Nanobody against alpha-synuclein

Description

NANOBODY AGAINST ALPHA- SYNUCLEINPRIORITY CLAIM AND CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 653,507 filed May 30, 2024, which is incorporated herein by reference in its entirety and relied upon.FIELD

[0002] This disclosure is related to nanobodies for treatment and / or diagnosis of synucleinopathies.BACKGROUND

[0003] Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are collectively referred to as synucleinopathies due to the pathological accumulation of alpha- synuclein (a-syn) aggregates in the affected regions of the brain. In PD and DLB, a-syn aggregates predominantly accumulate in neurons forming Lewy bodies (LBs) and Lewy neurites (LNs), whereas in MSA, aggregates localize primarily in oligodendrocytes as glial cytoplasmic inclusions (GCIs). These aggregated a-syn species are considered central to disease pathogenesis and are strongly associated with clinical progression and severity.

[0004] A growing body of evidence from human genetics, neuropathology, and experimental models supports a critical role for a-syn in the initiation and progression of synucleinopathies. Familial PD is associated with several missense mutations in the SNCA gene and SNCA gene duplications or triplications, all of which increase the propensity for a-syn aggregation and promote disease onset. Genome-wide association studies have confirmed that SNCA is a significant risk locus in sporadic PD. These findings demonstrate a dose-dependent and aggregation-driven mechanism in a-syn-mediated neurodegeneration.

[0005] Post-mortem analyses and in vivo models further reveal that pathological a-syn aggregates exhibit prion-like properties, including the capacity to seed and propagate across neural networks. Experimental transmission of a-syn seeds in animal models reproduces cardinal features of PD and MSA, including dopaminergic cell loss and motor deficits, underscoring a-syn’s pathogenic relevance.

[0006] Despite substantial progress in understanding disease mechanisms, there remains a lack of disease-modifying therapies and robust diagnostic tools for early and differential diagnosis of synucleinopathies. Immunotherapeutic approaches targeting misfolded a-syn aggregates offer a promising strategy. However, conventional monoclonal antibodies face limitations related to size, brain penetration, and intracellular target accessibility.

[0007] Nanobodies (Nbs), also known as single-domain antibodies, are derived from camelid heavychain-only antibodies and provide unique advantages over traditional antibodies. Due to their small size (~15 kDa), high solubility, and stability, Nbs can access cryptic epitopes, penetrate dense tissues, and be engineered for intracellular (intrabody) expression or extracellular delivery. These properties make Nbs ideal for selectively binding disease-relevant a-syn conformers (oligomers and fibrils) while sparing native monomeric a-syn.

[0008] Accordingly, a-syn aggregate-specific Nbs have broad potential utility across therapeutic and diagnostic domains, including: (a) Immunotherapy, Nbs may be used as direct therapeutic agents to neutralize toxic a-syn species, inhibit aggregate formation or propagation, and facilitate intracellular clearance via proteasomal or autophagic pathways. When delivered alone or via viral vectors or protein fusion formats, they may reduce pathological burden in affected brain regions, (b) Biomarker-based diagnostics, Nbs can be incorporated into high-affinity immunoassays (e.g., ELISA, SIMOA, MSD, flowcytometry or seeding amplification assays) to detect specific a-syn conformers in cerebrospinal fluid (CSF), plasma, or other biofluids or tissues, enabling early-stage and differential diagnosis of PD, DLB, and MSA. (c) Imaging agents, nanobodies conjugated to imaging reporters (e.g., PET, SPECT, or nearinfrared fluorophores) can serve as molecular imaging probes for non-invasive in vivo detection of a-syn aggregates. Their small size and high target specificity allow for superior brain penetration and signal-to- noise ratio compared to full-length antibodies, providing a valuable tool for both clinical diagnosis and therapeutic monitoring.

[0009] The development and application of Nbs targeting aggregated forms of a-syn thus represent a powerful and versatile platform for precision diagnostics, targeted therapeutics, and molecular imaging of synucleinopathies. These tools address key unmet needs in the management of PD, DLB, and MSA and are poised to significantly advance translational and clinical outcomes.SUMMARY

[0010] There is a need for therapies for synucleinopathies, including Parkinson’s Disease (PD), and reagents for their detection. Misfolded a-synuclein protein in the form of toxic amyloid fibrils is the hallmark of a range of synucleinopathies including, for example, Parkinson's disease (PD). Disclosed herein are nanobodies that bind aggregated a-synuclein protein, and methods of generating and using such nanobodies.

[0011] Methods for treating PD and other synucleopathies are described. The methods comprise administering a therapeutically effective amount of an a-syn nanobody described herein to a patient in need thereof. Provided are nanobodies (e.g., Nb-01, Nb-04 and Nb-40) having a high affinity for aggregatedforms of a-synuclein and a low affinity for monomeric forms of a-synuclein. The nanobodies are useful in the diagnosis and treatment of neurodegenerative diseases.

[0012] One aspect of the present disclosure provides a method of treating Parkinsons disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) in a subject in need thereof, which comprises, consists, or consists essentially of administering to the subject a therapeutically effective amount of an a-syn nanobody or engineered variant thereof.

[0013] In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of a VHH having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or in) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

[0014] In some embodiments, the VHH comprises, consists, or consists essentially of: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or in) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0015] In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto. In some embodiments, a-syn nanobody or engineered variant thereof is selected from Nb-04 (SEQ ID NO: 1), or Nb-40 (SEQ ID NO: 2). In some embodiments, a-syn nanobody or engineered variant thereof is selected from a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6.

[0016] In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of an amino acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid setforth in SEQ ID NO: 3. In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of an amino acid sequence having greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3.

[0017] In some embodiments, the a-syn nanobody or engineered variant thereof preferably recognizes oligomers and fibrils of a-synuclein protein and less the monomeric form of a-synuclein protein. In some embodiments, the a-syn nanobody or engineered variant thereof specifically recognizes oligomers and fibrils of a-synuclein protein and not monomeric a-synuclein protein.

[0018] In some embodiments, the a-syn nanobody or engineered variant thereof recognizes an epitope comprising, consisting, or consisting essentially of residues 113-126 of a-synuclein (SEQ ID NO: 44). In some embodiments, the a-syn or engineered variant thereof nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 127-140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the a-syn nanobody or engineered variant thereof recognizes an epitope comprising, consisting, or consisting essentially of residues 43-56 of a-synuclein (SEQ ID NO: 34).

[0019] One aspect of the disclosure provides a nanobody or engineered version thereof having high binding affinity for a-synuclein oligomers, high binding affinity to a-synuclein fibrils, and / or low binding affinity for a-synuclein monomers. In some embodiments, the nanobody or engineered version thereof comprises high binding affinity for a-synuclein oligomers. In some embodiments, the nanobody or engineered version thereof comprises a high binding affinity to a-synuclein fibrils. In some embodiments, the nanobody or engineered version thereof comprises a low binding affinity for a-synuclein monomers. In some embodiments, the nanobody or engineered version thereof comprises a high binding affinity for a-synuclein aggregates and low affinity binding for a-synuclein monomers.

[0020] In some embodiments, the nanobody or engineered version thereof comprises, consists, or consists essentially of a VHH having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or in) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

[0021] In some embodiments of the nanobody or engineered version thereof, the VHH comprises, consists, or consists essentially of: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or in) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0022] In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto. In some embodiments, a-syn nanobody or engineered variant thereof is selected from Nb-04 (SEQ ID NO: 1), or Nb-40 (SEQ ID NO: 2). In some embodiments, a-syn nanobody or engineered variant thereof is selected from a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6.

[0023] In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of an amino acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3. In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of an amino acid sequence having greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3.

[0024] In some embodiments, the a-synuclein aggregates comprise, consist, or consist essentially of protofibrils and / or soluble oligomers of a-synuclein. In some embodiments, the a-synuclein aggregates comprise, consist, or consist essentially of protofibrils of a-synuclein. In some embodiments, the a- synuclein aggregates comprise, consist, or consist essentially of low or high molecular weight soluble oligomers of a-synuclein. In some embodiments, the a-synuclein aggregates comprise, consist, or consist essentially of a-synuclein fibrils.

[0025] In some embodiments, the nanobody or engineered version thereof binds to an epitope comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 127- 140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the nanobody or engineered version thereof binds to an epitope comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44). In some embodiments, the nanobody or engineered version thereof binds to an epitope comprising, consisting, or consisting essentially of the N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34).

[0026] In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a monovalent nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a multi-valent nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a bi-valent nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a tri-valent nanobody.

[0027] In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a multi-specific nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a bi-specific nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a tri-specific nanobody.

[0028] In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a multi-paratopic nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a bi-paratopic nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a tri-paratopic nanobody.

