Antibodies to alpha-synuclein and uses thereof

Abstract

To provide human α-synuclein antibodies, and methods for preventing or treating diseases associated with α-synuclein.SOLUTION: The invention provides: antibodies that specifically bind to human α-synuclein with high affinity and reduce α-synuclein spreading in vivo: recombinant polypeptides comprising the antibodies or antigen-binding fragments thereof, and methods for generating such polypeptides; compositions and methods for generating the α-synuclein antibodies; and methods of using the α-synuclein antibodies for treatment of diseases of a central nervous system, particularly α-synucleinopathies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Patent Application No. 62 / 344,746, filed June 2, 2016. The foregoing application is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on May 30, 2017, is named 1848081-0002-091-WO1_SL.txt, and is 48,099 bytes in size.

[0003] The present invention relates to alpha-synuclein antibodies and their use in the prevention or treatment of diseases, particularly alpha-synucleinopathies, and more particularly Parkinson's disease (PD). [Background technology]

[0004] Alpha-synucleinopathies, also known as Lewy body diseases (LBD), are a family of neurodegenerative diseases that all have alpha-synuclein at their core as a key pathological feature (Jellinger, Mov Disord (2003), 18 Suppl 6: S2-12; and Spillantini and Goedert, Ann NY Acad Sci (2000), 920: 16-27; both of which are incorporated herein by reference). Alpha-synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).

[0005] PD is a slowly progressive, age-related movement disorder that affects more than 1% of people over the age of 65. PD is the second most common neurodegenerative condition after Alzheimer's disease.

[0006] The defining lesions of α-synucleinopathies are Lewy bodies and Lewy neurites, which are insoluble inclusions of aggregated protein found within neurons in the brain that become apparent upon postmortem histopathological examination.

[0007] The presence of Lewy pathology and neurological deficits in non-motor areas of the brain, such as the basal forebrain, mesopontine system, amygdala, neocortex, dorsal motor nucleus of the vagus nerve, olfactory bulb, locus coeruleus, and brainstem, can cause cognitive impairment and dementia, olfactory disorders, sleep disorders such as REM sleep behavior disorder (RBD), mood disorders such as depression and anxiety, autonomic dysfunction such as cardiovascular and gastrointestinal problems (such as constipation), and fatigue and somnolence.Some of these non-motor symptoms appear to be characteristic of the premotor or prodromal stage of the disease (Kalia et al. Lancet (2015), 386(9996): 896-912; incorporated herein by reference).

[0008] Neuronal deficits in the motor cortex of the brain, including the presence of Lewy pathology and cell death of dopaminergic neurons, most notably in the substantia nigra, can lead to resting tremor, rigidity, bradykinesia, and postural instability (Spillantini and Goedert, Ann NY Acad Sci (2000), 920: 16-27; incorporated herein by reference).

[0009] Alpha-synuclein (also referred to as "α-synuclein" or "α-syn") protein is the major structural component of Lewy bodies and Lewy neurites. α-Synuclein is a small, acidic protein composed of up to 140 amino acids (14 kDa). Human native wild-type α-synuclein has the amino acid sequence of SEQ ID NO: 1, as set forth under UniProtKB accession number P37840. Unless otherwise clear from the context, reference to α-synuclein or a fragment thereof includes the above native human wild-type amino acid sequence and its human allelic variants, particularly those associated with Lewy body disease (e.g., E46K, A30P, H50Q, G51D, and A53T, where the first letter indicates the amino acid in SEQ ID NO: 1, the number is the codon position in SEQ ID NO: 1, and the second letter is the amino acid in the allelic variant). Such variants may optionally exist individually or in any combination. The induced mutations E83Q, A90V, and A76T that promote α-synuclein aggregation can also occur individually or in combination with each other and / or with the human allelic variants E46K, A30P, H50Q, G51D, and A53T. At the structural level, α-synuclein contains three distinct regions: an amphipathic N-terminal α-helical domain (with lipid and membrane-binding properties) (residues 1-60), a central hydrophobic amyloid-binding domain (encoding the non-amyloid β component (NAC) of plaques) (residues 61-95), and an acidic proline-rich C-terminal tail (residues 96-140). Residues 71-82 of the NAC domain are thought to be important for the aggregation / fibrillation properties of α-synuclein by enabling the protein to switch from a random coil to a β-sheet structure (Bisaglia et al. FASEB J (2009), 23(2): 329-40; incorporated herein by reference). The C-terminal domain does not have significant secondary structure but contains a key phosphorylation site at residue Ser129 and multiple tyrosine residues that are nitrated in cytoplasmic α-synuclein inclusions. N- and C-terminal truncated forms of α-synuclein also exist.Post-translational modifications to the protein can affect alpha-synuclein aggregation and toxicity (Oueslati et al. Prog Brain Res (2010), 183: 115-45; incorporated herein by reference).

[0010] Alpha-synuclein is abundant in the central nervous system (CNS) / brain, being found both intracellularly in neurons and glial cells, and also extracellularly in the cerebrospinal fluid (CSF) (Mollenhauer et al. J Neural Transm (2012), 119(7): 739-46; incorporated herein by reference) and in the interstitial fluid (ISF) that bathes and surrounds brain cells (Emmanouilidou et al. PLoS One (2011), 6(7): e22225; incorporated herein by reference). Alpha-synuclein is a synaptic protein that is predominantly expressed in neurons of the neocortex, hippocampus, substantia nigra, thalamus, and cerebellum (Iwai et al. Neuron (1995), 14: 467-475; incorporated herein by reference). Under physiological conditions, α-synuclein is localized to neuronal synaptic terminals and is specifically upregulated at presynaptic terminals during acquisition-related synaptic reorganization (Fortin et al. J Neurosci (2005), 25: 10913-10921; incorporated herein by reference).

[0011] In vitro studies have shown that α-synuclein monomers can form the starting point of the aggregation process. Monomers can aggregate into various small oligomeric species, which are subsequently stabilized by β-sheet interactions, leading to the formation of fibrils that can then multimerize into insoluble fibrillar structures similar to those identified in Lewy bodies (Cremades et al. Cell (2012), 149(5): 1048-59; incorporated herein by reference).

[0012] In pathological conditions, abnormal α-synuclein aggregation may be important for the pathological changes seen in α-synucleinopathies (Lashuel et al. Nature (2002), 418: 291; and Tsigelny et al. FEBS Journal (2007), 274: 1862-1877; both of which are incorporated herein by reference). In vitro and in vivo studies have shown that the neurotoxic effects of α-synuclein appear to be caused by small soluble oligomeric conformers or fibrils (Winner et al. Proc Natl Acad Sci USA (2011), 108(10): 4194-9; and Danzer et al. J Neurosci (2007), 27(34): 9220-32; both of which are incorporated herein by reference). While fibrillar aggregates of α-synuclein are a hallmark of PD, oligomeric forms of α-synuclein are the toxic molecular species (Danzer et al. J Neurosci (2007), 27(34): 9220-32; Lashuel et al. Nature (2002), 418: 291; and Winner et al. Proc Natl Acad Sci USA (2011), 108: 4194-4199; each of which is incorporated herein by reference).

[0013] Alpha-synuclein oligomers can be released into the extracellular environment and taken up by neighboring cells by a "propagation" mechanism (Angot and Brundin, Parkinsonism Relat Disord (2009), 15 Suppl 3: S143-147; Desplats et al. Proc Natl Acad Sci USA (2009), 106: 13010-13015; and Lee et al. J Biol Chem (2010), 285: 9262-9272; each of which is incorporated herein by reference). α-synuclein aggregates can propagate misfolding through a prion-like spreading mechanism (Lee et al. Nat Rev Neurol (2010), 6: 702-706; Luk et al. J Exp Med (2012), 209(5): 975-86; and Luk et al. Science (2012), 338(6109): 949-53; each of which is incorporated herein by reference). Thus, α-synuclein can cause neurodegeneration either through oligomeric toxicity or propagation and prion-like spreading.

[0014] It is now well established and accepted that cells, including neurons, can secrete various forms of α-synuclein (monomers, oligomers, aggregates) under normal conditions and under conditions of cellular stress, that secretion of monomeric and aggregated α-synuclein is elevated under conditions of cellular stress, and that through this secretion of α-synuclein into the extracellular environment, lesion-propagating forms of α-synuclein can be propagated between neurons (Recasens and Dehay, Front Neuroanat (2014), 8: 159; incorporated herein by reference).

[0015] The effect of α-synuclein in PD can extend beyond immediate damage to vulnerable neurons. As with most neurodegenerative diseases, pro-inflammatory cellular responses have also been observed (Lee et al. J Biol Chem (2010), 285: 9262-9272; incorporated herein by reference). Circulating α-synuclein and / or activated astrocytes can activate microglia, which leads to increased production of reactive oxygen species, nitric oxide and cytokine production, and further to the aggravation of neurodegeneration (Lee et al. J Biol Chem (2010), 285: 9262-9272; incorporated herein by reference).

[0016] A variety of different experimental models have demonstrated the intercellular transmission of α-synuclein in cultured cells, or the in vivo spread and propagation of α-synuclein pathology. Lewy body pathology has been observed in embryonic midbrain nerve grafts more than 10 years after the grafts were therapeutically transplanted into the striatum of PD patients. Specifically, the transplanted neurons contained numerous Lewy body-like inclusions that stained positively for α-synuclein, indicating that α-synuclein pathology had been transmitted from the host to the graft (Li et al. Nat Med (2008), 14(5): 501-3; and Kordower et al. Nat Med (2008), 14(5): 504-6; both of which are incorporated herein by reference).

[0017] Furthermore, preformed recombinant α-synuclein fibers and α-synuclein oligomers can be internalized by cultured cells and neurons, and direct transfer of α-synuclein from donor to recipient cells has been demonstrated, accompanied by the formation of α-synuclein inclusions resembling Lewy pathologies (Danzer et al. J Neurosci (2007), 27(34): 9220-32; Volpicelli-Daley et al. Neuron (2011), 72(1): 57-71; and Luk et al. Proc Natl Acad Sci USA (2009), 106(47): 20051-6; each of which is incorporated herein by reference). Injection of preformed synthetic α-synuclein fibrils or Lewy body-like α-synuclein-containing material extracted from the brains of aged α-synuclein transgenic mice into the brains of asymptomatic recipient mice promotes the formation of Lewy body-like lesions in host neurons of the recipient animals, accompanied by neurodegeneration and neurological damage (Luk et al. J Exp Med (2012), 209(5): 975-86; and Luk et al. Science (2012), 338(6109): 949-53; both of which are incorporated herein by reference). Alpha-synuclein-containing Lewy body extracts isolated from PD brains inoculated into the substantia nigra or striatum of macaques and mice are rapidly taken up by host cells (within 24 hours), followed by a relatively slow loss of striatal dopaminergic terminals, with cell loss becoming evident after more than a year (Recasens et al. Ann Neurol (2014), 75(3): 351-62; incorporated herein by reference).Similarly, inoculation of mice with brain homogenates from patients with the synucleinopathies DLB or MSA results in α-synuclein Lewy-like pathology in the host mice (Watts et al. Proc Natl Acad Sci USA (2013), 110(48): 19555-60; and Masuda-Suzukake et al. Brain (2013), 136(Pt 4): 1128-38; both of which are incorporated herein by reference). Finally, the movement and transmission of both monomeric and oligomeric α-synuclein from the olfactory bulb to interconnected brain structures has been demonstrated in mice (Rey et al. Acta Neuropathol (2013), 126(4): 555-73; incorporated herein by reference).

[0018] Passive immunotherapy approaches using antibodies targeting α-synuclein have been tested in numerous preclinical α-synucleinopathy mouse models (Lawand et al. Expert Opin Ther Targets (2015): 1-10; incorporated herein by reference). Specifically, studies using a monoclonal antibody against α-synuclein (9E4) have shown clearance of α-synuclein aggregates and pathology in vivo, behavioral motor improvement, and neuroprotection (WO 2014 / 058924; incorporated herein by reference).

[0019] Further studies using passive immunization of α-synuclein transgenic mice, developed as an experimental model of PD / DLB, with the 9E4 monoclonal antibody have shown that the antibody abolishes α-synuclein pathology, reduces synaptic and axonal defects, reverses the loss of striatal tyrosine hydroxylase fibrils, and significantly reduces memory and motor function deficits (Games et al. J Neurosci (2014), 34(28): 9441-54; Bae et al. J Neurosci (2012), 32(39): 13454-69; and Masliah et al. PLoS One (2011), 6(4): e19338; each of which is incorporated herein by reference). Furthermore, passive administration of anti-α-synuclein monoclonal antibodies in wild-type mice injected intrastriatally with synthetic α-synuclein preformed fibrils (pff) resulted in a robust reduction of Lewy pathology, prevention of dopamine neuron loss in the substantia nigra, and a marked improvement in the motor deficits that are hallmarks of pff-treated mouse models (Tran et al. Cell Rep (2014), 7(6): 2054-65; incorporated herein by reference).

[0020] In addition, one of the major concerns associated with treating CNS disorders with macromolecular therapeutics, such as antibodies, is getting these drugs to the affected tissues. The passage of macromolecules into the brain and spinal cord is largely restricted by the blood-brain barrier (BBB). The BBB protects the brain, regulates brain homeostasis, and prevents free passage of molecules into large parts of the brain, thereby limiting the treatment of many brain diseases. Transport of essential molecules, such as nutrients, growth factors, and hormones, is achieved through a series of specific transporters and receptors that regulate their passage through brain endothelial cells. Therefore, delivery of biologics and other drugs to the brain presents significant challenges. Additionally, transport mechanisms likely exist to rapidly remove antibodies from the brain, presumably to prevent inflammatory responses resulting from Fc engagement with effector ligands that promote proinflammatory responses.

[0021] Over the past decade, there have been reports of antibody transport across the BBB, in which binding to the extracellular domain of transporter molecules facilitates transcytosis of receptor-antibody complexes across the endothelial cell layer.

[0022] The BBB is primarily composed of brain capillary endothelial cells, which have special features such as tight junctions that limit the transport of molecules into the brain (Reese et al. 1967, J. Cell Biol. 34: 207-217; Brightman et al. 1969, J. Cell Biol. 40: 648-677; Rubin et al. 1999, Ann. Rev. Neurosci. 22: 11-28). However, other cell types, such as pericytes, astrocytes, and neurons, also play important roles in BBB function. Typically, less than 0.1% of peripherally administered antibodies reach the brain (Boado et al. 2010, Mol. Pharm. 7: 237-244; Pepinsky et al. 2011, Nat. Neurosci. 8: 745-751). The BBB functions as a physiological, metabolic and immunological barrier (Gaillard et al. 2003, Microvasc. Res. 65: 24-31).

[0023] Antibody transport across the BBB can be enhanced by triggering receptor-mediated transcytosis in brain endothelial cells. Through this process, engagement of an antigen on the luminal side of the endothelial cell can induce internalization and shuttling of the antibody across the cell, followed by its release into the tissue. [Prior art documents] [Patent documents]

[0024] [Patent Document 1] International Publication No. 2014 / 058924 Brochure [Non-patent literature]

[0025] [Non-licensed document 1] Jellinger, Mov Disord (2003), 18 Suppl 6: S2-12 [Non-licensed document 2] Spillantini and Goedert, Ann NY Acad Sci (2000), 920: 16-27 [Non-licensed document 3] Kalia et al. Lancet (2015), 386(9996): 896-912

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

Non-licensed Document 25

Non-licensed Document 26

Non-licensed Document 27

Non-licensed Document 28

Non-licensed Document 29

Non-licensed Document 30

Non-licensed Document 31

Non-licensed Document 32

Non-licensed Document 33

Non-licensed Document 34

Non-licensed Document 35

[0026] Current drug therapies for PD focus primarily on treating the movement-related symptoms of the disease. There are currently no commercially available or available medications that can treat or prevent α-synucleinopathy. Thus, there is a need in the art for therapies to treat alpha-synucleinopathies, particularly in humans. [Means for solving the problem]

[0027] The present invention relates to isolated antibodies against human alpha-synuclein. The present invention provides antibodies or antigen-binding fragments thereof that have one or more of the functional properties of the aslo0452 ngl-3 antibody, such as:

[0028] The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to the C-terminal region of human alpha-synuclein. The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to a region comprised between about amino acid 102 and about amino acid 130 of human alpha-synuclein (e.g., SEQ ID NO: 1). In some embodiments, the antibodies or antigen-binding fragments thereof specifically bind to a region comprised between about amino acid 120 and about amino acid 130 of human alpha-synuclein (SEQ ID NO: 1). In some embodiments, the antibodies or antigen-binding fragments thereof bind to an epitope that is not the same as that bound by the 9E4 antibody.

[0029] The present invention provides antibodies or antigen-binding fragments thereof that bind to human α-synuclein but not to human β-synuclein or human γ-synuclein. The present invention provides antibodies or antigen-binding fragments thereof that bind to human, rat, and cynomolgus monkey α-synuclein.

[0030] The present invention provides antibodies or antigen-binding fragments thereof that bind to human alpha-synuclein with high affinity. In one embodiment, the antibodies or antigen-binding fragments thereof have a K of less than 500 picomolar (pM), less than 400 pM, less than 300 pM, less than 200 pM, less than 150 pM, less than 120 pM, less than 110 pM, or less than 10 pM, as measured, for example, using Octet analysis. D In one embodiment, an antibody of the invention, or antigen-binding fragment thereof, binds to alpha-synuclein with a K of less than 400 picomolar (pM), less than 300 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 120 pM, less than 110 pM, less than 100 pM, less than 80 pM, or less than 74 pM, as measured, for example, using KinExA analysis. D It binds to α-synuclein.

