Antibodies against α-synuclein and their use

Abstract

This invention provides therapeutic agents and methods for treating alpha-synucleinopathy in humans. [Solution] The present invention provides an antibody that binds specifically to human α-synuclein with high affinity and reduces α-synuclein diffusion in vivo, a recombinant polypeptide comprising the antibody or an antigen-binding fragment thereof, a method for producing such a polypeptide, a composition and method for producing an α-synuclein antibody, and a method for using the α-synuclein antibody for the treatment of central nervous system diseases, particularly α-synucleinopathy.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority from U.S. Provisional Patent Application No. 62 / 344,746, filed June 2, 2016. The aforementioned application is incorporated herein by reference in its entirety.

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

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

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

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

[0006] The defining characteristic lesions of α-synucleinopathy are Lewy bodies and Lewy neurites, which are aggregated protein insoluble inclusions found inside neurons in the brain, revealed during postmortem histopathological examination.

[0007] The presence of Lewy body lesions and neural defects 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 dysfunction, sleep disorders including REM sleep behavior disorder (RBD), mood disorders including depression and anxiety, autonomic dysfunction including 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 phase of the disease (Kalia et al. Lancet (2015), 386(9996): 896-912; incorporated herein by reference).

[0008] The presence of Lewy body lesions and neuronal defects in the motor cortex of the brain, most notably dopaminergic neuronal death in the substantia nigra, can cause 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 a major structural component of Lewy bodies and Lewy neurites. α-synuclein is a small, acidic protein (14 kDa) composed of up to 140 amino acids. Human natural wild-type α-synuclein has the amino acid sequence of Sequence ID No. 1 as described under UniProtKB registry number P37840. Unless otherwise evident from the context, references to α-synuclein or fragments thereof include the above-mentioned natural human wild-type amino acid sequence and its human allele 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 Sequence ID No. 1, the number indicates the codon position in Sequence ID No. 1, and the second letter indicates the amino acid in the allele variant). Such variants may exist individually or in any combination. E83Q, A90V, and A76T, which are induced mutations that promote α-synuclein aggregation, can also exist individually or in combination with each other and / or in combination with the human allele variants E46K, A30P, H50Q, G51D, and A53T. At the structural level, α-synuclein contains three distinct regions: an amphiphilic N-terminal α-helix domain (possessing 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 structure 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 a prominent secondary structure, but contains an important phosphorylation site at residue Ser129 and numerous tyrosine residues that are nitrated in cytoplasmic α-synuclein inclusions. N-terminal and C-terminal cleavage forms of α-synuclein also exist.Post-translational modifications to proteins can affect α-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, found intracellularly in both neuronal and glial cells, and extracellularly in 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 permeates and surrounds brain cells (Emmanouilidou et al. PLoS One (2011), 6(7): e22225; incorporated herein by reference). Alpha-synuclein is a synaptic protein 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 at the synaptic terminals of nerves and is specifically upregulated at the presynaptic terminals during synaptic reorganization associated with acquisition (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 initiation point for aggregation processes. The monomers can aggregate into various small oligomeric species, which are subsequently stabilized by β-sheet interactions and then lead to the formation of profibrils that can polymerize into insoluble fibrous structures very 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-synucleinopathy (Lashuel et al. Nature (2002), 418: 291; and Tsigelny et al. FEBS Journal (2007), 274: 1862-1877; both are incorporated herein by reference). In vitro and in vivo studies have shown that the neurotoxic effects of α-synuclein appear to be induced 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 are incorporated herein by reference). While fibrous aggregates of α-synuclein are characteristic of PD, the oligomeric form of α-synuclein is a 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 these is incorporated herein by reference).

[0013] α-synuclein oligomers can be released into the extracellular environment and taken up by neighboring cells through 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 these is incorporated herein by reference). α-synuclein aggregates can propagate misfolding through a prion-like diffusion 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 these is incorporated herein by reference). Thus, α-synuclein can induce neurodegeneration either through oligomeric toxicity or by propagation and prion-like diffusion.

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

[0015] The action of α-synuclein in PD can spread 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). Plasma α-synuclein and / or activated astrocytes can activate microglia, which leads to increased production of reactive oxygen species, nitric oxide, and cytokines, and further exacerbation of neurodegeneration (Lee et al. J Biol Chem (2010), 285: 9262-9272; incorporated herein by reference).

[0016] Cell-to-cell transmission of α-synuclein in cultured cells, or in vivo diffusion and propagation of α-synuclein lesions, have been demonstrated by various different experimental models. Lewy body lesions have been observed within embryonic midbrain neural grafts more than 10 years after therapeutic transplantation of grafts into the striatum of PD patients. Specifically, the transplanted neurons contained numerous Lewy body-like inclusions that stained positive for α-synuclein, indicating transmission of α-synuclein lesions from host to graft (Li et al. Nat Med (2008), 14(5): 501-3; and Kordower et al. Nat Med (2008), 14(5): 504-6; both incorporated herein by reference).

[0017] Furthermore, pre-formed 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, accompanied by the formation of α-synuclein inclusion bodies similar to those found in Lewy lesions, has been demonstrated (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 these is incorporated herein by reference). Injection of pre-formed synthetic α-synuclein fibers or Lewy body-like α-synuclein-containing materials 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 the host neurons of the recipient animals, accompanied by neurodegeneration and neurological injury (Luk et al. J Exp Med (2012), 209(5): 975-86; and Luk et al. Science (2012), 338(6109): 949-53; both of these are incorporated herein by reference). α-synuclein-containing Lewy body extracts isolated from PD brains inoculated into the substantia nigra or striatum of macaque monkeys and mice are rapidly taken up by host cells (within 24 hours), followed by relatively slow deletion of striatal dopaminergic terminals, accompanied by cellular deletions that become apparent more than one year later (Recasens et al. Ann Neurol (2014), 75(3): 351-62; incorporated herein by reference).Similarly, inoculation of mice with brain homogenates from patients with DLB or MSA, which are synucleinopathies, elicits α-synuclein Lewy-like lesions in the host mice (Watts et al. Proc Natl Acad Sci U S A (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, movement and transmission of both monomeric and oligomeric α-synuclein from the olfactory bulb to interconnected brain structures have 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 a number of preclinical α-synucleinopathy mouse models (Lawand et al. Expert Opin Ther Targets (2015): 1-10; incorporated herein by reference). Specifically, studies using monoclonal antibodies (9E4) against α-synuclein have shown disappearance of α-synuclein aggregates and lesions, behavioral motor improvement, and neuroprotective effects in vivo (International Publication No. WO 2014 / 058924; incorporated herein by reference).

[0019] Further studies using passive immunization with the 9E4 monoclonal antibody in α-synuclein transgenic mice, developed as an experimental model of PD / DLB, have shown that this antibody eliminates α-synuclein lesions, reduces synaptic and axonal defects, reverses tyrosine hydroxylase fiber deletions in the striatum, and significantly reduces memory and motor function defects (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 these is incorporated herein by reference). Furthermore, passive administration of anti-α-synuclein monoclonal antibodies to wild-type mice injected with pre-formed fibers (pff) of synthetic α-synuclein into the striatum resulted in a definite reduction in Lewy body lesions, prevention of dopamine neuron deficiency in the substantia nigra, and a significant improvement in motor deficiency, a major characteristic of the mouse model after pff treatment (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 those drugs to reach the affected tissue. 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 the free passage of molecules into most of the brain, thereby limiting the treatment of numerous brain diseases. The transport of essential molecules such as nutrients, growth factors, and hormones is achieved via a set of specific transporters and receptors that regulate their passage through brain endothelial cells. Therefore, the delivery of biologics and other drugs to the brain presents significant challenges. Furthermore, there appears to be a transport mechanism that rapidly removes antibodies from the brain, possibly to prevent inflammatory responses resulting from the engagement of Fc with effector ligands that promote pro-inflammatory responses.

[0021] Over the past decade, there have been reports of antibody transport across the blood-brain barrier (BBB), and these reports suggest that the ability of transporter molecules to bind to the extracellular domain facilitates the transcytosis of receptor-antibody complexes across the endothelial cell layer.

[0022] The blood-brain barrier (BBB) ​​is primarily composed of brain capillary endothelial cells, which possess special features such as tight junctions that restrict 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 antibodies administered peripherally reach the brain (Boado et al. 2010, Mol. Pharm. 7: 237-244; Pepinsky et al. 2011, Nat. Neurosci. 8: 745-751). The blood-brain barrier (BBB) ​​functions as a physiological, metabolic, and immunological barrier (Gaillard et al. 2003, Microvasc. Res. 65: 24-31).

[0023] Antibody transport across the blood-brain barrier (BBB) ​​can be enhanced by inducing receptor-mediated transcytosis in brain endothelial cells. Through this process, antigen engagement on the luminal side of endothelial cells can induce the internalization and reciprocal movement of antibodies across the cell, followed by their release into the tissue. [Prior art documents] [Patent Documents]

[0024] [Patent Document 1] International Publication No. 2014 / 058924 Pamphlet [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

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[0026] Current drug therapies for Parkinson's disease (PD) primarily focus on treating the exercise-related symptoms of the disease. There are currently no commercially available or commercially available medications that can treat or prevent alpha-synucleinopathy. Therefore, there is a need in this technology for therapies to treat alpha-synucleinopathy, particularly in humans. [Means for solving the problem]

[0027] This invention relates to an isolated antibody against human α-synuclein. The invention provides an antibody or its antigen-binding fragment having one or more of the functional properties of the aslo0452 ngl-3 antibody. For example:

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

[0029] The present invention provides an antibody or an antigen-binding fragment thereof that binds to human α-synuclein but does not bind 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 an antibody or its antigen-binding fragment that binds to human α-synuclein with high affinity. In one embodiment, the antibody or its antigen-binding fragment of the present invention, when measured by Octet analysis, has a K concentration of less than 500 picomoles (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 106 pM. D It has and binds to α-synuclein. In one embodiment, the antibody of the present invention, or its antigen-binding fragment, when measured using KinExA analysis, has a K concentration of less than 400 picomoles (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 74 pM or less. D Then, it binds to α-synuclein.

[0031] The present invention provides an antibody or an antigen-binding fragment thereof that binds to naturally occurring endogenous human α-synuclein. The present invention provides an antibody or an antigen-binding fragment thereof that binds to human α-synuclein in monomeric form. The present invention provides an antibody or an antigen-binding fragment thereof that binds to aggregates of human α-synuclein. The present invention provides an antibody or antigen-binding fragment thereof that binds to a disease-related lesion morphology α-synuclein.

[0032] The present invention provides an antibody or its antigen-binding fragment that reduces the level of α-synuclein in the interstitial fluid of the brain. In particular, the antibody or its antigen-binding fragment reduces the level of free, unbound α-synuclein in the interstitial fluid of the brain.

[0033] The present invention provides an antibody or its antigen-binding fragment that reduces the level of α-synuclein in cerebrospinal fluid. In particular, the antibody or its antigen-binding fragment reduces the level of free, unbound α-synuclein in cerebrospinal fluid.

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

[0035] This invention provides an antibody or its antigen-binding fragment that reduces α-synuclein diffusion in vivo. In one embodiment, the antibody of the present invention or its antigen-binding fragment competes with the antibody aslo0452 ngl-3 for binding to human α-synuclein. In one embodiment, the antibody of the present invention or its antigen-binding fragment binds to the same epitope on human α-synuclein as the antibody aslo0452 ngl-3.

[0036] In one embodiment, the antibody of the present invention or its antigen-binding fragment is derived from the antibody asyn0087, which includes 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 of the present invention is derived from the antibody asyn0087, in which case the antibody or antigen-binding fragment has a K content of less than 500 mM. D It has the following properties and binds to the same epitope as any one of the antibodies described herein: asyn0087, aslo0452 ngl-3, and aslo0543.

[0038] In this specification, "H-CDR" means the complementarity-determining region (CDR) of the heavy chain region of an antibody or its antigen-binding fragment, and "L-CDR" means the complementarity-determining region (CDR) of the light chain region. In one embodiment, the antibody or antigen-binding fragment of the present invention comprises at least one CDR selected from the following: (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of sequence number 6, (iii) H-CDR3 of sequence number 7, (iv) L-CDR1 of sequence number 9, (v) L-CDR2 of sequence number 10, (vi) L-CDR3 of sequence number 11.

