reference. Fully human frameworks can be selected from any of these functional germline genes. Generally, these frameworks differ from each other by a limited number of amino acid changes. These frameworks may be used with the CDRs described herein. Additional examples of human frameworks which may be used with the CDRs of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al., (1970), J. Exp. Med. 132:211-250, both of which are herein incorporated by reference).

[0087] In certain embodiments, the human aggregated alpha-synuclein binding molecules of the present invention comprise antibodies or antibody fragments (e.g., comprising one or more of the CDRs described herein, such as in Figure 6). An antibody, or antibody fragment, of the present invention can be prepared, for example, by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody recombinantly, a host cell may be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cell is cultured, from which medium the antibody can be recovered. Standard recombinant DNA methodologies may be used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al., all of which are herein incorporated by reference.

[0088] In certain antibodies, the anti-aggregated alpha-synuclein antibodies, or fragments, thereof prepared herein have an IgG isotype constant regions as shown in Table 1 below.

[0089] TABLE 1

[0090] To express an antibody with one or more of the CDRs herein, in certain embodiments, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of germline light and heavy chain variable sequences using the polymerase chain reaction (PCR).

[0091] Once the germline VH and VL fragments are obtained, these sequences can be mutated to encode one or more of the CDR amino acid sequences disclosed herein (see, Figure 6). The amino acid sequences encoded by the germline VH and VL DNA sequences may be compared to the CDRs sequence(s) desired to identify amino acid residues that differ from the germline sequences. Then the appropriate nucleotides of the germline DNA sequences are mutated such that the mutated germline sequence encodes the selected CDRs, using the genetic code to determine which nucleotide changes should be made. Mutagenesis of the germline sequences may be carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis. In other embodiments, the variable region is synthesized de novo (e.g., using a nucleic acid synthesizer).

[0092] Once DNA fragments encoding the desired VH and VL segments are obtained (e.g., by amplification and mutagenesis of germline VH and VL genes, or synthetic synthesis, as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH- encoding DNA fragment is operably linked to another DNA fragment encoding another polypeptide, such as an antibody constant region or a flexible linker. The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of mouse and human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be, for example, an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CHI constant region.

[0093] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of mouse and human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al., (1991) Sequences of Proteins of immunological Interest, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0094] To create a scFv gene, the VH- and VL-encoding DNA fragments may be operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)s, such that the VH and VL sequences can be expressed as a contiguous singlechain protein, with the VL and VH regions joined by the flexible linker (see e.g., Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and McCafferty et al., (1990) Nature 348:552-554), all of which are herein incorporated by reference). To express the antibodies, or antibody fragments of the invention, DNAs encoding partial or full-length light and heavy chains, (e.g. obtained as described above), may be inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are generally chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment(s) within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a nonimmunoglobulin protein).

[0095] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), herein incorporated by reference. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. In certain embodiments, regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma virus. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., all of which are herein incorporated by reference.

[0096] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634.665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neomycin gene (for G418 selection).

[0097] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains may be transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

[0098] In certain embodiments, the expression vector used to express the human aggregated alpha-synuclein binding molecules of the present invention are viral vectors, such as retroviral vectors. Such viral vectors may be employed to generate stably transduced cell lines (e.g. for a continues source of the aggregated alpha-synuclein binding molecules). In some embodiments, the GPEX gene product expression technology (from Catalent, Somerset, NJ) is employed to generate aggregated alpha-synuclein binding molecules (and stable cell lines expressing the aggregated alpha-synuclein binding molecules). In particular embodiments, the expression technology described in W00202783 and W00202738 to Bieck et al. (both of which are herein incorporated by reference in their entireties) is employed. Mammalian host cells for expressing the recombinant antibodies of the invention include, for example, PER. aggregated alpha-synuclein™ cells (Crucell, The Netherlands), Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In other embodiments, the host cells express GnT III as described in WO9954342 and U.S. Pat. Pub. 20030003097, both herein incorporated by reference, such that expressed aggregated alpha-synuclein binding molecules have increased ADCC activity. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are generally produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0099] Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to aggregated alpha-synuclein. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bi-functional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than aggregated alpha- synuclein (e.g., by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods).

[0100] In certain embodiments, the antibodies and antibody fragments of the present invention are produced in transgenic animals. For example, transgenic sheep and cows may be engineered to produce the antibodies or antibody fragments in their milk (see, e.g., Pollock DP, et al., (1999) Transgenic milk as a method for the production of recombinant antibodies. J. Immunol. Methods 231:147-157, herein incorporated by reference). The antibodies and antibody fragments of the present invention may also be produced in plants (see, e.g., Larrick et al., (2001) Production of secretory IgA antibodies in plants. Biomol. Eng. 18:87-94, herein incorporated by reference). Additional methodologies and purification protocols are provided in Humphreys et al., (2001) Therapeutic antibody production technologies: molecules applications, expression and purification, Curr. Opin. Drug Discov. Devel. 4:172- 185, herein incorporated by reference. In certain embodiments, the antibodies or antibody fragments of the present invention are produced by transgenic chickens (see, e.g., US Pat. Pub. Nos. 20020108132 and 20020028488, both of which are herein incorporated by reference).