[0029] In some embodiments, the bi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 113-126 (SEQ ID NO: 44) and residues 127-140 (SEQ ID NO: 46) of a-synuclein. In some embodiments, the bi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and a C-terminal region corresponding to residues 127-140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the biparatopic nanobody binds to epitopes comprising, consisting, or consisting essentially of an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and a C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44).

[0030] In some embodiments, the multi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 113-126 (SEQ ID NO: 44)and residues 127-140 (SEQ ID NO: 46) and an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) of a-synuclein.

[0031] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of a polypeptide comprising, consisting, or consisting essentially of two or more nanobodies or engineered variants thereof as described herein and a linker (e.g., a polypeptide linker) disposed between each of the two or more nanobodies or engineered variants thereof. In some embodiments, the each of the two or more nanobodies or engineered variants thereof are independently selected from any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto.

[0032] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more VHH domains each independently having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or in) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

[0033] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more VHH domains, each independently comprising, consisting, or consisting essentially of: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or in) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0034] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more VHH domains comprising, consisting, or consisting essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto. In some embodiments, the nanobody or engineered versionthereof is selected from Nb-04 (SEQ ID NO: 1), or Nb-40 (SEQ ID NO: 2). In some embodiments, a- syn nanobody or engineered variant thereof is selected from a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6.

[0035] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more (e.g., 2, 3, 4, 5, 6, or more) VHH domains comprising, consisting, or consisting essentially of an amino acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3. In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more (e.g., 2, 3, 4, 5, 6, or more) VHH domains comprising, consisting, or consisting essentially of an amino acid sequence having greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3.

[0036] In some embodiments, the multi-valent nanobody is bi-valent. In some embodiments, the multi-valent nanobody is tri-valent.

[0037] In some embodiments, the multi-valent nanobody comprises one or more linkers, wherein each of the one or more linkers are disposed between each of the two or more VHH domains. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an IgA linker (SPSTPPTPSPSTPPASSPSTPPTPSPSTPPAS; SEQ ID NO: 49).

[0038] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 50-59, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto.

[0039] In some embodiments, the nanobody or engineered version thereof, or the multi-valent nanobody, comprises one or more polypeptide tags or linkers (e.g., comprises an HA tag (SEQ ID NO: 47), a HIS tag (e.g., a multi-histidine tag, such as a 6x HIS tag (HHHHHH; SEQ ID NO: 48), and / or an IgA linker (SPSTPPTPSPSTPPASSPSTPPTPSPSTPPAS; SEQ ID NO: 49)).

[0040] In some embodiments, the nanobody or engineered version thereof preferably recognizes oligomers and fibrils of a-synuclein protein and less the monomeric form of a-synuclein protein.

[0041] One aspect of the disclosure provides a pharmaceutical composition comprising the nanobody or engineered version thereof set forth herein and a pharmaceutically acceptable diluent or carrier.

[0042] One aspect of the disclosure provides the nanobody or engineered version thereof set forth herein, or the pharmaceutical composition set forth herein, for use as a medicament.

[0043] One aspect of the disclosure provides a method for preventing or treating a neurodegenerative disorder with a-synuclein pathology in an subject in need thereof comprising, consisting, or consisting essentially of administration of the nanobody or engineered version thereof set forth herein or the pharmaceutical composition set forth herein, to the subject.

[0044] In some embodiments, the neurodegenerative disorder with a-synuclein pathology comprises, consists, or consists essentially of Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), or Alzheimer’s disease with Lewy bodies. In some embodiments, the neurodegenerative disorder with a-synuclein pathology consists of Alzheimer’s disease (AD).

[0045] One aspect of the disclosure provides a test kit for use in a method of determining whether or not an individual has a neurodegenerative disease, comprising, consisting, or consisting essentially of the nanobody or engineered version thereof as set forth herein. In some embodiments, the kit can include one or more other elements comprising, consisting, or consisting essentially of: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, the nanobody or engineered version thereof to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the nanobody or engineered version thereof for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0046] One aspect of the disclosure provides a method of detecting a-synuclein fibrils and aggregates comprising, consisting, or consisting essentially of the steps of: adding the nanobody or engineered version thereof as set forth herein to a biological sample; and detecting the presence of a complex formed between a-synuclein fibrils and / or aggregates and the nanobody or engineered version thereof.

[0047] One aspect of the disclosure provides a method for diagnosing a neurodegenerative disease associated with a-synuclein comprising, consisting, or consisting essentially of: adding the nanobody or engineered version thereof as set forth herein to a biological sample; and detecting the presence or absence of a complex formed between a-synuclein aggregates and the nanobody or engineered version thereof.

[0048] In some embodiments, the nanobody or engineered version thereof as set forth herein comprises a detectable label. In some embodiments, the detectable label is selected from the group comprising, consisting, or consisting essentially of a fluorescent label, a radioactive label, or a contrast agent.

[0049] One aspect of the disclosure provides the nanobody or engineered version thereof as set forth herein for use as an imaging agent of a-synuclein aggregates. In some embodiments, the a-synuclein aggregates are present in a biological sample or biological tissue. In some embodiments, the biological sample or biological tissue comprising, consisting, or consisting essentially of brain, gut, heart, skin, or eye.

[0050] One aspect of the disclosure provides a method for imaging a-synuclein aggregates comprising, consisting, or consisting essentially of: administering the nanobody or engineered version thereof as set forth herein to a subject; and detecting the nanobody or engineered version thereof.

[0051] In some embodiments, the nanobody or engineered version thereof comprises a detectable label.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is an image of SDS-PAGE showing the representative purity of monovalent and divalent forms of Nb-01, Nb-04 and Nb-40. 5 pg of each representative nanobody was loaded into an SDS-PAGE gel and stained.

[0053] FIGs. 2A-2C are graphs showing the results of epitope mapping by ELISA as described in Example 2. The plates were probed with nanobodies described herein, Nb-01 (FIG. 2A), Nb-04 (FIG. 2B), and Nb-n40 (FIG. 2C).

[0054] FIGs. 3A-3E are images depicting specificity of representative nanobodies as measured by filter retardation assay. FIGs. 3A-3C depict slot blot data with: different quantities of monomers (M) and fibrils (F) of a-synuclein (FIG. 3 A); different amyloid proteins including A(3, Tau and Islet Amyloid Polypeptide (IAPP) (FIG. 3B); and with (3- and y-synuclein (FIG. 3C). FIG. 3D depicts specificity of representative nanobodies with other truncated a-syn fibrils. FIG. 3E depicts the affinity of representative nanobodies to different quantities of a-syn fibrils and different oligomer preparations.

[0055] FIGs. 4A-4D depict the results of in vitro seed induced aggregation assay. Samples of a-syn monomers (25 pM) were seeded with 2 pM of seeds, which were incubated in the presence or absence ofNb-01 (FIG. 4A; from left to right, at each timepoint, M+S, 8 pM Nb-01 + S, and 10 pM Nb-01 + S) or Nb-04 (FIG. 4C) or Nb-04 (FIG. 4D) at different molar ratios for 6 hours continuous shaking at 37 °C. FIG. 4B shows electron microscopy (EM) images from in-vitro seeding assay with monomers and seeds in presence of Nb-01 at different time points as denoted. ‘M’ is monomeric form a-syn and ‘S’ is a-syn seeds. Scale bars = 500 nm.

[0056] FIGs. 5A-5B depict the effects of Nb-04 (FIG. 5A) and Nb-40 (FIG. 5B) on soluble and insoluble pSI29-a-syn and aggregation.

[0057] FIGs. 6A-6I depicts the results of an ITC experiment for the affinity interaction between Nb- 01 (FIG. 6A and FIG. 6B), Biv Nb-01 (FIG. 6C and FIG. 6D), Nb-04 (FIG. 6F and FIG. 6G) and Nb-40 (FIG. 6H) with a-syn monomers or a-syn fibrils. KD constant M of Nb-01 and Biv Nb-01 (FIG. 6E) Nb- 04 and Nb-40 (FIG. 61). AG, AH and TAS measured for the binding of Nb-01 and Biv Nb-01.

[0058] FIG. 7 is a graph showing the exemplary nanobodies Nb-01 and Biv Nb-01 inhibit the toxicity caused by a-syn seeds in SH-SY5Y cell model of PD. SH-SY5Y cell viability was evaluated using MTT assay. a-Syn seeds (2 pM) were incubated with (2 pM) Nb-01 or (2 pM) Biv Nb-01 and added to cells in two separate sets. After 1 h of incubation, 10 pM a-syn monomers were then added in Opti-MEM in one set and in another set Opti-MEM was added, incubation was further carried out for 48 h, prior to MTT addition. Newly formed formazan crystals were dissolved using solubilization buffer and the absorbance was measured, and percentage of the viable cells was plotted (average of 3 wells standard deviation). The results are expressed as percentages of the average of the control (untreated cells). Statistical analysis was performed using one-way ANOVA with Dunnet’s multiple comparison test (***P < 0.001; **P < 0.01; *P < 0.05).