[0039] In further embodiments, the CDR3 of the heavy chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of SEQ ID NO: 16 of the heavy chain of the antibody aslo0452 ngl-3; and / or the CDR3 of the light chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of SEQ ID NO: 21 of the light chain of the antibody aslo0452 ngl-3.

[0040] In one embodiment, the antibody or its antigen-binding fragment 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 the antibody aslo0452 ngl-3, that is, 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 its antigen-binding fragment 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 its antigen-binding fragment of the present invention is the CDR3 of the light chain of the antibody aslo0452 ngl-3. In one embodiment, the CDR3 of the heavy chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3. In one embodiment, the CDR3 of the light chain of the antibody or its antigen-binding fragment of the present 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 its antigen-binding fragment 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 its antigen-binding fragment of the present invention is the CDR3 of the light chain of the antibody aslo0452 ngl-3. The present invention provides an antibody having six CDRs of antibody aslo0452 ngl-3 or an antigen-binding fragment thereof.

[0043] In other words, in one embodiment, the antibody or antigen-binding fragment of the present invention comprises the following: (a) Three heavy chain CDRs having the following sequences: (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of Sequence ID No. 15; and (iii) H-CDR3 of Sequence ID No. 16, and (b) Three light chain CDRs having the following sequences: (i) L-CDR1 of sequence number 20, (ii) L-CDR2 of Sequence ID No. 10, and (iii) L-CDR3 of sequence number 21.

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

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

[0046] The present invention provides an antibody or an antigen-binding fragment thereof, 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 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 further specific embodiments, the antibody or antigen-binding fragment 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, with a K content of less than 500 pM. DIt binds to human α-synuclein and also binds to the same epitope asyn0087, aslo0452 ngl-3, or aslo0543.

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

[0050] In certain embodiments, the antibody or antigen-binding fragment 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 further embodiments, the antibody or antigen-binding fragment of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 with the sequence defined by SEQ ID NO: 19, and further comprises: (a) Three heavy chain CDRs having the following sequences: (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of Sequence ID No. 15; and (iii) H-CDR3 of Sequence ID No. 16, and (b) Three light chain CDRs having the following sequences: (i) L-CDR1 of sequence number 20, (ii) L-CDR2 of Sequence ID No. 10, and (iii) L-CDR3 of sequence number 21.

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

[0053] The present invention provides an antibody or an 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] The antibody of the present invention or an antigen-binding fragment thereof is also provided, 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, the antibody or antigen-binding fragment of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 with the sequence defined by SEQ ID NO: 30.

[0056] In certain embodiments, the antibody or antigen-binding fragment 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 further embodiments, the antibody or antigen-binding fragment of the present invention comprises a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 with the sequence defined by SEQ ID NO: 30, and further comprises: (c) Three heavy chain CDRs having the following sequences: (iv) H-CDR1 of sequence number 25, (v) H-CDR2 of Sequence ID No. 26; and (vi) H-CDR3 of Sequence ID No. 27, and (d) Three light chain CDRs having the following sequences: (iv) L-CDR1 of sequence number 31, (v) L-CDR2 of sequence number 32; and (vi) L-CDR3, sequence number 33.

[0058] The present invention also provides an antibody or an 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 an 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] The antibody or its antigen-binding fragment is also provided, 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 of the present invention as defined somewhere above is an antibody or antigen-binding fragment such as IgA, IgD, IgE, IgM, IgG, for example IgG1, IgG2, IgG3, or IgG4.

[0062] In another embodiment, the antibody or antigen-binding fragment of the present invention has a modified Fc region. Preferred modifications are well known to those skilled in the art and may include, among other things, modifications to increase or decrease the half-life, remove, reduce or enhance effector function, or provide a substituted cysteine ​​having a free thiol for conjugation. Examples of such modifications are YTE for increasing the half-life and / or TM for reducing effector function. In some embodiments, any of the antibodies or antigen-binding fragments disclosed herein contain the mutation 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 include a triple mutation in the Fc region (hereinafter abbreviated as "TM") corresponding to the L234F / L235E / P331S mutation disclosed in the literature by Oganesyan et al. (Acta Crystallogr D Biol Crystallogr, (2008) 64: 700-704). In one embodiment, the antibody or antigen-binding fragment of the present invention may be an IgG1 TM antibody or its antigen-binding fragment. In another embodiment, the antibody or antigen-binding fragment of the present invention may include an Fc region having a YTE mutation.

[0063] In another embodiment, the antibody or its antigen-binding fragment of the present invention can be coupled to a blood-brain barrier (BBB) ​​transporter portion, in which case the BBB transporter portion can pass through the BBB and transport the antibody or its antigen-binding fragment.

[0064] In one embodiment, the BBB transporter portion may be an antibody. In one embodiment, the BBB antibody may form a multispecific construct with an anti-α-syncuclein antibody or its antigen-binding fragment. The BBB transporter portion may include 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); in this case, VH-CDR1 includes SEQ ID NO: 40 or 49, VH-CDR2 includes SEQ ID NO: 41 or 50, VH-CDR3 includes SEQ ID NO: 42 or 51, VL-CDR1 includes SEQ ID NO: 36, 44 or 53, VL-CDR2 includes SEQ ID NO: 37, 45 or 54, and VL-CDR3 includes SEQ ID NO: 38, 46 or 55.

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

[0066] In addition, the transporter moiety can be selected from a complete antibody, Fv fragment, Fab fragment, Fab' fragment, F(ab')2 fragment, disulfide-bonded (dsFv) fragment, single-chain Fv(scFV) fragment, sc(Fv)2 fragment, diabody, triabody, tetrabody, minibody, and single-chain antibody. In certain embodiments, the transporter moiety includes an scFV fragment containing VH and VL domains fused via a linker. In some examples, the linker is (Gly4Ser) n (Sequence code 56) is possible, in which case 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 present invention can be combined with any α-synuclein-binding molecule of the present invention as described herein to provide a multispecific binding molecule of the present invention. The present invention provides an antibody or antigen-binding fragment thereof, as defined anywhere above, for use as a pharmaceutical.

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

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

[0070] The phrase "pharmaceutically acceptable excipients" includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, and absorption retarders that are suitable for drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may also contain other active compounds that provide auxiliary, additional, or enhanced therapeutic functions. The pharmaceutical composition may also be included in a container, pack, or dispenser, along with instructions for administration.

[0071] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Methods for administering them are known to those skilled in the art. Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous, or percutaneous. The present invention provides isolated nucleic acid molecules encoding the antibody of the present invention or its antigen-binding fragment, as defined somewhere above. In certain embodiments, the present invention provides isolated nucleic acid molecules comprising SEQ ID NO: 13 and / or SEQ ID NO: 18. In another specific embodiment, the present invention provides isolated nucleic acid molecules comprising SEQ ID NO: 23 and / or SEQ ID NO: 29.

[0072] With this information provided, those skilled in the art will be able to readily obtain nucleic acid molecules encoding the disclosed antibody or its antigen-binding fragment. Nucleic acids may include DNA or RNA and may be entirely or partially synthesized or recombinant. References to nucleotide sequences, unless the context requires otherwise, encompass DNA molecules containing the identified sequence and RNA molecules containing the identified sequence in which U replaces T. The nucleic acid molecule of the present invention comprises coding sequences for the CDR, VH domain, and / or VL domain as disclosed herein.

[0073] This disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcriptions, or expression cassettes, comprising at least one nucleic acid molecule encoding the antibody of the present invention or its antigen-binding fragment as defined anywhere above, particularly the CDR, VH domain, and / or VL domain as disclosed herein. This disclosure further provides a host cell comprising one or more constructs as described above.

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

[0075] Antigen-binding fragments, VH and / or VL domains, and coding 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, without or substantially without nucleic acids or genes of origin other than the sequence encoding the polypeptide having the required function.

[0076] Systems for the cloning and expression of polypeptides in various different host cells are well known in the art. For suitable cells for antibody production, 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 heterologous polypeptide expression 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 conforming to 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 include appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences where appropriate. The vector may be plasmid or viral (e.g., phage or phagemide) where appropriate. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. Numerous known techniques and protocols for nucleic acid construction, mutagenesis, sequencing, DNA introduction into cells and nucleic acid manipulation in gene expression, and protein analysis are described in detail in Current Protocols in Molecular Biology, 2nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992.

[0078] Further aspects of this disclosure provide a host cell comprising a vector containing nucleic acids as disclosed herein, in particular a nucleic acid molecule encoding an antibody of the present invention or an antigen-binding fragment thereof as defined anywhere above.

[0079] Further embodiments provide a method comprising the step of introducing such nucleic acids into host cells. The introduction can utilize any of the available techniques. With respect to eukaryotic cells, preferred techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia virus, or baculovirus in the case of insect cells). With respect to bacterial cells, preferred techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduction of nucleic acids into cells may be followed by a step of inducing or enabling expression from the nucleic acids (e.g., by culturing host cells under conditions for gene expression).

[0080] Brief description of the drawings and sequence list The present invention will be described in more detail with reference to the accompanying drawings and sequence listing, which show the following: JPEG2026086794000001.jpg247129JPEG2026086794000002.jpg181129 Table 1: Affinity measurements of major anti-α-synuclein antibodies against human α-syn performed on two affinity measurement platforms. [Table 1] [Brief explanation of the drawing]

