Agents, uses and methods for treatment of synucleinopathy

Abstract

To provide novel monoclonal anti-α-synuclein antibodies.SOLUTION: Disclosed is an antibody that can bind to the toxic α-synuclein fragment 1 to 119 / 122 and neutralize this truncated form of α-synuclein. The antibody can be used to treat synucleinopathies such as Parkinson's disease (including idiopathic and hereditary Parkinson's disease), diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV), combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel class of monoclonal antibodies that specifically bind to α-synuclein, including and the use of these molecules and their α-synucleolytic properties in the treatment and diagnosis of synucleinopathies. The present invention relates to a method of using in-binding fragments.

[0002] Sequence Listing Reference: This application is a division of one or more U.S. patent applications pursuant to 37 CFR 1.821. contains a number of sequence listings (see below), which are available on a computer readable medium (file name: 92_ST25.txt, created on June 22, 2016, with a size of 44kB ), which is incorporated herein by reference in its entirety. [Background technology]

[0003] Synucleinopathies, also known as Lewy body diseases (LBD), are characterized by the loss of the protein α- As Lewy bodies (LBs) and / or Lewy neurites, in which synuclein is the major component It is characterized by the accumulation of protein aggregates within cells that can be seen under a microscope (Jelli nger,Mov Disord.2012 Jan;27(1):8-30;McKe ith et al., Neurology (1996) 47:1113-24). Shinu Craniopathy includes Parkinson's disease (PD) (idiopathic and hereditary Parkinson's disease) dementia with Lewy bodies (DLB) and diffuse Lewy body disease (DLB) Also known as Lewy body variant Alzheimer's disease (LBV), combined Alzheimer's disease Marr's and Parkinson's Disease (CAPD), Pure Autonomic Failure (PAF) and Multisystem Atrophy (MSA; e.g., olivopontocerebellar atrophy, striatonigral degeneration and Shy-Dre Synucleinopathies are the main causes of Parkinson's disease. Dopaminergic nigrostriatal system causes movement disorders (rigidity, bradykinesia, resting tremor) Degeneration is often present in the central, peripheral and autonomic nervous systems and brain regions. and in other organs associated with non-motor dysfunction such as dementia and autonomic nervous system disorders. There is also widespread occurrence of cerebrospinal fluid (CBD) and degenerated Lewy neurites. Some non-motor signs and symptoms occurs prior to motor symptoms in Parkinson's disease and other synucleinopathies. Such early signs include, for example, REM sleep behavior disorder (RBD) and Symptoms include decreased sense of smell and constipation (Mahowald et al., Neuro Synucleinopathies are a growing problem in the aging population. It remains a common cause of motor and cognitive decline in et al., Arch. Neurol. (1994) 51:888-95).

[0004] α-synuclein is a member of the β- and γ-synucleins and synoretins. α-Synuclein is one of a family of proteins that contain the α-synuclein kinase inhibitor α-synuclein. It is normally expressed in synaptic neurons and regulates synaptic vesicle release, thereby promoting neurotransmission and It is believed to play a role in influencing plasticity, learning and memory.

[0005] Several studies suggest that α-synuclein plays a central role in PD pathogenesis It has been reported that, under pathological conditions, proteins aggregate to form insoluble fibrils within cells. For example, synuclein accumulates in LBs (Spillantini et al. al.,Nature(1997)388:839-40;Takeda et al. , J. Pathol. (1998) 152:367-72; t al., Neurosci. Lett. (1997) 239:45-8). In chronic Parkinson's disease, mutations in the α-synuclein gene and duplications of this gene are and triplications cosegregate (Kruger et al. ,Nature Gen.(1998)18:106-8;Polymeropoulo s, et al., Science (1997) 276: 2045-7). The important point is that insulin is secreted into extracellular fluid and can be present in plasma and cerebrospinal fluid (CSF). Important findings have been reported, for example, by Pacheco et al. (2015) and others (P acheco et al J Neurochem.2015 Mar;132(6) :731-4;Conway et al.,Proc Natl Acad Sci USA(2000)97:571-576;Volles et al., J.Bioc hem.42:7871-7878,2003) have demonstrated that extracellular synthase Nuclein has been suggested to play a pathogenic role in the brain. It has been shown that extracellular α-synuclein oligomers are neurotoxic to brain neuronal membranes. Another interesting hypothesis based on the synuclein secretion data is that α-synuclein Prion-like spread of cytoplasmic leukemia may underlie progression of Parkinson's disease and other synucleinopathies (Lee et al. 2014, Nat Rev Neur ol.2014 Feb;10(2):92-8;Hansen and Li 201 2,Trends Mol Med.2012 May;18(5):248-55). These findings raise the hope that extracellular synuclein may be targeted by immunotherapy. (Vekrellis et al. 2011, Lancet Neurol .2011 Nov;10(11):1015-25).