[0059] FIGs. 8A-8H depict immunohistochemical staining of nanobodies in post-mortem brain tissue. Representative images demonstrating staining in the dorsal motor nucleus of the vagal nerve (DMV; FIGs. 8A-8D) or entorhinal cortex (FIGs. 8E-8H). Cases with Lewy body disease (FIGs. 8B, 8D, 8F, 8H) are compared to control cases in the DMV (FIGs. 8A, 8C) or disease-control Alzheimer’s disease (AD) cases in the entorhinal cortex (FIGs. 8E, 8G). Scale bars = 100 uM.

[0060] FIG. 9A-9R are images depicting Lewy bodies immunoreactive for Nb-04 and Nb-40 in cingulate gyrus, Substantia nigra (S. Nigra), and Ventral Tegmental Area (VTA) from PD and DLB cases. Syn-O2 (as disclosed in US Patent No. 10,208,111, which is incorporated herein by reference in its entirety) was used as a control antibody. Scale bars = 100 mM.

[0061] FIG. 10 are images depicting specificity of representative nanobodies as measured by filter retardation assay. FIG. 10 depicts slot blot data with different quantities of monomers (M) and fibrils (F) of a-synuclein as specified and probed with specified nanobodies.

[0062] FIGs. 11A-11F are graphs depicting the results of in vitro aggregation inhibition assay. Samples of a-syn monomers (25 pM) were incubated in the presence or absence of Nb-04 (FIG. 11 A) or Nb-40 (FIG. 11B) or BivNb-04 (FIG. 11C) or BivNb-40 (FIG. 11D) or BivNb-04-40 (FIG. HE) or control nanobody Nb-65 (FIG. 1 IF) at different molar ratios as specified for 6 hours continuous shaking at 37 °C. The extent of fibrillation was monitored by Th-T binding assay.DETAILED DESCRIPTION

[0063] Parkinson’s disease (PD), dementia with Lewy bodies (DLB) ), and multiple system atrophy (MSA) are collectively referred to as synucleinopathies and idiopathic and familial forms of these diseases have been linked to abnormal expression of a-synuclein (a-syn). Provided herein are nanobodies and engineered versions thereof that specifically bind to a-synuclein oligomers and fibrils, compositions comprising the nanobody constructs, and methods and treatments using the same. The nanobodies described herein have an increased affinity to bind to a-synuclein aggregates compared to monomeric a- synuclein forms. The nanobody molecules disclosed herein may be used (e.g., alone or in combination with other agents or therapeutic modalities) to treat, prevent, and / or diagnose disorders, including Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), multiple system atrophy (MSA) or Alzheimer’s disease with Lewy bodies (AD-LB).

[0064] The aggregated forms of alpha synuclein (a-syn) protein are attractive target to produce antibodies for passive immunization against PD. Nanobodies (Nbs) or single-domain antigen-binding fragments of dromedary Heavy-chain antibodies (HCAb) are suitable candidate therapeutics for synucleinopathies because of their small size, solubility, and stability.Definitions

[0065] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0066] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0067] An “antigen -binding portion” or “antigen-binding fragment” of a nanobody refers to a molecule other than an intact or whole nanobody or antibody that comprises a portion of an intact nanobody or antibody that binds the antigen to which the intact nanobody or antibody binds (e.g., one or more fragments of a nanobody or antibody that retain the ability to specifically bind to an antigen).

[0068] “Nanobody” or “nanobodies” refers to the minimum known non-artificial antigen-specific binding functional fragment of an antibody. Nanobodies comprise a single variable antigen-binding (VHH) domain, and are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family. Such species in the Camelidae family include, for example camel, llama, dromedary, alpaca and guanaco, and other species besides Camelidae that may produce heavy chain antibodies naturally devoid of light chain.

[0069] “Engineered version of a nanobody” refers to recombinant and / or artificially generated nanobodies which may be bivalent, trivalent, bi-specific or tri-specific. In some embodiments, engineered nanobodies may be bi-paratopic or tri-paratopic. In some embodiments, engineered nanobodies may bind to more than one epitope. The more than one epitope may be more than one epitope of the same target protein.

[0070] The term “epitope” means a portion of an antigen capable of specific binding to an antibody. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which a nanobody binds can be determined using known techniques for epitope determination such as, for example, testing for nanobody binding to a-synuclein protein variants with different point-mutations or peptides comprising amino acid sequences identical to a portion or fragment of a-synuclein protein.

[0071] ‘Framework” or “FR” refers to variable domain residues other than complementarity determining region (CDR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence : FR1 -CD 1 -FR2-CDR2-FR3 -CDR3 -FR4.

[0072] Percent “identity” between a polypeptide sequence and a reference sequence, is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0073] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” By way of example, the following groups of amino acids are considered conservative substitutions for one another.

[0074] Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY.

[0075] The term “ka” (secas used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the kOff value.

[0076] The term “ka” (M Nsecas used herein, refers to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the konvalue.

[0077] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD = ka / ka.

[0078] The term “KA”as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction. KA = ka / ka.

[0079] With regard to the binding of a nanobody or engineered version thereof to a target molecule, the terms “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean bindingthat is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0080] The terms “treatment,” “treating”, etc., refer to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. Various embodiments may specifically include or exclude one or more of these modes of treatment. The herein disclosed anti-a-synuclein nanobodies may be used as medicaments for the treatment of a-synuclein-associated neurodegenerative disorders. Further embodiments are methods of treating a-synuclein-associated neurodegenerative disorders comprising administering an anti-a- synuclein nanobody to a subject in need thereof. In some embodiments, the a-synuclein-associated neurodegenerative disorder is Parkinson’s disease (PD), dementia with Lewy bodies, Alzheimer's disease, or multiple system atrophy. In some embodiments, the subject in need thereof is a human.

[0081] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a nanobody or composition that when administered to a subject is effective to treat a disease or disorder.Nanobodies

[0082] Provided herein are nanobodies and engineered variants thereof that preferably bind to aggregated forms of a-syn. In some embodiments, the nanobodies and engineered variants thereof selectively bind to aggregated forms of a-syn. In some embodiments, nanobodies and engineered variants thereof described herein comprise a low affinity for monomeric forms of a-syn.

[0083] One aspect of the present disclosure provides a method of treating Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Alzheimer’s disease with Lewybodies (AD-LB) in a subject in need thereof, which comprises, consists, or consists essentially of administering to the subject a therapeutically effective amount of an a-syn nanobody.

[0084] In some embodiments, the a-syn nanobody comprises, consists, or consists essentially of a VHH having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or in) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

[0085] In some embodiments, the VHH comprises, consists, or consists essentially of: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or in) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0086] In some embodiments, the VHH comprises, consists, or consists essentially of: i) an amino acid comprising one or more mutations relative to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 51, 54, and 56-59, and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) an amino acid comprising one or more mutations relative to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 52, and 55-59, and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; orHi) an amino acid comprising one or more mutations relative to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 6, 50, 53, 57, and 59, and a CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0087] In some embodiments, the VHH comprises, consists, or consists essentially of: i) an amino acid comprising at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 51, 54, and 56-59, and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) an amino acid comprising at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 52, and 55-59, and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or in) an amino acid comprising at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to an amino acid sequence set forth in any one of SEQ ID NOs: 3, 6, 50, 53, 57, and 59, and a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0088] In some embodiments, the a-syn nanobody comprises, consists, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto.

[0089] In some embodiments, a-syn nanobody is selected from nanobody Nb-04 (SEQ ID NO: 1), or nanobody Nb-40 (SEQ ID NO: 2). In some embodiments, a-syn nanobody or engineered variant thereof is selected from a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6.

[0090] In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of an amino acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3. In some embodiments, the a-syn nanobody or engineered variant thereof comprises, consists, or consists essentially of an amino acid sequence having greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3.

[0091] In some embodiments, the a-syn nanobody specifically recognizes oligomers and fibrils of a- synuclein protein and not monomeric a-synuclein protein.