[0081] [Figure 1] This is a schematic diagram of the HTRF® assay. [Figure 2] This figure shows a comparison of the amino acid sequences of the 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) of asyn0087, aslo0452ngl-3, and aslo0543. Underlined amino acids correspond to the CDR. [Figure 3A]The nucleotide and amino acid sequences of aslo0452 ngl-3 are shown. Figures 3A and 3B show the variable heavy chain and variable light chain nucleotides and amino acid sequences of aslo0452 ngl-3, respectively. Figure 3A discloses sequence numbers 13 and 14, respectively, in order of appearance. [Figure 3B] These are the nucleotide and amino acid sequences of aslo0452 ngl-3. Figures 3A and 3B show the variable heavy chain and variable light chain nucleotides and amino acid sequences of aslo0452 ngl-3, respectively. Figure 3B discloses sequence numbers 18 and 19, respectively, in order of appearance. [Figure 3C] These are the nucleotide and amino acid sequences of aslo0452 ngl-3. Figures 3C and 3D show the alignment of these sequences to the nearest human germline sequence. Figure 3C shows the alignment of the aslo0452 ngl-3 variable heavy chain domain amino acid sequence (SEQ ID NO: 14) with 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 bordered. All non-vernier residues in the light chain framework region are human germline amino acids. Vernier residues (*) have not been modified to match germline amino acids. [Figure 3D] These are the nucleotide and amino acid sequences of aslo0452 ngl-3. Figures 3C and 3D show the alignment of these sequences to the nearest human germline sequence. Figure 3D shows the alignment of three sequences: 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 bordered. All non-vernier residues in the light chain framework region are human germline amino acids. Vernier residues (*) have not been modified to match germline amino acids. [Figure 4] This figure shows the epitope binding of read isolate clones using a panel of α-syn truncates. The ELISA wells are coated with various commercially available α-syn truncates representing various defined regions of the following proteins: 1-140: full-length α-syn, 1-60: N-terminal region only, 61-140: plaque non-amyloid component (NAC) + C-terminal region, 1-95: N-terminal and NAC regions, 96-140: C-terminal region only, ΔNAC: NAC region deletion, NCAP: alternative splicing morphology 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 bar), aslo0543 (light gray bar), and NIP228 isotype-matched control (dark gray bar). Binding affinity 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] This figure shows the specificity of aslo0452 ngl-3 and aslo0543 to α-syn compared to synuclein family members using the DELFIA epitope competition assay. The specificity of affinity-optimized aslo0452 ngl-3 and aslo0543 clones to α-syn was measured using the epitope competition HTRF assay by titer measurement of unlabeled α-syn, β-syn, and γ-syn. The IC50 values ​​were then determined. [Figure 6] This figure shows the specificity of aslo0452 ngl-3 and aslo0543 to human, cynomolgus monkey, and rat α-syn using an HTRF epitope competition assay. The species cross-reactivity profiles of affinity-optimized clones were determined using the same assay by measuring the titer of unlabeled α-syn and inducing IC50 values ​​for α-syn of each species. [Figure 7A]This figure shows representative flow cytometry results demonstrating that affinity-optimized clones bind to native human α-syn in human neuroblastoma cell lines. Panels A, C, E, and G in Figures 7A and 7B show binding to BT20, an α-syn-negative human breast cancer cell line. Panels B, D, F, and H in Figures 7A and 7B show binding to SHSY5Y, an α-syn-positive human neuroblastoma cell line. Figure 7A: The primary human antibodies used in this study were asyn0087 and Hu IgG control. Human antibody binding was detected using secondary anti-human IgG-FITC (Jackson). [Figure 7B] This figure shows representative flow cytometry results demonstrating that affinity-optimized clones bind to native human α-syn in human neuroblastoma cell lines. Panels A, C, E, and G in Figures 7A and 7B show binding to BT20, an α-syn-negative human breast cancer cell line. Panels B, D, F, and H in Figures 7A and 7B show binding to SHSY5Y, an α-syn-positive human neuroblastoma cell line. Figure 7B: The primary human antibodies used in this study were aslo0452 ngl-3, aslo0543, and NIP228 isotype-matched IgG1™ control. Human antibody binding was detected using secondary anti-human IgG-FITC (Jackson). The primary mouse antibodies used were 4D6 (Covance) and isotype-matched negative control (R&D Systems). Mouse antibody binding was detected using secondary anti-mouse IgG-FITC (Sigma). [Figure 8] This figure shows the specificity of optimized anti-α-syn IgG against aggregated human α-syn using 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 did not detect captured monomeric α-syn. [Figure 9-1]This figure shows the specificity of affinity-optimized clones in disease-related tissues by immunohistochemistry. Figures 9A, 9B, and 9C show the staining properties using 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 shows low-level staining of α-syn in temporal cortical-derived cells 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 the isotype-matched control antibody does not stain the amygdala in PD brain tissue. [Figure 9-2] This figure shows the specificity of affinity-optimized clones in disease-associated tissues by immunohistochemistry. Figures 9A, 9B, and 9C show staining with aslo0452 ngl-3, asyn0087, and aslo0543, respectively. Panels I-M show staining of the locus coeruleus in PD brain tissue with asyn0087; identified pathological features are Lewy bodies (panels I and L), neural aggregates (panel J), Lewy neurites (panel K), and pale bodies (panel M). Panels N and O show that aslo0543 stains Lewy bodies and Lewy neurites in the substantia nigra of PD brain tissue. Panel P shows low-level staining of α-syn in temporal cortical-derived cells in normal brain sections with aslo0543. [Figure 10] This figure shows that systemic administration of aslo0452 ngl-3 rapidly reduces free asyn levels in the prefrontal cortex of rats. Free α-synuclein concentrations in the ISF of rats treated with aslo0452 ngl-3 (30 mg / kg intravenously; black circles) or vehicle (white circles), absolute (Figure 10A) or relative (Figure 10B) mean ± SEM. [Figure 11]This figure shows that aslo0452 ngl-3 reduces free asyn levels in rat CSF in a dose- and time-dependent manner upon systemic administration. Absolute (Figure 11A) or relative (Figure 11B) mean ± SEM free α-synuclein concentrations in CSF of rats treated with aslo0452 ngl-3 (3, 10, 30, 100 mg / kg intravenously; black circles) or vehicle (white circles). [Figure 12] This figure shows that the anti-α-synuclein antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A inhibit α-synuclein diffusion from the ipsilateral to the contralateral side. Figure 12A: Non-transient mice (black arrows) injected with LV-α-syn into the right hippocampus were passively immunized once a week for 13 weeks with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or NIP228 isotype control antibodies, and 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 from 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P<0.05 vs NIP228; One-way ANOVA using Dunnett's post-hoc test. Figure 12C: Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of contralateral hippocampal coronal sections shown in Panel A. Each column represents the mean ± SEM value from 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P<0.05 vs NIP228; One-way ANOVA using Dunnett's post-hoc test. [Figure 13]This figure shows that the anti-α-synuclein antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A reduce lentiviral-mediated α-synuclein deposition and dissemination along axons. Figure 13A: Non-tg mice injected with LV-α-syn into the right hippocampus were passively immunized once a week for 13 weeks with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or NIP228 isotype control antibodies, followed by immunocytochemical analysis of α-synuclein deposition (black arrows) along ipsilateral and contralateral interhippocampal axons. Figure 13B: Quantification of ipsilateral axonal α-synuclein deposition measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value from 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 measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value from 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]This figure 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 5 neocortical neurons. Figure 14A: Non-tg mice injected with LV-α-syn into the right hippocampus were passively immunized once a week for 13 weeks with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or NIP228 isotype control antibodies, and then immunocytochemical analysis was performed on α-synuclein deposition (black arrows) in ipsilateral CA1 hippocampal neurons and ipsilateral layer 5 neocortical neurons. Figure 14B: Quantification of α-synuclein deposition in ipsilateral layer 5 neocortical neurons measured by immunocytochemistry using SYN-1 and automated image analysis. The data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm². Each column is the mean ± SEM value from 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. The data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm². Each column is the mean ± SEM value from 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]This figure shows that the antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A inhibit 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 once a week for 13 weeks with anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or NIP228 isotype control antibodies, followed by immunocytochemistry measurements of α-synuclein diffusion using SYN-1 and automated image analysis. Figure 15B: Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of ipsilateral hippocampal coronal sections shown in Panel A. Each column represents the mean ± SEM value from 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P<0.05 vs NIP228; One-way ANOVA using Dunnett's post-hoc test. Figure 15C: Quantification of α-synuclein immunoreactivity data obtained from immunocytochemical analysis of contralateral hippocampal coronal sections shown in Panel A. Each column represents the mean ± SEM value from 10 independent antibody treatments (n = 10 mice per antibody treatment group). *P<0.05 vs NIP228; One-way ANOVA using Dunnett's post-hoc test. [Figure 16]This figure shows that the antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A reduce lentiviral-mediated α-synuclein deposition and dissemination along axons in transgenic mice. Figure 16A: α-syn tg mice, injected with LV-α-syn into the right hippocampus, were passively immunized once weekly for 13 weeks with the anti-α-synuclein mouse IgG1 antibodies aslo0452 ngl-3, aslo0452 ngl-3 D265A, 9E4, or NIP228 isotype control antibodies, followed by immunocytochemical analysis of α-synuclein deposition (black arrows) along ipsilateral and contralateral interhippocampal axons. Figure 16B: Quantification of ipsilateral axonal α-synuclein deposition measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value from 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 measured by immunocytochemistry using SYN-1 and automated image analysis. Each column represents the mean ± SEM value from 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]This figure shows that the antibodies aslo0452 ngl-3 and aslo0452 ngl-3-D265A reduce α-synuclein deposition in CA1 hippocampal neurons and layer 5 neocortical neurons in 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 NIP228 isotype control antibodies once a week for 13 weeks, and then immunocytochemical analysis was performed on α-synuclein deposition (black arrows) in ipsilateral CA1 hippocampal neurons and ipsilateral layer 5 neocortical neurons. Figure 17B: Quantification of α-synuclein deposition in ipsilateral layer 5 neocortical neurons, measured by immunocytochemistry using SYN-1 and automated image analysis. The data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm². Each column is the mean ± SEM value from 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, measured by immunocytochemistry using SYN-1 and automated image analysis. The data shown represents the number of α-synuclein-positive cells (neurons) per 0.1 mm². Each column is the mean ± SEM value from 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. The data shown represent the number of α-synuclein-positive cells (neurons) per 0.1 mm². Each column is 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]This figure demonstrates, using an HTRF assay, that epitope competition between BBBt0626gl-ScFv-Bs2-also0452-ngl-3-hIgG1TM and aslo452-ngl3-hIgG1TM does not alter the binding specificity of aslo452-ngl3-hIgG1TM due to the incorporation of the BBB moiety. The Dylight650-labeled anti-α-synuclein antibody aslo0452hgl3-hIgG1TM binds to biotinylated α-synuclein, which then binds to cyrptate-labeled streptavidin. After excitation of the cyrptate, an energy transfer (FRET) occurs, and in the presence of dylight650-labeled aslo0452hgl3-hIgG1TM, dylight650 is excited, producing fluorescence. If competing IgG is present, binding of dylight650-labeled aslo0452 is blocked, preventing excitation of dylight650-labeled aslo0452 and resulting in a reduced fluorescence signal. Both unlabeled aslo0452 and Bbbt0626-Bs2-also0452 hIgG1™ can similarly compete with dylight650-labeled aslo0452-ngl3-hIgG1™. [Figure 19]This figure demonstrates the efficient targeted engagement of the BBB region when BBBt0626gl-BS2-aslo452-ngl-3-hIgG1TM binds to mouse brain endothelial cells. Both FMAT (Fluorescence Micro-volume Assay Technology) and mirrorball assay techniques are 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 to Bbbt0626 hIgG1TM, Bbbt0626glscFv-Bs2-aslo0452-hIgG1TM, and Bbbt0626glscFv-Bs2-NIP228 hIgG1TM, but not to the control antibody NIP228 hIgG1TM. This binding affinity was detected using a mouse anti-Fc mAb (human-specific), followed by detection using Alexafluor647-labeled goat anti-mouse Fc. [Modes for carrying out the invention]

[0082] This invention is based on the surprising and unexpected discovery of the aslo0452 ngl-3 antibody and the aslo0543 antibody. This discovery resulted in a new group of antibodies sharing properties with the aslo0452 ngl-3 antibody and the aslo0543 antibody, as well as subgroups of antibodies possessing the properties of aslo0452 ngl-3 and aslo0543, respectively.

[0083] In one embodiment, the antibody of the present invention or its antigen-binding fragment is derived from the 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 of the present invention is derived from the antibody asyn0087, in which case the antibody or antigen-binding fragment has a K content of less than 500 nM. D It has the following properties and binds to the same epitope as any one of the antibodies asyn0087, aslo0452ngl-3, and aslo0543 described herein.

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

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

[0087] The aslo0452 ngl-3 antibody and the aslo0543 antibody 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 binding to different epitopes on human α-synuclein as compared to antibodies that do not bind to human, cynomolgus monkey and rat α-synuclein. That is, the aslo0452 ngl-3 antibody and the aslo0543 antibody can be used for in vivo safety evaluation and research in disease cynomolgus monkey and rat models.

[0088] The aslo0452 ngl-3 antibody and the aslo0543 antibody bind to human α-synuclein with high affinity. The aslo0452 ngl-3 antibody and the aslo0543 antibody bind to α-synuclein with a K of less than 500 picomolar concentration (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 106 pM or less, for example, when measured using Octet analysis (see, for example, Example 9). D The aslo0452 ngl-3 antibody and the aslo0543 antibody bind to α-synuclein with a K of less than 300 picomolar concentration (pM), less than 250 pM, less than 200 pM, less than 150 pM, less than 120 pM, less than 110 pM or 108 pM or less, less than 100 pM, less than 80 pM or 74 pM or less, for example, when measured using KinExA analysis (see, for example, Example 9 for the reference KinExA analysis protocol). D to bind to α-synuclein.

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

[0090] The aslo0452 ngl-3 antibody and the aslo0543 antibody bind to naturally occurring endogenous human α-synuclein. The aslo0452 ngl-3 antibody and the aslo0543 antibody bind to aggregates of human α-synuclein. Therefore, in particular, the antibodies bind to epitopes that are not required for aggregation. The aslo0452 ngl-3 antibody and the aslo0543 antibody can isolate both the monomeric and aggregated forms of α-synuclein. The antibodies of the present invention or their antigen-binding fragments can bind to both the monomeric and aggregated forms of α-synuclein.

[0091] The aslo0452 ngl-3 antibody and the aslo0543 antibody bind to disease-associated lesion morphologies of α-synuclein, such as Lewy bodies, Lewy neurites, and Lewy punctates in Parkinson's disease brain tissue. Minimal staining is observed in normal (non-disease) brain tissue.

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

[0093] The aslo0452 ngl-3 antibody reduces α-synuclein levels in cerebrospinal fluid. Specifically, it reduces levels of free, unbound α-synuclein in cerebrospinal fluid. The aslo0452 ngl-3 antibody also reduces α-synuclein diffusion in vivo. This novel function of inhibiting α-synuclein diffusion demonstrates binding affinity to different epitopes on human α-synuclein compared to antibodies that do not inhibit diffusion.