[0101] In certain embodiments, the human aggregated alpha-synuclein binding molecules of the present invention (e.g., as antibodies or antibody fragments) are useful for immunoassays which detect or quantify human aggregated alpha-synuclein in a sample (e.g., a purified blood sample from a subject). In some embodiments, an immunoassay for aggregated alpha- synuclein typically comprises incubating a biological sample in the presence of a detectably labeled antibody or antibody fragment of the present invention capable of selectively binding to aggregated alpha-synuclein, and detecting the labeled peptide or antibody which is bound in a sample. Various clinical assay procedures are well known in the art.

[0102] The present disclosure provides immunoassay methods for determining the presence, amount or concentration of human aggregated alpha-synuclein in a test sample. Any suitable assay known in the art can be used in such a method. Examples of such assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal- polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), an ARCHITECT assay (ABBOTT), a bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.

[0103] A human aggregated alpha-synuclein binding molecule can be captured on beads or nitrocellulose, or on any other solid support which is capable of immobilizing soluble proteins (e.g., magnetic beads). A human aggregated alpha-synuclein containing sample is then added to the support which is subsequently washed with suitable buffers to remove unbound proteins. A second, detectably labeled, molecule (e.g., antibody or peptide) that can bind to the human aggregated alpha-synuclein binding molecule is added to the solid phase support that can then be washed with the buffer a second time to remove unbound molecules. The amount of bound label on the solid support can then be detected by known methods. Detectably labeling the human aggregated alpha-synuclein binding molecule can be accomplished by coupling to an enzyme for use in an enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to generate a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the human aggregated alpha-synuclein binding molecules of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0104] In some embodiments of the present invention, human aggregated alpha-synuclein which is detected by the above assays can be present in a biological sample. Any sample containing human aggregated alpha-synuclein can be used. Preferably, the sample is a biological fluid such as, for example, blood, serum, lymph, urine, tissue sample, cerebrospinal fluid, amniotic fluid, synovial fluid, a tissue extract or homogenate, and the like. However, the invention is not limited to assays using only these samples, as it is possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.

[0105] In situ detection can be accomplished by removing a histological specimen from a patient, and providing the combination of labeled human aggregated alpha-synuclein binding molecules of the present disclosure to such a specimen. The human aggregated alpha- synuclein binding molecule is preferably provided by applying or by overlaying the labeled aggregated alpha-synuclein binding molecule to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of aggregated alpha- synuclein, but also the distribution of aggregated alpha-synuclein in the examined tissue.

[0106] In certain embodiments, provided here are kits for the detection of aggregated alpha- synuclein that include a human aggregated alpha-synuclein detection molecule. Such kits may include any of the immunodiagnostic reagents described herein and may further include instructions for the use of the immunodiagnostic reagents in immunoassays for determining the presence of human aggregated alpha-synuclein in a test sample. The kits may also include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and / or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.

[0107] The various components of the kit may be provided in suitable containers as necessary, e.g., a microliter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instrument for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.

[0108] EXAMPLES

[0109] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

[0110] EXAMPLE 1

[0111] Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils

[0112] This example describes an unexpectedly simple approach for performing quantitative sorting of an off-the-shelf, yeast-displayed human antibody library against amyloid fibril aggregates (Fig. 1). Our approach is to conjugate capture antibodies (e.g., a-synuclein antibodies) to quantum dots (QDs) and use the immunoconjugates to capture amyloid fibrils (e.g., a-synuclein fibrils). The resulting QD-amyloid conjugates are then used for quantitative, multi-parameter FACS sorting of a single, widely accessible human library to directly select antibodies with high affinity, strict conformational specificity and low polyreactivity. This example demonstrates that this methodology can be used to generate conformational antibodies in a robust and predictable manner with combinations of binding properties that are similar to or better than those of clinical- stage antibodies.

[0113] RESULTS Generation of human conformational antibodies specific for a-synuclein fibrils

[0114] We evaluated the feasibility of using procedure in Figure 1 , including using a human antibody library (14), to select conformational antibodies specific for a-synuclein fibrils linked to Parkinson’s disease. The yeast-displayed scFv library was first enriched via MACS against immobilized a-synuclein fibrils (four rounds). During these sorts, the percentage of collected cells increased from 0.02% in round 1 to 0.4% in round 4 (Fig. 5, initial discovery). We next performed a negative selection against a-synuclein monomer via FACS (round 5) and a positive selection against QD-a-synuclein fibril conjugates via FACS (round 6). The output of round 6 was sequenced, which led to the isolation of a lead clone (aS2.1; Fig. 6). We produced this antibody in the IgG format and found that it possessed intermediate affinity (78.2+4.3 nM) relative to the clinical-stage antibody cinpanemab (9.5+2.6 nM; Fig. 2A).

[0115] Encouragingly, aS2.1 displayed high conformational specificity for a-synuclein fibrils even when pre-incubated with 100-fold molar excess of a-synuclein monomer (-90% of fibril binding activity), which was much higher than the corresponding conformational specificity of cinpanemab (-44% of fibril binding activity; Fig. 2B).

[0116] We next affinity matured aS2.1 as summarized in the Methods section and Fig. 5. This led to the isolation of three antibody variants (H2.3, H2.4, and H2.7) with mutations in heavy chain CDR2 (Fig. 6). The IgGs were expressed in HEK-2936E cells, purified via Protein A chromatography, and their purity was examined by SDS-PAGE and SEC (Fig. 7). All antibodies showed high purity, including >90% monomer by SEC.