[0092] In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 113-126 of a-synuclein (SEQ ID NO: 44). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 127- 140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 43-56 of a-synuclein (SEQ ID NO: 34). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 1-14 of a-synuclein (SEQ ID NO: 28). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 8-21 of a- synuclein (SEQ ID NO: 29). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 15-28 of a-synuclein (SEQ ID NO: 30). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 22-35 of a-synuclein (SEQ ID NO: 31). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 29-42 of a-synuclein (SEQ ID NO: 32). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 36-49 of a-synuclein (SEQ ID NO: 33). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 50-63 of a-synuclein (SEQ ID NO: 35). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 57-70 of a-synuclein (SEQ ID NO: 36). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 64-77 of a-synuclein (SEQ ID NO: 37). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 71-84 of a-synuclein (SEQ ID NO: 38). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 78-91 of a-synuclein (SEQ ID NO: 39). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 85-98 of a-synuclein (SEQ ID NO: 40). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consistingessentially of residues 92-105 of a-synuclein (SEQ ID NO: 41). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 99-112 of a-synuclein (SEQ ID NO: 42). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 106-119 of a-synuclein (SEQ ID NO: 43). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 120-133 of a-synuclein (SEQ ID NO: 45).

[0093] One aspect of the disclosure provides a nanobody or engineered version thereof having high binding affinity for a-synuclein oligomers, high binding affinity to a-synuclein fibrils, and / or low binding affinity for a-synuclein monomers. In some embodiments, the nanobody or engineered version thereof comprises high binding affinity for a-synuclein oligomers. In some embodiments, the nanobody or engineered version thereof comprises a high binding affinity to a-synuclein fibrils. In some embodiments, the nanobody or engineered version thereof comprises a low binding affinity for a-synuclein monomers. In some embodiments, the nanobody or engineered version thereof comprises a high binding affinity for a-synuclein aggregates and low affinity binding for a-synuclein monomers.

[0094] In some embodiments, the nanobody or engineered version thereof comprises, consists, or consists essentially of a VHH having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; orHi) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

[0095] In some embodiments of the nanobody or engineered version thereof, the VHH comprises, consists, or consists essentially of: i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or iii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0096] In some embodiments, the nanobody or engineered version thereof comprises, consists, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto. In some embodiments, the nanobody or engineered version thereof is selected from Nb-04 (SEQ ID NO: 1), or Nb-40 (SEQ ID NO: 2). In some embodiments, a-syn nanobody or engineered variant thereof is selected from a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6.

[0097] In some embodiments, the nanobody or engineered version thereof comprises, consists, or consists essentially of a variant VHH comprising, consisting, or consisting essentially of an amino acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to any one of SEQ ID NOs: 1-6. In some embodiments, the nanobody or engineered version thereof comprises, consists, or consists essentially of a variant VHH comprising, consisting, or consisting essentially of an amino acid sequence having greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to any one of SEQ ID NOs: 1-6.

[0098] In some embodiments, the a-synuclein aggregates comprise, consist, or consist essentially of protofibrils and / or soluble oligomers of a-synuclein. In some embodiments, the a-synuclein aggregates comprise, consist, or consist essentially of protofibrils of a-synuclein. In some embodiments, the a- synuclein aggregates comprise, consist, or consist essentially of soluble oligomers of a-synuclein. In some embodiments, the a-synuclein aggregates comprise, consist, or consist essentially of a-synuclein fibrils.

[0099] In some embodiments, the nanobody or engineered version thereof binds to an epitope comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 127- 140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the nanobody or engineered version thereof binds to an epitope comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44). In some embodiments, the nanobody or engineered version thereof binds to an epitope comprising, consisting, or consisting essentially of the N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 1-14 of a-synuclein (SEQ ID NO: 28). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 8-21 of a- synuclein (SEQ ID NO: 29). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 15-28 of a-synuclein (SEQ ID NO: 30). Insome embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 22-35 of a-synuclein (SEQ ID NO: 31). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 29-42 of a-synuclein (SEQ ID NO: 32). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 36-49 of a-synuclein (SEQ ID NO: 33). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 50-63 of a-synuclein (SEQ ID NO: 35). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 57-70 of a-synuclein (SEQ ID NO: 36). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 64-77 of a-synuclein (SEQ ID NO: 37). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 71-84 of a-synuclein (SEQ ID NO: 38). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 78-91 of a-synuclein (SEQ ID NO: 39). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 85-98 of a-synuclein (SEQ ID NO: 40). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 92-105 of a-synuclein (SEQ ID NO: 41). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 99-112 of a-synuclein (SEQ ID NO: 42). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 106-119 of a-synuclein (SEQ ID NO: 43). In some embodiments, the a-syn nanobody recognizes an epitope comprising, consisting, or consisting essentially of residues 120-133 of a-synuclein (SEQ ID NO: 45).

[0100] In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a monovalent nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a multi-valent nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a bi-valent nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a tri-valent nanobody.

[0101] In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a multi-paratopic nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a bi-paratopic nanobody. In some embodiments, the engineered nanobody comprises, consists, or consists essentially of a tri-paratopic nanobody.

[0102] In some embodiments, the multi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 113-126 (SEQ ID NO: 44)and residues 127-140 (SEQ ID NO: 46) of a-synuclein. In some embodiments, the multi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and a C-terminal region corresponding to residues 127-140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the multiparatopic nanobody binds to epitopes comprising, consisting, or consisting essentially of an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and a C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44).

[0103] In some embodiments, the bi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of a C-terminal region corresponding to residues 113-126 (SEQ ID NO: 44) and residues 127-140 (SEQ ID NO: 46) of a-synuclein. In some embodiments, the bi-paratopic nanobody binds to epitopes comprising, consisting, or consisting essentially of an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and a C-terminal region corresponding to residues 127-140 of a-synuclein (SEQ ID NO: 46). In some embodiments, the biparatopic nanobody binds to epitopes comprising, consisting, or consisting essentially of an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and a C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44).

[0104] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of a polypeptide comprising, consisting, or consisting essentially of two or more nanobodies or engineered variants thereof as described herein and a linker (e.g., a polypeptide linker) disposed between each of the two or more nanobodies or engineered variants thereof. In some embodiments, the each of the two or more nanobodies or engineered variants thereof are independently selected from any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto.

[0105] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more VHH domains each independently having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or in) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

[0106] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more VHH domains, each independently comprising, consisting, or consisting essentially of: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or in) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0107] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more VHH domains comprising, consisting, or consisting essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto. In some embodiments, the nanobody or engineered version thereof is selected from Nb-04 (SEQ ID NO: 1), or Nb-40 (SEQ ID NO: 2). In some embodiments, a- syn nanobody or engineered variant thereof is selected from a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOs: 1-6.

[0108] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more (e.g., 2, 3, 4, 5, 6, or more) VHH domains comprising, consisting, or consisting essentially of an amino acid sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3. In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of two or more (e.g., 2, 3, 4, 5, 6, or more) VHH domains comprising, consisting, or consisting essentially of an amino acid sequence having greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to the amino acid set forth in SEQ ID NO: 3.

[0109] In some embodiments, the multi-valent nanobody is bi-valent. In some embodiments, the multi-valent nanobody is tri-valent.

[0110] In some embodiments, the multi-valent nanobody comprises one or more linkers, wherein each of the one or more linkers are disposed between each of the two or more VHH domains. In someembodiments, the linker is a peptide linker. In some embodiments, the linker is an IgA linker (SPSTPPTPSPSTPPASSPSTPPTPSPSTPPAS; SEQ ID NO: 49).[OHl] In some embodiments, the multi-valent nanobody comprises, consists, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 50-59, or a variant thereof having at least 55% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity thereto.

[0112] In some embodiments, the nanobody or engineered version thereof, or the multi-valent nanobody, comprises one or more polypeptide tags or linkers (e.g., comprises an HA tag (SEQ ID NO: 47), a HIS tag (e.g., a multi-histidine tag, such as a 6x HIS tag (HHHHHH; SEQ ID NO: 48), and / or an IgA linker (SPSTPPTPSPSTPPASSPSTPPTPSPSTPPAS; SEQ ID NO: 49)).

[0113] In some embodiments, the nanobody or engineered version thereof preferably recognizes oligomers and fibrils of a-synuclein protein and less the monomeric form of a-synuclein protein.

[0114] One aspect of the disclosure provides a nucleic acid molecule capable of expressing any of the nanobodies or engineered variants thereof provided herein. In some embodiments, the nucleic acid comprises an expression vector. Some embodiments provide a prokaryotic or eukaryotic host cell transformed with the one or more expression vectors. Some embodiments provide an oncolytic virus encoding the nucleic acid. Some embodiments provide a method for the production of a nanobody or engineered variant thereof as described herein comprising the steps of expressing a nucleic acid provided herein in a prokaryotic or eukaryotic host cell and recovering the protein from the cell or the cell culture supernatant.

[0115] For recombinant production of the nanobody or engineered variant thereof, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression.