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

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

[0096] Whether an antibody or its antigen-binding fragment binds to an epitope of a reference antibody or antigen-binding fragment as defined above can be easily determined. Such methods are common practice 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 incubated simultaneously with a given antigen. If a binding signal is obtained for the labeled antibody, the two antibodies are considered to recognize distinct, non-overlapping epitopes on the target protein. If no binding signal is obtained, conversely, they will be characterized as having overlapping epitopes on the protein sequence, since the binding of one antibody sterically prevents the binding of the second antibody. Furthermore, the amino acid localization of a given epitope can also be identified using modified proteins such as truncates, linear peptide sequences derived from the primary amino sequence of the antigen, species orthologues, and by proteolytic digestion and mass spectrometry of the antibody binding to the given protein. These methodologies serve to generate regions of antibody-antigen interaction.

[0097] Additional conventional experiments (such as peptide mutation and binding analysis) may be performed to confirm whether any observed binding deficiencies are actually due to binding to the epitope of the present invention, or to some other phenomenon (such as steric hindrance). Such experiments may be performed using ELISA, RIA, Biacore, flow cytometry, or other known antibody binding assays.

[0098] For example, for detailed mapping of specific epitopes, a mathematical model of the epitope-paratope interface can be derived from data generated by degrading 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 in identifying the key contact residues that define the epitope, point mutagenesis of the antigen and subsequent analysis of the effect of such established mutations on the strength of binding between the antigen and antibody must be performed. Using a combination of these methods, an accurate map of the key contact residues containing the epitopes can be established.

[0099] An antibody or antigen-binding fragment of the present invention that binds to an epitope of the antibody or antigen-binding fragment of the present invention can be generated by creating a variant of the antibody or antigen-binding fragment of the present invention. Such a variant antibody or antigen-binding fragment may have a CDR that shares a high level of identity with the CDR of the antibody or antigen-binding fragment 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 from 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 an antibody may have one or more mutations (e.g., conservative amino acid substitutions) in the framework region.

[0100] In one embodiment, the antibody or antigen-binding fragment of the present invention has mutations in the CDR amino acid sequence that maintain sequence identity of 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% with respect to the CDR of the antibody aslo0452 ngl-3.

[0101] In particular, conservative amino acid substitutions are intended. Conservative substitutions are those performed within the range of amino acid families that have 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: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. These families can be further classified: serine and threonine are aliphatic hydroxyl families; asparagine and glutamine are amide-containing families; alanine, valine, leucine, and isoleucine are aliphatic families; and phenylalanine, tryptophan, and tyrosine are aromatic families. In other words, generally speaking, independent substitutions such as leucine to isoleucine or valine, aspartic acid to glutamic acid, or threonine to serine, or similar substitutions from one amino acid to a structurally related amino acid, can be predicted to have little effect on the binding function or properties of the resulting antibody, especially if the substitution does not involve an amino acid within the CDR site.

[0102] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises at least one CDR selected from the following: (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of sequence number 6, (iii) H-CDR3 of sequence number 7, (iv) L-CDR1 of sequence number 9, (v) L-CDR2 of sequence number 10, (vi) L-CDR3 of sequence number 11.

[0103] In one embodiment, the antibody or its antigen-binding fragment of the present invention has at least one CDR selected from the CDRs of the 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 its antigen-binding fragment of the present invention is the CDR3 of the heavy chain of antibody aslo0452 ngl-3; and / or the CDR3 of the light chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of the light chain of antibody aslo0452 ngl-3. That is, in one embodiment, the CDR3 of the heavy chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of SEQ ID NO: 16 of the heavy chain of antibody aslo0452 ngl-3; and / or the CDR3 of the light chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of SEQ ID NO: 21 of the light chain of antibody aslo0452 ngl-3. In a further embodiment, the CDR3 of the heavy chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of the heavy chain of the antibody aslo0452 ngl-3. In one embodiment, the CDR3 of the light chain of the antibody or its antigen-binding fragment of the present invention is the CDR3 of the light chain of the antibody aslo0452 ngl-3.

[0105] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises six CDRs of the antibody aslo0452 ngl-3, namely 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, comprising a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 with the nucleotide sequence defined by SEQ ID NO: 18.

[0107] The present invention also provides an antibody or an 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, comprising a variable heavy chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 with the amino acid sequence defined by SEQ ID NO: 19.

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

[0110] The present invention provides an antibody or an antigen-binding fragment thereof, 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 of the present invention or its antigen-binding fragment has six CDRs of the antibody aslo0543.

[0111] In other words, in one embodiment, the antibody or antigen-binding fragment of the present invention comprises the following: (a) Three heavy chain CDRs having the following sequences: (i) H-CDR1 of sequence number 25, (ii) H-CDR2 of Sequence ID No. 26; and (iii) H-CDR3 of Sequence ID No. 27, and (b) Three light chain CDRs having the following sequences: (i) L-CDR1 of sequence number 31, (ii) L-CDR2 of Sequence ID No. 32, and (iii) L-CDR3 of sequence number 33.

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

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

[0114] The present invention also provides an antibody or antigen-binding fragment thereof, 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 further embodiments, the antibody or its antigen-binding fragment 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 region and CDR or antibody 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 mutations in the amino acid sequence of the antibody or its antigen-binding fragment of the present invention are considered to be included by the present invention, provided that such mutations maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, and most preferably at least 99% sequence identity with respect to the antibody or its antigen-binding fragment of the present invention as defined elsewhere herein. In particular, conservative amino acid substitutions are intended.

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

[0119] The 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 execution 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. This may be done by means of 53705) or by visual inspection (see Current Protocols in Molecular Biology, FM Ausbel et al, eds, Current Protocols, a joint venture between Greene Publishing Associates, In. And John Wiley & Sons, Inc. (1995 Supplement) Ausbel; incorporated herein by reference).

[0120] Examples of suitable algorithms for determining sequence similarity or identity (%) include 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 “http: / / www.ncbi.nlm.nih.gov / ”; both are incorporated herein by reference). In one embodiment, the antibody or its antigen-binding fragment of the present invention is isolated. In another embodiment, the antibody or its antigen-binding fragment of the present invention is purified.

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

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

[0123] In another embodiment, the antibody or antigen-binding fragment of the present invention has a reduced binding affinity to the IgG Fc receptor. That is, the antibody or antigen-binding fragment of the present invention has low immunogenicity. In one embodiment, the antibody or antigen-binding fragment is an IgG1™ antibody or antigen-binding fragment. IgG1™ is an IgG1 triple mutant containing three point mutations (L234F / L235E / P331S) in the Fc domain that reduce the binding affinity of the antibody or antigen-binding fragment to the Fc-γ receptor (FcγR) (Oganesyan et al. Acta Crystallogr D Biol Crystallogr, (2008) 64: 700-704; incorporated herein by reference). In some embodiments, that is, antibody-mediated prophylaxis of α-synuclein diffusion by the antibody of the present invention does not require Fc-related effector function as the 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 a sufficient framework for binding. Fab fragments may be monovalent fragments consisting of VL, VH, CL, and CH1 domains. F(ab')2 fragments may be bivalent fragments consisting of two Fab fragments linked via disulfide crosslinking in the hinge region. Fc fragments may consist of CH2 and CH3 domains. Fv fragments may consist of the VL and VH domains of a single arm of the antibody. dAb fragments (Ward et al. Nature (1989), 341: 544-546; incorporated herein by reference) may consist of a VH domain. Isolated complementarity-determining regions (CDRs) with a sufficient framework for binding may be antigen-binding portions of the variable region.

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

[0126] The antibody or its antigen-binding fragment of the present invention may have any or all of the advantageous properties defined above, or a combination thereof. In particular, the antibody or its antigen-binding fragment of the present invention may be selective for α-synuclein and may be able to delay or prevent the intercellular transfer and diffusion of α-synuclein in vivo.

[0127] The functionality of the obtained antibody of the present invention or its antigen-binding fragment, and in particular (i) its ability to bind to the epitope of α-synuclein; and (ii) its ability to delay or prevent intercellular transduction and diffusion of α-synuclein in vivo, can be readily determined by assaying its specific activity using the techniques described herein in the examples.

[0128] This disclosure provides compositions for the delivery of antibodies or antigen-binding fragments of the present invention across the blood-brain barrier (BBB) ​​using transporter molecules, the transporter molecules being able to pass through brain endothelial cells while associating with, for example, fusing with or conjugating to, the antibody or fragment. BBB sequences are provided herein.

[0129] As used herein, the term “payload” is an abbreviation for an antibody or its antigen-binding fragment as described herein, whose transport across the blood-brain barrier (BBB) ​​can be facilitated by the transporter molecules provided herein. In certain embodiments, “payload” includes the heavy chain variable region of the antibody of the present invention, more particularly the heavy chain variable region of also0452 ngl-3 or aslo0543.

[0130] The payload may be a portion of the transporter molecule, for example, as a fusion polypeptide, or it may be attached to the polypeptide via a disulfide bond or other covalent bond. Alternatively, the payload may associate with the transporter molecule in any manner that would enable the transporter molecule to facilitate its transport across the blood-brain barrier (BBB), as further described below. In certain embodiments, the payload remains a portion of the transporter molecule after BBB transport and retains central nervous system (CNS) activity in that form. Alternatively, the payload may be associated with the transporter molecule during BBB transport but may be able to separate from the transporter molecule after BBB transport.

[0131] This disclosure further provides methods for the treatment or diagnosis of diseases or disorders of the CNS, particularly alpha-synucleinopathy, comprising the use of such transporter molecules associated with the antibody or its antigen-binding fragment.

[0132] In certain embodiments, the disclosure provides isolated transporter molecules comprising immunoglobulin-derived polypeptides. In certain embodiments, the polypeptide is a mimetic or non-mimicking of the camel antibody FC5, identified and isolated using Fluorescence Micro-volume Assay Technology (FMAT) to detect binding affinity 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 that the transporter molecule can bind to BMVECs in one or more species (e.g., mouse BMVECs, rat BMVECs, cynomolgus monkey BMVECs, or human BMVECs), can be internalized into BMVECs in one or more species, and / or can cross 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 (which is incorporated herein by reference in its entirety). In certain embodiments, the transporter molecule includes 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 (which is 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 germline-treated; for example, Bbbt0626gl is a germline version of Bbbt0626, referred to as "Bbbt0626gl". In further specific embodiments, the BBB transporter molecule is BBBt0632gl or Bbbt0626gl.

[0135] In certain embodiments, transporter molecules do not bind to BMVECs but are still capable of transporting via the BBB, as demonstrated by in vitro transcytosis assays.

[0136] With respect to the BBB transporter molecule, the described VH CDR sequences 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 positions 23-34 of Kabat in the VL (in contrast, the classical L-CDR1 position according to the Kabat numbering system corresponds to positions 24-34).

[0137] In certain embodiments, the immunoglobulin-derived polypeptide includes immunoglobulin heavy chain complementarity-determining regions (CDRs). For example, the immunoglobulin-derived polypeptide may include immunoglobulin heavy chain complementarity-determining region-1 (H-CDR1), immunoglobulin heavy chain complementarity-determining region-2 (H-CDR2), and immunoglobulin heavy chain complementarity-determining region-3 (H-CDR3). In certain embodiments, the immunoglobulin-derived polypeptide may further include, or instead include, immunoglobulin light chain CDRs. For example, the immunoglobulin-derived polypeptide may include immunoglobulin light chain complementarity-determining region-1 (L-CDR1), immunoglobulin light chain complementarity-determining region-2 (L-CDR2), and immunoglobulin light chain complementarity-determining region-3 (L-CDR3).

[0138] In certain embodiments, the immunoglobulin-derived polypeptides include H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each having the following amino acid sequence: (a) Sequence ID 40 as H-CDR1, Sequence ID 41 as H-CDR2, Sequence ID 42 as H-CDR3, Sequence ID 36 as L-CDR1, Sequence ID 37 as L-CDR2, and Sequence ID 38 as L-CDR3 (in this case, the CDRs are similar to those of Bbbt0626 and Bbbt0626gl); (b) Sequence ID 40 as H-CDR1, Sequence ID 41 as H-CDR2, Sequence ID 42 as H-CDR3, Sequence ID 44 as L-CDR1, Sequence ID 45 as L-CDR2, and Sequence ID 46 as L-CDR3 (in this case, the CDR is the same as that of Bbbt0626 and Bbbt062gl).