[0006] Natural α-synuclein autoantibodies are present in both PD patients and healthy controls. It has been shown (Smith et al. 2012, PLoS One. 2012; 7(12):e52285;Maetzler et al.2014,PLoS On e.2014 Feb 21;9(2):e88604,Papachroni et al.2007 J Neurochem.2007 May;101(3):749- 56 and Woulfe et al. 2002, Neurology. 2002 Ma y 14;58(9):1435-6), and possibly α-synuclein in PD. Increased levels of autoantibodies against roimmunol.2011 Apr;233(1-2):221-7,Gruden et al.2012,Neuroimmunomodulation.2012;1 9(6):334-42 and Yanamandra 2011, PLoS One.2 011 Apr 25;6(4):e18513), or in PD patients compared with healthy controls Decreased autoantibodies against α-synuclein have been reported in patients with gbo et al 2013,Neurology.2013 Jan 8;80(2 ):169-75). Circulating anti-α-synuclein autoantibodies are involved in α-synuclein aggregation. The possibility that this autoantibody may play a protective role in the immune system was suggested very soon after its discovery (W oulfe et al.2002,Neurology.2002 May 14;5 8(9):1435-6).

[0007] Overexpression of α-synuclein in transgenic mice prevents the development of Lewy body disease Some of the pathological features of mice overexpressing α-synuclein are similar. Several different gene transfer systems have been developed in the past decade (described in the following publications: Koehler et al 2014,PLoS One.2013 May 31; 8(5):e64649;Fleming and Chesselet,2006,B ehav Pharmacol.2006 Sep;17(5-6):383-91;S pringer and Kahle 2006, Curr Neurol Neurol. sci Rep.2006 Sep;6(5):432-6). Thy-1 and PDG A mouse line carrying the F-β promoter develops motor and cognitive impairments in vivo. It has been used to demonstrate the neuroprotective effects of antibodies against α-synuclein. However, there are no gene transfer systems that demonstrate robust degeneration of dopaminergic neurons. In the case of Therefore, the positive outcomes of potential disease-modulating treatments are not expected to worsen in Parkinson's disease. mediated through effects on paminergic neurons or other central nervous system neurons It is not clear whether this will be the case.

[0008] One robust finding in transgenic mouse models is that human α-synuclein is Chronic overexpression of ribosomal kinase impairs synaptic function. Using both in vivo and in vivo studies, overexpression of wild-type (wt) human α-synuclein , which has been shown to impair synaptic transmission in the hippocampus (Nemani et al. 010,Neuron.2010 Jan 14;65(1):66-79;Paumi er et al.2013,PLoS One.2013 Aug 1;8(8):e 70274). This was shown in the CA1 region of the hippocampus, where both studies They found decreased basal synaptic transmission, and the underlying mechanism was a dysfunction of synaptic release. However, it was thought that the accumulation of α-synuclein in the cells led to synaptic Secretion of α-synuclein into the extracellular space and its effect on synaptic function in mice Recent findings regarding the toxic effects of klein oligomers may support the role of extracellular Klein in synaptic dysfunction. This opens the door to the possible role of α-synuclein and therefore the ability of therapeutic antibodies to rescue the disorder. It is something that happens.

[0009] Use of viral vectors to overexpress α-synuclein ameliorate PD in rodents This is an important way to model the The proportion of nigrostriatal neurons is significantly higher than that of non-nigrostriatal neurons, a feature that has not yet been reproduced by genetic mutations. This is because it causes relatively rapid progressive degeneration (Kirik and Bjorklund, 2013). 003,Trends Neurosci.2003 Jul;26(7):386-9 2) Furthermore, viral gene delivery demonstrated that wt α-synuclein induces nigrostriatal pathology. (Kirik et al. 2002, J Neurosci. 2 002 Apr 1;22(7):2780-91), which is due to α-synuclein duplication and These findings are consistent with evidence for familial PD with triple duplications (Lee and Trojanowski,2006,Neuron.2006 Oct 5;52(1 In one study, a pool of goat antibodies against α-synuclein was used to treat Protects the N-terminus from myelinergic cell death and inhibits AAV-α-synuclein-based signaling in Parkinson's disease It has been shown that it improves behavioral disorders in a rat model based on n et al 2015,PLoS One.2015 Feb 6;10(2):e 0116841).