[0116] Many different vectors are known in the art. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0117] Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells. Suitable prokaryotes include eubacteria, such as Gramnegative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia (E. coli). Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescans). Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa), and Streptomyces .One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli XI 776, and E. coli W3110 are suitable.

[0118] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody construct-encoding vectors. Saccharomyces ccrcvisiac. or common baker's yeast, is a commonly used lower eukaryotic host microorganism. However, a number of other genera, species, and strains are available and useful, such as Schizosaccharomyces pombc. Kluyveromyces (K. laclis. K. fragilis, K. bulgaricus K. wickcramii. K. waltii, K. drosophilarum, K. ihcrmololcrans. and K. marxianus). Yarrowia, Pichia pasloris. Candida (C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S. occidentalis), and filamentous fungi such as, for example Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A. niger).

[0119] Useful mammalian host cells include COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO); mouse sertoli cells; African green monkey kidney cells (VERO- 76), and the like.

[0120] The host cells used to produce the nanobody or engineered variant thereof described herein may be cultured in a variety of media. Commercially available media such as, for example, Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz., 1979, 58:44; Barnes et al., Anal. Biochem., 1980, 102:255; and U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469, or WO 90 / 03430 and WO 87 / 00195 may be used.

[0121] One aspect of the disclosure provides a pharmaceutical composition comprising the nanobody or engineered version thereof set forth herein and a pharmaceutically acceptable diluent or carrier.

[0122] One aspect of the disclosure provides the nanobody or engineered version thereof set forth herein, or the pharmaceutical composition set forth herein, for use as a medicament.

[0123] One aspect of the disclosure provides a method for preventing or treating a neurodegenerative disorder with a-synuclein pathology in an subject in need thereof comprising, consisting, or consisting essentially of administration of the nanobody or engineered version thereof set forth herein or the pharmaceutical composition set forth herein, to the subject.

[0124] In some embodiments, the neurodegenerative disorder with a-synuclein pathology comprises, consists, or consists essentially of Parkinson’s disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Alzheimer’s disease with Lewy bodies. In some embodiments, the neurodegenerative disorder with a-synuclein pathology consists of Alzheimer’s disease (AD).

[0125] One aspect of the disclosure provides a test kit for use in a method of determining whether or not an individual has a neurodegenerative disease, comprising, consisting, or consisting essentially of the nanobody or engineered version thereof as set forth herein. In some embodiments, the kit can include one or more other elements comprising, consisting, or consisting essentially of: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, the nanobody or engineered version thereof to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the nanobody or engineered version thereof for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0126] One aspect of the disclosure provides a method of detecting a-synuclein fibrils and aggregates comprising, consisting, or consisting essentially of the steps of: adding the nanobody or engineered version thereof as set forth herein to a biological sample; and detecting the presence of a complex formed between a-synuclein fibrils and / or aggregates and the nanobody or engineered version thereof.

[0127] One aspect of the disclosure provides a method for diagnosing a neurodegenerative disease associated with a-synuclein comprising, consisting, or consisting essentially of: adding the nanobody or engineered version thereof as set forth herein to a biological sample; and detecting the presence or absence of a complex formed between a-synuclein aggregates and the nanobody or engineered version thereof.

[0128] In some embodiments, the nanobody or engineered version thereof further comprises a detectable label. In some embodiments, the detectable label is selected from the group comprising, consisting, or consisting essentially of a fluorescent label, a radioactive label, or a contrast agent.

[0129] The detection of a complex formed between a-synuclein aggregates and the nanobody or engineered version thereof indicates the presence of a-synuclein aggregates in the sample.

[0130] The method can further comprise the step of measuring a level of complex formed and comparing the level to a reference level. The reference level will typically be calculated from a sample from an individual known not to have an a-synuclein pathology or from an earlier test of a sample from the same subject being tested.

[0131] The method can detect fibrils and oligomers of a-synuclein.

[0132] The method can be carried out in vitro in a tissue or biological fluid sample. The sample obtained from an individual to be tested, can for example be cerebrospinal fluid (CSF), blood, urine, saliva, brain, gut, colon, skin, or salivary gland tissues. In some embodiments, the sample is a CSF sample. In some embodiments, the sample is a brain tissue sample.

[0133] The sample may be combined with the nanobody or engineered version thereof under conditions effective to allow binding of the nanobody or engineered version thereof to a-synuclein aggregates in the sample.

[0134] The sample may be processed prior to being assayed using standard methods. In some embodiments, the tissue sample undergoes no pre-treatment (e.g., is not subjected to any treatment such as, autoclaving, formic acid, and / or proteinase K treatment) before testing.

[0135] One aspect of the disclosure provides the nanobody or engineered version thereof as set forth herein for use as an imaging agent of a-synuclein aggregates. In some embodiments, the a-synuclein aggregates are present in a biological sample or biological tissue. In some embodiments, the biological sample or biological tissue comprising, consisting, or consisting essentially of brain, gut, heart, skin, or eye.

[0136] One aspect of the disclosure provides a method for imaging a-synuclein aggregates comprising, consisting, or consisting essentially of: administering the nanobody or engineered version thereof as set forth herein to a subject; and detecting the nanobody or engineered version thereof.

[0137] In some embodiments, the nanobody or engineered version thereof comprises a detectable label.

[0138] In some embodiments, a nanobody as described herein does not bind one both of P-synuclein or y-synuclein. In some embodiments, binding specificity is determined by western blot, ELISA, slot blot, or any combination thereof.

[0139] In some embodiments, the antibody has high affinity for a-synuclein aggregates but low affinity for a-synuclein monomers. In some embodiments, the a-synuclein aggregates are protofibrils or soluble oligomers of a-synuclein and the antibody can have high affinity for the protofibrils, the soluble oligomers, or both. In some embodiments, the a-synuclein aggregates are a-synuclein fibrils.Pharmaceutical Compositions and Methods of Administration

[0140] Another aspect provides a pharmaceutical composition comprising one or more of the nanobodies or engineered versions thereof set forth herein. Any of the nanobodies or engineered versions thereof provided herein can be provided in any appropriate pharmaceutical composition and be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

[0141] In some embodiments, a pharmaceutical composition is one intended and suitable for the treatment of disease in humans. That is, it provides overall beneficial effect and does not contain amounts of ingredients or contaminants that cause toxic or other undesirable effects unrelated to the provision of the beneficial effect.

[0142] The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

[0143] In some embodiments, the pharmaceutical composition comprises an anti-foaming agent. Any suitable anti-foaming agent may be used. In some embodiments, the anti-foaming agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant, and combinations thereof. In some embodiments, the anti-foaming agent is selected from a mineral oil, a vegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.

[0144] In some embodiments, the pharmaceutical composition comprises a cosolvent. Illustrative examples of cosolvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, and propylene glycol.

[0145] In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.

[0146] In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, and guar gum.

[0147] In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include c / -alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol) succinate.

[0148] In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropyl cellulose, and magnesium oxide.

[0149] Other excipients that may be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, and sugars. Specific examples of each of these agents are described, for example, in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, incorporated by reference in its entirety.

[0150] In some embodiments, the pharmaceutical composition comprises a solvent. In some embodiments, the solvent is saline solution, such as a sterile isotonic saline solution or dextrose solution. In some embodiments, the solvent is water for injection.

[0151] In some embodiments, the pharmaceutical compositions are in a particulate form, such as a microparticle or a nanoparticle. Microparticles and nanoparticles may be formed from any suitable material, such as a polymer or a lipid. In some embodiments, the microparticles or nanoparticles are micelles, liposomes, or polymersomes. In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibody constructs.

[0152] Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising an antibody, since water can facilitate the degradation of some antibodies.

[0153] Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected.

[0154] An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0155] The amino acid sequences of the exemplary nanobodies are provided below:

[0156] One aspect is an immunoassay utilizing a nanobody or engineered variant thereof to detect or quantitate a-synuclein aggregates. An exemplary, non-limiting immunoassay is an enzyme-linked immunosorbent assay (ELISA). In other embodiments, the immunoassay is an immunohistochemical assay. Immunoassays measure substances, such as analytes, proteins, etc., using the specificity of an antibody to the substance.

[0157] One aspect is a sandwich ELISA. In such an assay, a capture antibody specific for the substance is associated with a solid support, such as a microtiter plate. A liquid containing the substance (or suspected of containing the substance, or a sample in need of determining not to include the substance) is allowed to bind to the capture antibody. Then a detection antibody, also specific for the substance, is added to allow detection of substance bound to the capture antibody.

[0158] Another aspect is an immunohistochemical assay. Immunohistochemistry involves the process of selectively imaging antigens (proteins) in a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Visualizing an antibody-antigen interaction canbe accomplished in a number of ways. In the most common instance, a detection antibody is used which allows the detection of the anti-a-synuclein nanobody engineered variant thereof and thus the substance to which the nanobody or engineered variant thereof is bound.