[0139] In certain embodiments, the immunoglobulin-derived polypeptides include H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each having the following amino acid sequence: (a) Sequence ID 49 as H-CDR1, Sequence ID 50 as H-CDR2, Sequence ID 51 as H-CDR3, Sequence ID 53 as L-CDR1, Sequence ID 54 as L-CDR2, and Sequence ID 55 as L-CDR3 (in this case, the CDR is similar to that of Bbbt0632gl); (b) Sequence ID 49 as H-CDR1, Sequence ID 50 as H-CDR2, Sequence ID 51 as H-CDR3, Sequence ID 53 as L-CDR1, Sequence ID 54 as L-CDR2, and Sequence ID 55 as L-CDR3 (in this case, the CDR is the same as that of Bbbt0632gl).

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

[0141] In certain embodiments, H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 can be arranged within an immunoglobulin framework region to generate antibodies VH and VL. In certain embodiments, the framework region may be a human-derived framework region. In certain embodiments, antibodies VH and VL are fused together, for example, via a flexible peptide linker, to form an scFv molecule. In certain embodiments, VH and VL further include 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, one or more immunoglobulin constant domains are derived from human immunoglobulin, e.g., human IgG1 immunoglobulin. In certain embodiments, the VH, VL, and / or constant domains may include mutations to enhance, for example, a longer or shorter half-life, increased or decreased effector function, or the ability to bind to the payload molecule via peptide fusion, disulfide bond, or chemical conjugate.

[0142] In certain aspects of the present invention, an antibody or antigen-binding fragment of the present invention is provided, which is associated with a transporter molecule that can pass through brain endothelial cells as described herein.

[0143] In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment 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 embodiments, the immunoglobulin-derived polypeptide comprises sequences 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 (where 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 (where 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 embodiments, the present disclosure provides an antibody or antigen-binding fragment 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, in which case: (a) The VH region contains sequence number 34 and the VL region contains sequence number 35; or (b) The VH region contains sequence number 39 and the VL region contains sequence number 43; or (c) The VH region contains sequence number 39 and the VL region contains sequence number 35; or (d) The VH region contains sequence number 47 and the VL region contains sequence number 43; or (e) The VH region contains sequence number 47 and the VL region contains sequence number 35; or (f) The VH region includes sequence number 48, and the VL region includes sequence number 52. In further embodiments, the VH and VL regions of the transporter molecule described above covalently bind to form a single-chain fragment (ScFv).

[0145] In certain embodiments, the transporter molecules provided herein have transporter activity and can, for example, bind to BMVECs derived from one or more species (e.g., mouse, rat, cynomolgus monkey, or human BMVECs), be internalized in BMVECs of one or more species, or cross the blood-brain barrier.

[0146] In certain embodiments, the 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] Where used herein, “BBB-permeable fragment” means a fragment of a transporter molecule that can specifically bind to BMVECs of one or more species and can pass through BMVECs from the peripheral vascular system to the CNS vascular system 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, diuretic assays as described in US 62 / 094,503, and other in vivo assays. Other assays that may be used to measure the in vivo delivery of payloads across the blood-brain barrier (BBB) ​​include, but are not limited to, chronic ligation injury (CCI); spared nerve injury model (SNI); or spinal nerve ligation (SNL), all of which can be measured via paw flick or Hargreaves' method (Hargreaves K, et al., Pain; 1988; 32; 77-88). In certain embodiments, the transporter molecules provided herein may bind to BMVECs (e.g., human, cynomolgus monkey, mouse, rat, or bovine BMVECs) derived from one or more species. Binding can be demonstrated in various ways known to those skilled in the art, for example, by the FMAT assay as described in US 62 / 094,503. In certain embodiments, BMVECs are cerebral capillary endothelial cells (BCECs). In certain embodiments, transporter molecules as 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 visualization of the transporter molecule in the CNS. For example, a tritium-labeled transporter molecule can be delivered to a subject (e.g., peripherally in a mouse, e.g., intravenously) and subsequently visualized in the CNS via quantitative whole-body radiography.In certain embodiments, transporter molecules are localized to specific regions of the CNS, such as the cerebellar cortex, cerebral gray matter, spinal gray matter, pons, or a combination thereof.

[0148] In certain embodiments, the transporter molecule as described herein comprises an antibody or a BBB-permeable fragment thereof, comprising or consisting of two or more subunits (for example, a heavy chain or a fragment thereof and a light chain or a fragment thereof, wherein the heavy chain and light chain associate, 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 present invention provides an antibody or antigen-binding fragment thereof associated with a blood-brain barrier transporter molecule as described herein.

[0149] In certain embodiments, the antibody or antigen-binding fragment of the present invention is associated with a blood-brain barrier transporter molecule, in which case the transporter molecule is a single-chain fragment (scFv) comprising the following: i. The heavy chain variable region (VH) of BBBt0626gl in SEQ ID NO: 39 and the light chain variable region (VL) of BBBt0626gl in SEQ ID NO: 43, or ii. The heavy chain variable region (VH) of BBBt0626 in sequence number 47 and the light chain variable region (VL) of BBBt0626 in sequence number 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 heavy chain constant domains, e.g., a CH1 domain, a hinge, a CH2 domain, and / or a CH3 domain, or fragments thereof. In certain embodiments, the heavy chain constant domain is an IgG constant domain or 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 fragment thereof comprises altered glycosylation and / or one or more amino acid substitutions compared to a wild-type IgG constant domain, in which case the modified IgG has certain properties, e.g., an increased or decreased half-life compared to IgG having a wild-type IgG constant domain, increased or decreased effector function compared to a wild-type IgG constant domain, or the ability to bind to heterologous moieties, e.g., via peptide bonds, disulfide bonds, or chemical conjugates. In certain embodiments, the IgG constant domain or a fragment thereof has altered glycosylation compared to the wild-type IgG constant domain, in which case the modified IgG has certain properties, such as an increased or decreased half-life compared to 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, the antibody of the present invention or its antigen-binding fragment associates with BBBt0626 or BBBt0626gl as defined herein to form a bispecific antibody molecule.

[0152] In other embodiments, the bispecific antibody of the present invention comprises a human IgG1™ skeleton (i.e., the CH1, CH2, CH3 regions of the IgG1™ heavy chain) associated with a single-chain fragment (scFv) containing the VH and VL regions of BBBt0626 or BBBt0626gl, grafted onto the N-terminus ("BiS2 form") or C-terminus ("BiS3 form") or VL-terminus ("BiS1 form") of the anti-α-synuclein antibody of the present invention. References to the BiS forms are disclosed in DiMasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92. In some embodiments, the bispecific antibody of the present invention further comprises a light chain including a κ or λCL region associated with the VL of the anti-α-synuclein antibody of the present invention.

[0153] In certain embodiments, the bispecific antibody of the present invention comprises a human IgG1™ skeleton associated with the following: (i) A single-chain fragment (scFv) of BBBt0626gl containing the heavy chain variable region (VH) of SEQ ID NO: 39 and the light chain variable region (VL) of SEQ ID NO: 43; or (ii) A single-chain fragment (scFv) of Bbbt0626 containing 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 form) or C-terminus (BiS3 form) of the heavy chain of aslo0452 ngl-3 of sequence number 12, or the N-terminus ("BiS1 form") of the light chain of sequence number 17.

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

[0156] In other specific embodiments, the bispecific antibody of the present invention comprises a human IgG1™ skeleton associated with: (i) A single-chain fragment (scFv) of BBBt0626gl containing the heavy chain variable region (VH) of SEQ ID NO: 39 and the light chain variable region (VL) of SEQ ID NO: 43; or (ii) A single-chain fragment (scFv) of Bbbt0626 containing 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 form) or C-terminus (BiS3 form) of the heavy chain of aslo0543 in sequence number 22, or the N-terminus ("BiS1 form") of the light chain of sequence number 28.

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

[0158] The present invention also provides the use of antibodies or antigen-binding fragments thereof for the manufacture of pharmaceuticals for the prevention or treatment of central nervous system diseases, particularly alpha-synucleinopathy. In one embodiment, the alpha-synucleinopathy is selected from Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). In a preferred embodiment, the alpha-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 the step of administering an antibody or antigen-binding fragment of the present invention to the patient. In one embodiment, the alpha-synucleinopathy is selected from Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). In a preferred embodiment, the alpha-synucleinopathy is Parkinson's disease (PD).

[0160] When used, the antibody or its antigen-binding fragment of the present invention can treat or prevent disease progression by inhibiting the propagation and diffusion of α-synuclein in vivo. The antibody or its antigen-binding fragment of the present invention offers advantages that differentiate it from other therapeutic agents. The present invention also provides a method for delaying or preventing disease progression in a subject requiring it, comprising the step of administering the antibody or its antigen-binding fragment of the present invention to a patient.

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

[0162] The dosage range for administering the antibody or its antigen-binding fragment of the present invention is intended to produce the desired therapeutic effect. The required dosage range depends on the exact properties of the antibody or its antigen-binding fragment or composition, the route of administration, the properties of the formulation, the patient's age, the nature, degree, or severity of the patient's condition, any contraindications, and the judgment of the attending physician. Changes to these dosage levels can be adjusted using standard empirical methods for optimization.

[0163] The preferred dosage is within the range of 1 to 50 mg per kg of body weight. The dosage may be within the range of 5 to 30 mg / kg, 10 to 25 mg / kg, or 15 to 20 mg / kg. The unit dose can be administered once a day or less, for example, once a week or once a month.

[0164] Administration can be carried out by repeated administration of the antibody or antigen-binding fragment of the present invention over a long period of time. Administration may be simultaneous or sequential, and can be carried out in any order.

[0165] The prevention or treatment provided herein may be administered as a single therapy, or may include, in addition to the antibody or antigen-binding fragment of the present invention, the administration of other agents or established therapeutic agents commonly used in the treatment of alpha-synucleinopathy (such as L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (receptor) agonists, catechol-O-methyltransferase (COMT) inhibitors, and / or monoamine oxidase (MAO-B) inhibitors). The administration of other agents or established therapeutic agents may be carried out in combination with, or as an adjunct to, the antibody or antigen-binding fragment of the present invention, and may be by simultaneous, sequential, or separate administration of the individual components of the treatment.

[0166] Combination therapy may be carried out in any manner deemed necessary or convenient by those skilled in the art, and for the purposes of this specification, no restrictions are intended with respect to the order, quantity, repetition or relative quantity of the compounds used in combination.

[0167] A therapeutically effective dose means the amount of an antibody or its antigen-binding fragment that, when administered to a patient alone or in combination to treat at least one of the disease or clinical symptoms of the disease, is sufficient to have an effect on such treatment of the disease or symptom. The therapeutically effective dose may vary, for example, depending on the antibody and / or symptoms of the disease, the age, weight, and / or health status of the patient being treated, and the judgment of the prescribing physician. An appropriate therapeutically effective dose in any given example may be determined by a person skilled in the art or by conventional experimental procedures. The therapeutically effective dose is also such that the beneficial effects of the antibody or the antibody or its antigen-binding fragment outweigh any toxic or adverse effects.

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

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

[0170] The pharmaceutical compositions of the present invention can be formulated for parenteral delivery (e.g., intramuscular, subcutaneous, or intravenous). Suitable compositions for intramuscular, subcutaneous, or intravenous injection include sterile aqueous solutions.

[0171] The pharmaceutical composition may take the form of an aqueous solution and may contain a physiologically compatible buffer (such as Hanks' solution, Ringer's solution, or buffered saline). The pharmaceutical composition may additionally or alternatively contain a substance that increases the viscosity of the suspension (such as sodium carboxymethylcellulose, sorbitol, or dextran). The pharmaceutical composition may be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include oils and fats (such as sesame oil), synthetic fatty acid esters (such as ethyl oleate or triglycerides), or liposomes. Optionally, the pharmaceutical composition may contain a suitable stabilizer or an agent that increases the solubility of the compound to enable the preparation of a highly concentrated solution.

[0172] The present invention provides isolated nucleic acid molecules encoding the antibody or its antigen-binding fragment. The present invention also provides vectors comprising the isolated nucleic acid molecules of the present invention. The present invention further provides host cells comprising the vector of the present invention.