[0031] The present invention provides antibodies or antigen-binding fragments thereof that bind to native endogenous human alpha-synuclein. The present invention provides antibodies or antigen-binding fragments thereof that bind to the monomeric form of human alpha-synuclein. The present invention provides an antibody or an antigen-binding fragment thereof that binds to aggregates of human α-synuclein. The present invention provides antibodies or antigen-binding fragments thereof that bind to disease-associated, pathological forms of alpha-synuclein.

[0032] The present invention provides an antibody or antigen-binding fragment thereof that reduces α-synuclein levels in brain interstitial fluid, particularly, an antibody or antigen-binding fragment thereof that reduces free, unbound α-synuclein levels in brain interstitial fluid.

[0033] The present invention provides an antibody or antigen-binding fragment thereof that reduces α-synuclein levels in cerebrospinal fluid, particularly, an antibody or antigen-binding fragment thereof that reduces free, unbound α-synuclein levels in cerebrospinal fluid.

[0034] As used herein, the term "free, unbound α-synuclein" refers to α-synuclein that is not bound to an antibody or antigen-binding fragment thereof of the present invention. The term can apply to α-synuclein in its monomeric or oligomeric form, or in aggregated form. The term applies broadly to any pathological form of α-synuclein.

[0035] The present invention provides antibodies or antigen-binding fragments thereof that reduce alpha-synuclein diffusion in vivo. In one embodiment, the antibody or antigen-binding fragment thereof of the invention competes with antibody aslo0452 ngl-3 for binding to human alpha-synuclein. In one embodiment, the antibody or antigen-binding fragment thereof of the invention binds to the same epitope on human alpha-synuclein as the antibody aslo0452 ngl-3.

[0036] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is derived from antibody asyn0087, which comprises a variable heavy chain region (VH) of the amino acid sequence of SEQ ID NO: 2 and a variable light chain region (VL) of the amino acid sequence of SEQ ID NO: 3, as disclosed herein.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is derived from the antibody asyn0087, wherein the antibody or antigen-binding fragment has a K of less than 500 mM. D and binds to the same epitope as any one of antibodies asyn0087, aslo0452 ngl-3 and aslo0543 described herein.

[0038] As used herein, "H-CDR" refers to the complementarity-determining region (CDR) of the heavy chain region of an antibody or an antigen-binding fragment thereof, and "L-CDR" refers to the complementarity-determining region (CDR) of the light chain region. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises at least one CDR selected from the following: (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 6; (iii) H-CDR3 of SEQ ID NO: 7; (iv) L-CDR1 of SEQ ID NO: 9; (v) L-CDR2 of SEQ ID NO: 10; (vi) L-CDR3 of SEQ ID NO: 11.

[0039] In a further embodiment, the CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3 as set forth in SEQ ID NO: 16; and / or the CDR3 of the light chain of the antibody or antigen-binding fragment thereof of the present invention is the CDR3 of the light chain of the antibody aslo0452 ngl-3 as set forth in SEQ ID NO: 21.

[0040] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has at least one, at least two, at least three, at least four, at least five, or all of the CDRs selected from the CDRs of antibody aslo0452 ngl-3, i.e., at least one CDR selected from any one of SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 10, and SEQ ID NO: 21.

[0041] In one embodiment, the CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3; and / or the CDR3 of the light chain of the antibody or antigen-binding fragment thereof is the CDR3 of the light chain of the antibody aslo0452 ngl-3. In one embodiment, the CDR3 of the heavy chain of an antibody or antigen-binding fragment thereof of the invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3. In one embodiment, the CDR3 of the light chain of an antibody or antigen-binding fragment thereof of the invention is the CDR3 of the light chain of the antibody aslo0452 ngl-3.

[0042] In one embodiment, the CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3, and the CDR3 of the light chain of the antibody or antigen-binding fragment thereof is the CDR3 of the light chain of the antibody aslo0452 ngl-3. The present invention provides an antibody or an antigen-binding fragment thereof having six CDRs of the antibody aslo0452 ngl-3.

[0043] Thus, in one embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises: (a) three heavy chain CDRs having the following sequences: (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 15; and (iii) H-CDR3 of SEQ ID NO: 16, and (b) three light chain CDRs having the following sequences: (i) L-CDR1 of SEQ ID NO: 20; (ii) L-CDR2 of SEQ ID NO: 10, and (iii) L-CDR3 of SEQ ID NO: 21.

[0044] The present invention provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence defined by SEQ ID NO:13, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence defined by SEQ ID NO:18.

[0045] The present invention provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence defined by SEQ ID NO: 14, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence defined by SEQ ID NO: 19.

[0046] The present invention provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having the amino acid sequence of SEQ ID NO:14, and a variable light chain having the amino acid sequence of SEQ ID NO:19.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having the sequence defined by SEQ ID NO:4 and a variable light chain having the sequence defined by SEQ ID NO:8.

[0048] In a further specific embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having the sequence defined by SEQ ID NO: 4 and a variable light chain having the sequence defined by SEQ ID NO: 8, and has a K DIt binds to human α-synuclein at 100 kJ / s and binds to the same epitope as asyn0087, aslo0452 ngl-3 or aslo0543.

[0049] In another embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 14, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 19.

[0050] In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having the sequence defined by SEQ ID NO:14 and a variable light chain having the sequence defined by SEQ ID NO:19.

[0051] In a further embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 14 and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 19, and further comprises: (a) three heavy chain CDRs having the following sequences: (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 15; and (iii) H-CDR3 of SEQ ID NO: 16, and (b) three light chain CDRs having the following sequences: (i) L-CDR1 of SEQ ID NO: 20; (ii) L-CDR2 of SEQ ID NO: 10, and (iii) L-CDR3 of SEQ ID NO: 21.

[0052] The present invention also provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain having the nucleotide sequence defined by SEQ ID NO:13 and a variable light chain having the nucleotide sequence defined by SEQ ID NO:18.

[0053] The present invention provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain having the amino acid sequence defined by SEQ ID NO:14 and a variable light chain having the amino acid sequence defined by SEQ ID NO:19.

[0054] Also provided is an antibody or antigen-binding fragment thereof of the present invention, comprising a heavy chain having the amino acid sequence defined by SEQ ID NO:12 and a light chain having the amino acid sequence defined by SEQ ID NO:17.

[0055] In another embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO:24, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO:30.

[0056] In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having the sequence defined by SEQ ID NO:24 and a variable light chain having the sequence defined by SEQ ID NO:30.

[0057] In a further embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 24, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 30, and further comprising: (c) three heavy chain CDRs having the following sequences: (iv) H-CDR1 of SEQ ID NO: 25; (v) H-CDR2 of SEQ ID NO: 26; and (vi) H-CDR3 of SEQ ID NO: 27, and (d) three light chain CDRs having the following sequences: (iv) L-CDR1 of SEQ ID NO: 31; (v) L-CDR2 of SEQ ID NO: 32; and (vi) L-CDR3 of sequence number 33.

[0058] The present invention also provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain having the nucleotide sequence defined by SEQ ID NO:24, and a variable light chain having the nucleotide sequence defined by SEQ ID NO:30.

[0059] The present invention provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain having the amino acid sequence defined by SEQ ID NO:24, and a variable light chain having the amino acid sequence defined by SEQ ID NO:30.

[0060] Also provided is an antibody or antigen-binding fragment thereof of the present invention, comprising a heavy chain having the amino acid sequence defined by SEQ ID NO:22 and a light chain having the amino acid sequence defined by SEQ ID NO:28.

[0061] In one embodiment, the antibody or antigen-binding fragment thereof of the invention as defined anywhere above is an antibody or antigen-binding fragment thereof, such as an IgA, IgD, IgE, IgM, IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0062] In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention have a modified Fc region. Suitable modifications are well known to those skilled in the art and can include, among others, modifications to increase or decrease half-life, eliminate, reduce, or enhance effector function, or provide a substituted cysteine ​​with a free thiol for conjugation. Examples of such modifications are YTE to increase half-life and / or TM to decrease effector function. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein contain the mutations M252Y / S254T / T256E (YTE) in the Fc region of the antibody (Dall'Acqua et al., 2006, J. Biol. Chem, 281:23514-23524). In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein comprises a triple mutation (abbreviated herein as "TM") in the Fc region corresponding to the L234F / L235E / P331S mutations disclosed in Oganesyan et al. (Acta Crystallogr D Biol Crystallogr, (2008) 64: 700-704). In one embodiment, an antibody or antigen-binding fragment thereof of the present invention may be an IgG1 TM antibody or antigen-binding fragment thereof. In another embodiment, an antibody or antigen-binding fragment thereof of the present invention may comprise an Fc region with a YTE mutation.

[0063] In another embodiment, an antibody or antigen-binding fragment thereof of the present invention can be coupled to a blood-brain barrier (BBB) ​​transporter moiety, wherein the BBB transporter moiety is capable of transporting the antibody or antigen-binding fragment thereof across the BBB.

[0064] In one embodiment, the BBB transporter moiety can be an antibody, which can form a multispecific construct with an anti-α-synuclein antibody or antigen-binding fragment thereof. The BBB transporter portion can comprise immunoglobulin variable heavy chain complementarity determining region-1 (VH-CDR1), immunoglobulin variable heavy chain complementarity determining region-2 (VH-CDR2), immunoglobulin variable heavy chain complementarity determining region-3 (VH-CDR3), immunoglobulin variable light chain complementarity determining region-1 (VL-CDR1), immunoglobulin variable light chain complementarity determining region-2 (VL-CDR2), and immunoglobulin variable light chain complementarity determining region-3 (VL-CDR3); wherein VH-CDR1 comprises SEQ ID NO: 40 or 49, VH-CDR2 comprises SEQ ID NO: 41 or 50, VH-CDR3 comprises SEQ ID NO: 42 or 51, VL-CDR1 comprises SEQ ID NO: 36, 44, or 53, VL-CDR2 comprises SEQ ID NO: 37, 45, or 54, and VL-CDR3 comprises SEQ ID NO: 38, 46, or 55.

[0065] In some embodiments, the transporter portion comprises an immunoglobulin variable heavy chain (VH) region comprising SEQ ID NO: 47 or SEQ ID NO: 39. In some embodiments, the transporter portion comprises an immunoglobulin variable light chain (VL) region comprising SEQ ID NO: 43.

[0066] In addition, the transporter moiety can be selected from a complete antibody, an Fv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a disulfide-linked (dsFv) fragment, a single-chain Fv (scFv) fragment, a sc(Fv)2 fragment, a diabody, a triabody, a tetrabody, a minibody, and a single-chain antibody. In certain embodiments, the transporter moiety comprises an scFv fragment comprising a VH domain and a VL domain fused via a linker. In some examples, the linker is (Gly4Ser) n (SEQ ID NO: 56), where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0067] In some embodiments, any transporter molecule of the invention can be combined with any alpha-synuclein binding molecule of the invention as described herein to provide a multispecific binding molecule of the invention. The present invention provides an antibody or antigen-binding fragment thereof of the present invention as defined anywhere above for use as a medicament.

[0068] The present invention also provides an antibody or antigen-binding fragment thereof of the invention as defined anywhere above for use in the prevention or treatment of an alpha-synucleinopathy. In one embodiment, the alpha-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). In one embodiment, the alpha-synucleinopathy is Parkinson's disease (PD).

[0069] The present invention provides a method of treating or preventing a disease, particularly a disease associated with the central nervous system, in a patient, the method comprising the step of administering to the patient an antibody or antigen-binding fragment thereof of the invention as defined anywhere above. In one embodiment, the disease is an alpha-synucleinopathy. In one embodiment, the alpha-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). In one embodiment, the alpha-synucleinopathy is Parkinson's disease (PD). The present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention as defined anywhere above, and a pharmaceutically acceptable excipient.

[0070] The phrase "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition can also contain other active compounds that provide supplementary, additional, or enhanced therapeutic functions. The pharmaceutical composition can also be included in a container, pack, or dispenser together with instructions for administration.

[0071] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods for accomplishing administration are known to those skilled in the art. Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. The present invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention as defined anywhere above. In certain embodiments, the present invention provides an isolated nucleic acid molecule comprising SEQ ID NO:13 and / or SEQ ID NO:18. In another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising SEQ ID NO:23 and / or SEQ ID NO:29.

[0072] Armed with this information, one of skill in the art could readily obtain nucleic acid molecules encoding the disclosed antibodies or antigen-binding fragments thereof. Nucleic acids can comprise DNA or RNA, and can be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence encompasses DNA molecules containing the specified sequence, unless the context requires otherwise, and also encompasses RNA molecules containing the specified sequence in which U replaces T. The nucleic acid molecules of the invention comprise coding sequences for the CDRs, VH domains, and / or VL domains disclosed herein.

[0073] The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes comprising at least one nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention as defined anywhere above, in particular encoding the CDRs, VH domains, and / or VL domains disclosed herein. The present disclosure further provides a host cell comprising one or more constructs as described above.

[0074] Also provided are nucleic acids encoding any one or more CDRs (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, or L-CDR3), VH or VL domains disclosed herein, and methods for producing the encoded products. The methods include expressing the encoded product from the encoding nucleic acid. Expression may be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, the VH or VL domain, or specific binding member, may be isolated and / or purified using any suitable technique and then used as appropriate.

[0075] Antigen-binding fragments, VH and / or VL domains and encoding nucleic acid molecules and vectors can be isolated and / or purified from their natural environment in substantially pure or homogeneous form, or, in the case of nucleic acids, free or substantially free of nucleic acids or genes of other origin than the sequence encoding a polypeptide having the required function.

[0076] Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. For cells suitable for producing antibodies, see Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999. Briefly, suitable host cells include bacteria, plant cells, mammalian cells, as well as yeast and baculovirus systems. Mammalian cell lines available in the art for expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cells, and many others. A common bacterial host is Escherichia coli. Any protein expression system compatible with the present invention can be used to produce the disclosed antibodies. Suitable expression systems include the transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0077] Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences, as appropriate. Vectors can be plasmid or viral (e.g., phage or phagemid) as appropriate. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. For example, numerous known techniques and protocols for creating nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and manipulating nucleic acids for gene expression, and analyzing proteins are described in detail in Current Protocols in Molecular Biology, 2nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992.

[0078] A further aspect of the present disclosure provides a host cell comprising a vector comprising a nucleic acid as disclosed herein, in particular a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention as defined anywhere above.

[0079] A still further aspect provides a method comprising introducing such a nucleic acid into a host cell. Introduction can utilize any available technique. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia virus, or, for insect cells, baculovirus). For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. Introduction of the nucleic acid into the cell can be followed by a step of causing or allowing expression from the nucleic acid (e.g., by culturing the host cells under conditions for expression of the gene).

[0080] BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING The present invention will now be described in more detail with reference to the accompanying drawings and sequence listing, in which: JPEG0007823118000001.jpg247129JPEG0007823118000002.jpg181129 Table 1: Affinity measurements of leading anti-α-synuclein antibodies against human α-syn performed on two affinity measurement platforms. [Table 1] [Brief explanation of the drawings]

[0081] [Figure 1] FIG. 1 is a schematic diagram of an HTRF® assay. [Figure 2]