[0159] In some embodiments, the detection antibody is a labeled antibody. In other embodiments, the anti-a-synuclein nanobody or engineered variant thereof is labeled. The label can include a radioactive label, an enzyme label, a colorimetric label, a fluorescent label, a chemiluminescent label, or other labels known to persons of skill in the art. In some such embodiments, the detection antibody is biotinylated so that it can bind an enzyme-linked avidin molecule, such as streptavidin conjugated with horseradish peroxidase or alkaline phosphatase. In alternative embodiments, an avidin molecule is conjugated with another detectable label, for example, a fluorescent dye or quantum dot. In some embodiments, the detection antibody is directly labeled. Still further alternatives are familiar to one of skill in the art.

[0160] In further embodiments in which the nanobody or engineered variant thereof is labeled, the nanobody or engineered variant thereof is used for in vivo imaging by administering the labeled nanobody or engineered variant thereof to a subject and detecting the label.

[0161] In some embodiments, the label is an enzymatic label such as a peroxidase (e.g., horseradish peroxidase), a galactosidase (e.g., P-D-galactosidase), or a phosphatase (e.g., alkaline phosphatase). For enzymatic labels, a substrate is needed which is cleaved by the enzyme to produce a color, fluorescence, or luminescence, which is measured spectrophotometrically. Exemplary colorimetric substrates for peroxidase include, but are not limited to, 3,3 ’,5,5 ’-tetramethylbenzidine (TMB), 3, 3', 4, 4' diaminobenzidine (DAB), 4-chloro-l -naphthol (4CN), 2,2'-azino-di [3-ethylbenzthiazoline] sulfonate (ABTS), and o-phenylenediamine (OPD). In some embodiments, when the assay is an ELISA, the substrate is TMB which produces a blue color which is measured at a wavelength of 650 nm. The reaction can be halted by addition of acid or another stop reagent. Using a sulfuric acid stop solution turns TMB yellow and the color can then be read at 450 nm. Exemplary colorimetric substrates for phosphatase include, but are not limited to, 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium (BCIP / NBT) and p-nitrophenylphosphate (p-NPP). Exemplary colorimetric substrates for galactosidase include, but are not limited to, 5-dodecanoylaminofluorescein di-P-D-galactopyranoside (C12FDG), 9H-(l,3-dichloro-9,9-dimethylacridin-2-one-7-yl), and P-D-galactopyranoside (DDAO galactoside). Exemplary fluorescent substrates include, but are not limited to, 4-methylumbelliferyl phosphate (4-MUP; for phosphatase), and 4-methylumbelliferyl galactoside (MUG; for galactosidase), fluorescein di-P-D-galactopyranoside (FDG; for galactosidase), hydroxyphenylacetic acid (HPA; for peroxidase), and 3-p-hydroxyphenylproprionic acid (HPPA; for peroxidase). Exemplary luminescentsubstrates include, but are not limited to, luminol, polyphenols (e.g., pyrogallol, pupurogallin, gallic acid, and umbelliferone) and acridine esters, and luciferin for peroxidase; 3-(2'-spiroadamantane)-4- methyl-4-(3 '-phosphoryloxyphenyl- 1, 2-dioxetane, disodium salt) (AMPPD) for phosphatase; and (3- (2'-spiroadamantane)-4-methoxy-4-(3'-P-D-galactopyranosyloxyphenyl-l,2-dioxetane (AMPGD) for galactosidase.

[0162] In some embodiments, the label is horseradish peroxidase and the substrate is TMB.

[0163] In some embodiments, the label is a colorimetric label, a fluorescent label, or a luminescent label. An exemplary colorimetric label includes, but is not limited to, nanoparticulate gold. Exemplary fluorescent labels include, but are not limited to, ethidium bromide, fluorescein and its derivatives, rhodamine and its derivatives, green fluorescent protein, Texas Red, Cascade Blue, Oregon Green, Marina Blue, an atto label, a CF™ dye, an Alexa Fluor, and a cyanine dye. Exemplary luminescent labels include, but are not limited to, luciferin and firefly luciferase.

[0164] With respect to the assay aspects, some embodiments the assay is used as a diagnostic assay to determine whether or not an individual has a neurodegenerative disease associated with a-synuclein pathology, for example, Parkinson’s disease, dementia with Lewy bodies, multiple system atrophy or Alzheimer's disease with Lewy bodies. In some embodiments, the diagnostic assay comprises adding the nanobody or engineered variant thereof to a biological sample (e.g., a biological sample from a subject), and detecting the presence or absence of a complex formed between a-synuclein aggregates and the nanobody or engineered variant thereof.

[0165] With respect to the assay aspects, some embodiments comprise a test kit comprising an anti-a- synuclein antibody and other reagents or equipment needed to carry out the assay.

[0166] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherentlycontains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0167] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0168] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of’ excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0169] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0170] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.EXAMPLESExample 1. a-synuclein nanobodies

[0171] Here, we constructed immune nanobody libraries by immunizing camels with a-syn fibrils. Using phage display technology, we screened and identified three nanobodies; Nb-01 (SEQ ID NO: 3), Nb- 04 (SEQ ID NO: 1), and Nb-40 (SEQ ID NO: 2). Nb-01 was found to recognize preferentially a-syn fibrils compared to the monomeric form whereas, Nb-04 and Nb-40 were found to specifically recognize oligomers and fibrils of a-syn and not the monomeric form. As described in Example 2, below, epitope mapping using peptide scanning revealed that Nb-01 recognizes a region in the N-terminal of a-syn thatcontains many familial mutations involved in early onset of Parkinson’s Disease (PD) cases. Nb-04 and Nb-40 recognized a region in the C-terminal region of a-syn. (FIGs. 2A-2C)).

[0172] The nanobodies were further engineered to produce tandem linked bivalent constructs named BivNb-01 (SEQ ID NO: 53), BivNb-04 (SEQ ID NO: 54), and BivNb-40 (SEQ ID NO: 55). Without wishing to be bound by theory, producing multi-valent constructs increases the nanobody’s avidity. The three nanobodies were able to block a-syn seeded aggregation in vitro and a-syn seed-induced toxicity. All nanobody formats were able to recognize Lewy body pathology in human post-mortem brain tissue from Parkinson’s Disease (PD) and dementia with Lewy bodies (DLB) cases (data not shown).Example. 2: Epitope Mapping for nanobodies

[0173] The affinity of the nanobodies was tested against a peptide library covering portions of the a- syn sequence to identify linear or conformational epitopes. Fourteen amino acid long peptides, with 7 amino acid overlap, covering the entire sequence of a-syn (Table 1) or recombinant a-syn, were synthesized and subsequently coated onto a 384 well black MaxiSorp ELISA plate (Nunc, Denmark). The wells were dried overnight at 37°C and then blocked with 2.25% gelatin in PBST (PBS containing 0.05% Tween 20) for 1 h at room temperature (RT). After washing with PBST, 50 pl / wcll (1 pg / ml) of Nb-01 (SEQ ID NO: 6) was added to the plate and incubated for Ih at RT followed by washing and adding anti-His antibody (Abeam) at 1 / 1000 dilution. The plate was washed and incubated with HRP conjugated goat anti-mouse IgG secondary antibody for Ih at RT. The bound HRP was detected by adding 50 pl / well of substrate (Super Signal ELISA Femto Maximum Sensitivity Substrate, Pierce Biotechnology, Rockford, USA) and the chemiluminescence in relative light units measured with a Victor X3 2030 multi-label plate reader (Perkin Elmer, USA). For preabsorption experiment, Nb-01 was mixed with either 500, 250 or 125 pM of the peptides for 2 h at RT and then added to a 384 well plate coated with recombinant a-syn. The plate was washed and incubated with HRP-conjugated secondary antibody for 1 h at RT and developed as previously described.Table 1. The amino acid sequences of a-syn peptides used for epitope mapping nanobodies described herein.Example. 3: Filter retardation assay-slot blot