[0173] The antibodies or antigen-binding fragments of the present invention are not limited to any particular method of preparation or production. In other words, the present invention provides antibodies produced from antibody-secreting hybridomas, as well as antibodies produced from recombinantly generated cells transformed or transfected with one or more nucleic acids encoding antibodies. Such hybridomas, recombinantly generated cells, and nucleic acids form part of the present invention. [Examples]

[0174] Example 1: Antibody Production Anti-α-syn specific antibodies were isolated from phage display libraries using a series of selection cycles against recombinant human α-syn ("hu α-syn"), both passively immobilized on microtiter wells and free in solution. Naive human single-chain Fv (scFv) phage display libraries cloned into phagemid vectors based on linear phage M13 were 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 are incorporated herein by reference).

[0175] A representative number of individual clones from the selected 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 Escherichia coli extract (Kipriyanov et al. J Immunol Methods (1997) 200: 69-77; incorporated herein by reference) using a homogeneous FRET (fluorescence resonance energy transfer) HTRF® (Homogeneous Time-Resolved Fluorescence, Cisbio International) assay.

[0176] The HTRF® assay (Figure 1) is a homogeneous assay technique that utilizes the fluorescence resonance energy transfer between adjacent donor and acceptor fluorophores (Mathis G Clin Chem (1995) 41: 1391-1397; incorporated herein by reference). This assay involves using a donor fluorophore (e.g., europium (Eu)) as one of the target molecules. 3+ This assay was used to measure polymer interactions by directly or indirectly coupling a cryptotate molecule with the acceptor fluorophore XL665 (stable, cross-linked allophycocyanin). Excitation of the cryptotate molecule (337 nm) resulted in fluorescence emission at 620 nm. The energy from this emission was transferred to XL665 adjacent to the cryptotate, resulting in specific long-lived fluorescence emission from XL665 (665 nm). By measuring the specific signals of both the donor (620 nm) and acceptor (665 nm), it was possible to calculate the 665 / 620 nm ratio that offsets the presence of colored compounds in the assay.

[0177] Unpurified anti-α-syn scFv samples were tested for binding to biotinylated α-syn. A 5-microliter solution containing 40 nM biotinylated human α-syn combined 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 were added to the plate. Finally, a 5-microliter 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). Assay plates were incubated at room temperature for 3 hours, followed by incubation at 4°C for 16 hours. Time-resolved fluorescence at emission wavelengths of 620 nm and 665 nm was then read using an EnVision plate reader (Perkin Elmer).

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

[0179]

number

[0180]

number

[0181]

number

[0182] Single-chain Fv clones binding to human α-syn as unpurified periplasmic extract 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 are incorporated herein by reference). Intrinsic scFv were reexpressed in bacteria and purified by affinity chromatography (as described in International Publication No. 01 / 66754; incorporated herein by reference). The potency of these samples was determined by titer measurement of the purified preparations for binding to biotinylated human α-syn using the HTRF assay described above. The purified scFv preparation showing the strongest α-syn interaction was selected for conversion to IgG format.

[0183] The clones were ranked in terms of their binding strength to α-syn by measuring antibody titer in an HTRF assay. The binding kinetics of the strongest α-syn-binding substance to α-syn as IgG were measured using an Octet Red biosensor (see the method described in Example 9). offFurther analysis was conducted on the selectivity of synuclein family members (human α-syn, β-syn, γ-syn) (see the method described in Example 4) and cross-reactivity with mouse α-syn (see the method described in Example 5).

[0184] Example 2: Derivation of aslo0452 ngl-3 The C-terminally reactive α-syn-specific clone asyn0087 was identified by screening its binding affinity to human α-syn using the 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 nearest possible human germline sequence (Tomlinson VBASE. MRC Centre of Protein Engineering, Cambridge, UK. 1997; incorporated herein by reference), and this did not affect potency with standard mutagenesis techniques prior to optimization. Following germline re-evaluation, the clone was re-evaluated for its binding affinity to α-syn. No adverse effects were observed.

[0185] Large scFv-phage libraries derived from lead clones were constructed 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 libraries were subjected to affinity-based phage display selection against soluble biotinylated human α-syn to select mutants with relatively high affinity for human α-syn. The selection was carried out essentially as described above, except that the concentration of soluble biotinylated human α-syn was decreased for each round of selection performed.

[0186] Representative clones from each selected output were initially screened by HTRF assay as soluble scFv fragments in periplasmic E. coli extracts for their ability to compete with the parental α-syn-binding clone asyn0087 for binding to soluble α-syn. The performance of each library in these population screenings provided information on which CDR mutagenesis libraries to genetically combine or “recombinate” to create novel libraries, and these recombinant libraries were subjected to a further round of affinity-driven selection against soluble biotinylated human α-syn.

[0187] For both clones derived from individual libraries and recombinant mutagenesis libraries, after sequence analysis of positive binding agents, the clones were expressed as both scFv fragments and IgG, purified, and their binding affinity to soluble α-syn was reconfirmed by epitope-competitive HTRF assay. By titrating the antibodies in the HTRF epitope-competitive assay, the clones were compared to the parental IgG asyn0087 in terms of their IC (Impulse Control Value) and relative improvement in their binding affinity to α-syn. 50 The substances were ranked by their values. The strongest α-syn binding substances 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 using either a direct binding HTRF assay or an epitope-competitive HTRF assay.

[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, which showed positive improvement in potency, was performed on aslo0452 ngl-1 for each CDR. Each selected CDR site was individually mutated through all 20 possible amino acid residues, resulting in improved IC compared to aslo0452 ngl-1 IgG. 50 The following were screened again using an epitope-competitive HTRF assay. Multiple residues were identified across four CDRs (H2, H3, L1, and L3) and combined with both aslo0452 ngl-1 and aslo0467, resulting in improved IC compared to aslo0452 ngl-1. 50 The substances were re-evaluated using an epitope-competitive HTRF assay. From these experiments, two of the most improved binding substances 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 scFv to IgG1™ Single-chain Fv clones possessing the desired α-syn binding properties were converted into an effector-free full immunoglobulin G1™ (IgG1™) antibody format (Oganesyan et al. Acta Crystallogr D Biol Crystallogr. (2008), 64(Pt 6):700-4; incorporated herein by reference), which was essentially as described by Persic et al. (Persic et al, Gene (1997) 187: 9-18; incorporated herein by reference), with the following modifications. An OriP fragment was included in the expression vector to facilitate use with CHO transient cells and to enable episomal replication. The variable heavy chain (VH) domain was cloned into a vector (pEU1.4) containing the human heavy chain constant domain and regulatory elements for expressing the full IgG1™ heavy chain in mammalian cells. Similarly, the variable light chain (VL) domain was cloned into a vector (pEU4.4) for the expression of the human light chain (λ) constant domain and regulatory elements for the expression of the complete IgG light chain in mammalian cells. To obtain IgG, the heavy chain IgG expression vector and the light chain IgG expression vector were transfected into CHO transient mammalian cells (Daramola et al, Biotechnol Prog (2014) 30: 132-141; incorporated herein by reference). IgG was expressed and secreted into the culture medium. The recovered material 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 appropriate size and washed with 50 mM Tris-HCl pH 8.0 and 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 IgG concentration was determined spectrophotometrically using the attenuation coefficient based on the amino acid sequence of IgG (Mach et al, Anal Biochem (1992) 200: 74-80; incorporated herein by reference). The 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 collateral killing resulting from activation of immune cells and complement (i.e., overproduction of C3a, which can cause inflammation). Collateral 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 selected 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 has been brought as close as possible to the human germline amino acid sequence without affecting efficacy. This means that some amino acids in aslo0452 ngl-3, including the Burnier 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 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). Also, in the V domain of aslo0452 ngl-3 L all framework residues match the human germline IGLV5-45 and IGJL2 or IGJL3 sequences (Figure 3D).

[0193] Example 3: Confirmation of affinity-optimized anti-α-syn IgG epitopes Recombinant human α-, β-, and γ-synuclein, recombinant truncates of human α-synuclein (aa1-60, aa1-95, aa61-140, 96-140, ΔNAC, and NCAP), and mouse α-synuclein were obtained from rPeptide. 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 at 4°C overnight. After rinsing the wells with PBS, 1 μg / mL dilutions of each anti-α-syn antibody were added. After 1 hour of incubation and washing, the bound antibody was detected by the addition of anti-human IgG conjugated to either HRP or Eu 3+ After incubation and washing, the appropriate detection substrate was added (TMB or DELFIA enhancer solution, respectively), and the plates were 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 (Figure 4A). Both aslo0452 ngl-3 and aslo0543 maintain the recognition of an epitope located in the C-terminal region of the α-syn protein from amino acids 102 to 130, the same as their parental lead isolate asyn0087 (Figure 4B).

[0196] Example 4: Specificity of aslo0452 ngl-3 and aslo0543 to α-syn compared with synuclein family members using an epitope-competitive HTRF assay. Specificity to human α-syn is important for antibodies intended for therapeutic use in order 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 to other synuclein family members, β-syn and γ-syn, was determined using an HTRF epitope competition assay that measures the binding affinity of biotinylated human α-syn to antibodies in solution.

[0198] α-syn, β-syn, and γ-syn were titrated for assays, and their selectivity for aslo0452 ngl-3 IgG and aslo0543 IgG was evaluated by measuring the degree of inhibition of biotinylated human α-syn binding to aslo0452 ngl-3 / aslo0543. 50 The values ​​were determined by curve fitting the data to a four-parameter logical equation using PRISM 6® software (Graphpad). A more sensitive HTRF assay, which measures the direct binding affinity 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, the antibody test sample was replaced with only the isotype control antibody or buffer.

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

[0200] Example 5: Specificity of aslo0452 ngl-3 against human, cynomolgus monkey, and rat α-syn using an HTRF epitope competition assay. For therapeutic applications, it is important that the antibody cross-reactive to cynomolgus monkey α-synuclein to within 10 times the reactivity observed to human α-syn, and preferably to rat α-synuclein. This is because it allows safety studies to be conducted in both cynomolgus monkey and rat species.

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

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

[0203] Representative ICs obtained using human, cynomolgus monkey, and rat α-syn proteins in HTRF epitope competition assays. 50 The values ​​are shown in Figure 6. The aslo0452 ngl-3 ICs have impedances of 5.7 nM and 6.8 nM, respectively. 50 It binds to human and cynomolgus monkey α-syn at a value of 19.6 nM (within 4 times) IC2. 50The values ​​indicate binding to rat α-syn. aslo0543 has IC2.0 nM and 2.1 nM IC2. 50 It binds to human and cynomolgus monkey α-syn at a value of 3.8 nM (within 2x) IC 50 It binds to rat α-syn at a certain value.

[0204] The ability of aslo0452 ngl-3 to bind to human, cynomolgus monkey, and rat α-syn demonstrates its ability to bind to different epitopes 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 natural α-syn as measured by flow cytometry The specificity of affinity-optimized anti-α-syn IgGs, aslo0452 ngl-3 and aslo0543, for binding to naturally occurring endogenous human α-syn was determined by flow cytometry using α-syn-positive and α-syn-negative cell lines.

[0206] In short, SHSY5Y neuroblastoma cells (α-syn positive) and BT-20 breast cancer cells (α-syn negative) were fixed in 0.01% formaldehyde, then permeabilized with 0.5% (v / v) Tween20, and subsequently incubated with anti-α-syn antibody, positive control, or isotype control antibody. After thorough washing, the bound antibody was detected by incubation with anti-human or anti-mouse IgG-FITC secondary antibody. After further washing, the 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] The data are plotted as a histogram showing the difference between cells stained alone and cells stained with the primary antibody. The results in Figure 7A show a shift in the fluorescence signal in α-syn-positive SH-SY5Y cells in the presence of asyn0087 (Panel D) compared to isotype control and secondary antibody alone (Panel B), which indicates the recognition of endogenously expressed α-syn. asyn0087 does not bind to BT-20, an α-syn-negative human breast cancer cell line (Panel C). The results in Figure 7B show a strong shift in the fluorescence signal in the presence of either aslo0452 ngl-3 or aslo0543 (Panel H), comparable to the positive control antibody 4D6 against α-syn-positive SH-SY5Y cells (Panel F), while no shift is shown in α-syn-negative BT-20 cells (Panel G). This demonstrates that both aslo0452 ngl-3 and aslo0543 bind to naturally endogenously expressed intracellular human α-syn.