[0010] Prion-like spreading of α-synuclein pathology manifests as α-synuclein pathology and leads to It has recently been shown that it also induces paminergic cell death (Luk et al. 2012). ,Science.2012 Nov 16;338(6109):949-53). This model shows that α-synuclein antibodies can ameliorate this pathology. Used (Tran et al. 2014, Cell Rep. 2014 Jun. 26;7(6):2054-65). In this model, antibody treatment increased the expression of β-glucose in several brain regions. Accumulation of phosphorylated α-synuclein in the substantia nigra (including dopaminergic neurons) The study was able to reduce the volume of the blood vessels and reduce the incidence of movement disorders.

[0011] In addition to mutations, alternative splicing of the α-synuclein gene and protein Post-translational modifications of α-synuclein, including phosphorylation, ubiquitination, nitration, and cleavage, mediate the expression of α-synuclein. α-Synuclein with enhanced ability to form aggregated and / or toxic forms of α-synuclein (Beyer and Ariza, Mol Neurobiol. ol.2013 Apr;47(2):509-24). However, α-synuclein The exact pathological species of the IL-1 virus remains unclear. Misfolded / aggregated / secreted species and various post-translational modifications are associated with toxicity. But even if there is in fact one toxic species, there is a great deal of opinion as to which is most important. There is no match.

[0012] Overall, similar morphological findings in various animal models, such as humans, mice, and flies, The accumulation of α-synuclein with associated neurological changes suggests that this molecule may be involved in the pathogenesis of Lewy body disease. These results suggest that it plays a central role in the disease.

[0013] Several different antibodies against α-synuclein have demonstrated therapeutic efficacy in preclinical animal models. It has been shown to have the effect of targeting an epitope containing α-synuclein residues 91-99. Antibodies targeting α-synuclein and epitopes including residues 118-126 Both antibodies have an effect on motor and cognitive impairment in transgenic mice. It has been shown that this can be achieved (Games et al. 2014, J Neurosci. 2014 Jul 9;34(28):9441-54). These state-of-the-art antibodies are It targets an epitope containing -synuclein residues 118-126 and is currently undergoing clinical trials. It is a humanized antibody based on the mouse monoclonal antibody 9E4, which is in Phase I. -C-terminal antibody 274 (B ae et al.2012,J Neurosci.2012 Sep 26;32( 39):13454-69) also demonstrated cell-to-cell spread of the lesion in preclinical models. In addition, it has been shown to have an effect on α-synuclein oligomers. Antibodies targeting conformational species such as α-synuclein and fibrils may be useful in preventing the formation of these potentially toxic α-synuclein proteins. It has been shown that the levels of rhein species can be at least reduced (Lindstr oem et al.2014,Neurobiol Dis.2014 Sep;69 :134-43 and Spencer et al. 2014, Mol Ther. 20 14 Oct;22(10):1753-67). These conformational antibodies that reduce nuclein oligomer levels also target amino acids 121-1 25, which has been shown to target an epitope in the C-terminus of α-synuclein. (U.S. Patent Application Publication No. 20120308572). Other conformations, fibrils and Oligomer-specific antibodies also target C-terminal sequences (Vaikath et al. Urobiol Dis.2015;79:81-99).

[0014] Because the toxic form of α-synuclein is unknown, therapeutic antibodies would ideally be selectively targeted to The majority of α-synuclein species are formed by splicing or post-translational modifications (e.g., cleavage). The antibody should be capable of binding to the nucleotide moiety, as well as to oligomeric and fibrillar forms. One problem with current antibodies being tested as therapeutics in preclinical models is that Many of them contain C-terminal fragments that are not found in some of the major truncated forms of α-synuclein. For example, the amino acids important for 9E4 binding are: (U.S. Patent Application Publication No. 20140127131, as shown by alanine scanning) If so, the asparagine 122 and tyrosine 125 are asparagine 122 and tyrosine 125, which indicates that the antibody is capable of binding to parkin At amino acids 119 and 122, which are some of the major truncations in Son's disease brain tissue This means that it cannot bind to α-synuclein, which is then cleaved (Kellie et al. Sci Rep. 2014;4:5797). The same was true for antibodies 274 and This may also be the case for the antibody mab47 (U.S. Pat. No. 8,632,776). Also, the amino-terminal antibody probably targets α-synuclein cleaved at amino acids 5–140. It binds to some of the major truncated forms of α-synuclein that lack the first amino acid, such as In the case of the 9E4 antibody, the antibody inhibits the protease from blocking α-synuclein. The mechanism of action is to bind to the same region that cleaves ribonuclease, i.e., amino acids 119–120 in the extracellular space. 22 (Games et al. 2014, J Neurosci.2014 Jul 9;34(28):9441-54). The mechanism of action of the antibody is also seen in close proximity to this site, and therefore the Many antibodies would be expected to have this activity.