[0117] Notably, the three antibody mutants displayed both higher affinity (Fig. 2A) and conformational specificity (Fig. 2B) than cinpanemab. Immunoblot analysis revealed the matured antibodies also selectively recognized a-synuclein transgenic (A53T) mouse brain lysates relative to those for wild-type mice, especially for H2.7, in a manner similar to that for cinpanemab (Fig. 8). We also confirmed that H2.7 selectively immunostains human brain samples associated with Parkinson’ s and Diffuse Lewy Body diseases relative to nondiseased brains (Fig. 3). Immunofluorescence analysis revealed co-staining of H2.7 with antibodies specific for phosphorylated a-synuclein (pSynl29) and total a-synuclein (LB509).

[0118] Biophysical characterization of human conformational antibodies

[0119] To be used as therapeutic or diagnostic agents, conformational antibodies should also possess a combination of favorable biophysical properties, including i) high stability, ii) high specificity (i.e., low off-target binding), and iii) low self-association. Therefore, we evaluated the biophysical properties of our antibodies using a panel of assays. First, we examined the specificity of the a-synuclein antibodies by assaying their interactions with a polyspecificity reagent, namely soluble membrane proteins from CHO cells17, in comparison to clinical-stage antibodies which have been shown to have high (emibetuzumab) and low (elotuzumab) nonspecific binding17 18.

[0120] For the a-synuclein antibodies, the lead antibody (aS2.1) and its affinity matured variants (H2.3, H2.4 and H2.7) demonstrated moderate to high non-specific binding, which was modestly higher than the level of non-specific binding for cinpanemab.

[0121] Next, we analyzed the folding stability of the a-synuclein antibodies to evaluate whether the lead or affinity-matured antibodies possess sub-optimal stabilities, such as melting temperatures (Tm) below 65 °C (Fig. 4B). We observed encouraging results for the a -synuclein antibodies, as the lead antibody (aS2.1, formatted as an IgG) displayed high stability (Tmof 72.2+ 0.1 °C), and the affinity-matured variants (Tmvalues of 71 .7-72.4 °C) and cinpanemab (Tmof 74.3+0.7 °C) displayed similar stabilities.

[0122] Finally, we examined the level of self-association for the a-synuclein antibodies in a common formulation condition used for therapeutic antibodies, namely pH 6 and 10 mM histidine (Fig. 4C)19. High levels of antibody self- association are linked to high antibody viscosity and opalescence in concentrated formulations, such as those required for therapeutic formulations20 22Therefore, we analyzed this property using charge-stabilized selfinteraction nanoparticle spectroscopy (CS-SINS), which identifies antibodies with low selfassociation and low risk for high viscosity and opalescence in concentrated formulations as those with CS-SINS scores <0.3523. Encouragingly, three affinity matured a-synuclein antibodies (H2.3, H2.4 and H2.7) display CS-SINS scores <0.35. In contrast, the clinical- stage antibodies display mixed results, as zagotenemab displays low self-association and cinpanemab displays a high self-association. Overall, these results reveal that our a-synuclein antibodies possess a combination of biophysical properties that are similar or superior to those for clinical- stage antibodies, which demonstrates the high-quality nature of the antibodies generated in our work against different amyloid targets from a single human library without the need for immunization.

[0123] We have demonstrated a quantitative method for generating and affinity maturing conformational antibodies against an amyloid- forming protein using an off-the-shelf human library. While the majority of conformational antibodies have previously been isolated using in vivo discovery methods, such approaches provide poor control over the selection process and do not reliably produce the desired conformational specificity. Our approach builds on previous work using nonimmune in vitro libraries to perform library panning10,11,32,33,24-31and MACS13,3435to isolate conformational antibodies. Both techniques provide some control over antigen presentation and concentration during sorting. The majority of previous studies using these methods employed either alternating rounds of positive selections against immobilized aggregates and negative selections against immobilized monomers24,34or selections against immobilized aggregates in the presence of a competitive form of the amyloid protein (e.g., monomers) in solution11,25. Despite some success, these approaches yield variable and hard- to-predict results. We find that multiparameter selections for both antibody expression and either QD-amyloid binding or a lack of monomer (e.g., a synuclein) binding, which cannot be simultaneously performed using library panning or MACS, are critical for reliably identifying conformational antibodies with both high affinity and specificity.

[0124] We have demonstrated the strength of this FACS-based technique by isolating high- affinity conformational antibodies directed toward a-synuclein fibrils. The properties of the antibodies isolated using this method should be considered in the context of previously reported anti-amyloid antibodies. For this purpose, we have directly compared the properties of our antibodies to those of clinical-stage antibodies directed toward the same targets. The properties of our a-synuclein antibodies should be compared to those for cinpanemab, a conformational a-synuclein antibody that was evaluated in clinical trials37. Three of our best a-synuclein antibodies (H2.3, H2.4 and H2.7) displayed higher affinity and conformational specificity than cinpanemab (Fig. 2). Our a-synuclein antibodies also showed high conformational specificity. For immunodot blot analysis of brain samples isolated from wildtype or a-synuclein transgenic (A53T) mouse models, aS2.1 and the affinity-matured variants, like cinpanemab, show high specificity for samples isolated from A53T mice (Fig. 8). Our antibodies were also stably folded like cinpanemab (Tmvalues >70 °C; Fig. 4B), and they show much lower levels of self-association (Fig. 4C) and moderately higher levels of non-specific binding (Fig. 4A) than cinpanemab. These generally favorable biophysical properties of our antibodies relative to cinpanemab are also encouraging because our best antibody variants, such as H2.7, display highly specific binding to pathological a-synuclein species in human brain samples from both Parkinson’s and diffuse Lewy body disease samples (Fig. 3). MATERIALS AND METHODS