[0023] Figure 1 shows a comparison of the amino acid sequences of the asyn0087, aslo0452ngl-3, and aslo0543 heavy chain variable regions (VH) (SEQ ID NOs: 2, 14, and 24, respectively) and light chain variable regions (VL) (SEQ ID NOs: 3, 19, and 30, respectively). The underlined amino acids correspond to the CDRs. [Figure 3A]Figures 3A and 3B show the nucleotide and amino acid sequences of the variable heavy and variable light chains, respectively, of aslo0452 ngl-3. Figure 3A discloses SEQ ID NOs: 13 and 14, respectively, in order of appearance. [Figure 3B] Figures 3A and 3B show the nucleotide and amino acid sequences of the variable heavy and variable light chains, respectively, of aslo0452 ngl-3. Figure 3B discloses SEQ ID NOs: 18 and 19, respectively, in order of appearance. [Figure 3C] Figures 3C and 3D show the nucleotide and amino acid sequences of aslo0452 ngl-3. Figures 3C and 3D show alignments of these sequences to the closest human germline sequences. Figure 3C shows alignments of the aslo0452 ngl-3 variable heavy chain domain amino acid sequence (SEQ ID NO: 14) with the germline IGHV3-23 (SEQ ID NO: 58) and JH6 sequences (SEQ ID NO: 59). Complementarity-determining regions (CDRs) are underlined and labeled. Differences from the germline are highlighted in bold and outlined. All non-Vernier residues in the light chain framework regions are human germline amino acids. Vernier residues (*) were not changed to match the germline amino acid. [Figure 3D] Figures 3C and 3D show the nucleotide and amino acid sequences of aslo0452 ngl-3. Figures 3C and 3D show alignments of these sequences to the closest human germline sequence. Figure 3D shows a triple sequence alignment of the aslo0452 ngl-3 variable light chain domain amino acid sequence (SEQ ID NO: 19) with germline IGLV5-45 (SEQ ID NO: 60) and JL2 (SEQ ID NO: 61). Complementarity-determining regions (CDRs) are underlined and labeled. Differences from the germline are highlighted in bold and outlined. All non-Vernier residues in the light chain framework regions are human germline amino acids. Vernier residues (*) were not altered to match the germline amino acid. [Figure 4] Figure 4 shows epitope binding of lead isolate clones using a panel of α-syn truncations. ELISA wells were coated with various commercially available α-syn truncations representing various defined regions of the protein: 1-140: full-length α-syn, 1-60: N-terminal region only, 61-140: non-amyloid component of plaque (NAC) + C-terminal region, 1-95: N-terminal and NAC regions, 96-140: C-terminal region only, ΔNAC: NAC region deletion, NCAP: alternatively spliced ​​form of α-syn lacking amino acids 103-129 (rPeptide). The primary antibodies used for detection were (Figure 4A) Asyn087; and (Figure 4B) Aslo0452 ngl-3 (black bars), aslo0543 (light gray bars), and NIP228 isotype-matched control (dark gray bars). Binding is detected using either an anti-human IgG Eu3+ secondary antibody (Figure 4A) or an anti-human IgG-HRP secondary antibody (Figure 4B). [Figure 5] Figure 1 shows the specificity of aslo0452 ngl-3 and aslo0543 for α-syn relative to other synuclein family members using a DELFIA epitope competition assay. Using an epitope competition HTRF assay, the specificity of affinity-optimized aslo0452 ngl-3 and aslo0543 clones for α-syn was measured by titration of unlabeled α-syn, β-syn, and γ-syn. IC50 values ​​were determined. [Figure 6] Figure 1 shows the specificity of aslo0452 ngl-3 and aslo0543 for human, cynomolgus monkey, and rat α-syn using HTRF epitope competition assays. Using epitope competition HTRF assays, the species cross-reactivity profiles of affinity-optimized clones were determined in similar assays by titrating unlabeled α-syn and deriving IC50 values ​​for α-syn in each species. [Figure 7A]Representative flow cytometry results demonstrate that affinity-optimized clones bind to native human α-syn in human neuroblastoma cell lines. Panels A, C, E, and G of Figures 7A and 7B show binding to BT20, an α-syn-negative human breast cancer cell line. Panels B, D, F, and H of Figures 7A and 7B show binding to SHSY5Y, an α-syn-positive human neuroblastoma cell line. Figure 7A: The primary human antibody used in this study was asyn0087 and Hu IgG control. Human antibody binding was detected using a secondary anti-human IgG-FITC (Jackson). [Figure 7B] Representative flow cytometry results demonstrate that affinity-optimized clones bind to native human α-syn in human neuroblastoma cell lines. Panels A, C, E, and G of Figures 7A and 7B show binding to BT20, an α-syn-negative human breast cancer cell line. Panels B, D, F, and H of Figures 7A and 7B show binding to SHSY5Y, an α-syn-positive human neuroblastoma cell line. Figure 7B: Primary human antibodies used in this study were aslo0452 ngl-3, aslo0543, and the NIP228 isotype-matched IgG1™ control. Human antibody binding was detected using a secondary anti-human IgG-FITC (Jackson). Primary mouse antibodies used were 4D6 (Covance) and an isotype-matched negative control (R&D Systems). Mouse antibody binding was detected using a secondary anti-mouse IgG-FITC (Sigma). [Figure 8] Figure 1 shows the specificity of optimized anti-α-syn IgG for aggregated human α-syn by DELFIA ELISA. The graph shows that two high-affinity α-syn-specific clones, aslo0452 ngl-3 and aslo0543, and the lead antibody asyn0087, detected captured aggregated α-syn (black bars), but not captured monomeric α-syn. [Figure 9-1]Figures 9A, 9B, and 9C show the specificity of affinity-optimized clones in disease-relevant tissues by immunohistochemistry. Figures 9A, 9B, and 9C show staining with aslo0452 ngl-3, asyn0087, and aslo0543, respectively. Panels A, B, and C show that aslo0452 ngl-3 stains both Lewy bodies (panels A and B) and Lewy neurites (panel C) in the substantia nigra of PD brain tissue. Panel D shows that aslo0452 ngl-3 exhibits low-level staining of α-syn in cells derived from the temporal cortex in normal brain sections. Panels E, F, and G show that aslo0452 ngl-3 stains Lewy bodies, Lewy neurites, and Lewy dots in the amygdala of PD brain tissue. Panel H shows that an isotype-matched control antibody shows no staining in the amygdala of PD brain tissue. [Figure 9-2] Immunohistochemistry demonstrates the specificity of affinity-optimized clones in disease-relevant tissues. Figures 9A, 9B, and 9C show staining with aslo0452 ngl-3, asyn0087, and aslo0543, respectively. Panels I–M show asyn0087 staining of the locus coeruleus in PD brain tissue; pathological features identified are Lewy bodies (panels I and L), neuronal aggregates (panel J), Lewy neurites (panel K), and pale bodies (panel M). Panels N and O show aslo0543 staining of Lewy bodies and Lewy neurites in the substantia nigra in PD brain tissue. Panel P shows low-level staining of α-syn in cells derived from the temporal cortex in normal brain sections with aslo0543. [Figure 10] Figure 10 shows that systemic administration of aslo0452 ngl-3 rapidly reduces free asyn levels in the rat prefrontal cortex. Absolute (Figure 10A) or relative (Figure 10B) mean ± SEM free α-synuclein concentrations in the ISF of rats treated with aslo0452 ngl-3 (30 mg / kg i.v.; closed circles) or vehicle (open circles). [Figure 11]Figure 11 shows that aslo0452 ngl-3 reduces free asyn levels in the CSF of rats upon systemic administration in a dose- and time-dependent manner. Absolute (Figure 11A) or relative (Figure 11B) mean ± SEM free α-synuclein concentrations in the CSF of rats treated with aslo0452 ngl-3 (3, 10, 30, 100 mg / kg i.v.; filled circles) or vehicle (open circles). [Figure 12] Figure 12A shows that the anti-α-synuclein antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A block α-synuclein diffusion from the ipsilateral to the contralateral side. (Figure 12A) Non-tg mice (black arrows) injected with LV-α-syn into the right hippocampus were passively immunized with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or the NIP228 isotype control antibody once weekly for 13 weeks. α-synuclein diffusion was subsequently measured by immunocytochemistry using SYN-1 and automated image analysis. (Figure 12B) Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of ipsilateral hippocampal coronal sections shown in (A). Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 12C: Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of contralateral coronal hippocampal sections represented in panel A. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. [Figure 13]Figure 13A shows that the anti-α-synuclein antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A reduce the deposition and dissemination of lentivirus-mediated expressed α-synuclein along axons. (Figure 13A) Non-tg mice injected with LV-α-syn into the right hippocampus were passively immunized with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or the NIP228 isotype control antibody once weekly for 13 weeks, followed by immunocytochemical analysis of α-synuclein deposition (black arrows) along ipsilateral and contralateral interhippocampal axons. (Figure 13B) Quantification of α-synuclein deposition in ipsilateral axons measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 13C: Quantification of α-synuclein deposition in contralateral axons as measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. [Figure 14]Figure 14A shows that the anti-α-synuclein antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A reduce α-synuclein deposition in CA1 hippocampal neurons and layer V neocortical neurons. (Figure 14A) Non-tg mice injected with LV-α-syn into the right hippocampus were passively immunized with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or the NIP228 isotype control antibody once weekly for 13 weeks, followed by immunocytochemical analysis of α-synuclein deposition (black arrows) in ipsilateral CA1 hippocampal neurons and ipsilateral layer V neocortical neurons. (Figure 14B) Quantification of α-synuclein deposition in ipsilateral layer V neocortical neurons measured by immunocytochemistry using SYN-1 and automated image analysis. Data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm2. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs. NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 14C: Quantification of α-synuclein deposition in ipsilateral CA1 hippocampal neurons measured by immunocytochemistry using SYN-1 and automated image analysis. Data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm2. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs. NIP228; one-way ANOVA with Dunnett's post-hoc test. [Figure 15]Figure 15 shows that the aslo0452 ngl-3 and aslo0452 ngl-3-D265A antibodies block α-synuclein diffusion in α-synuclein transgenic mice. Figure 15A: α-syn tg mice (black arrows) injected with LV-α-syn into the right hippocampus were passively immunized with the anti-α-synuclein mouse IgG1 antibody aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or the NIP228 isotype control antibody once weekly for 13 weeks, followed by measurement of α-synuclein diffusion by immunocytochemistry using SYN-1 and automated image analysis. Figure 15B: Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of ipsilateral hippocampal coronal sections depicted in panel A. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 15C: Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of contralateral coronal hippocampal sections represented in panel A. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. [Figure 16]Figure 16 shows that the aslo0452 ngl-3 and aslo0452 ngl-3-D265A antibodies reduce the deposition and dissemination of lentiviral-mediated expressed α-synuclein along axons in transgenic mice. (Figure 16A) α-syn tg mice injected with LV-α-syn into the right hippocampus were passively immunized with the anti-α-synuclein mouse IgG1 antibody aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or the NIP228 isotype control antibody once weekly for 13 weeks, followed by immunocytochemical analysis of α-synuclein deposition (black arrows) along ipsilateral and contralateral interhippocampal axons. (Figure 16B) Quantification of α-synuclein deposition in ipsilateral axons measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 16C: Quantification of α-synuclein deposition in contralateral axons as measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs NIP228; one-way ANOVA with Dunnett's post-hoc test. [Figure 17]Figure 17A: The aslo0452 ngl-3 and aslo0452 ngl-3-D265A antibodies reduce α-synuclein deposition in CA1 hippocampal and layer V neocortical neurons of α-synuclein transgenic mice. (Figure 17A) α-syn tg mice injected with LV-α-syn into the right hippocampus were passively immunized with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or the NIP228 isotype control antibody once weekly for 13 weeks, followed by immunocytochemical analysis of α-synuclein deposits (black arrows) in ipsilateral CA1 hippocampal and ipsilateral layer V neocortical neurons. Figure 17B: Quantification of α-synuclein deposition in ipsilateral layer 5 neocortical neurons, as measured by immunocytochemistry using SYN-1 and automated image analysis. Data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm2. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P < 0.05 vs. NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 17C: Quantification of α-synuclein deposition in ipsilateral CA1 hippocampal neurons, as measured by immunocytochemistry using SYN-1 and automated image analysis. Data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm2. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P<0.05 vs. NIP228; one-way ANOVA with Dunnett's post-hoc test. Figure 17D: Quantification of α-synuclein deposition in contralateral CA1 hippocampal neurons measured by immunocytochemistry using SYN-1 and automated image analysis. Data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm2. Each column represents the mean ± SEM value of 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P<0.05 vs. NIP228; one-way ANOVA with Dunnett's post-hoc test. [Figure 18]Figure 1 shows an HTRF assay demonstrating that epitope competition between aslo452-ngl3-hIgG1™ and BBBt0626gl-ScFv-Bs2-also0452-ngl-3-hIgG1™ does not alter the binding specificity of aslo452-ngl3-hIgG1™ due to the incorporation of the BBB moiety. The Dylight650-labeled anti-α-synuclein antibody, aslo0452hgl3-hIgG1™, binds to biotinylated α-synuclein, which in turn binds to cryptate-labeled streptavidin. Following excitation of the cryptate, energy transfer (FRET) occurs, and in the presence of Dylight650-labeled aslo0452hgl3-hIgG1™, Dylight650 is excited, resulting in fluorescence. In the presence of competing IgG, binding of Dylight650-labeled aslo0452 is blocked, preventing excitation of Dylight650-labeled aslo0452 and resulting in a decrease in the fluorescent signal. Both unlabeled aslo0452 and Bbbt0626-Bs2-also0452 hIgG1™ can similarly compete with Dylight650-labeled aslo0452-ngl3-hIgG1™. [Figure 19]Figure 1 shows the mouse brain endothelial cell binding of BBBt0626gl-BS2-aslo452-ngl-3-hIgG1™, demonstrating efficient target engagement of the BBB when conjugated with aslo452-ngl3-hIgG1™. FMAT (Fluorescence Micro-volume Assay Technology) or mirror ball assay techniques are both used to measure the specific binding of antibodies to brain endothelial cells. This assay measures the binding of human IgG to mouse brain endothelial cells (b.End3). B.End3 cells are similarly bound by Bbbt0626 hIgG1™, Bbbt0626glscFv-Bs2-aslo0452-hIgG1™, and Bbbt0626glscFv-Bs2-NIP228 hIgG1™, but not by the control antibody, NIP228 hIgG1™. This binding is detected using a mouse anti-Fc mAb (human specific) followed by detection with Alexafluor647-labeled goat anti-mouse Fc. DETAILED DESCRIPTION OF THE INVENTION

[0082] The present invention is based on the surprising and unexpected discovery of the aslo0452 ngl-3 and aslo0543 antibodies, which has resulted in a new group of antibodies that have properties shared by the aslo0452 ngl-3 and aslo0543 antibodies, as well as a subgroup of antibodies that have the properties of aslo0452 ngl-3 and aslo0543, respectively.

[0083] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is derived from antibody asyn0087 having a variable heavy chain region (VH) of the amino acid sequence of SEQ ID NO: 2 and a variable light chain region (VL) of the amino acid sequence of SEQ ID NO: 3, as disclosed herein.

[0084] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is derived from the antibody asyn0087, wherein the antibody or antigen-binding fragment has a K of less than 500 nM. D and binds to the same epitope as any one of antibodies asyn0087, aslo0452ngl-3 and aslo0543 described herein.

[0085] Like asyn0087, the aslo0452 ngl-3 and aslo0543 antibodies bind to the C-terminal region of human α-synuclein (residues 96-140). More specifically, the aslo0452 ngl-3 and aslo0543 antibodies, or antigen-binding fragments thereof, bind to a region comprised between about amino acid 102 and about amino acid 130 of human α-synuclein (e.g., SEQ ID NO: 1). In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein bind to a region comprised between about amino acid 120 and about amino acid 130 of human α-synuclein (e.g., SEQ ID NO: 1). In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein bind to an epitope that is not the same epitope bound by the 9E4 antibody.

[0086] The aslo0452 ngl-3 antibody and the aslo0543 antibody are selective for α-synuclein. The antibodies or antigen-binding fragments thereof do not bind to other synuclein family members, such as β-synuclein or γ-synuclein. More specifically, the antibodies or antigen-binding fragments thereof are specific for human α-synuclein.

[0087] The aslo0452 ngl-3 and aslo0543 antibodies bind to human, rat, or cynomolgus monkey α-synuclein. The ability of aslo0452 ngl-3 and aslo0543 to bind to human, cynomolgus monkey, and rat α-synuclein indicates that they bind to different epitopes on human α-synuclein compared to antibodies that do not bind to human, cynomolgus monkey, or rat α-synuclein. Thus, the aslo0452 ngl-3 and aslo0543 antibodies can be used for in vivo safety evaluation and research in cynomolgus monkey and rat models of disease.

[0088] The aslo0452 ngl-3 and aslo0543 antibodies bind to human α-synuclein with high affinity. The aslo0452 ngl-3 and aslo0543 antibodies have a K of less than 500 picomolar (pM), less than 400 pM, less than 300 pM, less than 150 pM, less than 120 pM, less than 115 pM, less than 110 pM, or less than 106 pM, as measured, for example, using Octet analysis (see, e.g., Example 9). D The aslo0452 ngl-3 and aslo0543 antibodies bind to alpha-synuclein at a K of less than 300 picomolar (pM), less than 250 pM, less than 200 pM, less than 150 pM, less than 120 pM, less than 110 pM, or less than 108 pM, less than 100 pM, less than 80 pM, or less than 74 pM, as measured, for example, using KinExA analysis (see, e.g., Example 9 for a reference KinExA analysis protocol). D It binds to α-synuclein.

[0089] The aslo0452 ngl-3 Fab fragment binds to human α-synuclein with high affinity, e.g., a K of less than 300 picomolar (pM), less than 200 pM, less than 180 pM, or less than 174 pM, as measured using KinExA analysis (see, e.g., Example 9.3). D It binds to α-synuclein.

[0090] The aslo0452 ngl-3 and aslo0543 antibodies bind to native, endogenous human α-synuclein. The aslo0452 ngl-3 and aslo0543 antibodies bind to aggregates of human α-synuclein. Thus, in particular, the antibodies bind to epitopes that are not required for aggregation. The aslo0452 ngl-3 and aslo0543 antibodies are capable of sequestering both the monomeric and aggregated forms of α-synuclein. The antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both the monomeric and aggregated forms of α-synuclein.

[0091] The aslo0452 ngl-3 and aslo0543 antibodies bind to disease-associated pathological forms of alpha-synuclein, such as Lewy bodies, Lewy neurites, and Lewy puncta, in Parkinson's disease brain tissue. Minimal staining is observed in normal (non-diseased) brain.

[0092] The aslo0452 ngl-3 antibody reduces α-synuclein levels in brain interstitial fluid, particularly the free, unbound α-synuclein levels in brain interstitial fluid.

[0093] The aslo0452 ngl-3 antibody reduces α-synuclein levels in cerebrospinal fluid. Specifically, the aslo0452 ngl-3 antibody reduces free, unbound α-synuclein levels in cerebrospinal fluid. The aslo0452 ngl-3 antibody reduces α-synuclein diffusion in vivo. This novel ability to inhibit α-synuclein diffusion indicates binding to a distinct epitope on human α-synuclein compared to antibodies that do not inhibit diffusion.

[0094] In some embodiments, any of the antibodies or antigen-binding fragments thereof disclosed herein have any one or more of the functional characteristics of aslo0452 ngl-3, e.g., any of the aslo0452 ngl-3 functional characteristics specified herein. In some embodiments, any of the antibodies or antigen-binding fragments thereof disclosed herein have any one or more of the functional characteristics of aslo0543, e.g., any of the aslo0543 functional characteristics specified herein.

[0095] In one embodiment, an antibody or antigen-binding fragment of the invention competes with antibody aslo0452 ngl-3 and / or aslo0543 for binding to human alpha-synuclein. In another embodiment, an antibody or antigen-binding fragment of the invention binds to the same epitope on human alpha-synuclein as antibody aslo0452 ngl-3 and / or aslo0543.