[0015] There is some support for a toxic role for truncated α-synuclein species in animal models. Expression of truncated α-synuclein under the tyrosine hydroxylase promoter was Transgenic α-synuclein model leads to unusual nigrostriatal pathology It has been shown that the GABAergic receptor agonist (Tofaris et al. 2006, J Neurosci. 2006 Apr 12;26(15):3942-50;Wakamatsu et al.2006,Neurobiol Aging.2008 Apr;29(4):5 74-85) For example, the α-synuclein protein of human having the A53T mutation Expression of amino acids 1-130 prevents embryonic loss of dopaminergic neurons in the substantia nigra pars compacta expression of the full-length protein did not cause this (Wakama tsu et al.2006, Neurobiol Aging.2008 Apr; 29(4):574-85). Calcium / calmodulin-dependent protein kinase I Expression of the 120 amino acid α-synuclein molecule in the Iα (CamKII-α) promoter We investigated α-synuclein aggregation and cortico-hippocampal memory including the Barnes maze and novel object recognition. associated with progressive impairment in memory tests (Hall et al. 2015, Exp Neurol. 2015 Feb;264:8-13). In addition, In mice, co-expression of C-terminally truncated α-synuclein suppressed the induction of full-length α-synuclein. (Ulusoy et al. 2010, Eur J Neuros ci.2010 Aug;32(3):409-22). Summary of the Invention [Problem to be solved by the invention]

[0016] In the present invention, the toxic α-synuclein fragment 1-119 / 122 is bound to An antibody capable of neutralizing the truncated form of α-synuclein ("GM37" described in the Examples) Antibodies of the invention, such as GM37 and GM285, were produced. It binds to other oligomeric forms of α-synuclein and interacts with other CNs to limit the spread of disease. It is possible to alter the uptake of S by resident cells. The antibodies of the invention, such as 285 and 286, bind to different α-synuclein species in the human brain. It was unexpectedly found to be superior to prior art antibodies such as 9E4 in binding to extracellular Removes α-synuclein and inhibits the expression of α-synuclein induced by the presence of abnormal α-synuclein in vivo. They have an unexpectedly excellent effect in normalizing synaptic transmission disorders. To further illustrate, the antibodies of the present invention, such as GM37 and 285, are useful in treating Parkinson's disease. In rat models, it is possible to prevent the development of disease-associated motor phenotypes. The proteins GM37 and GM285 inhibited the synthesis of extracellularly added proteins in primary mouse neurons. Aggregation of endogenous α-synuclein induced by recombinant pathological α-synuclein seeds Antibodies such as GM37 and 285 can inhibit the seeding and phosphorylation of . The body is able to upregulate dopaminergic neurons in vivo using a mouse model of Parkinson's disease. The seeding of α-synuclein lesions into the nucleoid matrix can also be inhibited, which may contribute to the intercellular These data further support the therapeutic potential of these antibodies in preventing the transmission of In addition, the researchers believe that blocking the mechanism by which disease lesions spread between neurons in Parkinson's disease patients may be a solution to the disease. These novel antibodies, GM37 and GM37, are novel therapeutic agents capable of modifying This strongly supports the use of 285.

[0017] In a further embodiment of the invention, three amino acid variants of the GM37 antibody are provided. All mutants had the same functional readout as the parent antibody, GM37. These variations have a low yield rate but have improved manufacturability properties. The variant reduces the risk of post-translational modifications occurring within the binding domain of the GM37 antibody, thus improving antibody production. This provides some improvement over the previously described method, because large-scale clinical or commercial production of antibodies is complicated. It is difficult to provide a homogeneous product for drugs, especially for immunoglobulins and proteins. This is advantageous because it is important for protein synthesis. [Means for solving the problem]

[0018] The present invention relates to the amino acids 112 to 117 of α-synuclein (SEQ ID NO: 9 (ILED A novel monoclonal antibody capable of specifically binding to an epitope in the MP), and antigen-binding fragments thereof. An exemplary antibody "GM37," or "GM 285, or other epitopes bound by the antibodies or antibody-binding fragments thereof of the present invention. The epitope is referred to herein as the "112-117 epitope." In one embodiment, the antibody specifically binds to an epitope within the 112-117 epitopes. Antibodies GM37 or GM285 for binding to an epitope within amino acids 112-117 For example, the antibody or antigen-binding fragment thereof of the present invention may be capable of competing with SEQ ID NO: 7 and a light chain consisting of a variable region of SEQ ID NO: 8. It can compete for binding to an epitope within amino acids 112-117 of nuclein. Such competitive inhibition of binding can be determined using assays and methods well known in the art. can be determined using a non-labeled binding assay, such as surface plasmon resonance (SPR). For example, human α-synuclein can be immobilized on a surface and the antibody or binding fragment to be tested can be attached to the surface. The incubation was performed with or without the reference antibody 'GM37' before incubation with the Alternatively, a pairwise mapping approach can be used, in which the reference The control antibody 'GM37' is immobilized on the surface and the human α-synuclein antigen is bound to the immobilized antibody. The secondary antibody is then tested for its simultaneous binding ability to human α-synuclein (see, e.g., 'BIAcore® Assay H', the contents of which are incorporated herein by reference. andbook',GE Healthcare Life Sciences,29- (see 0194-00 AA 05 / 2012).