[0125] Preparation of antibody-quantum dot conjugates

[0126] Site-click Qdot 655 antibody labeling kit (Invitrogen, S 10453) was used to conjugate an anti-a-synuclein antibody (MJFR1, Abeam, ab 138501) to dibenzocyclooctyne (DIBO) modified QDs. Briefly, 125 pg of capture IgG (MJFR1) was buffer exchanged into antibody preparation buffer provided in the kit to a final volume of ~80 pL and then mixed with 10 pL of P-galactosidase for 4 h at 37 °C. Following the incubation, 80 pL of GalT enzyme together with UDP-GalNAz were added and the reaction was carried out in Tris buffer (pH 7.0) overnight at 30 °C to generate IgG with azide-functionalized glycans. Following extensive washing steps using Tris buffer (pH 7.0), the azide-modified IgG (in a final volume of 100 pL) was mixed with 50 pL of QD solution for overnight incubation at room temperature. The resultant antibody conjugates were further purified with a Nanosep centrifugal filter (MWCO, 300 kDa) and resuspended into 250 pL of Tris buffer (pH 7.0). This final solution was further centrifuged at 4000x g for 5 min, and the supernatant was collected and stored at 4 °C until further use.

[0127] Library screening for isolating and affinity maturing a-synuclein antibodies

[0128] The screening for conformational antibodies that recognize a-synuclein fibrils was performed as follows, a-synuclein fibrils (100 ug; Stressmarq, SPR-317) were sonicated on ice for 5 min (30 sec on, 30 sec off). Next, 8xl07tosyl beads were washed twice with PBS by placing them on the magnet. The fibrils were added to beads and incubated with end-over- end mixing at room temperature for 2-3 d. Beads immobilized with fibrils were moved to 4 °C prior to use.

[0129] The same human scFv library was used for selections against a-synuclein fibrils. In round 1, MACS was performed in the largely the same way as the tan selections. Yeast cells (109) were incubated with fibril-coated beads (107). In rounds 2-4, MACS was performed with a reduced number of cells (107) without reducing the number of beads (107). In round 5, a negative FACS selection was performed for a lack of binding to disaggregated a-synuclein monomer. Yeast cells ( 107) were incubated with 10 nM biotinylated a-synuclein monomer and mouse anti-Myc antibody (lOOOx dilution) at room temperature for 3 h with end-over- end mixing. Post primary incubation, the cells were washed once with ice-cold PBSB and incubated with a 200x dilution of goat anti-mouse Alexa Fluor 488 and lOOOx dilution of streptavidin Alexa Fluor 647 on ice for 4 min. After incubation with secondary reagents, cells were washed once with ice-cold PBSB and sorted on a Beckman Coulter MoFlo Astrios sorter. Yeast cells that displayed antibodies and showed minimal binding to a-synuclein monomer were collected via FACS. In rounds 6-8, positive FACS selections were performed against lx a-synuclein fibril-QD conjugates (high antigen condition).

[0130] The lead a-synuclein antibody aS2.1 identified during initial discovery was cloned into a modified form of the pCTCON2 yeast-surface display plasmid in the format of a single-chain Fab (scFab). The antibody was cloned as an N-terminal fusion to Aga2 (VL-CL- Iinker-VH-Cnl-Aga2) and sequence confirmed. Next, an HCDR2 library was prepared using soft randomization for affinity maturation by randomizing the sites in aS2. 1 indicated in Fig. 6. The library was prepared as described previously13,34. In round I, MACS was performed with 109cells and 107beads. In rounds 2, 4 and 6, FACS positive selections were performed against a-synuclein fibril-QD conjugates (high antigen condition). In rounds 3 and 5, FACS negative selections were performed against a polyspecificity reagent (soluble membrane proteins in round 3), prepared as described previously18,40, and disaggregated a-synuclein monomer (round 5).

[0131] Antibody cloning and production

[0132] Antibody genes were isolated from the terminal rounds of sorting against a-synuclein fibrils using a yeast miniprep kit (Zymo, D2004). For a-synuclein, VH genes were amplified by PCR and cloned into an IgG heavy chain expression plasmid (pTT5) using restriction sites EcoRI and Nhel, and the VL genes were amplified by PCR and cloned into an IgG light chain expression plasmid (pTT5) using restriction sites EcoRI and BsiWI. The ligation mixtures were next transformed into competent DH5a cells, plated on LB-agar plates (supplemented with 100 pg / mL ampicillin) and incubation at 37 °C for 16-20 h. The following day, individual colonies were picked from these plates, grown overnight in LB media (supplemented with 100 pg / mL of ampicillin), mini -prepped (Qiagen, 27106) and sequenced.