[0096] Whether an antibody or its antigen-binding fragment binds to the epitope of a reference antibody or antigen-binding fragment, as defined above, can be readily determined. Such methods are routine in the art. For example, an antibody can be compared to another antibody by a biochemical competition assay in which two antibodies (one labeled for detection purposes and the other unlabeled) are simultaneously incubated with a given antigen. If a binding signal is obtained with the labeled antibody, the two antibodies are considered to recognize distinct, non-overlapping epitopes on the protein of interest. If no binding signal is obtained, conversely, they would be characterized as having overlapping epitopes on the protein sequence because binding of one antibody sterically prevents binding of the second antibody. Furthermore, the amino acid location of a given epitope can also be identified using modified proteins, such as truncated versions, linear peptide sequences derived from the primary amino acid sequence of the antigen, or species orthologs, and by proteolytic digestion and mass spectrometry of antibodies binding to a given protein. These methodologies function to generate regions of antibody-antigen interaction.

[0097] Additional routine experiments (such as peptide mutations and binding analyses) can be performed to confirm whether any observed binding defects are indeed due to binding to the epitopes of the invention or whether they are due to some other phenomenon (such as steric hindrance). Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or other known antibody binding assays.

[0098] For example, for detailed mapping of specific epitopes, mathematical models of the epitope:paratope interface can be derived from data generated through resolving the structure of the antigen:antibody complex using high-resolution imaging methods such as cocrystallization, including X-ray diffraction. To validate the derived mathematical model for identifying the key contact residues that define the epitope, point mutagenesis of the antigen must be performed, followed by analysis of the effect on the strength of binding between the antigen and antibody caused by such established mutations. Using this combination of methods, an accurate map of the key contact residues that comprise the epitope can be established.

[0099] Antibodies or antigen-binding fragments thereof that bind to the epitope of the antibody or antigen-binding fragment thereof of the present invention can be generated by creating variants of the antibody or antigen-binding fragment thereof. Such variant antibodies or antigen-binding fragments thereof may have CDRs that share a high level of identity with the CDRs of the antibody or antigen-binding fragment thereof of the present invention. For example, in some embodiments, any of the CDRs disclosed herein of any of the antibodies or antigen-binding fragments disclosed herein may differ by one or two amino acid residues compared to any one or more of the specific CDR sequences referenced herein (e.g., any one or more of the CDRs having SEQ ID NOs: 5, 15, 16, 20, 10, and 21). In addition, such antibodies may have one or more mutations (e.g., conservative amino acid substitutions) in the framework regions.

[0100] In one embodiment, the antibody or antigen-binding fragment thereof of the invention has mutations in the CDR amino acid sequence that maintain at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and up to 99% sequence identity to the CDRs of antibody aslo0452 ngl-3.

[0101] Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are made within a family of amino acids with related side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic: aspartic acid, glutamic acid; (2) basic: lysine, arginine, histidine; (3) nonpolar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. These families can be further classified: serine and threonine are the aliphatic hydroxy family; asparagine and glutamine are the amide-containing family; alanine, valine, leucine, and isoleucine are the aliphatic family; and phenylalanine, tryptophan, and tyrosine are the aromatic family. That is, in general, independent substitutions of leucine for isoleucine or valine, aspartic acid for glutamic acid, threonine for serine, or similar substitutions of one amino acid for a structurally related amino acid would not be expected to have a significant effect on the binding function or properties of the resulting antibody, particularly if the substitution does not involve an amino acid within a CDR site.

[0102] In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises at least one CDR selected from the following: (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 6; (iii) H-CDR3 of SEQ ID NO: 7; (iv) L-CDR1 of SEQ ID NO: 9; (v) L-CDR2 of SEQ ID NO: 10; (vi) L-CDR3 of SEQ ID NO: 11.

[0103] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has at least one CDR selected from the CDRs of antibody aslo0452 ngl-3, i.e., at least one CDR selected from any one of SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 10, and SEQ ID NO: 21.

[0104] In another embodiment, the CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3; and / or the CDR3 of the light chain of the antibody or antigen-binding fragment thereof is the CDR3 of the light chain of the antibody aslo0452 ngl-3. That is, in one embodiment, the CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3 set forth in SEQ ID NO: 16; and / or the CDR3 of the light chain of the antibody or antigen-binding fragment thereof is the CDR3 of the light chain of the antibody aslo0452 ngl-3 set forth in SEQ ID NO: 21. In a further embodiment, the CDR3 of the heavy chain of an antibody or antigen-binding fragment thereof of the invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3. In one embodiment, the CDR3 of the light chain of an antibody or antigen-binding fragment thereof of the invention is the CDR3 of the light chain of the antibody aslo0452 ngl-3.

[0105] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has six CDRs of antibody aslo0452 ngl-3, i.e., three heavy chain CDRs having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:16; and three light chain CDRs having the amino acid sequences of SEQ ID NO:20, SEQ ID NO:10, and SEQ ID NO:21.

[0106] The present invention provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence defined by SEQ ID NO:13, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence defined by SEQ ID NO:18.

[0107] The present invention also provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain having the nucleotide sequence defined by SEQ ID NO:13, and a variable light chain having the nucleotide sequence defined by SEQ ID NO:18.

[0108] The present invention provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence defined by SEQ ID NO: 14, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence defined by SEQ ID NO: 19.

[0109] In one embodiment, the antibody or antigen-binding fragment thereof comprises (i) a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence defined by SEQ ID NO: 14, and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence defined by SEQ ID NO: 19, and (ii) the six CDRs of antibody aslo0452 ngl-3.

[0110] The present invention provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having the amino acid sequence of SEQ ID NO:14, and a variable light chain having the amino acid sequence of SEQ ID NO:19. In one embodiment, the antibody or antigen-binding fragment thereof of the invention has the six CDRs of antibody aslo0543.

[0111] Thus, in one embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises: (a) three heavy chain CDRs having the following sequences: (i) H-CDR1 of SEQ ID NO: 25; (ii) H-CDR2 of SEQ ID NO: 26; and (iii) H-CDR3 of SEQ ID NO: 27, and (b) three light chain CDRs having the following sequences: (i) L-CDR1 of SEQ ID NO: 31; (ii) L-CDR2 of SEQ ID NO: 32, and (iii) L-CDR3 of SEQ ID NO: 33.

[0112] In a further embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 14 and a variable light chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence defined by SEQ ID NO: 19, and further comprises: (a) three heavy chain CDRs having the following sequences: (vii) H-CDR1 of SEQ ID NO: 25; (viii) H-CDR2 of SEQ ID NO: 26; and (ix) H-CDR3 of SEQ ID NO: 27, and (b) three light chain CDRs having the following sequences: (vii) L-CDR1 of SEQ ID NO: 31; (viii) L-CDR2 of SEQ ID NO: 32, and (ix) L-CDR3 of sequence number 33.

[0113] The present invention also provides an antibody or antigen-binding fragment thereof comprising a variable heavy chain having the nucleotide sequence defined by SEQ ID NO:23 and a variable light chain having the nucleotide sequence defined by SEQ ID NO:29.

[0114] The present invention also provides an antibody or antigen-binding fragment thereof of the present invention, comprising a variable heavy chain having the amino acid sequence of SEQ ID NO:24 and a variable light chain having the amino acid sequence of SEQ ID NO:30.

[0115] In a further embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence defined by SEQ ID NO:22 and a light chain having the amino acid sequence defined by SEQ ID NO:28.

[0116] The framework regions and CDRs or antibodies can be precisely defined (Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services (1991), 91-3242, 1991; and Chothia et al. J. MoI. Biol. (1987), 196:901-917, both of which are incorporated herein by reference).

[0117] Minor variations in the amino acid sequence of the antibodies or antigen-binding fragments thereof of the invention are considered to be encompassed by the present invention, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, and most preferably at least 99% sequence identity to the antibodies or antigen-binding fragments thereof of the invention as defined anywhere herein. In particular, conservative amino acid substitutions are contemplated.

[0118] The present invention also provides a single-chain amino acid sequence comprising the light chain of an antibody or antigen-binding fragment thereof of the present invention as defined anywhere herein. The present invention also provides a single-chain amino acid sequence comprising the heavy chain of an antibody or antigen-binding fragment thereof of the present invention as defined anywhere herein.

[0119] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology alignment algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2 (1981), 484; incorporated herein by reference), by the algorithm of Needleman and Wunsch (Needleman & Wunsch, J. Mol. Biol. (1970), 48: 443; incorporated herein by reference), by the similarity search method of Pearson and Lipman (Pearson & Lipman, Proc Natl Acad Sci USA (1988), 85: 2444; incorporated herein by reference), or by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA - Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) or by visual evaluation (see Current Protocols in Molecular Biology, F. M. Ausbel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, In. And John Wiley & Sons, Inc. (1995 Supplement) Ausbubel; incorporated herein by reference).

[0120] Examples of suitable algorithms for determining percent sequence similarity or identity are the BLAST and BLAST 2.0 algorithms (see Altschul et al. J. Mol. Biol. (1990), 215(3): 403-410; and the National Center for Biotechnology Information at "http: / / www.ncbi.nlm.nih.gov / "; both of which are incorporated herein by reference). In one embodiment, the antibodies or antigen-binding fragments thereof of the invention are isolated, hi another embodiment, the antibodies or antigen-binding fragments thereof of the invention are purified.

[0121] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody. In another embodiment, the antibody or antigen-binding fragment thereof of the present invention is a humanized antibody. In yet another embodiment, the antibody or antigen-binding fragment thereof of the present invention is a human antibody.

[0122] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is an IgA, IgD, IgE, IgM or IgG (such as IgG1, IgG2, IgG3, and IgG4) antibody or antigen-binding fragment thereof.

[0123] In another embodiment, the antibody or antigen-binding fragment thereof of the present invention has reduced binding affinity to IgG Fc receptors. That is, the antibody or antigen-binding fragment thereof has reduced immunogenic effect. In one embodiment, the antibody or antigen-binding fragment thereof is an IgG1 TM antibody or antigen-binding fragment thereof. IgG1 TM is an IgG1 triple mutant that contains three point mutations (L234F / L235E / P331S) in the Fc domain that reduce the binding affinity of the antibody or antigen-binding fragment thereof to Fc-gamma receptors (FcγR) (Oganesyan et al. Acta Crystallogr D Biol Crystallogr, (2008) 64: 700-704; incorporated herein by reference). In some embodiments, that is, antibody-mediated prevention of α-synuclein spreading by the antibody of the present invention does not require Fc-related effector function as a primary mechanism of action.

[0124] Antigen-binding fragments include Fab, Fv, scFv, dAb, Fd, Fab', F(ab')2, or isolated complementarity-determining regions (CDRs) with sufficient framework for binding. Fab fragments can be monovalent fragments consisting of the VL, VH, CL, and CH1 domains. F(ab')2 fragments can be bivalent fragments consisting of two Fab fragments linked via a disulfide bridge at the hinge region. Fc fragments can consist of the CH2 and CH3 domains. Fv fragments can consist of the VL and VH domains of a single antibody arm. dAb fragments (Ward et al. Nature (1989), 341: 544-546; incorporated herein by reference) can consist of the VH domain. Isolated complementarity-determining regions (CDRs) with sufficient framework for binding can be the antigen-binding portion of the variable region.

[0125] The antigen-binding portion of the light chain variable region and the antigen-binding portion of the heavy chain variable region (for example, the two domains of the Fv fragment, VL and VH) can be combined using recombinant techniques with a synthetic linker, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. Science (1988), 242(4877): 423-426; and Huston et al. Proc Natl Acad Sci USA (1988), 85: 5879-5883; both of which are incorporated herein by reference). These are obtained using conventional techniques known to those skilled in the art, and the portions are screened for utility in the same manner as whole antibodies.

[0126] The antibodies or antigen-binding fragments thereof of the present invention may have any or all of the advantageous properties as defined above, or a combination thereof. In particular, the antibodies or antigen-binding fragments thereof of the present invention may be selective for alpha-synuclein and may be able to slow or prevent the intercellular transmission and spread of alpha-synuclein in vivo.

[0127] The functionality of the resulting antibodies or antigen-binding fragments thereof of the present invention, and in particular (i) their ability to bind to an epitope of alpha-synuclein; and (ii) their ability to slow or prevent the intercellular transmission and spread of alpha-synuclein in vivo, can be readily determined by assaying their specific activity using the techniques described herein in the Examples.

[0128] The present disclosure provides compositions for delivery of an antibody of the invention or an antigen-binding fragment thereof across the blood-brain barrier (BBB) ​​using a transporter molecule that can cross brain endothelial cells while associated with, e.g., fused or conjugated to, the antibody or fragment. BBB sequences are provided herein.

[0129] As used herein, the term "payload" is used as shorthand for an antibody or antigen-binding fragment thereof as described herein, the transport of which across the BBB can be facilitated by a transporter molecule as provided herein. In certain embodiments, the "payload" encompasses the heavy chain variable region of an antibody of the invention, more particularly the heavy chain variable region of also0452 ngl-3 or aslo0543.

[0130] The payload can be a part of the transporter molecule, for example, as a fusion polypeptide, or can be bound to the polypeptide via a disulfide bond or other covalent bond. Alternatively, the payload can be associated with the transporter molecule in any manner that will allow the transporter molecule to facilitate its transport across the BBB, as described further below. In certain embodiments, the payload remains part of the transporter molecule after BBB transport and retains central nervous system (CNS) activity in that form. Alternatively, the payload can be associated with the transporter molecule during BBB transport, but in a manner that allows it to separate from the transporter molecule after BBB transport.

[0131] The present disclosure further provides methods for the treatment or diagnosis of diseases or disorders of the CNS, particularly alpha-synucleinopathies, comprising the use of such transporter molecules in association with the antibodies or antigen-binding fragments thereof of the present invention.

[0132] In certain embodiments, the present disclosure provides isolated transporter molecules comprising immunoglobulin-derived polypeptides. In certain embodiments, the polypeptides are mimetics or non-mimetics of the camelid antibody FC5, identified and isolated using Fluorescence Micro-volume Assay Technology (FMAT), which detects binding to brain microvascular endothelial cells (BMVECs), e.g., mouse B.End3 cells. In certain embodiments, the immunoglobulin-derived polypeptide is an antibody or an active fragment thereof, where "active" means, for example, that the transporter molecule is capable of binding to BMVECs in one or more species (e.g., mouse BMVECs, rat BMVECs, cynomolgus monkey BMVECs, or human BMVECs), being internalized into BMVECs of one or more species, and / or crossing the blood-brain barrier, either alone or in association with a payload.

[0133] In some embodiments, the BBB transporter molecule is a BBB transporter molecule described in U.S. Provisional Patent Application No. 62 / 094,503 (incorporated herein by reference in its entirety). In certain aspects, the transporter molecule comprises one or more of Bbbt0241, Bbbt0626, Bbbt0626gl, Bbbt0632, BBBt0632gl Bbbt0654, Bbbt0726, Bbbt0727, Bbbt0732, Bbbt0754, Bbbt0674, Bbbt0755, Bbbt0643, Bbbt0579, or Bbbt0671, as described in U.S. Provisional Patent Application No. 62 / 094,503 (incorporated herein by reference in its entirety). In certain embodiments, the BBB transporter molecule is Bbbt0626 or BBBt0632.

[0134] In certain embodiments, the BBB transporter molecule is germlined, for example, Bbbt0626gl is the germline version of Bbbt0626 referred to as "Bbbt0626gl." In a further specific embodiment, the BBB transporter molecule is BBBt0632gl or Bbbt0626gl.

[0135] In certain embodiments, the transporter molecule does not bind to BMVEC but is still capable of transporting across the BBB as shown in an in vitro transcytosis assay.

[0136] For BBB transporter molecules, the VH CDR sequences described correspond to classical Kabat numbering positions, i.e., Kabat H-CDR1 is at positions 31-35, H-CDR2 is at positions 50-65, and H-CDR3 is at positions 95-102. L-CDR2 and L-CDR3 also correspond to classical Kabat numbering positions, i.e., positions 50-56 and 89-97, respectively. As used herein, the term "L-CDR1" or "light chain CDR1" corresponds to the sequence located at Kabat positions 23-34 in the VL (in contrast, the classical L-CDR1 position according to Kabat numbering corresponds to positions 24-34).

[0137] In certain embodiments, the immunoglobulin-derived polypeptide comprises an immunoglobulin heavy chain complementarity-determining region (CDR). For example, the immunoglobulin-derived polypeptide can comprise an immunoglobulin heavy chain complementarity-determining region-1 (H-CDR1), an immunoglobulin heavy chain complementarity-determining region-2 (H-CDR2), and an immunoglobulin heavy chain complementarity-determining region-3 (H-CDR3). In certain embodiments, the immunoglobulin-derived polypeptide can further comprise, or instead comprise, an immunoglobulin light chain CDR. For example, the immunoglobulin-derived polypeptide can comprise an immunoglobulin light chain complementarity-determining region-1 (L-CDR1), an immunoglobulin light chain complementarity-determining region-2 (L-CDR2), and an immunoglobulin light chain complementarity-determining region-3 (L-CDR3).

[0138] In certain embodiments, the immunoglobulin-derived polypeptide comprises H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each having the following amino acid sequences: (a) SEQ ID NO: 40 as H-CDR1, SEQ ID NO: 41 as H-CDR2, SEQ ID NO: 42 as H-CDR3, SEQ ID NO: 36 as L-CDR1, SEQ ID NO: 37 as L-CDR2, and SEQ ID NO: 38 as L-CDR3 (wherein the CDRs are similar to those of Bbbt0626 and Bbbt0626gl); (b) SEQ ID NO: 40 as H-CDR1, SEQ ID NO: 41 as H-CDR2, SEQ ID NO: 42 as H-CDR3, SEQ ID NO: 44 as L-CDR1, SEQ ID NO: 45 as L-CDR2, and SEQ ID NO: 46 as L-CDR3 (wherein the CDRs are identical to those of Bbbt0626 and Bbbt062gl).