[0019] More specifically, the GM285 antibody binds to residues 112-115 (ILED) of α-synuclein. 19) which binds to an epitope within residues 112-117 of alpha-synuclein do.

[0020] In one embodiment, the present invention provides monoclonal antibody GM37, variants thereof (e.g., GM37 mutant 1, GM37 mutant 2, and GM37 mutant 3), or GM285 do.

[0021] In particular, the present invention relates to the monoclonal antibody GM37, its variants (e.g., GM37 variants 1, GM37 mutant 2 and GM37 mutant 3), or GM285, and A sufficient number (e.g., 1, 2, or 3) light chain CDRs and a sufficient number (e.g., 1, 2, or 3) heavy chain CDRs. The present invention includes such antibodies having the CDRs of the 2-amino acid sequence and derivatives thereof. Such an antibody has three light chain CDRs and three heavy chain CDRs, as defined below. The numbering of amino acid residues in this region is based on the IMGT® International ImMunoG eneTics Information System (registered trademark) or Kabat, EA, Wu, TT .,Perry, HM, Gottesmann, KS & Foeller, C. (1991).Sequences of Proteins of Immunolo gical Interest,5th edit.,NIH Publication No. 91-3242, U.S. Department of Health and Human Services alth and Human Services);Chothia, C. & Les k,AM(1987).Canonical structures For th e Hypervariable domains Of Immunogloblin sJMol.Biol.196,901-917.

[0022] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is human α - Capable of specifically binding to synuclein (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; and / or (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:2; and / or (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; and / or (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; and / or (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and / or (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO:6 The synuclein antigen-binding fragment comprises or consists of:

[0023] In another embodiment, the monoclonal antibody or antigen-binding fragment thereof is human Capable of specifically binding to α-synuclein (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, 34, or 35; (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO:6; The synuclein antigen-binding fragment comprises or consists of:

[0024] In yet another embodiment, the monoclonal antibody or antigen-binding fragment thereof is , capable of specifically binding to human α-synuclein (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20; and / or (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21; and / or (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22; and / or (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 23; and / or (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 24; and / or (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 25 The synuclein antigen-binding fragment comprises or consists of:

[0025] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is a natural antigen. α-synuclein antibodies and (such natural anti-α-synuclein antibodies) Compare: (i) Differences in binding affinity (KD) for α-synuclein; (ii) differential ability to inhibit protease cleavage of α-synuclein fibrils; (iii) Impairment of basal synaptic transmission in F28-snca transgenic mice differences in ability to ameliorate harm; (iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis and / or differences in the ability to reduce the levels of (v) When administered chronically, it improves motor function in a rat model of Parkinson's disease. Differences in ability to recover, (vi) α-synuclein seeding (in vitro and / or in Parkinson's disease) The ability of the compound to prevent the accumulation of insoluble phosphorylated α-synuclein in mouse models of Alzheimer's disease Differences; and / or (vii) Differential ability to bind truncated α-synuclein in human brain The amino acid sequence showing the The synuclein antigen-binding fragment comprises a synuclein antigen-binding fragment having a constant region of

[0026] The antibodies and antigen-binding fragments thereof of the present invention are useful in treating Parkinson's disease (PD), idiopathic and hereditary Parkinson's disease), diffuse Lewy body disease (DLBD), Lewy body Somatic variant Alzheimer's disease (LBV), Gaucher disease (GD), combined Alzheimer's Symptoms of Parkinson's disease (CAPD), pure autonomic failure and multiple system atrophy It may be used in methods for treating, diagnosing or imaging nucleinopathies. [Brief description of the drawings]