[0133] Antibodies were expressed with HEK293-6E cells (National Research Council of Canada). Cells were maintained and passaged in F17 media (Invitrogen, A1383502) supplemented with 0.1% w / v kolliphor (Fisher, NC0917244), 30 mL / L L-glutamine (Gibco, 25030081) and 0.5 mL / L geneticin (Gibco, 10131035) at a density of -1.7-2 million cells / mL, as described previously41. Cells were transfected at a density of -1.7-2 million cells / mL in F17 media (un- supplemented) with 15 pg of plasmid (15 pg for tau scFv-Fc plasmid or 7.5 pg each for a-synuclein IgG heavy and light chain plasmids) and 45 pg of PEI. Yeastolate (BD Sciences, 292804) was added at 20% w / v 24-48 h post transfection and cells were allowed to grow for additional 3-4 d. Post expression, cells were spun down at 3500xg for 40 min and media was moved to a new tube. Protein A agarose beads (300-500 L of slurry; Fisher, 20334) were added to the tubes followed by gentle shaking overnight at 4 °C. The following day, the beads were collected in filter columns (Fisher, 89898) under vacuum and washed with 50 mL of PBS. Beads were next incubated with 1 mL 0.1 M glycine (pH 3.0) for 15 min to elute protein, and the eluate was collected by centrifugation. Proteins were next buffer exchanged into 20 mM acetate (pH 5.0) with Zeba desalting columns (Fisher, 89890), passed thorough 0.22 pm filters (EMD Millipore, SLGV004SL), aliquoted, and stored at -80 °C. Protein concentrations were evaluated by measuring the absorbance at 280 nm, and the purity was analyzed by SDS-PAGE (Invitrogen, WG1203BOX).

[0134] Antibody affinity and conformational specificity analysis

[0135] Antibody affinity was analyzed via flow cytometry using a bead-based assay1342. Dynabeads coated with a-synuclein aggregates (prepared as described above) were blocked with 10 mM glycine (pH 7.4) at room temperature for 1 h with end-over-end mixing followed by one wash with PBSB. Antibodies were thawed at room temperature, centrifuged at 21000xg for 5 min, and the supernatant was moved to a new tube. Antibody concentrations were next evaluated by measuring the absorbance at 280 nm. In each well of a 96- well plate (Greiner, 650261), 10sfibril-coated beads were incubated with varying concentration of antibodies in PBSB supplemented with 1% milk for 3 h at room temperature with mild agitation. Post primary incubation, the beads were washed once with ice-cold PBSB and incubated with a 300x dilution of goat anti-human Fc, Alexa Fluor 647 (Jackson Immunoresearch, 109-605-098) on ice for 4 min. The beads were washed one more with ice- cold PBSB and analyzed on a Bio-Rad ZE5 flow cytometer, and the mean fluorescent values were measured.

[0136] Antibody conformational specificity was measured using a related bead-based assay13'42. First, antibodies at fixed concentration (10 nM) were incubated with varying concentrations (0.1-1000 nM) of disaggregated a-synuclein monomer in PBSB supplemented with 1% milk in a 96-well plate for 1 h at room temperature with mild agitation. Next, 105a- synuclein aggregate-coated DynaBeads beads, which were blocked with 10 mM glycine as described above, were added to the antibody-monomer mixtures, and beads were allowed to incubate with the antibody-monomer mixtures for an additional 3 h. Post primary incubation, the beads were washed once with ice-cold PBSB and incubated with a 300x dilution of goat anti-human Fc Alexa Fluor 647 on ice for 4 min. The beads were washed once more and analyzed on Bio-Rad ZE5 flow cytometer. The mean a-synuclein fluorescent measurements of antibodies binding to fibrils were recorded.

[0137] Immunostaining and western blotting of mouse brain samples

[0138] This Example was conducted in a facility approved by the American Association for the Accreditation of Laboratory Animal Care, and all experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Michigan. Mice were housed at the University of Michigan animal care facility and maintained according to U.S. Department of Agriculture standards (12 h light / dark cycle with food and water available ad libitum). Hemizygous P301S tau mice (B6;C3-Tg-Prnp-MAPT- P301S PS19Vle / J; The Jackson laboratory stock #008169)43, homozygous A53T mice (B6;C3-Tg(Pmp-SNCA*A53T)83Vle / J; The Jackson laboratory stock #004479)44and non- transgenic littermates were bred at the University of Michigan and euthanized at 9, 10 and 11 months for brain collection.

[0139] Animals were deeply anesthetized with isofluorane and perfused transcardially with lx PBS. Brains were divided sagittally. One half was immediately placed on dry ice and stored at -80 °C for biochemical studies while the other half was fixed in 4% paraformaldehyde at 4 °C for 24 h, and cryoprotected in a series of 10% and 30% sucrose in lx PBS at 4 °C until saturated. Fixed hemispheres were snap frozen in OCT medium and sectioned at 12 pm sagittally using a cryostat and sections were stored at -20 °C for immunofluorescence.

[0140] The 11 -month-old P301S, 10-month-old A53T and non-transgenic littermate brain samples were homogenized in PBS with a protease inhibitor cocktail (Sigma Aldrich, 1 1873580001) using a 1:3 dilution of tissue:PBS (w / v). Samples were centrifuged at 9300xg for 10 min at 4 °C. Supernatants (soluble fraction) were snap frozen and stored at -80 °C for Western blot analysis. Pellets were resuspended in PBS with protease inhibitor cocktail (Roche, 11836170001), centrifuged at 9300xg for 10 min (4 °C), and supernatants were discarded. Hie pellet was resuspended in 1% sarkosyl with protease inhibitor, vortexed (1 min), and incubated at room temperature (1 h). Samples were sonicated (water bath sonicator) for 5 min and centrifuged for 30 min (16000xg at 4 °C). Sarkosyl (insoluble) fractions of brain extracts (7 |ig of total protein) were spotted directly onto nitrocellulose membranes and allowed to dry (1 h). Control dot blots (loading controls) were stained with Ponceau S (5 min) and washed 3x with distilled water. The other dot blots were blocked with 10% nonfat dry milk in Tris Buffered Saline with 0.1% Tween-20 (TBST) buffer at room temperature (1 h).