[0174] A bloting system using vacuum filtration method (Fisher scientific) was assembled with a prewet 0.2 pm nitrocellulose membrane using the manufacturer's protocol. Specified quantities of a-syn monomer (M) and a-syn fibrils (F) (FIG. 3A) or monomers and fibrils of a-syn and other amyloid proteins including Abeta, Tau and IAPP (FIG. 3B) or - and y-syn (FIG. 3C), or fibrils of truncated a- syn as specified (FIG. 3D); or a-syn fibrils, 4-oxo-2-noenal (ONE), 4-hydroxy-2-nonenal (HNE) a-synoligomers, and dopamine oligomers (DA) (FIG. 3E) were prepared in 50 pl PBS and applied to each slot. The wells were subsequently washed with 1000 pl PBS and the membrane was then air dried for 45 minutes. The dried membrane was blocked with 5% skimmed milk in 0.05% PBST for Ih at RT. After blocking, the membranes were incubated overnight in nanobodies described herein or control antibodies diluted in 0.05% PBST at the following specified concentrations: Nanobodies as specified (250 ng / ml); 82E1 ( Ms mAb for Abeta, IBL America, 50 ng / ml), 5E2 (Ms mAb for Tau, 50 ng / ml,), Anti-ABri ( Rabbit anti-ABri antiserum, 1 / 3000); FL140 (Rb pAb for a-syn, from Santa Cruz Biotech, 1 :5000); Fl 1 ( Ms mAb for a / f> syn, Santa Cruz Biotech (discontinued), 1 :2000) and E20 (Gt pAb for y-syn, Santa Cruz Biotech, 1 :2000). For blots probed with nanobodies of the disclosure, after overnight incubation with respective nanobody, an additional Anti-His (Ms mAb from Abeam, 1:2000) was applied for 2 hrs at RT. Following primary Ab incubation, blots were washed three times and incubated in respective HRP conjugated secondary Ab (1 :20k for mAbs and 1: 100k for pAbs) for 1 hour at RT and detected using west pico (mAb) or west femto (for pAbs) chemiluminescent substrate and imaged using BioRad Imaging System.Example. 4: In-vitro seeded aggregation assay

[0175] Samples of a-syn monomers (25 pM) were seeded with 2 pM of seeds, which were incubated in the presence or absence of Nb-01 (FIG. 4A; from left to right, at each timepoint, M+S, 8 pM Nb-01 + S, and 10 pM Nb-01 + S) or Nb-04 (FIG. 4C), or Nb-04 (FIG. 4D) at different molar ratios for 6 hours continuous shaking at 800 rpm at 37 °C. The extent of fibrillation was monitored by Th-S binding assay. The extent of fibrillation was further monitor by transmission electron microscopy (TEM) (FIG. 4B). Protein samples for electron microscopy images were prepared by depositing the samples on Formvar-coated 400 mesh copper grids followed by fixing briefly with 0.5% gluteraldehyde and negatively staining with 2% uranyl acetate. Images were acquired using FEI Talos 200 C electron microscope (FIG. 4B).Example. 5: Effects of Nb-04 and Nb-40 on soluble and insoluble pS129-a-syn and aggregation

[0176] The pS129-a-syn and aggregation of a-syn were assessed in 10 pg and 15 pg of insoluble and soluble proteins from cell lysates of untransfected (control) and transfected HEK cells by immunoblotting proteins using antibodies specific to pS129-a-syn and total a-syn (Syn-1; BD Bioscienses). One group of transfected HEK cells were simultaneously transfected with seeds and Nb- 04 / Nb-40 at 1: 1 or 1:5 molar ratio after an incubation at 37°C for one hour, the other group was transfected with seeds to be followed by Nb-04 / Nb-40 treatment at 1 : 1 or 1 : 5 molar ratio in OptiMEMfor 48 hours. Recombinant pS129 a-syn (rpS129-a-syn) and recombinant a-syn (r-a-syn) proteins were loaded (50 ng) as positive controls. Re-immunoblotting with (3-actin antibody was performed to normalize the amount of loaded proteins. The results for Nb-04 and Nb-40 are shown in FIG. 5A and FIG. 5B, respectively.Example. 6: Affinity measurement by Isothermal titration calorimetry

[0177] The binding of exemplary nanobodies as described herein to a-synuclein was carried out by ITC using a MicroCai Auto-iTC200 microcalorimeter (Malvern, USA) at 25 °C. (Nb-01 (FIG. 6A and FIG. 6B), Biv Nb-01 (FIG. 6C and FIG. 6D), Nb-04 (FIG. 6 J and FIG. 6K) and Nb-40 (FIG. 6L)). The Nanobodies as specified were titrated into the sample cell, containing a-synuclein (400 pil of 10 pM of monomeric alpha syn or 40 pM of Fibrils), sequentially by injecting a 2 pL aliquot at each titration point with a time interval of 120s. A total of 20 injections of the exemplary Nanobody was mixed into a-synuclein cell with stirring speed of 750 rpm. First, an initial small injection was used to minimize the impact of equilibration artifacts and was disregarded during evaluation of the data. As a control experiment, buffer solution was used in sample cell with the same concentration of the exemplary nanobodies within the syringe. The heats of mixing and dilution of control experiment was subtracted from the heat of binding per injection. To determine the equilibrium dissociation constant (Kd) and the enthalpic change (AH) associated with binding, the ITC isotherms were iteratively fit to a one-site binding model by non-linear least squares regression analysis using the MicroCai PEAQ-ITC analysis software (Malvern instruments). (KD constant M of Nb-01 and Biv NbasynOl (FIG. 6E) Nb-04 and Nb-40 (FIG. 61). AG, AH and TAS measured for the binding of Nb-01 and Biv Nb-01).Example. 7: MTT assay

[0178] This example shows exemplary nanobodies as disclosed herein inhibit the toxicity caused by a-syn seeds in SH-S75Y cell model of Parkinson’s Disease (PD). To perform MTT assay, SH-SY5Y cells were plated in 96-well MaxiSorp plate (Nunc), 15,000 cells (200 pL / well) and incubated for 24h in CO2 incubator at 37°C. a-Syn pure seeds (2 pM) were mixed with either Nb-01 (2pM) or BivNb-01 (2pM) using lOOpL Opti-MEM for 30min at 37°C with 300rpm shaking. The media was removed and lOOpL Opti-MEM containing seeds and antibodies was added to the cells. a-Syn monomers (10 pM) were added in another lOOpL Opti-MEM to the cells after Ih and incubated for further 48h. Wells containing Opti-MEM were control wells and had no treatment, and blank wells consisted of no cells. The 96-well plate was further incubated at 37C for 4.5h after addition of 20 pL of 6 mg / mL MTT to each well. lOOpL / well of 15% (w / v) SDS, 50% (v / v) N,N-dimethylformamide, pH 4.7, was added after the removal of medium-MTT solution and the plate was again incubated overnight at 37°C. The resultant absorbance was detected using Nano Quant instrument by TECAN at 590nm wavelength.Example. 8: Immunohistochemistry

[0179] Post-mortem brain tissue from DMV of one control and one DLB case, and entorhinal cortex of one AD case and one DLB case, was obtained from Newcastle Brain Tissue Resource, Newcastle University, UK and stained with nanobodies. After dewaxing and epitope unmasking using boiling citrate pH 6 and formic acid, tissue sections were incubated with the two nanobody formats for one hour at room temperature (Nb-01, 1 pg / ml; Biv Nb-01, 250 ng / ml). To determine the exemplary nanobody binding, we used an antibody against the polyhistidine tag (abl8184, Abeam, Cambridge, UK; 0.5 mg / ml) and incubated for one hour at room temperature. Primary antibody binding was then visualized with Menarini MenaPath kits (Menarini Diagnostics, UK) according to the manufacturer’s instructions, and counterstained with hematoxylin. Donors or next of kin provided informed consent to donate tissue and all procedures were approved by Newcastle Brain Tissue Resource Ethics Committee and the local UK National Health Service Research Ethics Committee. Sections were visualized and images obtained using a Nikon Eclipse 90i microscope coupled to a computer with NIS Elements software (Nikon, Tokyo, Japan). Representative images demonstrating staining in the dorsal motor nucleus of the vagal nerve (DMV; FIGs. 8A-8D) or entorhinal cortex (FIGs. 8E-8H). Cases with Lewy body disease (FIGs. 8B, 8D, 8F, 8H) are compared to control cases in the DMV (FIGs. 8A, 8C) or disease-control Alzheimer’s disease (AD) cases in the entorhinal cortex (FIGs. 8E, 8G).Example. 9:

[0180] Formalin-fixed, paraffin-embedded slides were heated at 60°C for 30 minutes, followed by dewaxing in xylene and rehydration through graded ethanol solutions. After rehydration in distilled water, antigen retrieval was performed by microwaving the slides in boiling citrate buffer, followed by cooling for 10 minutes. The slides were washed, incubated in formic acid, and washed again in distilled water. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, followed by blocking in normal goat serum. Primary antibodies (Nb-04 and Nb-40, or control mouse a-syn antibody, Syn-1; BD Biosciences) were incubated, washed, and secondary antibodies (anti- polyhistidine and biotinylated anti-mouse) were applied, with subsequent washing. ABC solution was incubated on the slides before the substrate (e.g., DAB) was applied for visualization. After a final wash, the slides were dehydrated, air-dried, cleared in xylene, and mounted with DPX.Example. 10: Exemplary nanobodies analyzed via filter retardation assay-slot blot