[0208] Example 7: Specificity of optimized anti-α-syn IgG against aggregated human α-syn by DELFIA ELISA Fibrous preparations or aggregates of human α-syn were prepared 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 Saalstat tubes and placed in a 37°C shaking incubator at 280 rpm for 3 days. The presence of aggregated α-syn was determined by the uptake of thioflavin T, which was added to a final concentration of 10 μM, incubated in the dark at room temperature for 1 hour, and whose fluorescence was read using an Envision microplate reader at excitation wavelengths of 450 nm and emission wavelengths of 485 nm.

[0209] The specificities of aslo0452 ngl-3 and aslo0543, which are affinity-optimized anti-α-syn IgGs, and the lead antibody asyn0087 for aggregated human α-syn were 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, the mouse IgG1 version of the anti-α-syn antibody was immobilized onto the wells of a 96-well microtiter plate (Nunc). After blocking, aggregated or monomeric human α-syn was incubated in the wells. After washing, the captured human α-syn was detected by the addition of the human IgG1 TM version of the same anti-α-syn antibody and subsequent addition of 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 on a microtiter plate reader.

[0210] In this assay, only aggregated human α-syn should be captured and detected because multiple copies of the same epitope are present on a single aggregate. In monomeric α-syn, there is only one copy of the epitope, and thus the secondary antibody for detection cannot bind 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. That is, the epitope to which asyn0087 binds is not itself involved in the aggregation of α-syn. 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 affinity-optimized anti-α-syn IgGs aslo0452 ngl-3 and aslo0543, as well as the lead antibody asyn0087, for disease-related forms of human α-syn was determined by immunohistochemical staining of Parkinson's disease brain tissue. The results are shown in Figure 9, demonstrating that both aslo0452 ngl-3 and aslo0543, as well as asyn0087, can recognize disease-related lesion forms of human α-syn in Parkinson's disease brain tissue sections, including Lewy bodies, Lewy neurites, neural aggregates, Lewy nodules, and background brain tissue. No nonspecific staining was observed in normal or healthy brain tissue.

[0212] Example 9: Measurement of affinity of anti-α-syn antibody Equilibrium dissociation constant (K) for anti-α-syn IgG against human α-syn D The following two platform technologies were used to determine the optimal configuration: Octet Red (Forte Bio) and KinExA (Sapidyne Instruments).

[0213] Both assay systems showed good agreement, indicating that the aslo0452 ngl-3 affinity was within the sub-nanomolecal range. Table 1 shows affinity measurements derived from the major anti-α-syn clones generated through the read isolation and read optimization processes.

[0214] Example 9.1: Affinity of aslo0452 ngl-3 by Octet The affinity of aslo0452 ngl-3 IgG for recombinant bacterial monomer-type human avi tag α-syn-Flag-His was evaluated using an Octet Red instrument. aslo0452 ngl-3 was pre-mixed with various ligand concentrations until equilibrium was reached. Subsequently, the amount of free antibody was measured using Octet by capturing the 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 The following 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 by KinExA In addition, the solution affinity (K) of aslo0452 ngl-3 IgG to recombinant bacterial monomer-type human biotinylated α-syn D The equilibrium dissociation constant (K) was determined using a KinExA instrument (Sapidyne Instruments). aslo0452 ngl-3 was pre-mixed with various ligand concentrations until equilibrium was reached. Subsequently, the amount of free antibody was measured using KinExA by capturing the free aslo0452 ngl-3 with α-syn coated beads, washing away unbound material, and detecting the bound antibody using a fluorescently labeled species-specific antibody. The amount of free antibody detected at each α-syn concentration was plotted against the ligand concentration, and the equilibrium dissociation constant (K) was determined using KinExA software. D The affinity 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 shows good agreement with the Octet solution-phase affinity assay described above.

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

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

[0218] One day before the experiment, a push-pull probe (4 mm 1-3 MDa polyethylene membrane) was implanted in the prefrontal cortex using a stereotactic frame (coordinates relative to the probe: AP = -3.4 mm (relative to bregma), +0.8 mm laterally (relative to the midline), -5.0 mm ventrally (relative to the dura mater), and the incisor bur was set to -3.3 mm (all coordinates follow 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, a push-pull microdialysis probe was connected to a microperfusion pump (Harvard) using a flexible PEEK tube (Western Analytical Products Inc. USA; PK005-020), and artificial CSF (perfusion fluid) (containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2 + 0.2% BSA) was perfused at a flow rate of 0.5 μL / min. The probe outlet was connected to a flexible FEP tube. After a minimum of 2 hours of pre-stabilization, aslo0452 ngl-3 formulated in PBS or PBS alone (vehicle) was administered at 30 mg / kg or 0 mg / kg, respectively. The compound was administered intravenously at 2 mL / kg. Microdialysis samples were collected at 120-minute intervals. The samples were collected in mini vials (Microbiotech / se AB, Sweden; 4001029). All samples were stored at -80°C.

[0220] To determine the free α-synuclein concentration in rat ISF, first, microdialysis samples were subjected to immunoprecipitation to remove aslo0452 ngl-3. Immunoprecipitation coprecipitates α-synuclein bound to the therapeutic antibody, but unbound "free" α-synuclein remains in the supernatant. A solution of Protein A beads (DynaBeads® Protein A) was added to a 96-well skirtless plate (0.2 mL polypropylene), and a magnet (DynaMag) was used to separate the beads from the solution. TM The samples were washed twice with TBST (50 mM TBS + 0.1% Tween 20) using a 96 side). Thawed rat ISF microdialysis samples (10 or 20 μL) were added to each well and mixed with the beads by pipetting up and down, then incubated at 4°C for 10 minutes with tilt rotation. Subsequently, the beads were pelleted twice using a magnet to completely remove them. The immunoprecipitated ISF samples were subjected to an anti-α-synuclein ELISA kit (Sensolyte). TM The sample was transferred to a 96-well plate pre-filled with sample dilution buffer from the 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 two replicates, 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 protection 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 in the dark at room temperature for 10–15 minutes. To stop the reaction, 50 μL of stop solution was added to each well, and the plate was read at 450 nm absorbance within 2 hours. Quantification was performed by plotting the response of the standard curve 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 ISF after a single intravenous administration of aslo0452 ngl-3 at 30 mg / kg (Figure 10).

[0221] Example 11: Effect of aslo0452 ngl-3 on free unbound α-synuclein levels in CSF of male Sprague Dawley rats Adult male Sprague Dawley rats were anesthetized, and a catheter was placed in the cisterna magna to allow for 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 secured in place using dental acrylic cement and attached to the skull with three stainless steel screws. The animals were allowed to recover for at least two days before administering the compound.

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

[0223] After collecting clean CSF samples at least four times over a minimum of two days, the compounds were administered. All animals were administered either aslo0452 ngl-3 or the vehicle on day "0". CSF samples were collected at each indicated time point. All samples were stored at -80°C until shipment.

[0224] To measure the free α-synuclein in CSF, α-synuclein bound to aslo0452 ngl-3 was removed by immunoprecipitation (IP) before analysis. IP coprecipitates α-synuclein bound to the therapeutic antibody, but unbound "free" α-synuclein remains in the supernatant. The level of free α-synuclein in the supernatant was determined using a commercially available ELISA kit obtained from Anaspec. The 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 CSF was demonstrated after a single intravenous administration of aslo0452 ngl-3 in the dose range of 3–100 mg / kg (Figure 11).

[0226] Example 12: Functional characterization of aslo0452 ngl-3 by reduction of α-synuclein diffusion in a lentiviral in vivo model of α-synucleinopathy. The ability of the high-affinity anti-α-synuclein antibody aslo0452 ngl-3 to inhibit α-synuclein diffusion was investigated using a lentiviral in vivo mouse model of α-synucleinopathy. For this purpose, both non-transgenic wild-type mice (non-tg) and α-synuclein-overexpressing transgenic mice (α-syn tg) were injected into the right hippocampus with a lentiviral vector expressing α-synuclein (LV-α-syn), and subsequently passively immunized weekly for 13 weeks with an anti-α-synuclein mouse IgG1 antibody containing aslo0452 ngl-3 and isotype control mouse IgG1 NIP228. At the end of the immunization period, the mice were euthanized, their brains were fixed in 4% PFA, and then coronal sections were prepared. The levels of ipsilateral and contralateral α-synuclein immunoreactivity relative to the LV-α-syn injection site were analyzed by immunocytochemistry using automated image analysis.

[0227] Surgery and passive immunization Non-transgenic wild-type mice (non-tg; n=40) and α-synuclein transgenic mice (α-syn tg; n=40) aged 3-4 months were subjected to a single unilateral injection of an α-synuclein-expressing lentiviral vector (LV-α-syn) into the right hippocampus (bregma -2.0, 1.5, -1.3). Two weeks after LV-α-syn injection surgery, mice were administered either 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), or 9E4 (non-tg n=10; α-syn tg n=10), or with NIP228 isotype control mouse IgG1 (non-tg n=10; α-syn tg n=10).

[0228] All mice were administered IgG at a dose of 20 mg / kg via the intraperitoneal (IP) route for 13 weeks. Animals were housed in groups of the maximum number per cage or in groups of four. Animals were housed in a 12 / 12 light-dark cycle with free access to food and water. Cages were changed weekly and animals were monitored daily. No adverse events were reported. All animals tolerated surgical procedures and immunization. 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 along the coronal axis and evaluated by immunocytochemistry for neuropathological analysis of α-synuclein diffusion.

[0229] α-Synuclein Immunocytochemistry Brains were removed, fixed in 4% paraformaldehyde, and sections were prepared at 40 μm intervals along the coronal axis using a vibratome. These sections were stored at -30°C in a cryoprotective medium (30% glycerin, 30% ethylene glycol, 40% PBS). After PBS washing and blocking buffer steps, the sections were incubated overnight at 4°C with a primary antibody (anti-α-synuclein mAb SYN-1 (BD), 1:500 dilution), washed in PBS, and incubated with a secondary antibody (biotinized anti-mouse IgG (Vector Laboratories), 1:100 dilution) at room temperature for 1 hour. After the final PBS washing step, α-synuclein staining was localized using an avidin / biotin-peroxidase complex detection system (Elite ABC, Vector Laboratories). Next, the sections were analyzed using automated image analysis to determine the level of α-synuclein on the ipsilateral and contralateral sides relative to the lentiviral vector (LV-α-syn) injection site.

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

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

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

[0233] In contrast, in non-transient (TG) mice passively immunized with either the aslo0452 ngl-3 antibody or the aslo0452 ngl-3 no-effector mutant (aslo0452-ngl-3-D265A) (a substitution of aspartate to alanine at position 265 in mouse IgG1 (D265A) eliminates the interaction between this isotype and low-affinity IgG Fc receptors (FcγRIIB and FcγRIII) found in microglia), lentivirally expressed α-synuclein levels in both the ipsilateral and contralateral hippocampal regions were significantly lower than in non-transient (TG) mice passively immunized with NIP228 isotype control mouse IgG1 (Figure 12A, B, C; aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared to NIP228). This indicates that passive immunization of mice using either aslo0452-ngl-3 or the uneffector D265A mutant of aslo0452-ngl-3 reliably inhibits α-synuclein diffusion 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 using aslo0452-ngl-3 or aslo0452-ngl-3-D265A reliably and statistically significantly reduced ipsilateral and contralateral levels of α-synuclein immunoreactivity in the hippocampus compared to NIP228-treated α-syn tg mice, but not in 9E4 (Figure 15A, B, C).

[0234] At higher magnification, lentiviral-expressed α-synuclein immunoreactive deposits could be observed along both the ipsilateral axon on the injected side and the contralateral axon on the uninjected side of non-transient mice (Figure 13A; black arrows indicate interhippocampal axons), suggesting that α-synuclein diffusion to the contralateral hippocampus can, in principle, occur along the axon (transaxonal diffusion). Ipsilateral and contralateral levels of α-synuclein deposits on axons in non-transient mice passively immunized with either 9E4 antibody or NIP228 isotype control mouse IgG1 did not differ significantly (Figures 13A, B, C; 9E4 compared to 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 seeding along axons in this lentiviral α-synucleinopathy diffusion model.