[0027] [Figure 1] The immunization protocol for the generation of hybridomas is shown. The table summarizes the differences in immunogens and mouse strains used to identify GM37 and GM285. Different HCo17-Balb / c and HCo12 / Balb / c mice were immunized separately (description of these mice is shown below). Hybridoma expressing GM37 was identified from mice immunized with full-length α-synuclein containing amino acids 1-140 fibrils and boosted with truncated α-synuclein fragments 1-60 and 1-119 of full-length (FL) α-synuclein (SEQ ID NO: 10). Hybridoma expressing antibody GM285 was obtained from an immunization protocol (Example 1) in which HCo12-Balb / c mice were immunized with full-length monomeric α-synuclein, amino acids 1-140, followed by full-length fibrillar α-synuclein. [Figure 2-1](Panels A-C) show screening of GM37 for binding to alpha-synuclein, alpha-synuclein homologs and orthologs. A) Binding of antibody GM37 to alpha-synuclein using a no-wash solution-based ELISA (FMAT). B) Using SPR (Fortebio), binding of antibody GM37 is specific to alpha-synuclein (alpha panel) and does not bind to other related synuclein family proteins, beta-synuclein (beta panel) and gamma-synuclein (gamma panel). Measurements were performed using SPR (Fortebio Octetred) and GM37 shows similar binding to alpha-synuclein from cynomolgus monkeys (cynomolgus panel) and mice (mouse panel) (Example 1). C) Using SPR (Fortebio Octetred), binding of antibody GM285 is specific to alpha-synuclein and does not bind to other related synuclein family proteins, beta-synuclein and gamma-synuclein. Measurements were performed using SPR (Fortebio Octetred) and show similar binding of GM285 to α-synuclein from cynomolgus monkeys (Macaca fascicularis) and mice (Mouse) (Example 1). [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Diagram 2-7] Same as above. [Figure 2-8] Same as above. [Figure 2-9] Same as above. [Figure 3-1](Panels A-C) show the real-time binding affinity of GM37. A) Binding of antibody GM37 to α-synuclein measured in RU (Relative Units) (y-axis) versus time (x-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on a CM5 chip. GM37 was captured on a goat anti-human IgG immobilized chip and a series of concentrations of human α-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. The sensor surface was regenerated between each cycle. B) Signal from binding at different concentrations converted to a binding curve. C) Calculated binding constant (Example 2) of antibody GM37 (designated hlgG1-6004-037-C106S). [Figure 3-2] Same as above. [Figure 4-1] (Panels A-C) show the real-time binding affinity of GM285. A) Binding of antibody GM285 to α-synuclein measured in RU (y-axis) versus time (x-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on a CM5 chip. GM285 was captured on the goat anti-human IgG immobilized chip and a series of concentrations of human α-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. The sensor surface was regenerated between each cycle. B) Signal from binding at different concentrations converted to a binding curve. C) Calculated binding constant (Example 2) of antibody GM285 (denoted hlgG1-6004-285). [Figure 4-2] Same as above. [Figure 5-1](Panels A-C) show real-time binding of comparative antibody 9E4. A) Binding of 9E4 to α-synuclein measured in RU (y-axis) versus time (x-axis) as determined by SPR (BIAcore® 3000). Goat anti-human IgG was immobilized on a CM5 chip. 9E4 was captured on the chip by its binding to the goat anti-human IgG immobilized on the chip. A range of concentrations of human α-synuclein (3.125, 6.25, 12.5, 25, 50, 100 nM) were tested for binding to the surface. The sensor surface was regenerated between each cycle. B) Signal from binding at different concentrations converted to a binding curve. C) Calculated binding constant for antibody 9E4 (Example 2). [Figure 5-2] Same as above. [Figure 6] The amino acid sequence of alpha-synuclein is shown. Major cleavage sites (indicated by arrows) identified by mass spectrometry in human brain tissue (Kellie JF, Higgs RE, Ryder JW, Major A, Beach TG, Adler CH, Merchant K, Knierman MD. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by mass spectrometry. Sci Rep. 2014 Jul 23;4:5797. doi:10.1038 / srep05797). [Figure 7] (Panels A-B) show epitope mapping of antibodies GM37 and GM285. ELISA data showing the relative levels of antibody binding to a contiguous peptide (20-mer) derived from α-synuclein amino acid sequence 95-132 (other non-binding peptides not shown). A) The GM37 epitope requires the peptide sequence ILEDMP (SEQ ID NO: 9) for full binding. B) GM285 requires the peptide ILED (SEQ ID NO: 19) for full binding (Example 3). [Figure 8](Panels A-B) show schematic diagrams of truncated forms of α-synuclein. A) The binding epitopes of GM37 / 285 (ILEDMP; SEQ ID NO: 9) and 9E4 (NEAYE; SEQ ID NO: 36) are shown in bold in the