[0139] In certain embodiments, the immunoglobulin-derived polypeptide comprises H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each having the following amino acid sequences: (a) SEQ ID NO: 49 as H-CDR1, SEQ ID NO: 50 as H-CDR2, SEQ ID NO: 51 as H-CDR3, SEQ ID NO: 53 as L-CDR1, SEQ ID NO: 54 as L-CDR2, and SEQ ID NO: 55 as L-CDR3 (wherein the CDRs are similar to those of Bbbt0632gl); (b) SEQ ID NO: 49 as H-CDR1, SEQ ID NO: 50 as H-CDR2, SEQ ID NO: 51 as H-CDR3, SEQ ID NO: 53 as L-CDR1, SEQ ID NO: 54 as L-CDR2, and SEQ ID NO: 55 as L-CDR3 (wherein the CDRs are identical to those of Bbbt0632gl).

[0140] In certain alternative embodiments, one or more CDRs as described above are identical to the specified CDR except for, for example, one, two, three, four, or five single amino acid deletions, substitutions, or insertions. In certain embodiments, the transporter molecules as provided above are capable of crossing the blood-brain barrier.

[0141] In certain embodiments, H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 can be arranged into immunoglobulin framework regions to generate an antibody VH and an antibody VL. In certain embodiments, the framework regions can be human-derived framework regions. In certain embodiments, the antibody VH and antibody VL are fused together, e.g., via a flexible peptide linker, to form an scFv molecule. In certain embodiments, the VH and VL further comprise one or more immunoglobulin constant domains, e.g., a CH1 domain, a hinge region, a CH3 domain, a CH3 domain, a CL-κ domain, and / or a CL-λ domain. In certain embodiments, the one or more immunoglobulin constant domains are derived from a human immunoglobulin, e.g., a human IgG1 immunoglobulin. In certain embodiments, the VH, VL, and / or constant domains can contain mutations to, for example, enhance longer or shorter half-life, increased or decreased effector function, or ability to bind payload molecules either via peptide fusion, disulfide bonds, or chemical conjugation.

[0142] In certain aspects of the invention, there is provided an antibody or antigen-binding fragment thereof of the invention associated with a transporter molecule capable of crossing brain endothelial cells as described herein.

[0143] In certain aspects, the present disclosure provides an antibody or antigen-binding fragment thereof of the present invention associated with a transporter molecule comprising an immunoglobulin-derived polypeptide, wherein the polypeptide comprises an immunoglobulin heavy chain variable region (VH) region and an immunoglobulin light chain variable region (VL) region. In certain aspects, the immunoglobulin-derived polypeptide comprises a sequence provided herein, including the following: (a) a VH amino acid sequence that is at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39, and a VL amino acid sequence that is at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43, wherein SEQ ID NO: 39 and SEQ ID NO: 43 encode the VH and VL regions of Bbbt0626gl; (b) a VH amino acid sequence that is at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 47, and a VL amino acid sequence that is at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43, wherein SEQ ID NO: 47 and SEQ ID NO: 43 encode the VH and VL regions of Bbbt0626; (c) a VH amino acid sequence that is at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 48 and a VL amino acid sequence that is at least 80%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43, where SEQ ID NO: 48 and SEQ ID NO: 43 encode the VH and VL regions of Bbbt0632.

[0144] In certain aspects, the present disclosure provides an antibody or antigen-binding fragment thereof of the present invention associated with a transporter molecule comprising an immunoglobulin-derived polypeptide, wherein the immunoglobulin-derived polypeptide comprises a VH region and a VL region, wherein: (a) the VH region comprises SEQ ID NO: 34 and the VL region comprises SEQ ID NO: 35; or (b) the VH region comprises SEQ ID NO: 39 and the VL region comprises SEQ ID NO: 43; or (c) the VH region comprises SEQ ID NO: 39 and the VL region comprises SEQ ID NO: 35; or (d) the VH region comprises SEQ ID NO: 47 and the VL region comprises SEQ ID NO: 43; or (e) the VH region comprises SEQ ID NO: 47 and the VL region comprises SEQ ID NO: 35; or (f) the VH region comprises SEQ ID NO: 48 and the VL region comprises SEQ ID NO: 52. In a further embodiment, the VH and VL regions of the transporter molecules as described above are covalently linked to form a single chain fragment (ScFv).

[0145] In certain embodiments, the transporter molecules provided herein have transporter activity, e.g., are capable of binding to BMVECs from one or more species (e.g., mouse, rat, cynomolgus monkey, or human BMVECs), being internalized into BMVECs from one or more species, or being able to cross the blood-brain barrier.

[0146] In certain aspects, a transporter molecule as provided herein comprises an immunoglobulin-derived polypeptide, wherein the immunoglobulin-derived polypeptide comprises an antibody or a BBB-permeable fragment thereof.

[0147] As used herein, a "BBB-permeable fragment" is a fragment of a transporter molecule that can specifically bind to BMVECs of one or more species and pass through BMVECs from the peripheral vasculature to the CNS vasculature in vitro or in vivo. Whether a given fragment is a BBB-permeable fragment can be tested by various in vitro or in vivo assays known to those skilled in the art. For example, transporter molecules can be tested by in vitro transcytosis assays, in vivo assays such as diuresis assays as described in US 62 / 094,503, and the like. Other assays that may be used to measure in vivo delivery of payloads across the BBB include, but are not limited to, chronic constriction injury (CCI); spared nerve injury (SNI) or spinal nerve ligation (SNL), all of which can be measured via paw flick or the Hargreaves method (Hargreaves K, et al., Pain; 1988; 32; 77-88). In certain embodiments, the transporter molecules provided herein can bind to BMVECs from one or more species (e.g., human, cynomolgus monkey, mouse, rat, or bovine BMVECs). Binding can be demonstrated by various methods known to those skilled in the art, for example, by the FMAT assay as described in US 62 / 094,503. In certain embodiments, the BMVECs are brain capillary endothelial cells (BCECs). In certain embodiments, the transporter molecules provided herein can pass through a monolayer of BCEC in an in vitro transcytosis assay. In certain embodiments, transporter molecule activity can be demonstrated by visualizing the transporter molecule in the CNS. For example, tritium-labeled transporter molecules can be delivered to a subject (e.g., into the mouse periphery, e.g., intravenously), and then visualized in the CNS via quantitative whole-body radiography.In certain embodiments, the transporter molecule is localized to a particular region of the CNS, for example, the cerebellar cortex, the cerebral gray matter, the spinal gray matter, the pons, or a combination thereof.

[0148] In certain embodiments, a transporter molecule as described herein comprises an antibody or BBB-permeable fragment thereof, comprising or consisting of two or more subunits (e.g., a heavy chain or fragment thereof and a light chain or fragment thereof, wherein the heavy and light chains are associated, for example, as a single fusion protein (e.g., scFv) or as two subunits held together by one or more disulfide bonds). In certain embodiments, the heavy chain comprises a VH domain or region, and the light chain comprises a VL domain or region. In one embodiment, the invention provides an antibody or antigen-binding fragment thereof of the invention associated with a blood-brain barrier transporter molecule as described herein.

[0149] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is associated with a blood-brain barrier transporter molecule, wherein the transporter molecule is a single chain fragment (scFv) comprising: i. the heavy chain variable region (VH) of BBBt0626gl of SEQ ID NO: 39 and the light chain variable region (VL) of BBBt0626gl of SEQ ID NO: 43, or ii. the heavy chain variable region (VH) of BBBt0626 of SEQ ID NO: 47 and the light chain variable region (VL) of BBBt0626 of SEQ ID NO: 43; iii. The heavy chain variable region (VH) of BBBt0632gl of SEQ ID NO: 48 and the light chain variable region (VL) of BBBt0632gl of SEQ ID NO: 52.

[0150] In certain embodiments, the heavy chain further comprises a heavy chain constant domain, e.g., a CH1 domain, hinge, CH2 domain, and / or CH3 domain, or a fragment thereof. In certain embodiments, the heavy chain constant domain is an IgG constant domain or a fragment thereof, e.g., a human IgG constant domain, e.g., a human IgG1, IgG2, IgG3, or IgG4 constant domain. In certain embodiments, the IgG constant domain or a fragment thereof comprises altered glycosylation and / or one or more amino acid substitutions compared to a wild-type IgG constant domain, such that the modified IgG has particular properties, e.g., an increased or decreased half-life compared to the half-life of an IgG having the wild-type IgG constant domain, an increased or decreased effector function compared to the wild-type IgG constant domain, or the ability to bind to a heterologous moiety, e.g., via a peptide bond, disulfide bond, or chemical conjugation. In certain embodiments, the IgG constant domain or fragment thereof has altered glycosylation compared to the wild-type IgG constant domain, such that the modified IgG has particular properties, such as an increased or decreased half-life compared to the half-life of an IgG having the wild-type IgG constant domain, or an increased or decreased effector function compared to the wild-type IgG constant domain.

[0151] In some embodiments, an antibody or antigen-binding fragment thereof of the present invention associates with BBBt0626 or BBBt0626gl as defined herein to form a bispecific antibody molecule.

[0152] In other embodiments, the bispecific antibodies of the invention comprise a human IgG1 TM scaffold (i.e., the IgG1 TM heavy chain CH1, CH2, CH3 regions) associated with a single-chain fragment (scFv) comprising the VH and VL regions of BBBt0626 or BBBt0626gl, grafted onto the N-terminus ("BiS2 format") or C-terminus ("BiS3 format") of the heavy chain or the N-terminus ("BiS1 format") of an anti-α-synuclein antibody of the invention. Reference to BiS formats is as disclosed in DiMasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92. In some embodiments, the bispecific antibody of the invention further comprises a light chain comprising a κ or λ CL region associated with the VL of an anti-α-synuclein antibody of the invention.

[0153] In a particular embodiment, the bispecific antibody of the invention comprises a human IgG1 TM backbone associated with: (i) a single-chain fragment (scFv) of BBBt0626gl comprising a heavy chain variable region (VH) of SEQ ID NO: 39 and a light chain variable region (VL) of SEQ ID NO: 43; or (ii) a single-chain fragment (scFv) of Bbbt0626 comprising the heavy chain variable region (VH) of SEQ ID NO: 47 and the light chain variable region (VL) of SEQ ID NO: 43;

[0154] In this case, the scFv is grafted onto the N-terminus (BiS2 format) or C-terminus (BiS3 format) of the heavy chain of aslo0452 ngl-3 of SEQ ID NO: 12 or onto the N-terminus of the light chain of SEQ ID NO: 17 ("BiS1 format").

[0155] In yet a further specific embodiment, the bispecific antibody of the invention comprises a human IgG1 TM scaffold associated with a single chain fragment (scFv) of BBBt0626gl comprising: (i) a heavy chain variable region (VH) of SEQ ID NO: 39 and (ii) a light chain variable region (VL) of SEQ ID NO: 43; wherein the scFv is grafted onto the N-terminus (BiS2 format) or C-terminus (BiS3 format) of the heavy chain of aslo0452 ngl-3 of SEQ ID NO: 12 or the N-terminus of the light chain of SEQ ID NO: 17 ("BiS1 format").

[0156] In other specific embodiments, the bispecific antibody of the invention comprises a human IgG1 TM backbone associated with: (i) a single-chain fragment (scFv) of BBBt0626gl comprising a heavy chain variable region (VH) of SEQ ID NO: 39 and a light chain variable region (VL) of SEQ ID NO: 43; or (ii) a single-chain fragment (scFv) of Bbbt0626 comprising the heavy chain variable region (VH) of SEQ ID NO: 47 and the light chain variable region (VL) of SEQ ID NO: 43; In this case, the scFv is grafted onto the N-terminus (BiS2 format) or C-terminus (BiS3 format) of the heavy chain of aslo0543 of SEQ ID NO: 22 or onto the N-terminus of the light chain of SEQ ID NO: 28 ("BiS1 format").

[0157] The present invention also provides the antibody or antigen-binding fragment thereof of the present invention for use as a pharmaceutical. The present invention also provides the antibody or antigen-binding fragment thereof of the present invention for use in the prevention or treatment of central nervous system diseases, particularly α-synucleinopathy. In one embodiment, the α-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). In a preferred embodiment, the α-synucleinopathy is Parkinson's disease (PD).

[0158] The present invention also provides use of the antibody or antigen-binding fragment thereof of the present invention for the manufacture of a medicament for preventing or treating a central nervous system disease, particularly an α-synucleinopathy. In one embodiment, the α-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). In a preferred embodiment, the α-synucleinopathy is Parkinson's disease (PD).

[0159] The present invention also provides a method for treating or preventing a disease in a patient, the method comprising administering to the patient an antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the α-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). In a preferred embodiment, the α-synucleinopathy is Parkinson's disease (PD).

[0160] In use, the antibodies or antigen-binding fragments thereof of the present invention can treat or prevent disease progression by inhibiting the propagation and spread of alpha-synuclein in vivo. The antibodies or antigen-binding fragments thereof thus offer distinct advantages over other therapeutic agents. The present invention also provides a method for slowing or preventing disease progression in a subject in need thereof, comprising administering to the patient an antibody or antigen-binding fragment thereof of the present invention.

[0161] In one embodiment, the method for treating the disease comprises administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention, hi other embodiments, the method for preventing the disease or delaying or preventing disease progression comprises administering a prophylactically effective amount of an antibody or antigen-binding fragment thereof of the invention.

[0162] The dosage ranges for administration of the antibodies or antigen-binding fragments thereof of the present invention are those to produce the desired therapeutic effect. The required dosage range depends on the exact nature of the antibody or antigen-binding fragment thereof or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent, or severity of the patient's condition, any contraindications, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimization.

[0163] A suitable dosage is in the range of 1-50 mg / kg body weight. Dosages can be in the range of 5-30 mg / kg, 10-25 mg / kg, or 15-20 mg / kg. Unit dosages can be administered once daily or less frequently, for example, once weekly or once monthly.

[0164] Administration can be by repeated administration of the antibodies or antigen-binding fragments thereof of the invention over an extended period of time. Administration can be simultaneous or sequential, and can be in any order.

[0165] The prevention or treatment provided herein can be administered as the sole therapy or can involve, in addition to the antibody or antigen-binding fragment of the invention, the administration of other drugs or established therapeutic agents commonly used in the treatment of α-synucleinopathies, such as L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (receptor) agonists, catechol-O-methyltransferase (COMT) inhibitors, and / or monoamine oxidase type B (MAO-B) inhibitors. The administration of other drugs or established therapeutic agents can be in combination with, as an adjunct to, or in conjunction with the antibody or antigen-binding fragment thereof of the invention, and can be by simultaneous, sequential, or separate administration of the individual components of the treatment.

[0166] Combination treatment can be carried out in any manner deemed necessary or convenient by one of skill in the art, and for purposes of this specification, no limitations are intended regarding the order, amount, repetition, or relative amounts of the compounds that are to be used in combination.

[0167] A therapeutically effective amount refers to the amount of an antibody or antigen-binding fragment thereof that, when administered alone or in combination to a patient to treat a disease or at least one clinical symptom of the disease, is sufficient to affect such treatment of the disease or symptom. A therapeutically effective amount may vary depending, for example, on the antibody and / or the symptoms of the disease, the age, weight, and / or health of the patient being treated, and the judgment of the prescribing physician. The appropriate therapeutically effective amount for any given case may be ascertained by one of ordinary skill in the art or may be determined by routine experimentation. A therapeutically effective amount is also one in which any toxic or adverse effects of the antibody or antibody or antigen-binding fragment thereof are outweighed by the beneficial effects.

[0168] A "prophylactically effective amount" is an amount of an antibody or either of the antibodies or antigen-binding fragments thereof that, when administered alone or in combination to a patient, inhibits or delays the onset or recurrence of at least one of the disease or clinical symptoms of the disease. In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of the disease. "Inhibiting" onset means reducing the likelihood of the disease developing or preventing the disease from developing altogether.

[0169] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention. Thus, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention together with a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients can facilitate the processing of the active compound into a preparation suitable for pharmaceutical administration.

[0170] The pharmaceutical compositions of the present invention can be formulated for parenteral delivery (e.g., intramuscular, subcutaneous, or intravenous), without limitation. Compositions suitable for intramuscular, subcutaneous, or intravenous injection include sterile aqueous solutions.

[0171] The pharmaceutical composition can be in the form of an aqueous solution and can contain physiologically compatible buffers (such as Hank's solution, Ringer's solution, or buffered saline). The pharmaceutical composition can additionally or alternatively contain substances that increase the viscosity of the suspension (such as sodium carboxymethylcellulose, sorbitol, or dextran). The pharmaceutical composition can be prepared as an appropriate oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil), or synthetic fatty acid esters (such as ethyl oleate or triglycerides), or liposomes. Optionally, the pharmaceutical composition can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0172] The present invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the present invention. The present invention also provides a vector comprising the isolated nucleic acid molecule of the present invention. The present invention further provides a host cell comprising the vector of the present invention.

[0173] The antibodies or antigen-binding fragments of the present invention are not limited to a particular method of production or manufacturing. That is, the present invention provides antibodies produced from hybridomas that secrete the antibodies, as well as antibodies produced from recombinantly produced cells that have been transformed or transfected with nucleic acid(s) encoding the antibodies. Such hybridomas, recombinantly produced cells, and nucleic acids form part of the present invention. [Example]

[0174] Example 1: Antibody production Anti-α-syn specific antibodies were isolated from a phage display library using a series of selection cycles against recombinant human α-syn ("hu α-syn") both passively immobilized on microtiter wells and free in solution. A naive human single-chain Fv (scFv) phage display library cloned into a phagemid vector based on the filamentous phage M13 was used for selection (Lloyd et al., Protein Eng Des Sel. (2009), 22(3):159-68; and Vaughan et al., Nat Biotechnol. (1996), 14(3); 309-14; both of which are incorporated herein by reference).