[0235] In contrast, the ipsilateral and contralateral levels of α-synuclein deposits on axons in non-transient mice passively immunized with either the aslo0452-ngl-3 antibody or the aslo0452-ngl-3 non-effector mutant (aslo0452-ngl-3-D265A) were significantly lower than the levels of α-synuclein deposits on axons in non-transient mice treated with NIP228 isotype control mouse IgG1 (Figure 13A, B, C; NIP2 Compared to aslo0452-ngl-3 and aslo0452-ngl-3-D265A, this indicates that passive immunization of mice using either aslo0452-ngl-3 or the non-effector D265A mutant of aslo0452-ngl-3 eliminates α-synuclein deposits on axons and reliably prevents ipsilateral-contralateral migration of α-synuclein along axons in this lentiviral α-synucleinopathy mouse model.

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

[0237] In LV-α-syn-injected non-transient mice, stronger α-synuclein immunoreactivity was detected at higher magnification in the ipsilateral hippocampal network and at a lower degree in the ipsilateral neocortical network (Figure 14A). In addition, strong α-synuclein deposition was detected in a large number of identifiable neuronal somas (SOMAs) in the CA1 region of the ipsilateral hippocampus, and weaker α-synuclein immunoreactivity was detected in layer 5 neurons of the ipsilateral neocortex (Figure 14; black arrows). Treatment with the 9E4 antibody did not significantly alter the number of ipsilateral CA1 hippocampal neurons or ipsilateral layer 5 neocortical neurons containing α-synuclein deposits compared to non-g mice immunized with NIP228 isotype control mouse IgG (Figure 14A, B, C; 9E4 compared to NIP228). However, non-g mice treated with either the aslo0452-ngl-3 antibody or the aslo0452-ngl-3 uneffector mutant (aslo0452-ngl-3-D265A) had a significantly reduced number of ipsilateral CA1 neurons and ipsilateral layer 5 neurons containing α-synuclein deposits compared to NIP228 isotype control mouse IgG-treated non-g mice (Figure 14A, B, C; aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared to NIP228).

[0238] Furthermore, the intensity of α-synuclein immunoreactivity in neurons within the neural network and in the ipsilateral CA1 region of the hippocampus and the ipsilateral layer 5 region of the neocortex was significantly reduced in aslo0452-ngl-3-treated and aslo0452-ngl-3-D265A-treated non-transient mice compared to NIP228 isotype control mice treated with IgG (Figure 14A; aslo0452-ngl-3 and aslo0452-ngl-3-D265A compared to 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 resulted in a statistically significant reduction in the number of neurons, including strong α-synuclein immunoreactivity, in the ipsilateral CA1 hippocampal region and layer 5 neocortex, as well as the contralateral CA1 hippocampal region, compared to NIP228-treated α-syn tg mice, but not in 9E4 (Figure 17A, B, C, D).

[0240] Passive immunization of either non-TG wild-type mice or α-syn TG mice, either stereotactically immobilized with a lentiviral vector driving human α-synuclein expression into one hippocampus using the high-affinity anti-α-synuclein mouse IgG1 antibody aslo0452-ngl-3, reliably reduced ipsilateral-contralateral transaxonal diffusion of lentivirally expressed α-synuclein observed in this mouse model of α-synuclein propagation. This newly disclosed property of the anti-α-synuclein antibody to inhibit α-synuclein in vivo diffusion demonstrates its ability to bind to different epitopes of human α-synuclein compared to antibodies that do not inhibit diffusion in the tested model (e.g., 9E4 antibody).

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

[0242] In summary, the antibodies of the present invention that target α-synuclein, and their antigen-binding fragments, have the ability to act as disease-altering factors in PD, DLB, or MSA by inhibiting or delaying the pathological uptake of α-synuclein into recipient cells, and by preventing the dissemination and transmission of α-synuclein lesions between anatomically connected brain regions. Thus, antibodies targeting α-synuclein may be able to treat or prevent disease progression and may provide therapeutic benefits to patients with synucleinopathy such as PD, DLB, or MSA.

[0243] Example 13: Preparation of a bispecific antibody 0452 ngl-3-BBBt0626gl The exemplary bispecific antibody of the present invention, comprising a human IgG1™ skeleton conjugated to a single-chain fragment (scFv) of BBBt0626gl grafted to the N-terminus (Bis2 form) or C-terminus (Bis3 form) of the heavy chain of aslo0452 ngl-3, was prepared as described below.

[0244] The Bis2-format bispecific antibodies of the present invention were prepared by synthetically generating DNA fragments encoding Bbbt0626glscFv-(G4S)x2-aslo0452 ngl-3 VH or Bbbt0626wt-(G4S)x2-aslo452 ngl-3 VH, respectively, which contained BssHII and BstEII flanking endonuclease restriction sites upstream of Bbbt0626glscFv or Bbbt0626wt and downstream of aslo0452 ngl-3 VH. Subsequently, the digested DNA fragments were targeted cloning to the hIgG1™ vector backbone.

[0245] The Bis3-formatted bispecific antibody of the present invention was prepared by PCR amplification followed by directed cloning using restriction endonuclease sites (SfiI and XbaI). Two PCR fragments were generated: (1) an overlapping PCR fragment containing a forward PCR oligo that incorporates the C-terminus of the CH3-(G4S)x3 linker (SEQ ID NO: 57) and the N-terminus of Bbbt0626gl, along with an oligo that amplifies the vector sequence immediately downstream of the C-terminus of Bbbt0626gl scFv and beyond the XbaI restriction site. Both PCR fragments were joined together by pull-through PCR and subsequently directed to 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 the mouse brain endothelial cell line (b.end3) to confirm binding activity to the BBB transporter moiety. Furthermore, competition for binding to the also0452 ngl-3 epitope was tested using an HTRF-based epitope competition assay to confirm binding to the also0452 ngl-3 epitope.

Claims

1. K below 500 pM D An antibody or its antigen-binding fragment that binds to human α-synuclein, specifically to a region within the C-terminal region of human α-synuclein between amino acid 102 and amino acid 130, and reduces α-synuclein diffusion in vivo.

2. The antibody or its antigen-binding fragment according to claim 1, 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 claim 1 or 2, which binds to human, rat, and cynomolgus monkey α-synuclein.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which binds to naturally occurring endogenous human α-synuclein.

5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to aggregates of human α-synuclein.

6. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to disease-associated lesion forms of α-synuclein.

7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which reduces α-synuclein levels, particularly free unbound α-synuclein, in the interstitial fluid and / or cerebrospinal fluid.

8. (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of sequence number 6, (iii) H-CDR3 of sequence number 7, (iv) L-CDR1 of sequence number 9, (v) L-CDR2 of sequence number 10, (vi) L-CDR3 of sequence number 11 An antibody or antigen-binding fragment according to any one of claims 1 to 7, having at least one CDR selected from

9. (a) The CDR3 of the heavy chain of the antibody or its antigen-binding fragment 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 its antigen-binding fragment is the CDR3 of SEQ ID NO: 21 of the light chain of antibody aslo0452 ngl-3. The antibody or its antigen-binding fragment according to claim 8.

10. (a) Three heavy chain CDRs having the following sequences: (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of Sequence ID No. 15; and (iii) H-CDR3 of Sequence ID No. 16, and (b) Three light chain CDRs having the following sequences: (i) L-CDR1 of sequence number 20, (ii) L-CDR2 of Sequence ID No. 10; and (iii) L-CDR3 of sequence number 21 An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising:

11. The antibody or antigen-binding fragment according to claim 10, comprising a variable heavy chain having at least 90% identity with the sequence defined by SEQ ID NO: 14, and a variable light chain having at least 90% identity with the sequence defined by SEQ ID NO:

19.

12. The antibody or antigen-binding fragment 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 sequence number 25, (ii) H-CDR2 of Sequence ID No. 26; and (iii) H-CDR3 of Sequence ID No. 27, and (b) Three light chain CDRs having the following sequences: (i) L-CDR1 of sequence number 31, (ii) L-CDR2 of Sequence ID No. 32; and (iii) L-CDR3 of sequence number 33 The antibody or antigen-binding fragment thereof according to claim 11, comprising:

14. The antibody or antigen-binding fragment according to any one of claims 1 to 13, which is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody or an antigen-binding fragment thereof.

15. The antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is an IgG1™ antibody or an antigen-binding fragment thereof.

16. An antibody or its antigen-binding fragment according to any one of claims 1 to 15, which competes with the antibody aslo0452 ngl-3 in terms of its binding affinity to human α-synuclein.

17. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, which binds to the same epitope on human α-synuclein as antibody aslo0452 ngl-3.

18. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, which is associated with a transporter molecule for delivery across the blood-brain barrier (BBB).

19. The aforementioned transporter molecule: a. Immunoglobulin-derived polypeptides containing BBBt0626gl or its blood-brain barrier-penetrating fragment, or b. BBBt0626 or its blood-brain barrier-penetrating fragment, c. BBBt0632gl or its blood-brain barrier-penetrating fragments The antibody or antigen-binding fragment thereof according to claim 18.

20. The antibody or antigen-binding fragment according to claim 19, wherein the transporter molecule is a single-chain fragment (scFv) comprising the following: (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 in SEQ ID NO: 47 and the light chain variable region (VL) of BBBt0626 in 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 that binds to human α-synuclein or an antigen-binding fragment thereof, (a) Three heavy chain CDRs having the following sequences: (i) H-CDR1 of sequence number 5, (ii) H-CDR2 of Sequence ID No. 15; and (iii) H-CDR3 of Sequence ID No. 16, and (b) Three light chain CDRs having the following sequences: (i) L-CDR1 of sequence number 20, (ii) L-CDR2 of Sequence ID No. 10; and (iii) L-CDR3 of sequence number 21 The above antibody or its antigen-binding fragment, including the above antibody.

22. K below 500 pM D The antibody or antigen-binding fragment thereof according to claim 21, which binds to human α-synuclein.

23. The antibody or antigen-binding fragment thereof according to claim 21 or 22, which reduces α-synuclein diffusion in vivo.

24. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 23, which binds to human α-synuclein but does not bind to human β-synuclein or human γ-synuclein.

25. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 24, which binds to human, rat, and cynomolgus monkey α-synuclein.

26. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 25, which binds to naturally occurring endogenous human α-synuclein.

27. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 26, which binds to aggregates of human α-synuclein.

28. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 27, which binds to disease-associated lesion forms of α-synuclein.

29. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 28, which reduces α-synuclein levels, particularly free unbound α-synuclein, in the interstitial fluid and / or cerebrospinal fluid.

30. The antibody or antigen-binding fragment thereof according to any one of claims 21 to 29, comprising a variable heavy chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

14.

31. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 30, comprising a variable heavy chain having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

14.

32. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 31, comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO:

14.

33. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 32, comprising a variable light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

19.

34. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 33, comprising a variable light chain having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

19.

35. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 34, comprising a variable light chain containing the amino acid sequence of SEQ ID NO:

19.

36. An antibody, as described in any one of claims 1 to 35, or an antigen-binding fragment thereof.

37. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 36, comprising the L234F / L235E / P331S triple mutation in the Fc region.

38. The antibody or antigen-binding fragment thereof according to any one of claims 21 to 29, comprising a heavy chain containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

12.

39. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 29, comprising a heavy chain containing the amino acid sequence of SEQ ID NO:

12.

40. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 29 or 38 to 39, comprising a light chain having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

17.

41. An antibody or antigen-binding fragment thereof according to any one of claims 21 to 29 or 38 to 39, comprising a light chain containing the amino acid sequence of SEQ ID NO:

17.

42. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 41, for use as a pharmaceutical.

43. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 42, for use in the prevention or treatment of alpha-synucleinopathy.

44. An antibody or its antigen-binding fragment for use according to claim 43, wherein the alpha-synucleinopathy is selected from Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA).

45. An antibody or its antigen-binding fragment for use according to claim 44, wherein the α-synucleinopathy is Parkinson's disease (PD).

46. A method for treating or preventing a central nervous system (CNS) disease in a patient, comprising the step of administering to the patient an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 41.

47. The method according to claim 46, wherein the disease is α-synucleinopathy.

48. The method according to claim 47, wherein the alpha-synucleinopathy is selected from Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA).

49. The method according to claim 48, wherein the alpha-synucleinopathy is Parkinson's disease (PD).

50. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 41, and a pharmaceutically acceptable excipient.

51. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 41.

52. An 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. An 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 a nucleic acid molecule according to any one of claims 51 to 53.

55. Subject matter as described in the specification.

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