[0181] Specificity of representative nanobodies compared to control (mouse anti-a-syn antibody, 11D12) as measured by filter retardation assay (FIG 10). The blotting system using vacuum filtration method (Fisher scientific) was assembled with a pre-wet 0.2 pm nitrocellulose membrane using themanufacturer's protocol. Different concentration of either a-syn fibrils or monomers (500 ng to 31.25 ng / well in 50 pil PBS) were prepared and applied to each slot. Following this, the wells were washed with 1000 pl PBS and the membrane was then air dried for 45 minutes. The dried membrane was blocked with 5% skimmed milk in 0.05% PBST for Ih at RT. After blocking, the membranes were incubated overnight in generated nanobodies and its engineered formats or control antibodies diluted in 0.05% PBST. Following washing, the membranes were probed with Anti-His (Ms mAb from Abeam, 1:2000), applied for 2 hrs at RT. Following primary Ab incubation, blots were washed three times and incubated in respective HRP conjugated secondary Ab (1 : 15k in PBST) for 1 hour at RT and detected using west pico chemiluminescent substrate and imaged using BioRad Imaging System.Example. 11: In-vitro aggregation inhibition assay

[0182] a-Syn monomer (25 pM) was incubated alone or with exemplary nanobody at different molar ratios (a-syn : nanobody; 1:0.5, 1:1 or 1:2) in a total volume of 500 pL at 37°C with continuous shaking at 800 rpm for 3 days. Time point sample aliquots were taken out at day 0, 1, 2 and 3 and a-Syn aggregation was determined using Th-T fluorescence assay. Each time point sample was mixed with Th-T reagent (sample : ThT; 5uM :20uM) in PBS and the resultant fluorescence was detected in a 384- well, non-treated, black micro well plate (Nunc) with a Perkin Elmer EnVision Multimode Plate Reader using 440 nm and 485 nm excitation and emission wavelengths respectively. The results are shown in FIGs. 11A-11F.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating Parkinsons disease (PD) or dementia with Lewy bodies (DLB) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an a-syn nanobody or engineered variant thereof.

2. The method of claim 1, wherein the a-syn nanobody or engineered variant thereof comprises a VHH having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or iii) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

3. The method of claim 2, wherein the VHH comprises: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or iii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

4. The method of any one of claims 1-3, wherein the a-syn nanobody or engineered variant thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or a variant thereof having at least 55% identity thereto.

5. The method of any one of claims 1-4, wherein the a-syn nanobody or engineered variant thereof is selected from Nb-04 (SEQ ID NO: 1), or Nb-40 (SEQ ID NO: 2).

6. The method of any one of claims 1-5, wherein the a-syn nanobody or engineered variant thereof specifically recognizes oligomers and fibrils of a-synuclein protein and not monomeric a- synuclein protein.

7. The method of any one of claims 1-4, wherein the a-syn nanobody comprises an amino acid sequence having at least 85%.

8. The method of claim 7, wherein the a-syn nanobody specifically binds fibrils of a-syn compared to the monomeric form.

9. The method of any one of claims 1-6, wherein the a-syn nanobody or engineered variant thereof recognizes an epitope comprising residues 113-126 of a-synuclein (SEQ ID NO: 44).

10. The method of any one of claims 1-6, wherein the a-syn nanobody or engineered variant thereof recognizes an epitope comprising residues 127-140 of a-synuclein (SEQ ID NO: 46).

11. The method of any one of claims 1-4, 7, or 8, wherein the a-syn nanobody or engineered variant thereof recognizes an epitope comprising residues 43-56 of a-synuclein (SEQ ID NO: 34).

12. A nanobody or engineered version thereof having high binding affinity for a-synuclein oligomers, high binding affinity to a-synuclein fibrils, and / or low binding affinity for a-synuclein monomers, wherein the nanobody or engineered version thereof comprises a VHH having: i) a CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-12; ii) a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 16-18; or iii) a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 22-24.

13. The nanobody or engineered version thereof according to claim 9, wherein the VHH comprises: i) a CDR1 comprising the amino acid sequence setforth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or iii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24.

14. The nanobody or engineered version thereof according to claim 12 or 13, wherein the nanobody or engineered version thereof comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof having at least 55% identity thereto.

15. The nanobody or engineered version thereof according to any one of claims 12-14, wherein the nanobody or engineered version thereof is selected from Nb-04 (SEQ ID NO: 1) and Nb-40 (SEQ ID NO: 2).

16. The nanobody or engineered version thereof according to any one of claims 12-15, having high affinity for a-synuclein aggregates and low affinity binding for a-synuclein monomers.

17. The nanobody or engineered version thereof according to any one of claims 12-16, wherein the a- synuclein aggregates comprise protofibrils and / or soluble oligomers of a-synuclein.

18. The nanobody or engineered version thereof according to any one of claims 12-16, wherein the a- synuclein aggregates comprise a-synuclein fibrils.

19. The nanobody or engineered version thereof according to any one of claims 12-18, wherein the nanobody or engineered version thereof binds to an epitope comprising a C-terminal region corresponding to residues 127-140 of a-synuclein (SEQ ID NO: 46).

20. The nanobody or engineered version thereof according to any one of claims 12-18, wherein the nanobody or engineered version thereof binds to an epitope comprising a C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44).

21. The nanobody or engineered version thereof according to claim 12, wherein the nanobody or engineered version thereof binds to an epitope comprising the N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34).

22. The engineered nanobody of claim 12, wherein the engineered nanobody comprises a biparatopic nanobody.

23. The engineered nanobody of claim 22, wherein the bi-paratopic nanobody binds to epitopes comprising a C-terminal region corresponding to residues 113-126 (SEQ ID NO: 44) and residues 127-140 of a-synuclein (SEQ ID NO: 46).

24. The engineered nanobody of claim 22, wherein the bi-paratopic nanobody binds to epitopes comprising an N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and the C-terminal region corresponding to residues 127-140 of a-synuclein (SEQ ID NO: 46).

25. The engineered nanobody of claim 22, wherein the bi-paratopic nanobody binds to epitopes comprising the N-terminal region corresponding to residues 43-56 of a-synuclein (SEQ ID NO: 34) and the C-terminal region corresponding to residues 113-126 of a-synuclein (SEQ ID NO: 44).

26. The nanobody or engineered version thereof according to claim 12, wherein the nanobody or engineered version thereof is a conformation-specific nanobody.

27. A pharmaceutical composition comprising the nanobody or engineered version thereof according to any one of claims 12-26 and a pharmaceutically acceptable diluent or carrier.

28. The nanobody or engineered version thereof according to any one of claims 12-26, or the pharmaceutical composition according to claim 27, for use as a medicament.

29. A method for preventing or treating a neurodegenerative disorder with a-synuclein pathology in an individual comprising administration of the nanobody or engineered version thereof according to any one of claims 12-26 or the pharmaceutical composition according to claim 27, to the individual.

30. The method according to claim 29, wherein the neurodegenerative disorder with a-synuclein pathology is Parkinson’s disease, dementia with Lewy Bodies, multiple system atrophy, or Alzheimer’s disease.

31. A test kit for use in a method of determining whether or not an individual has a neurodegenerative disease, comprising the nanobody or engineered version thereof according to any one of claims 12-26.

32. A method of detecting a-synuclein fibrils and aggregates comprising the steps of: adding the nanobody or engineered version thereof according to any one of claims 12-26 to a biological sample; and detecting the presence of a complex formed between a-synuclein fibrils and / or aggregates and the nanobody or engineered version thereof.

33. A method for diagnosing a neurodegenerative disease associated with a-synuclein comprising: adding the nanobody or engineered version thereof according to any one of claims 12-26 to a biological sample; and detecting the presence or absence of a complex formed between a-synuclein aggregates and the nanobody or engineered version thereof.

34. The nanobody or engineered version thereof according to any one of claims 12-26, further comprising a detectable label, wherein the detectable label is selected from the group consisting of a fluorescent label, a radioactive label or a contrast agent.

35. The nanobody or engineered version thereof according to any one of claims 12-26 or claim 34, for use as an imaging agent of alpha-synuclein aggregates in brain, gut, heart, skin, or eye.

36. A method for imaging a-synuclein aggregates comprising: administering the nanobody or engineered version thereof according to any one of claims 12-26 or claim 34 to a subject; and detecting the nanobody or engineered version thereof.

37. The method according to claim 36, wherein the nanobody or engineered version thereof comprises a detectable label.

Images