[0141] Tau dot blots were next incubated with antibodies at 10 nM (1% nonfat dry milk in TBST) overnight at 4 °C. Next, the blots were washed with TBST and incubated with a 5000x diluted solution of HRP-conjugated goat anti-human IgG or a lOOOx diluted solution of HRP-conjugated goat anti-mouse or goat anti-rabbit at room temperature for 1 h. Afterward, the blots were washed with TBST and developed using Ecobright Pico HRP Substrate (Innovative Solutions) and visualized with the Genesys G:Box imaging system (Syngene). Three independent repeats were performed.

[0142] Immunostaining of human brain samples

[0143] Paraffin-embedded brain tissue sections from the midbrain was attained from subjects with Parkinson’s disease, dementia with Lewy bodies, and age-matched control subjects, as well as mid-frontal cortex from progressive supranuclear palsy, Alzheimer’s disease and age- matched control subjects from the Michigan Brain Bank (University of Michigan, Ann Arbor, MI, USA). Brain tissue was collected with patient consent and protocols were approved by the Institutional Review Board of the University of Michigan and abide by the Declaration of Helsinki principles. Samples were examined at autopsy by neuropathologists for diagnosis.

[0144] Analytical size-exclusion chromatography

[0145] Antibodies were analyzed via size-exclusion chromatography (SEC) with a Shimadzu Prominence HPLC System. After purification by Protein A, antibodies were buffer exchanged into lx PBS. 100 L of 0.1 mg / mL antibody was then injected into a SEC column (Superdex 200 Increase 10 / 300 GL column; GE, 28990944) and analyzed at 0.75 mL / min using a lx PBS running buffer with 200 mM arginine (pH 7.4). Absorbance was monitored at 280 nm, and the percentage of antibody monomer was calculated by analyzing absorbance peaks between the void volume and buffer elution times.

[0146] Polyspecificity analysis The polyspecificity reagent (soluble membrane proteins, SMP) was prepared as previously described18,40. For biotinylation, sulfo-NHS-LC -biotin (Thermo Fisher, PI21335) was dissolved in distilled water at -11.5 mg / mL. Stock solution of Sulfo-NHS-LC-biotin (150 pL) and the SMP reagent (4.5 mL at 1.0 mg / mL) were mixed end-over-end mixing at room temperature (45 min). The reaction was quenched (10 pL of 1.5 M hydroxylamine at pH 7.2), and biotinylated SMP was aliquoted and stored at -80 °C. The specificity assay was performed as previously described13,18. Three independent repeats were performed and were normalized between high (emibetuzumab) and low (elotuzumab) polyspecificity control mAbs at the highest evaluated antibody concentrations.

[0147] Antibody melting temperature analysis

[0148] The stability of antibodies was evaluated by differential scanning fluorimetry. The antibodies were diluted to 0. 12 mg / mL and then mixed with Protein Thermal Shift Dye (Applied Biosystems 4461164) to achieve a final concentration of lx in final volume of 20 pL. Samples were submitted to the University of Michigan Advanced Genomics Core for analysis. The measurements were taken on ABI Prism 7900HT Sequence Detection System (Applied Biosystems), and the temperature range examined spanned between 25 and 98 °C. Three independent repeats were performed for this study.

[0149] Charged-stabilized self-interaction nanoparticle spectroscopy (CS-SINS)

[0150] CS-SINS was measured as reported previously23. Briefly, capture antibody (Jackson ImmunoResearch, 109-005-008) and polylysine (90%: 10% w / w ratio respectively; Fisher Scientific, ICN19454405) were immobilized on concentrated gold nanoparticles and incubated overnight. Dilute mAb solutions (11.1 pg / mL, 45 pL) were incubated with 5 pL of gold conjugates for 4 h at room temperature. Absorbance spectra was measured on a Biotek Synergy Neo plate reader (Biotek, Winooski, VT) in 1 nm increments between 450 and 650 nm. The inflection of a quadratic equation fit describing the forty data points surrounding the maximum absorbance was calculated to determine the plasmon wavelength. Plasmon wavelengths were normalized between two parameters and calibrated against a panel of five antibodies (NIST, ibalizumab, mepolizumab, trastuzumab, and romosozumab). This calibration panel was used to rescale the measurements to the same scale as reported in the original study23. REFERENCES

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[0193] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, medicine, and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS:We claim:

1. A composition comprising a human aggregated alpha-synuclein binding molecule, or one or more nucleic acid molecules encoding said human aggregated alpha-synuclein binding molecule, wherein said human aggregated alpha-synuclein binding molecule comprises: a) a heavy chain variable region, wherein said heavy chain variable region comprises: i) a CDRH1 amino acid sequence comprising SEQ ID NO: 3, 12, 21, 30,39, 48, 57, 66, 75, 84, 93, 102, 1 11, 120, 129, 138, or 147 or any of the preceding with one with one or two conservative amino acid changes, ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 4, 13, 22, 31,40, 49, 58, 67, 76, 85, 94, 103, 112, 121, 130, 139, or 148, or any of the preceding with one or two conservative amino acid changes, and iii) a CDRH3 amino acid sequence comprising SEQ ID NO: 5, 14, 23, 32,41, 50, 59, 68, 77, 86, 95, 104, 113, 122, 131, 140, or 149 or any of the preceding with one with one or two conservative amino acid changes, and / or; b) a light chain variable region, wherein said light chain variable region comprises; i) a CDRL1 amino acid sequence comprising SEQ ID NO: 7, 16, 25, 34, 43, 52, 61, 70, 79, 88, 97, 106, 115, 124, 133, 142, or 151 or any of the preceding with one with one or two conservative amino acid changes, ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 8, 17, 26, 35,44, 53, 62, 71, 80, 89, 98, 107, 1 16, 125, 134, 143, or 152, or any of the preceding with one with one or two conservative amino acid changes, and iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 9, 18, 27, 36,45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, or 153, or any of the preceding with one with one or two conservative amino acid changes.

2. The composition of claim 1, wherein: i) said CDRH1 amino acid sequence comprises SEQ ID NO:12, 21, 30, 39, 48, 57, 66, 75, 84, 93, 102, 111, 120, 129, 138, or 147;ii) said CDRH2 amino acid sequence comprises SEQ ID NO: 13, 22, 31, 40, 49, 58,67, 76, 85, 94, 103, 112, 121, 130, 139, or 148; iii) said CDRH3 amino acid sequence comprises SEQ ID NO: 14, 23, 32, 41, 50, 59,68, 77, 86, 95, 104, 113, 122, 131, 140, or 149 ; iv) said CDRL1 amino acid sequence comprises SEQ ID NO:16, 25, 34, 43, 52,61, 70, 79, 88, 97, 106, 115, 124, 133, 142, or 151; v) said CDRL2 amino acid sequence comprises SEQ ID NO: 17, 26, 35, 44, 53,62, 71 , 80, 89, 98, 107, 1 16, 125, 134, 143, or 152; and vi) said CDRL3 amino acid sequence comprises SEQ ID NO: 18, 27, 36, 45, 54,63, 72, 81, 90, 99, 108, 117, 126, 135, 144, or 153.

3. The composition of Claim 1, wherein said human aggregated alpha-synuclein binding molecule is an antibody, minibody, diabody, scFv, or antibody fragment capable of binding human aggregated alpha-synuclein fibrils.

4. The composition of Claim 3, wherein said antibody fragment is a Fab, F(ab')2 or Fv antibody fragment.

5. The composition of Claim 2, wherein said antibody or antibody fragment comprises at least an antigen binding portion of: 1) H2.3 antibody, 2) H2.4 antibody, 3) H2.7 antibody; 4) Ll. l antibody; 5) LI.2 antibody; 6) LI.5 antibody; 7) LI.6 antibody; 8) LI.8 antibody; 9) LI.9 antibody; 10) LI.10 antibody; 11) L2.1 Antibody; 12) L2.2 antibody; 13) L2.3 antibody; 14) L2.6 antibody; 15) L2.8 antibody; or 16) L2.9 antibody.

6. The composition of Claim 1 , wherein said heavy chain and / or light chain variable region comprises a human framework region.

7. The composition of Claim 1 , wherein said human aggregated alpha-synuclein binding molecule further comprises a light chain constant region and a CHI heavy chain constant region.

8. The composition of Claim 7, wherein said alpha-synuclein binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

9. The composition of Claim 8, wherein said light chain constant region is human or a humanized murine, and / or wherein said CHI, CH2, and CH3 heavy chain constant regions are human or are humanized murine.

10. The composition of Claim 1 , wherein said human aggregated alpha- synuclein binding molecule comprises an antibody, wherein the light chain constant region of said antibody is selected from: IgG Kappa and IgG Lambda, and wherein the heavy chain constant region of said antibody is selected from: TgGl , IgG2, IgG3, and lgG4.

11. The composition of Claim 1 , wherein said human aggregated alpha-synuclein binding molecule comprises an antibody, or antigen binding portion thereof, which is glycosylated or non-glycosylated.

12. The composition of Claim 1 , further comprising a physiologically tolerable buffer.

13. The composition of Claim 1 , wherein said heavy chain variable regions comprises SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74, 83, 92, 101, 110, 119, 128, 137, or 146 or any of the preceding with one or more conservative amino acid changes.

14. The composition of Claim 1 , wherein said light chain variable region comprises SEQ ID NO: 6, 15, 24, 33, 42, 51, 60, 69, 78, 87, 96, 105, 114, 123, 132, 141, or 150, or any of the preceding with one or more conservative amino acid changes.

15. The composition of Claim 1, wherein said composition comprises said one or more nucleic acid molecules.

16. The composition of Claim 1 , wherein said one or more nucleic acid molecules comprise: i) a first nucleic acid sequence encoding said heavy chain variable region, and ii) a second nucleic acid sequence encoding said light chain variable region.