α-synuclein amino acid sequence (SEQ ID NO: 10). The arrow indicates the c-terminal cleavage site from FIG. 6. B) The main truncated forms of α-synuclein identified from human brain material. The sizes based on the number of amino acids are shown on the right. Full-length α-synuclein is 140 amino acids. As can be inferred from the epitopes, GM37, its variants 1-3, and GM285 should bind to the full-length and 1-119 / 122, 1-135 fragments. Antibody 9E4 will bind only to the full-length and 1-135 fragments. The specificity of the smaller c-terminal fragments remaining after cleavage is not shown. [Figure 9] Figure 2 shows that antibodies GM37 and GM285 immunoprecipitate full-length and truncated α-synuclein from human brain. Crude homogenates of human DLB brain were incubated with test antibodies (beads (No Ab), B12-human IgG1 control antibody that does not bind α-synuclein, GM-37, GM37 variant 2, GM-285 and mouse (m)9E4) and immunodepleted supernatants, and immunoprecipitated material was resolved in SDS-PAGE. Western blot shows bands representing full-length and various truncated forms of α-synuclein depleted from the supernatant and immunoprecipitated with antibodies (IP). As can be seen, GM37, GM37v2 and GM285 antibodies deplete the major α-synuclein species from the supernatant, and IP shows these species, truncated species 1-135, 1-119 / 122 and full-length α-synuclein. 9E4 does not affect the 1-119 / 122 species, but only affects IP full length and 1-135 (Example 4). [Figure 10]Schematic diagram of the proteolysis of α-synuclein fibrils cleaved by calpain at amino acids 119 / 122. α-Synuclein fibrils (PFF) are added to cultures with (PFF+) or without (PFF) test antibodies. The presence of GM-37 / 285 inhibits the formation of cleaved α-synuclein within the cells and its secretion into the cell culture medium. [Figure 11-1] FIG. 11A shows that GM37 inhibits the formation of a cleavage band (12KD) in the medium and in cell lysates of primary mouse cortical cultures treated with PFF. Proteins were separated by SDS-PAGE and Western blotted to detect various species of α-synuclein. In cells treated with PFF alone or with a control antibody (B12), two monomeric α-synuclein bands were detected at 12 and 14 kDa, representing cleavage and full-length α-synuclein, respectively. In the presence of GM-37, there is only a faint band at 12Kd, indicating that most of the cleavage is inhibited. This effect is also reflected in the medium of the cells. The relative level of accumulation may also be inhibited by the presence of GM-37, as reflected by the reduced relative intensity of the 14Kd band. Alternatively, there may be a reduced amount of the 14Kd band available for uptake by the cells (Example 5). [Figure 11-2]FIG. 11B shows dose-dependent inhibition of proteolysis of α-synuclein fibrils by antibodies GM37, GM37 variant 2 and GM285. In cell lysates from primary mouse cortical cultures at low antibody concentrations (0.1ug / ml), there are both bands representing full-length (FL) α-synuclein and C-terminally truncated (CT) α-synuclein (indicated by arrows). Increasing the antibody concentration to 1, 5 and 10ug / ml leads to a decrease in the proteolysis of α-synuclein fibrils in the cells. This is observed with both antibodies GM37, GM37v2 and GM285. Control samples are treated with human IgG1 antibody B12, which does not recognize α-synuclein. There are also controls with no antibody added (No Antibody) and cells with no α-synuclein fibrils added (No Asyn). Additionally, the total amount of α-synuclein was reduced in samples treated with 37, 37v2 and 285 compared to B12 or the "no antibody" control, indicating that all three antibodies reduce intracellular α-synuclein accumulation in a concentration-dependent manner. The actin bands at the top of the gel indicate equal loading of the samples (Example 5). [Figure 12-1]Figure 12 shows the effect of GM37 and GM285 on seeding of α-synuclein aggregation and α-synuclein phosphorylation in mouse primary cortical neurons. 12A) Example images of primary neurons stained for phosphorylated α-synuclein, which appears as intracellular spots or punctate staining, when cells are seeded with either 1 ng pure or crude seeds of α-synuclein. 12B) Western blot of proteins from primary cortical neurons separated into soluble and insoluble fractions. Blots stained with human α-synuclein specific antibody (4B12 / H a-syn), phospho-Ser-129-α-synuclein specific antibody (ab51253 / pS-a-Syn) and mouse α-synuclein specific antibody (D37A2 / M a-syn) show that addition of crude seeds to primary neurons results in the accumulation of endogenous mouse α-synuclein and phosphorylated α-synuclein and higher molecular weight multimers of α-synuclein in the insoluble fraction. 