[0175] A representative number of individual clones from the selection output after two or three of the above selection rounds were initially screened for binding to soluble human α-synuclein as soluble scFv fragments in periplasmic E. coli extracts (Kipriyanov et al. J Immunol Methods (1997) 200: 69-77; incorporated herein by reference) in a homogeneous FRET (fluorescence resonance energy transfer) HTRF® (Homogeneous Time-Resolved Fluorescence, Cisbio International) assay.

[0176] The HTRF® assay (FIG. 1) is a homogeneous assay technique that utilizes fluorescence resonance energy transfer between closely spaced donor and acceptor fluorophores (Mathis G Clin Chem (1995) 41: 1391-1397; incorporated herein by reference). The assay involves coupling one of the molecules of interest to a donor fluorophore (e.g., europium (Eu 3+ This assay was used to measure macromolecular interactions by coupling a donor (620 nm) or acceptor (665 nm) fluorophore directly or indirectly to a donor fluorophore (cryptate) and another molecule of interest to the acceptor fluorophore XL665 (stably cross-linked allophycocyanin). Excitation of the cryptate molecule (337 nm) resulted in fluorescence emission at 620 nm. Energy from this emission was transferred to XL665 in close proximity to the cryptate, resulting in specific, long-lived fluorescence (665 nm) emission from XL665. Measuring the specific signals of both the donor (620 nm) and acceptor (665 nm) allowed for the calculation of the 665 / 620 nm ratio, which compensates for the presence of colored compounds in the assay.

[0177] Unpurified anti-α-syn scFv samples were tested for binding to biotinylated α-syn. Five microliters of a solution containing 40 nM biotinylated human α-syn in combination with 0.8 nM streptavidin terbium (Cisbio International, 610SATLB) was added to a 384-well low-volume assay plate (Costar, 3676). Next, 10 microliters of each dilution of the antibody test sample was added to the plate. Finally, 5 microliters of a solution containing DC anti-myc (Cisbio International, 661MYCDAB) was added to the assay plate. All dilutions were performed in assay buffer containing 0.8 M potassium fluoride (BDH 103444T) and 0.1% bovine serum albumin (BSA, Sigma A9576) in Dulbecco's PBS (Invitrogen, 14190185). The assay plate was incubated at room temperature for 3 hours, followed by 16 hours at 4°C, after which time-resolved fluorescence was read at emission wavelengths of 620 nm and 665 nm using an EnVision plate reader (Perkin Elmer).

[0178] Data was analyzed by calculating the 665 / 620 nm ratio for each sample, followed by the % Delta F value. The 665 / 620 nm ratio was used to correct for sample interference using the following equation:

[0179]

number

[0180]

number

[0181]

number

[0182] Single-chain Fv clones that bound human α-syn as crude periplasmic extracts were subjected to DNA sequencing (Osbourn et al., Immunotechnology (1996), 2: 181-196; and Vaughan et al., Nat Biotechnol. (1996), 14(3); 309-14; both of which are incorporated herein by reference). Unique scFvs were again expressed in bacteria and purified by affinity chromatography (as described in WO 01 / 66754; incorporated herein by reference). The potency of these samples was determined by titrating the purified preparations for binding to biotinylated human α-syn in the HTRF assay as described above. Purified scFv preparations that showed the strongest α-syn interaction were selected for conversion to IgG format.

[0183] Clones were ranked for binding strength to α-syn by titrating the antibodies in an HTRF assay. The strongest α-syn binders were identified by their binding kinetics (k) to α-syn as IgG on an Octet Red biosensor (see methods described in Example 9). off), as well as synuclein family member selectivity (human α-syn, β-syn, γ-syn) (see methods described in Example 4) and cross-reactivity with murine α-syn (see methods described in Example 5).

[0184] Example 2: Induction of aslo0452 ngl-3 A C-terminally reactive α-syn-specific clone, asyn0087, was identified by screening for binding to human α-syn in a DELFIA assay (see the method described in Example 4). Asyn0087 specifically binds to human, cynomolgus monkey, and rodent α-syn (see the method described in Example 5). Asyn0087 was reverted to the closest possible human germline sequence (Tomlinson VBASE. MRC Centre of Protein Engineering, Cambridge, UK. 1997; incorporated herein by reference), which did not affect potency by standard mutagenesis techniques prior to optimization. Following germlining, the clone was re-evaluated for binding to α-syn. No adverse effects were observed.

[0185] A large scFv-phage library derived from the lead clone was generated by oligonucleotide-targeted mutagenesis of variable heavy chain (VH) complementarity-determining regions (CDRs) 2 and 3 and light chain (VL) CDRs 1 and 3 using standard molecular biology techniques as described in Clarkson and Lowman (2004) (Phage display: A practical approach. Oxford: Oxford University Press; incorporated herein by reference). The library was subjected to affinity-based phage display selection performed against soluble biotinylated human α-syn to select variants with relatively high affinity for human α-syn. Selection was performed essentially as described above, except that the concentration of soluble biotinylated human α-syn was decreased with each round of selection performed.

[0186] Representative clones from each selection output were initially screened in HTRF assays as soluble scFv fragments in periplasmic E. coli extracts for their ability to compete against the parent α-syn-binding clone asyn0087 for binding to soluble α-syn. The performance of each library in these population screens was used to inform which CDR mutagenesis libraries were genetically added together or "recombined" to create new libraries, and these recombined libraries were subjected to further rounds of affinity-driven selection against soluble biotinylated human α-syn.

[0187] After sequence analysis of positive binders for both the individual and recombinant mutagenesis library derived clones, the clones were expressed and purified as both scFv fragments and IgG, and their binding to soluble α-syn was reconfirmed by epitope competition HTRF assay. By titrating the antibodies in the HTRF epitope competition assay, the clones were compared for their IC in terms of relative improvement in binding to α-syn compared to the parental IgG asyn0087. 50 The strongest α-syn binders were further analyzed for synuclein family member selectivity (human α-syn, β-syn, γ-syn) and cross-reactivity with cynomolgus monkey and rat α-syn by either direct binding or epitope competition HTRF assays.

[0188] These iterative rounds of library recombination and screening identified two potent α-syn-specific cynomolgus monkey and rat α-syn cross-reactive clones, aslo0452 ngl-1 and aslo0467.

[0189] I C 50Single-point mutagenesis was performed on aslo0452 ngl-1 for each of the CDRs, where a positive improvement in potency was observed. Each position in the selected CDR was individually mutated through all 20 possible amino acid residues, resulting in improved IC compared to aslo0452 ngl-1 IgG. 50 The peptides were again screened by epitope competition HTRF assay. Multiple residues across the four CDRs (H2, H3, L1, and L3) were identified and combined in both aslo0452 ngl-1 and aslo0467, resulting in improved IC compared to aslo0452 ngl-1. 50 were again evaluated by epitope competition HTRF assay. From these experiments, the two most improved binders were identified as aslo0452 ngl-3 and aslo0543. Figure 2 compares the amino acid sequences of the VH and VL regions of asyn0087, aslo0452 ngl-3 and aslo0543.

[0190] Example 2.1: Reformatting of scFv into IgG1 TM Single-chain Fv clones with the desired α-syn binding properties were converted into an effector-free, complete immunoglobulin G1™ (IgG1™) (Oganesyan et al. Acta Crystallogr D Biol Crystallogr. (2008), 64(Pt 6):700-4; incorporated herein by reference) antibody format, essentially as described by Persic et al. (Persic et al., Gene (1997) 187:9-18; incorporated herein by reference), with the following modifications: To facilitate use with CHO transient cells and to enable episomal replication, an OriP fragment was included in the expression vector. The variable heavy (VH) domain was cloned into a vector (pEU1.4) containing human heavy chain constant domains and regulatory elements for expression of the complete IgG1™ heavy chain in mammalian cells. Similarly, the variable light (VL) domain was cloned into a vector (pEU4.4) for expression of the human light chain (λ) constant domain and regulatory elements for expressing a complete IgG light chain in mammalian cells. To obtain IgG, the heavy and light chain IgG expression vectors were transfected into CHO transient mammalian cells (Daramola et al., Biotechnol Prog (2014) 30: 132-141; incorporated herein by reference). The IgG was expressed and secreted into the culture medium. The harvest was filtered before purification, and the IgG was subsequently purified using Protein A chromatography. The culture supernatant was loaded onto a ceramic Protein A (BioSepra) column of an appropriate size and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl. The bound IgG was eluted from the column with 0.1 M sodium citrate (pH 3.0) and neutralized by the addition of Tris-HCl (pH 9.0).The eluted material was buffer exchanged into PBS using a Nap10 column (Amersham, #17-0854-02), and the concentration of IgG was determined spectrophotometrically using an extinction coefficient based on the amino acid sequence of IgG (Mach et al., Anal Biochem (1992) 200: 74-80; incorporated herein by reference). Purified IgG was analyzed for aggregation and degradation purity using SEC-HPLC and by SDS-PAGE.

[0191] The rationale for using IgG1 TM as a candidate drug format is to minimize third-party killing due to immune cell and complement activation (i.e., excessive production of C3a, which can cause inflammation). Third-party cell killing may be caused by the possible accumulation of immune complexes formed by the candidate drug and extracellular α-synuclein, which has been demonstrated to interact with lipid membranes (Bartels et al., Biophys. J. (2010), 99: 2116-2124; incorporated herein by reference). The IgG1 TM format was chosen because it has been demonstrated to have negligible binding to Fcγ receptors (FcγR) and reduced C1q-mediated complement activation by immune complexes (Oganesyan et al. Acta Crystallogr D Biol Crystallogr. (2008), 64(Pt 6):700-4; incorporated herein by reference).

[0192] To minimize any potential risk of immunogenicity, the framework of aslo0452 ngl-3 is as close to the human germline amino acid sequence as possible without affecting efficacy. This means that some amino acids in aslo0452 ngl-3, including Vernier residues (Foote and Winter, J Mol Biol. (1992), 224(2): 487-99; incorporated herein by reference), have not been mutated to the closest human germline sequence. The V of aslo0452 ngl-3 HIn the V domain, there is one Vernier residue in the V region that is not mutated in the human germline IGVH3-23 and IGJH6 sequences (Figure 3C). L In the domain, all framework residues match the human germline IGLV5-45 and IGJL2 or IGJL3 sequences (Fig. 3D).

[0193] Example 3: Identification of affinity-optimized anti-α-syn IgG epitopes Recombinant human α-, β-, and γ-synuclein, recombinant truncated human α-synuclein (aa1-60, aa1-95, aa61-140, 96-140, ΔNAC, and NCAP), and mouse α-synuclein were obtained from rPeptide, Inc. Approximate epitope mapping was performed using commercially available α-syn truncates.

[0194] Briefly, 1 microgram per milliliter of each truncate was coated onto microtiter wells overnight at 4°C. After rinsing the wells with PBS, a 1 μg / mL dilution of each anti-α-syn antibody was added. After a 1-hour incubation and wash, the bound antibody was lysed using either HRP or Eu 3+ Detection was by addition of anti-human IgG conjugated to either TMB or DELFIA enhancer solution. After incubation and washing, the appropriate detection substrate was added (TMB or DELFIA enhancer solution, respectively) and the plate was read on a microtiter plate reader.

[0195] These epitope binding studies revealed that the lead isolate asyn0087 recognizes an epitope located in the C-terminal region of the α-syn protein, from amino acids 102 to 130 (Fig. 4A). Both aslo0452 ngl-3 and aslo0543 maintain recognition of the same epitope located in the C-terminal region of the α-syn protein, from amino acids 102 to 130, as their parent lead isolate asyn0087 (Fig. 4B).

[0196] Example 4: Specificity of aslo0452 ngl-3 and aslo0543 for α-syn compared to synuclein family members using an epitope competition HTRF assay Specificity for human α-syn is important for antibodies intended for therapeutic use to minimize any potential safety risks associated with off-target interactions with other synucleins (β-synuclein and γ-synuclein).

[0197] The specificity of aslo0452 ngl-3 and aslo0543 for α-syn compared with other synuclein family members, β-syn and γ-syn, was determined using an HTRF epitope competition assay, which measures the binding of biotinylated human α-syn to the antibody in solution.

[0198] α-syn, β-syn, and γ-syn were titrated into the assay, and the selectivity of aslo0452 ngl-3 IgG and aslo0543 IgG was assessed by measuring the inhibition of biotinylated human α-syn binding to aslo0452 ngl-3 / aslo0543. IC 50 Values ​​were determined by curve fitting the data to a four-parameter logistic equation using PRISM 6® software (Graphpad). A more sensitive HTRF assay measuring direct binding of IgG to human α-syn, β-syn, and γ-syn was also used to confirm α-syn specificity (data not shown). For negative controls, antibody test samples were replaced with an isotype control antibody or buffer alone.

[0199] Representative ICs obtained with α-syn, β-syn, and γ-syn proteins in the aslo0452 ngl-3 and aslo0543 HTRF epitope competition assays 50 Values ​​are shown in Figure 5. No binding was observed to β-syn and γ-syn at the concentrations tested (up to 5 μM), demonstrating that aslo0452 ngl-3 and aslo0543 are selective for α-syn.

[0200] Example 5: Specificity of aslo0452 ngl-3 for human, cynomolgus monkey, and rat α-syn using HTRF epitope competition assays Considering therapeutic use, it is important that the antibody be cross-reactive to cynomolgus monkey α-synuclein to within 10-fold of the reactivity observed to human α-syn, and preferably cross-reactive to rat α-synuclein, to allow safety studies to be performed in both cynomolgus monkey and rat species.

[0201] The specificity of aslo0452 ngl-3 and aslo0543 for human, cynomolgus monkey, and rat α-syn was determined using an HTRF epitope competition assay that measures the binding of biotinylated human α-syn to aslo0452 ngl-3 in solution.

[0202] Human, cynomolgus monkey, and rat α-syn were titrated into the assay, and antibody selectivity was assessed by measuring the degree of inhibition of antibody binding to biotinylated human α-syn. IC 50 Values ​​were determined by curve fitting the data to a four-parameter logistic equation using PRISM 6® software (Graphpad). The species cross-reactivity of aslo0452 ngl-3 and aslo0543 was also confirmed using a direct-binding HTRF assay format (not shown). Aslo0452 ngl-3 (or aslo0543) was titrated into the assay to compete for human, cynomolgus monkey, or rat α-syn binding with aslo0452 ngl-3 (or aslo0543) by HTRF assay. For negative controls, antibody test samples were replaced with an isotype control antibody or buffer alone.

[0203] Representative ICs obtained using human, cynomolgus monkey, and rat α-syn proteins in an HTRF epitope competition assay 50 The values ​​are shown in Figure 6. aslo0452 ngl-3 had IC values ​​of 5.7 nM and 6.8 nM, respectively. 50 Binds to human and cynomolgus monkey α-syn with an IC of 19.6 nM (within 4-fold) 50aslo0543 binds to rat α-syn with IC values ​​of 2.0 nM and 2.1 nM, respectively. 50 Binds to human and cynomolgus monkey α-syn with an IC of 3.8 nM (within 2-fold) 50 It binds to rat α-syn at 1000kJ / kg / day.

[0204] The ability of aslo0452 ngl-3 to bind to human, cynomolgus monkey, and rat α-syn indicates that it binds to a different epitope on human α-synuclein compared to antibodies that do not bind to human, cynomolgus monkey, and rat α-syn.

[0205] Example 6: Specificity of affinity-optimized clones for native α-syn measured by flow cytometry The binding specificity of the affinity-optimized anti-α-syn IgGs aslo0452 ngl-3 and aslo0543 to native endogenous human α-syn was determined by flow cytometry using α-syn-positive and -negative cell lines.

[0206] Briefly, SHSY5Y neuroblastoma cells (α-syn positive) and BT-20 breast cancer cells (α-syn negative) were fixed in 0.01% formaldehyde, followed by permeabilization with 0.5% (v / v) Tween 20, and then incubated with anti-α-syn antibody, positive control, or isotype control antibody. After extensive washing, bound antibody was detected by incubation with anti-human or anti-mouse IgG-FITC secondary antibody. After further washing, cells were analyzed using a FACS Canto II instrument (Becton Dickinson, Franklin Lakes, NJ), and data analysis was performed using FlowJo software (Tree Star, Ashland, OR).

[0207] Data are plotted as histograms showing the difference between cells stained alone and cells stained with the primary antibody. Results in Figure 7A show a shift in the fluorescent signal in the presence of asyn0087 (panel D) in α-syn-positive SH-SY5Y cells compared to the isotype control and secondary antibody alone (panel B), indicating recognition of endogenously expressed α-syn. Asyn0087 does not bind to the α-syn-negative human breast cancer cell line, BT-20 (panel C). Results in Figure 7B show a strong shift in the fluorescent signal in the presence of either aslo0452 ngl-3 or aslo0543 (panel H), comparable to the positive control antibody 4D6 (panel F) in α-syn-positive SH-SY5Y cells, but no shift in α-syn-negative BT-20 cells (panel G). This demonstrates that both aslo0452 ngl-3 and aslo0543 bind to native, endogenously expressed intracellular human α-syn.

[0208] Example 7: Specificity of optimized anti-α-syn IgG for aggregated human α-syn by DELFIA ELISA Human α-syn fibrillar preparations or aggregates were generated as described by Emadi et al. (Emadi et al., Biochemistry (2004), 43: 2871-2878; incorporated herein by reference). Briefly, 200 μL of 50 μM recombinant α-syn was dispensed into 1.8 mL Sarstedt tubes and placed in a shaking incubator at 280 rpm at 37°C for 3 days. The presence of aggregated α-syn was determined by incorporation of thioflavin T, which was added to a final concentration of 10 μM, incubated in the dark at room temperature for 1 hour, and fluorescence was read using an Envision microplate reader at excitation wavelengths of 450 nm and emission wavelengths of 485 nm.