17. The composition of Claim 16, further comprising an expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.

18. A method of treating or preventing a synucleinopathy disease or condition comprising: treating a subject with a human aggregated alpha- synuclein binding molecule, or an expression vector comprising said one or more nucleic acid molecules encoding said human aggregated alpha- synuclein binding molecule, as recited in any of Claims 1-17 or 42, and wherein said subject has, or is suspected to develop, a synucleinopathy disease or condition.

19. The method of Claim 18, wherein said neurodegenerative disease or condition is selected from the group consisting of: Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and Alzheimer's Disease with Amygdalar Restricted Lewy Bodies (AD / ALB) .

20. The method of Claim 18, wherein said human aggregated alpha-synuclein binding molecule is an antibody or antigen binding portion thereof.

21. The method of Claim 18, wherein said antibody or antigen binding portion thereof is a human antibody or antigen binding portion thereof.

22. The method of Claim 18, wherein said antibody or antigen binding portion thereof is a humanized antibody or antigen binding portion thereof.

23. The method of Claim 18, wherein said heavy chain variable regions comprises SEQ ID NO: 2, 11, 20, 29, 38, 47, 56, 65, 74, 83, 92, 101, 110, 119, 128, 137, or 146 or any of the preceding with one or more conservative amino acid changes.

24. The method of Claim 18, wherein said light chain variable region comprises SEQ ID NO: 6, 15, 24, 33, 42, 51, 60, 69, 78, 87, 96, 105, 114, 123, 132, 141, or 150, or any of the preceding with one or more conservative amino acid changes.

25. A method of detecting human aggregated alpha-synuclein a sample comprising: a) contacting a sample with the human aggregated alpha-synuclein binding molecule of any of Claims 1-17 or 42, wherein said sample is suspected of containing human aggregated alpha-synuclein fibril, and wherein said human aggregated alpha-synuclein binding molecule forms a complex with said human aggregated alpha-synuclein if present in said sample; and b) detecting the presence or absence of said complex in said sample.

26. The method of Claim 25, wherein said sample is from a subject that has, or is suspected to develop, a synucleinopathy disease or condition.

27. The method of Claim 25, wherein said human aggregated alpha-synuclein binding molecule comprises a detectable label.

28. The method of Claim 25, further comprising contacting said sample with a conjugate molecule capable of binding to said human aggregated alpha-synuclein binding molecule, wherein said conjugate molecule comprises a detectable label.

29. A method of generating an enriched cell fraction comprising target cells expressing antibodies, or fragments thereof, that bind an aggregated conformation of a target protein comprising: a) contacting a plurality of candidate cells that surface-display an antibody library with an aggregated conformation of a target peptide such that a positive cell subpopulation, but not a negative cell sub-population, binds said aggregated conformation of said target protein; b) separating said positive cell sub-population from said negative cell subpopulation; c) contacting said positive cell sub-population with a disaggregated conformation of said target peptide such that a positive cell fraction, but not a negative cell fraction, binds said disaggregated conformation of said target protein; d) separating said negative cell fraction from said positive cell fraction; and e) sorting said negative cell fraction against a plurality of QD-AgTP conjugates such than an enriched cell fraction is generated comprising target cells expressing antibodies, or fragments thereof, that bind said aggregated conformation of said target protein,wherein each of said QD-AgTP conjugates comprises a quantum dot attached to, or operably linked, to an aggregated conformation of said target protein.

30. The method of claim 29, further comprising: f) treating said target cells such that target nuclei acid molecules are isolated encoding said antibodies, or fragments thereof, that bind said aggregated conformation of said target protein. 1 . The method of claim 30, further comprising: g) sequencing at least some of said target DNA to obtain sequencing data comprising the nucleic acid sequences encoding one or more antibodies, or antigen binding fragments thereof, that are specific for said aggregated conformation of said target protein.

32. The method of claim 29, wherein said target protein is selected from: amyloid beta, tau, aggregated alpha- sy nuclein, TAR DNA binding protein 43, a C9orf72 related protein, islet amyloid polypeptide, and transthyretin.

33. The method of claim 29, wherein said aggregated conformation of a target peptide in step a) is conjugated to a solid support.

34. The method of claim 33, wherein said solid support comprises magnetic beads.

35. The method of claim 29, wherein said plurality of candidate cells comprise yeast cells.

36. The method of claim 29, wherein said antibody library comprises at least 1 million different antibodies or fragments thereof.

37. The method of claim 29, wherein said antibody library comprises at least 100 million or 1 billion different antibodies or fragments thereof.

38. The method of claim 29, wherein said sorting comprises flow cytometry, wherein said flow cytometry is optionally fluorescence-activated cell sorting (FACS).

39. The method of claim 29, wherein said antibody library comprises a single-chain antibody library.

40. The method of claim 30, further comprising: h) affinity maturing at least one of said target nuclei acid molecules to generate at least one matured target nucleic acid molecule.

41. The method of claim 40, further comprising: i) expressing said at least one matured target nucleic acid molecule in a host cell to generate an antibody, or antibody fragment.

42. A composition comprising a human aggregated alpha-synuclein binding molecule or one or more nucleic acid molecules encoding said human aggregated alpha-synuclein binding molecule, wherein said human aggregated alpha-synuclein binding molecule comprises a human aggregated alpha-synuclein antibody, antibody fragment, or nanobody that specifically binds a first epitope of human aggregated alpha-synuclein, wherein said first epitope comprises 33- TKEGVLYVGS-42.

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