[0209] The specificity of the affinity-optimized anti-α-syn IgGs aslo0452 ngl-3 and aslo0543, as well as the lead antibody asyn0087, for aggregated human α-syn was determined using a DELFIA® antibody capture assay. This assay measured the capture of aggregated human α-syn by aslo0452 ngl-3, aslo0543, or asyn0087 in a pairwise ELISA. Briefly, a mouse IgG1 version of the anti-α-syn antibody was immobilized in the wells of a 96-well microtiter plate (Nunc). After blocking, aggregated or monomeric human α-syn was incubated in the wells. After washing, captured human α-syn was detected by adding the human IgG1™ version of the same anti-α-syn antibody followed by an anti-human IgG europium conjugate (Perkin Elmer) or an anti-human IgG-HRP conjugate. Following incubation and washing, the appropriate detection substrate was added (TMB or DELFIA enhancer solution, respectively) and the plate was read in a microtiter plate reader.

[0210] This assay should capture and detect only aggregated human α-syn because multiple copies of the same epitope are present on a single aggregate. Monomeric α-syn has only one copy of the epitope, and therefore the secondary antibody used for detection cannot bind to it in the presence of the capture antibody. The data are summarized in Figure 8. The lead isolate, asyn0087, can bind to aggregated recombinant human α-syn. This means that the epitope bound by asyn0087 is not itself involved in α-syn aggregation. Both aslo0452 ngl-3 and aslo0543 retained their ability to bind to aggregated recombinant human α-syn.

[0211] Example 8: Specificity of optimized anti-α-syn IgG in disease-related tissues by immunohistochemistry The specificity of the affinity-optimized anti-α-syn IgGs aslo0452 ngl-3 and aslo0543, as well as the lead antibody asyn0087, for disease-associated forms of human α-syn was determined by immunohistochemical staining of Parkinson's disease brain tissue. The results are shown in Figure 9 and demonstrate that, like asyn0087, both aslo0452 ngl-3 and aslo0543 can recognize disease-associated pathological forms of human α-syn in Parkinson's disease brain tissue sections, including Lewy bodies, Lewy neurites, neuronal aggregates, Lewy dots, and background brain tissue. No nonspecific staining was observed in normal or healthy brain tissue.

[0212] Example 9: Anti-α-syn antibody affinity measurement The equilibrium dissociation constant (K) for anti-α-syn IgG against human α-syn D ) was determined using two platform technologies: Octet Red (Forte Bio) and KinExA (Sapidyne Instruments).

[0213] Both assay systems showed good agreement, indicating that aslo0452 ngl-3 affinity was in the subnanomolar range. Table 1 shows affinity measurements derived from key anti-α-syn clones generated through the lead isolation and lead optimization process.

[0214] Example 9.1: Affinity of aslo0452 ngl-3 with Octet The affinity of aslo0452 ngl-3 IgG for recombinant, bacterially expressed, monomeric human avi-tagged α-syn-Flag-His was assessed using an Octet Red instrument. Aslo0452 ngl-3 was premixed with various concentrations of each ligand until equilibrium was reached. The amount of free antibody was then measured using the Octet Red instrument by capturing free aslo0452 ngl-3 with biotinylated α-syn immobilized on a streptavidin-coated sensor. The amount of free antibody detected at each α-syn concentration was plotted against the ligand concentration, and the equilibrium dissociation constant (K) was calculated using KinExA software.D ) was calculated. The results, shown in Table 1, demonstrate that aslo0452 ngl-3 IgG binds to human α-syn with an affinity of 106 pM.

[0215] Example 9.2: Affinity of aslo0452 ngl-3 with KinExA Additionally, the solution-phase affinity (K ) of aslo0452 ngl-3 IgG for recombinant bacterially expressed monomeric human biotinylated α-syn was measured. D The equilibrium dissociation constant (K) was determined using a KinExA instrument (Sapidyne Instruments). Aslo0452 ngl-3 was premixed with various concentrations of each ligand until equilibrium was reached. The amount of free antibody was then measured using KinExA by capturing free Aslo0452 ngl-3 with α-syn-coated beads, washing away unbound material, and detecting bound antibody with a fluorescently labeled species-specific antibody. The amount of free antibody detected at each α-syn concentration was plotted against the concentration of ligand, and the KinExA software was used to calculate the equilibrium dissociation constant (K). D ) was calculated. The results, shown in Table 1, demonstrate that aslo0452 ngl-3 IgG binds to α-syn with an affinity of 74 pM, which is in good agreement with the Octet solution-phase affinity assay described above.

[0216] Example 9.3: Affinity of aslo0452 ngl-3 Fab fragment with KinExA The aslo0452 ngl-3 Fab fragment binds to α-synuclein with high affinity. The K D The value is 174 pM (95% CI: 15-177 pM) as determined by KinExA analysis (as described in the examples above for the full-length antibody).

[0217] Example 10: Effect of aslo0452 ngl-3 on free unbound α-synuclein levels in the prefrontal cortex interstitial fluid (ISF) of male Sprague Dawley rats Adult male Sprague Dawley rats (293–417 g; Harlan, the Netherlands) were anesthetized and implanted with guides into the prefrontal cortex.

[0218] One day before the experiment, a push-pull probe (4 mm, 1–3 MDa polyethylene membrane) was implanted into the prefrontal cortex using a stereotaxic frame (probe coordinates: AP = −3.4 mm (relative to bregma), lateral +0.8 mm (relative to midline), ventral −5.0 mm (relative to dura), incisor bar set at −3.3 mm (all coordinates according to Paxinos and Watson, *The rat brain in stereotaxic coordinates*, Academic Press, New York, 6th edition 2008). The probe was attached to the skull using stainless steel screws and dental cement.

[0219] On the day of the experiment, the push-pull microdialysis probe was connected to a microperfusion pump (Harvard) using flexible PEEK tubing (Western Analytical Products Inc., USA; PK005-020) and perfused with artificial CSF (perfusate) (containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl, and 1.2 mM MgCl + 0.2% BSA) at a flow rate of 0.5 μL / min. The probe outlet was connected to flexible FEP tubing. After a minimum of 2 h of prestabilization, aslo0452 ng / 3 formulated in PBS or PBS alone (vehicle) was administered at 30 mg / kg or 0 mg / kg, respectively. Compounds were administered intravenously at 2 mL / kg. Microdialysis samples were collected at 120-min intervals. Samples were collected into minivials (Microbiotech / se AB, Sweden; 4001029). All samples were stored at -80°C.

[0220] To determine the free α-synuclein concentration in rat ISF, microdialysis samples were first subjected to immunoprecipitation to remove aslo0452 ngl-3. The immunoprecipitation co-precipitates α-synuclein bound to the therapeutic antibody, while unbound "free" α-synuclein remains in the supernatant. A solution of Protein A beads (Dynabeads® Protein A) was added to a 96-well unskirted polypropylene plate (0.2 mL) and placed under a magnet (DynaMag) to separate the beads from the solution. TM The beads were washed twice with TBST (50 mM TBS + 0.1% Tween 20) using a 96-well plate. Thawed rat ISF microdialysis samples (10 or 20 μL) were added to each well, mixed with the beads by pipetting up and down, and incubated at 4°C for 10 minutes with tilt rotation. The beads were then pelleted twice using a magnet to completely remove them. The immunoprecipitated ISF samples were analyzed using an anti-α-synuclein ELISA kit (Sensolyte TM The samples were transferred to a 96-well plate containing sample dilution buffer (derived from a quantitative ELISA kit, human / mouse / rat, AnaSpec, US, AS-55550) to a total volume of 100 μL. 100 μL of calibration sample was added to the plate in duplicate per well, and 50 μL of detection antibody working solution was added to each well. The plate was incubated overnight at +4-8°C with agitation and protected from light, followed by six washes with 350 μL of wash buffer. Finally, 100 μL of TMB chromogenic substrate was added to each well, and the plate was incubated for 10-15 minutes at room temperature in the dark. To terminate the reaction, 50 μL of stop solution was added to each well, and the plate was read at an absorbance of 450 nm within 2 hours. Quantification was performed by plotting the standard curve response as absorbance units on a linear scale against concentration on a logarithmic scale. A four-parameter function was used for curve fitting. A time-dependent decrease in free α-synuclein was demonstrated in the ISF after a single intravenous aslo0452 ngl-3 dose at 30 mg / kg (FIG. 10).

[0221] Example 11: Effect of aslo0452 ngl-3 on free unbound alpha-synuclein levels in the CSF of male Sprague Dawley rats Adult male Sprague Dawley rats were anesthetized and a catheter was placed in the cisterna magna to accommodate CSF collection. A 0.8 cm indwelling cannula was inserted into the cisterna magna and exposed through an incision at the top of the skull. The end of the CSF catheter was fixed in place with dental acrylic cement and attached to the skull with three stainless steel screws. Animals were allowed to recover for a minimum of two days before compound administration.

[0222] Aslo0452 ngl-3 was formulated in buffer for administration at 3, 10, 30, or 100 mg / kg. Compound or vehicle alone was administered intravenously at 2 mL / kg.

[0223] Compounds were administered after collection of at least four clean CSF samples over a minimum of two days. All animals received either aslo0452 ngl-3 or vehicle on day "0." CSF samples were collected at each indicated time point. All samples were stored at -80°C until shipping.

[0224] To measure free α-synuclein in CSF, α-synuclein bound to aslo0452 ngl-3 was removed by immunoprecipitation (IP) prior to analysis. IP co-precipitates α-synuclein bound to the therapeutic antibody, while unbound "free" α-synuclein remains in the supernatant. Determination of free levels of α-synuclein in the supernatant was performed using a commercially available ELISA kit obtained from Anaspec. Analysis was performed as described for the ISF results (as described in Example 10).

[0225] A dose- and time-dependent decrease in free α-synuclein in the CSF was demonstrated after a single intravenous aslo0452 ngl-3 administration in the dose range of 3-100 mg / kg (Figure 11).

[0226] Example 12: Functional characterization of aslo0452 ngl-3 by reducing alpha-synuclein diffusion in a lentiviral in vivo model of alpha-synucleinopathy The ability of the high-affinity anti-α-synuclein antibody aslo0452 ngl-3 to block α-synuclein diffusion was investigated using a lentiviral in vivo mouse model of α-synucleinopathy. To this end, both non-transgenic wild-type mice (non-tg) and transgenic mice overexpressing α-synuclein (α-syn tg) were injected with an α-synuclein-expressing lentiviral vector (LV-α-syn) into the right hippocampus. Subsequently, they were passively immunized weekly for 13 weeks with anti-α-synuclein mouse IgG1 antibody containing aslo0452 ngl-3 and isotype control mouse IgG1 NIP228. At the end of the immunization period, mice were euthanized, and their brains were fixed in 4% PFA. Subsequently, they were coronally sectioned and analyzed by immunocytochemistry for the level of α-synuclein immunoreactivity ipsilaterally and contralaterally to the LV-α-syn injection site using automated image analysis.

[0227] Surgery and passive immunization Three- to four-month-old nontransgenic wild-type mice (non-tg; n = 40) and α-synuclein transgenic mice (α-syn tg; n = 40) received a single unilateral injection of a lentiviral vector expressing α-synuclein (LV-α-syn) into the right hippocampus (−2.0, 1.5, and −1.3 from bregma). Two weeks after LV-α-syn injection, mice were treated weekly with anti-α-synuclein mouse IgG1 antibodies: aslo0452 ngl-3 (non-tg n = 10; α-syn tg n = 10), aslo0452 ngl-3 D265A (non-tg n = 10; α-syn tg n = 10), 9E4 (non-tg n = 10; α-syn tg n = 10), or NIP228 isotype control mouse IgG1 (non-tg n = 10; α-syn tg n = 10).

[0228] All mouse IgGs were administered at 20 mg / kg via the intraperitoneal (IP) route for 13 weeks. Animals were group-housed with a maximum number of animals per cage or four per cage. Animals were housed under a 12 / 12 light / dark cycle with free access to food and water. Cages were changed weekly and monitored daily. Any adverse events were reported. All animals tolerated surgical procedures and immunizations. At the end of the antibody treatment period, mice were euthanized in accordance with guidelines for the humane treatment of animals, and their brains were serially sectioned coronally and evaluated for neuropathological analysis of α-synuclein distribution by immunocytochemistry.

[0229] α-synuclein immunocytochemistry Brains were removed, fixed in 4% paraformaldehyde, and sectioned coronally at 40 μm intervals using a vibratome. They were then stored at -30°C in cryoprotectant medium (30% glycerin, 30% ethylene glycol, 40% PBS). After a PBS wash and a blocking buffer step, sections were incubated overnight at 4°C with primary antibody (anti-α-synuclein mAb SYN-1 (BD), 1:500 dilution), washed in PBS, and incubated for 1 hour at room temperature with secondary antibody (biotinylated anti-mouse IgG (Vector Laboratories), 1:100 dilution). After a final PBS wash, α-synuclein staining was localized using an avidin / biotin-peroxidase complex detection system (Elite ABC, Vector Laboratories). Sections were subsequently analyzed for levels of α-synuclein ipsilateral and contralateral to the lentiviral vector (LV-α-syn) injection site using automated image analysis.

[0230] statistics Data generated by automated image acquisition of α-synuclein levels across non-tg and α-syn tg treatment groups were analyzed using GraphPad Prism software (San Diego, California, USA). One-way analysis of variance was performed with Dunnett's multiple comparison post-hoc test. Data shown in figures are expressed as mean ± standard error of the mean (SEM). Differences between groups were considered statistically significant when p<0.05. All analyses were performed blinded to the assessor. The antibody treatment groups were also blinded to the assessor.

[0231] result In both non-tg and α-syn tg mice, α-synuclein immunoreactivity was strong in the neuropil on the ipsilateral side of the LV-α-syn injection, covering most of the hippocampal surface (Figures 12A and 15A; NIP228-ipsilateral). The contralateral hippocampi of non-tg and α-syn tg mice also showed high levels of α-synuclein immunoreactivity, indicating that lentivirally expressed α-synuclein spread from the right to the left hippocampus on the injected side (Figures 12A and 15A; NIP228-contralateral). Previous experiments have shown that in this lentiviral α-synuclein-injected mouse model, only the expressed α-synuclein protein spreads to the contralateral side, without any sign of lentivirus migration, as determined by PCR analysis (data not shown).

[0232] The ipsilateral and contralateral hippocampal levels of lentiviral-expressed α-synuclein in non-tg mice passively immunized with the 9E4 antibody (9E4: the mouse version of PRX002 (Prothena)) were nearly identical to the ipsilateral and contralateral hippocampal levels of lentiviral-expressed α-synuclein in non-tg mice passively immunized with the NIP228 isotype control mouse IgG1 (Figure 12A, B, C; 9E4 compared to NIP228), indicating that 9E4 does not block α-synuclein dissemination in this α-synuclein diffusion model when administered via the IP route at 20 mg / kg once weekly for 13 weeks.

[0233] In contrast, the ipsilateral and contralateral hippocampal levels of lentiviral-expressed α-synuclein in non-tg mice passively immunized with either the aslo0452 ngl-3 antibody or an effector-free mutant form of aslo0452 ngl-3 (aslo0452-ngl-3-D265A) (the aspartic acid-to-alanine substitution at position 265 in mouse IgG1 (D265A) deficients this isotype from interacting with the low-affinity IgG Fc receptors (FcγRIIB and FcγRIII) found on microglia) were significantly lower than the ipsilateral and contralateral hippocampal levels of lentiviral-expressed α-synuclein in non-tg mice passively immunized with the NIP228 isotype control mouse IgG1 (Fig. 12A, B, C; aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared with NIP228). This indicates that passive immunization of mice with either aslo0452-ngl-3 or the effector-free D265A mutant of aslo0452-ngl-3 robustly blocks α-synuclein spread in this mouse model of α-synucleinopathy. Similar results were obtained when the LV-α-syn vector was injected into the right hippocampus of α-syn tg mice; passive immunization with aslo0452-ngl-3 or aslo0452-ngl-3-D265A robustly and statistically significantly reduced both ipsilateral and contralateral levels of α-synuclein immunoreactivity in the hippocampus compared with NIP228-treated α-syn tg mice, but not with 9E4 (Figure 15A, B, C).

[0234] At higher magnification, lentivirus-expressed α-synuclein immunoreactive deposits could be observed along both ipsilateral axons of the injected side and contralateral axons of the uninjected side in non-tg mice (Fig. 13A; black arrows indicate interhippocampal axons), suggesting that α-synuclein diffusion to the contralateral hippocampus could, in principle, occur along axons (transaxonal diffusion). The ipsilateral and contralateral levels of α-synuclein deposits on axons in non-tg mice passively immunized with either the 9E4 antibody or the NIP228 isotype control mouse IgG1 were not significantly different (Fig. 13A, B, C; 9E4 compared with NIP228), indicating that under our experimental conditions, 9E4 does not affect the level of α-synuclein deposits on axons and does not reduce α-synuclein dissemination along axons in this lentiviral α-synucleinopathy diffusion model.

[0235] In contrast, the levels of α-synuclein deposits on both the ipsilateral and contralateral sides of axons in non-tg mice passively immunized with either the aslo0452-ngl-3 antibody or the effector-free mutant form of aslo0452-ngl-3 (aslo0452-ngl-3-D265A) were significantly lower than the levels of α-synuclein deposits on axons in non-tg mice treated with the NIP228 isotype control mouse IgG1 (Figure 13A, B, C; NIP2 aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared with 28), demonstrating that passive immunization of mice with either aslo0452-ngl-3 or the effector-free D265A mutant of aslo0452-ngl-3 eliminates α-synuclein deposits on axons and robustly blocks ipsilateral to contralateral movement of α-synuclein along axons in this lentiviral α-synucleinopathy mouse model.

[0236] Very similar results were obtained when the LV-α-syn vector was injected into the right hippocampus of α-syn tg mice; passive immunization with aslo0452-ngl-3 or aslo0452-ngl-3-D265A, but not 9E4, robustly and statistically significantly reduced both ipsilateral and contralateral levels of α-synuclein immunoreactivity along axons compared to NIP228-treated α-syn tg mice (Figure 16A, B, C).

[0237] In LV-α-syn-injected non-tg mice, strong α-synuclein immunoreactivity was detected in the neuropil of the ipsilateral hippocampus and, to a lesser extent, in the neuropil of the ipsilateral neocortex under higher magnification (Fig. 14A). In addition, strong α-synuclein deposits were detected in many identifiable neuronal somas in the CA1 region of the ipsilateral hippocampus, and weaker α-synuclein immunoreactivity was detected in layer 5 neurons of the ipsilateral neocortex (Fig. 14; black arrows). Treatment with the 9E4 antibody did not significantly alter the number of ipsilateral CA1 hippocampal neurons or ipsilateral layer V neocortical neurons containing α-synuclein deposits compared with non-tg mice immunized with NIP228 isotype control mouse IgG (Fig. 14A, B, C; 9E4 compared with NIP228). However, non-tg mice treated with either the aslo0452-ngl-3 antibody or an effector-free mutant form of aslo0452-ngl-3 (aslo0452-ngl-3-D265A) had significantly reduced numbers of ipsilateral CA1 neurons and ipsilateral layer V neurons containing α-synuclein deposits compared with NIP228 isotype control mouse IgG-treated non-tg mice (Fig. 14A, B, C; aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared with NIP228).

[0238] Furthermore, the intensity of α-synuclein immunoreactivity in the neuropil and in neurons in the ipsilateral CA1 region of the hippocampus and the ipsilateral layer 5 region of the neocortex was significantly reduced in aslo0452-ngl-3- and aslo0452-ngl-3-D265A-treated non-tg mice compared with NIP228 isotype control mouse IgG-treated non-tg mice (Figure 14A; aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared with NIP228).

[0239] Similar results were obtained when the LV-α-syn vector was injected into the right hippocampus of α-syn tg mice; treatment with aslo0452-ngl-3 or aslo0452-ngl-3-D265A, but not 9E4, resulted in a statistically significant reduction in the number of neurons containing strong α-synuclein immunoreactivity in the ipsilateral CA1 hippocampal and layer V neocortical regions, as well as in the contralateral CA1 hippocampal region, compared with NIP228-treated α-syn tg mice (Figure 17A, B, C, D).

[0240] Passive immunization of either non-tg wild-type or α-syn tg mice, both stereotaxically injected with a lentiviral vector driving expression of human α-synuclein in one hippocampus, with the high-affinity anti-α-synuclein mouse IgG1 antibody aslo0452-ngl-3 demonstrated a robust reduction in the ipsilateral-to-contralateral transaxonal spread of lentiviral-expressed α-synuclein observed in this mouse model of α-synuclein propagation. The newly disclosed ability of this anti-α-synuclein antibody to inhibit in vivo α-synuclein spread, compared with antibodies (e.g., 9E4 antibody) that do not inhibit spreading in the model tested, indicates binding to a distinct epitope on human α-synuclein.

[0241] Furthermore, the data showing that the effector-free D265A mutant form of aslo0452-ngl-3 is equally effective as aslo0452-ngl-3 in reducing α-synuclein diffusion in this model indicate that antibody-mediated prevention of α-synuclein diffusion does not require Fc-associated effector function as a primary mechanism of action, and in particular, there appears to be no requirement or role for Fc receptors (FcγRIIB and FcγRIII) present on microglia in antibody-mediated blockade of α-synuclein diffusion.

[0242] In summary, the antibodies of the present invention, and antigen-binding fragments thereof, that target α-synuclein have the potential to be disease-modifying agents in PD, DLB, or MSA by blocking or slowing the pathological uptake of α-synuclein into recipient cells and by preventing the dissemination and transmission of α-synuclein pathology between anatomically connected brain regions. Thus, antibodies that target α-synuclein can treat or prevent disease progression and may be of therapeutic benefit to patients with synucleinopathies such as PD, DLB, or MSA.

[0243] Example 13: Generation of 0452 ngl-3-BBBt0626gl bispecific antibody Exemplary bispecific antibodies of the invention comprising a human IgG1 TM backbone coupled to a single-chain fragment (scFv) of BBBt0626gl grafted onto the N-terminus (Bis2 format) or C-terminus (Bis3 format) of the heavy chain of aslo0452 ngl-3 were generated as described below.

[0244] Bispecific antibodies of the present invention in the Bis2 format were generated by synthetically generating DNA fragments encoding Bbbt0626glscFv-(G4S)x2-aslo0452 ngl-3 VH or Bbbt0626wt-(G4S)x2-aslo452 ngl-3 VH, which contained BssHII and BstEII flanking endonuclease restriction sites upstream of Bbbt0626glscFv or Bbbt0626wt, respectively, and downstream of aslo0452 ngl-3 VH. The digested DNA fragments were then directionally cloned into the hIgG1™ vector backbone.

[0245] Bispecific antibodies of the present invention in Bis3 format were generated by PCR amplification followed by directional cloning using restriction endonuclease sites (SfiI and XbaI). Two PCR fragments were generated: (1) amplifying the hIgG1™-CH3 domain from the SfiI restriction site to the C-terminus of the CH3 domain, and (2) an overlapping PCR fragment containing forward PCR oligos incorporating the C-terminus of the CH3-(G4S)x3 linker (SEQ ID NO: 57) and the N-terminus of Bbbt0626gl, along with oligos amplifying the C-terminus of the Bbbt0626gl scFv and vector sequences immediately downstream beyond the XbaI restriction site. Both PCR fragments were joined in a pull-through PCR reaction and subsequently directional cloned into pEU1_4 (human IgG1™ vector) via the SfiI and XbaI restriction endonuclease sites.

[0246] These bispecific antibodies were expressed in a CHO-based expression system, and the resulting antibodies were purified via Protein A column purification. All Bbbt0626-derived bispecific antibodies were tested for in vitro binding to a mouse brain endothelial cell line (b.end3) to confirm the binding activity of the BBB transporter moiety, and also for binding competition with also0452 ngl-3 in an HTRF-based epitope competition assay to confirm binding to the aslo0452 ngl-3 epitope. The present invention provides the following: 1. K less than 500 pM D An antibody or antigen-binding fragment thereof that binds to human alpha-synuclein, specifically binds to a region contained between approximately amino acid 102 and approximately amino acid 130 in the C-terminal region of human alpha-synuclein, and reduces alpha-synuclein diffusion in vivo. 2. The antibody or antigen-binding fragment thereof according to 1 above, which binds to human α-synuclein but does not bind to human β-synuclein or human γ-synuclein. 3. An antibody or antigen-binding fragment thereof according to 1 or 2 above, which binds to human, rat, and cynomolgus monkey α-synuclein. 4. An antibody or antigen-binding fragment thereof according to any one of 1 to 3 above, which binds to natural endogenous human alpha-synuclein. 5. An antibody or antigen-binding fragment thereof according to any one of 1 to 4 above, which binds to aggregates of human α-synuclein. 6. An antibody or antigen-binding fragment thereof according to any one of 1 to 5 above, which binds to a disease-related pathological form of α-synuclein. 7. An antibody or antigen-binding fragment thereof according to any one of 1 to 6 above, which reduces alpha-synuclein levels, particularly free, unbound alpha-synuclein, in brain interstitial fluid and / or cerebrospinal fluid. 8. (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 6; (iii) H-CDR3 of SEQ ID NO: 7; (iv) L-CDR1 of SEQ ID NO: 9; (v) L-CDR2 of SEQ ID NO: 10; (vi) L-CDR3 of SEQ ID NO: 11 8. The antibody or antigen-binding fragment thereof according to any one of 1 to 7 above, which has at least one CDR selected from the following: 9. (a) the CDR3 of the heavy chain of the antibody or antigen-binding fragment thereof is the CDR3 of SEQ ID NO: 16 of the heavy chain of antibody aslo0452 ngl-3; and / or (b) the CDR3 of the light chain of the antibody or antigen-binding fragment thereof is the CDR3 of the light chain of antibody aslo0452 ngl-3 as set forth in SEQ ID NO: 21; 9. The antibody or antigen-binding fragment thereof according to 8 above. 10. (a) three heavy chain CDRs having the following sequences: (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 15; and (iii) H-CDR3 of SEQ ID NO: 16, and (b) three light chain CDRs having the following sequences: (i) L-CDR1 of SEQ ID NO: 20; (ii) L-CDR2 of SEQ ID NO: 10; and (iii) L-CDR3 of SEQ ID NO: 21 10. The antibody or antigen-binding fragment thereof according to any one of 1 to 9 above, comprising: 11. An antibody or antigen-binding fragment thereof according to claim 10, comprising a variable heavy chain having at least 90% identity to the sequence defined by SEQ ID NO: 14, and a variable light chain having at least 90% identity to the sequence defined by SEQ ID NO: 19. 12. The antibody or antigen-binding fragment thereof according to claim 11, comprising a variable heavy chain having the sequence defined by SEQ ID NO: 14 and a variable light chain having the sequence defined by SEQ ID NO: 19. 13. (a) three heavy chain CDRs having the following sequences: (i) H-CDR1 of SEQ ID NO: 25; (ii) H-CDR2 of SEQ ID NO: 26; and (iii) H-CDR3 of SEQ ID NO: 27, and (b) three light chain CDRs having the following sequences: (i) L-CDR1 of SEQ ID NO: 31; (ii) L-CDR2 of SEQ ID NO: 32; and (iii) L-CDR3 of SEQ ID NO: 33 12. The antibody or antigen-binding fragment thereof according to claim 11, comprising: 14. The antibody or antigen-binding fragment thereof according to any one of 1 to 13 above, which is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody or antigen-binding fragment thereof. 15. The antibody or antigen-binding fragment thereof according to any one of 1 to 14 above, which is an IgG1 TM antibody or antigen-binding fragment thereof. 16. An antibody or antigen-binding fragment thereof according to any one of 1 to 15 above, which competes with antibody aslo0452 ngl-3 for binding to human α-synuclein. 17. An antibody or antigen-binding fragment thereof according to any one of 1 to 16 above, which binds to the same epitope on human α-synuclein as antibody aslo0452 ngl-3. 18. The antibody or antigen-binding fragment thereof according to any one of 1 to 17 above, which is associated with a transporter molecule for delivery across the blood-brain barrier (BBB). 19. The transporter molecule is: a. an immunoglobulin-derived polypeptide comprising BBBt0626gl or a blood-brain barrier-permeable fragment thereof; or b. BBBt0626 or a blood-brain barrier-permeable fragment thereof, or c. BBBt0632gl or its blood-brain barrier-permeable fragment 19. The antibody or antigen-binding fragment thereof according to 18 above, 20. The antibody or antigen-binding fragment thereof according to claim 19, wherein the transporter molecule is a single-chain fragment (scFv) comprising: (i) the heavy chain variable region (VH) of BBBt0626gl of SEQ ID NO: 39 and the light chain variable region (VL) of BBBt0626gl of SEQ ID NO: 43, or (ii) the heavy chain variable region (VH) of BBBt0626 of SEQ ID NO: 47 and the light chain variable region (VL) of BBBt0626 of SEQ ID NO: 43, or (iii) the heavy chain variable region (VH) of BBBt0632gl of SEQ ID NO: 48 and the light chain variable region (VL) of BBBt0632gl of SEQ ID NO: 52. 21. An antibody or antigen-binding fragment thereof that binds to human alpha-synuclein, (a) three heavy chain CDRs having the following sequences: (i) H-CDR1 of SEQ ID NO: 5; (ii) H-CDR2 of SEQ ID NO: 15; and (iii) H-CDR3 of SEQ ID NO: 16, and (b) three light chain CDRs having the following sequences: (i) L-CDR1 of SEQ ID NO: 20; (ii) L-CDR2 of SEQ ID NO: 10; and (iii) L-CDR3 of SEQ ID NO: 21 The antibody or antigen-binding fragment thereof, comprising: 22. K<500pM D 22. The antibody or antigen-binding fragment thereof according to claim 21, which binds to human alpha-synuclein at 23. An antibody or antigen-binding fragment thereof according to 21 or 22 above, which reduces alpha-synuclein diffusion in vivo. 24. The antibody or antigen-binding fragment thereof according to any one of 21 to 23 above, which binds to human α-synuclein but does not bind to human β-synuclein or human γ-synuclein. 25. The antibody or antigen-binding fragment thereof according to any one of 21 to 24 above, which binds to human, rat, and cynomolgus monkey α-synuclein. 26. The antibody or antigen-binding fragment thereof according to any one of 21 to 25 above, which binds to natural endogenous human alpha-synuclein. 27. The antibody or antigen-binding fragment thereof according to any one of 21 to 26 above, which binds to aggregates of human α-synuclein. 28. The antibody or antigen-binding fragment thereof according to any one of 21 to 27 above, which binds to a disease-related pathological form of α-synuclein. 29. An antibody or antigen-binding fragment thereof according to any one of 21 to 28 above, which reduces alpha-synuclein levels, particularly free, unbound alpha-synuclein, in brain interstitial fluid and / or cerebrospinal fluid. 30. The antibody or antigen-binding fragment thereof according to any one of 21 to 29 above, which comprises a variable heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. 31. The antibody or antigen-binding fragment thereof according to any one of 21 to 30 above, which comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14. 32. The antibody or antigen-binding fragment thereof according to any one of 21 to 31 above, which comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 14. 33. The antibody or antigen-binding fragment thereof according to any one of 21 to 32 above, which comprises a variable light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19. 34. The antibody or antigen-binding fragment thereof according to any one of 21 to 33 above, which comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 19. 35. The antibody or antigen-binding fragment thereof according to any one of 21 to 34 above, which comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 19. 36. The antibody or antigen-binding fragment thereof according to any one of 1 to 35 above, which is an antibody. 37. An antibody or antigen-binding fragment thereof according to any one of 1 to 36 above, comprising a triple mutation L234F / L235E / P331S in the Fc region. 38. The antibody or antigen-binding fragment thereof according to any one of 21 to 29 above, which comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. 39. The antibody or antigen-binding fragment thereof according to any one of 21 to 29 above, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12. 40. The antibody or antigen-binding fragment thereof according to any one of 21 to 29 or 38 to 39 above, which comprises a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17. 41. The antibody or antigen-binding fragment thereof according to any one of 21 to 29 or 38 to 39 above, which comprises a light chain comprising the amino acid sequence of SEQ ID NO: 17. 42. The antibody or antigen-binding fragment thereof according to any one of 1 to 41 above, for use as a pharmaceutical. 43. The antibody or antigen-binding fragment thereof according to any one of 1 to 42 above, for use in the prevention or treatment of α-synucleinopathy. 44. The antibody or antigen-binding fragment thereof for use according to 43 above, wherein the α-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). 45. The antibody or antigen-binding fragment thereof for use according to 44 above, wherein the α-synucleinopathy is Parkinson's disease (PD). 46. ​​A method for treating or preventing a disease of the central nervous system (CNS) in a patient, comprising the step of administering to the patient the antibody or antigen-binding fragment thereof described in any one of 1 to 41 above. 47. The method according to claim 46, wherein the disease is alpha-synucleinopathy. 48. The method according to claim 47, wherein the α-synucleinopathy is selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). 49. The method according to 48 above, wherein the α-synucleinopathy is Parkinson's disease (PD). 50. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of 1 to 41 above, and a pharmaceutically acceptable excipient. 51. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in any one of 1 to 41 above. 52. The isolated nucleic acid molecule according to claim 51, comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 13. 53. The isolated nucleic acid molecule according to claim 51, comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 18. 54. A host cell comprising a vector containing the nucleic acid molecule according to any one of 51 to 53 above.

Claims

1. A method for producing an antibody or antigen-binding fragment thereof that binds to human alpha-synuclein, the method comprising: (i) H-CDR1 of SEQ ID NO: 5; (ii) the H-CDR2 of SEQ ID NO: 15, and (iii) H-CDR3 of SEQ ID NO: 16 and three heavy chain CDRs having the sequences: (i) L-CDR1 of SEQ ID NO: 20; (ii) the L-CDR2 of SEQ ID NO: 10, and (iii) L-CDR3 of SEQ ID NO: 21 Three light chain CDRs having the sequences wherein the method comprises: (a) expressing in vitro in a host cell the encoded product from a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof; or (b) producing the antibody or antigen-binding fragment thereof by culturing under appropriate conditions a host cell containing a vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof; The above method, comprising:

2. The method of claim 1, further comprising isolating and / or purifying the antibody or antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof has a K of less than 500 pM. D The method of claim 1 or 2, wherein the antibody binds to human alpha-synuclein at

4. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof reduces intercellular transmission of alpha-synuclein in vivo.

5. The method of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof binds to human α-synuclein but does not bind to human β-synuclein or human γ-synuclein.

6. The method of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof binds to human, rat, and cynomolgus monkey α-synuclein.

7. The method of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

14.

8. The method of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

14.

9. The method of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

14.

10. The method of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

11. The method of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

19.

12. The method of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

19.

13. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

12.

14. The method of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:

12.

15. The method of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

17.

16. The method of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:

17.

17. The method of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14, and further comprises a light chain variable region comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

19.

18. The method of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

19.

19. 19. The method of any one of claims 1 to 12, 17, or 18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and further comprises a light chain comprising the amino acid sequence of SEQ ID NO:

17.

20. The method of any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof is an antibody.

21. 21. The method of claim 20, wherein the antibody comprises a L234F / L235E / P331S triple mutation in the Fc region.

Images