12C) GM37, GM37 variant 2 and GM285 inhibit the appearance of phosphorylated α-synuclein, quantified by a Cellomics ARRAYSCAN™ automated microscope as the number of α-synuclein phosphoserine 129 positive spots in cells. GM37, GM37v2 and GM285 dose-dependently reduce the amount of phosphorylated α-synuclein spots in cells. 12D) Western blots of homogenates from primary cortical neurons treated with the highest dose of antibody (133 nM) and stained for actin, human α-synuclein, phosphorylated α-synuclein and mouse α-synuclein show that antibodies 37, 37v2 and 285 inhibit the cleavage of α-synuclein crude seeds taken up by cells in the insoluble fraction. All antibodies also inhibit the accumulation of phosphorylated, endogenous mouse α-synuclein and higher molecular weight multimers of phosphorylated mouse α-synuclein in the insoluble fraction. The actin bands at the top of the gel indicate equal loading of the samples (Example 6). [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above. [Figure 13] Basal synaptic transmission at Schaffer collateral-CA1 synapses in the hippocampus of F28-snca transgenic mice and age-matched control mice. Excitatory field postsynaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the slope of the fEPSP as a function of stimulus intensity. Short-term synaptic plasticity was assessed by eliciting paired-pulse facilitation. Stimuli of various intensities were applied in succession from lowest to highest, with each intensity repeated 2-3 times. Basal synaptic transmission was found to be significantly reduced in F28-snca transgenic mice overexpressing wild-type α-synuclein compared to age-matched control mice (Example 7). [Figure 14] Figure 1 shows the effect of systemic administration of a single dose of human 9E4 (15 mg / kg, intraperitoneally (ip)) on the impairment of basal synaptic transmission at Schaffer collateral-CA1 synapses in the hippocampus of F28-snca transgenic mice. Excitatory field postsynaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the slope of the fEPSP as a function of stimulus intensity. Acute treatment with h9E4 induced a significant improvement of the impairment of basal synaptic transmission in F28-snca transgenic mice (Tg-snca+h9E4 vs. Tg-snca+PBS, p=0.002). However, the improvement by h9E4 was only partial, as shown by a significantly lower basal synaptic transmission compared to PBS-treated littermates (p=0.007) (Example 7). [Figure 15]Figure 1 shows the effect of systemic administration of a single dose of human GM37 (15mg / kg, intraperitoneally (ip)) or isotype control antibody (B12) on the impairment of basal synaptic transmission at Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic mice. Excitatory field postsynaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the slope of the fEPSP as a function of stimulus intensity. Acute treatment with GM37 induced a complete amelioration of the impairment of basal synaptic transmission in F28-snca transgenic mice (Tg-snca+GM37 vs. Tg-snca+B12, p=0.004) (Example 7). [Figure 16] Figure 1 shows the effect of systemic administration of a single dose of human GM285 (15mg / kg, intraperitoneally (ip)) on the impairment of basal synaptic transmission at Schaffer collateral-CA1 synapse in the hippocampus of F28-snca transgenic mice. Excitatory field postsynaptic potentials (fEPSPs) were evoked by a single stimulus applied to the Schaffer collateral, and basal synaptic transmission was assessed by measuring the slope of fEPSPs as a function of stimulus intensity. Acute treatment with GM285 induced a complete amelioration of the impairment of basal synaptic transmission in F28-snca transgenic mice (Tg-snca+GM285 vs. Tg-snca+PBS, p=0.001) (Example 7). [Figure 17](Panels A-B) show the effect of systemic administration (15 mg / kg, intraperitoneally (ip)) of human 9E4, GM37 or isotype control antibody (anti-HEL) on the levels of human α-synuclein in the interstitial fluid (ISF) in the hippocampus of freely moving F28-snca transgenic mice. The average of two to three basal values ​​(4-6 h) before antibody treatment was taken as baseline and set to 100% for each animal. Differences were analyzed using a two-way analysis of variance (ANOVA) with repeated measures. The basal level of human α-synuclein in the hippocampus was 8.1 ± 1.1 ng / ml (mean ± SEM, n = 25, not corrected for the recovery rate of the in vitro dialysis probe). Administration of GM37 induced a greater decrease of human α-synuclein in the hippocampus of F28 mice compared to both the comparison antibody, human 9E4, and the control isotype, anti-HEL. Time points showing significant differences in the levels of α-synuclein between animals treated with GM37 or control antibody are indicated by an asterisk (Example 8). [Figure 18] FIG. 20 shows a schematic diagram of the timeline for antibody treatment (downward arrows), virus injection and behavioral assessment (Example 9) in the rat AAV human α-synuclein model shown in FIG. [Figure 19] This shows that antibody GM37 can reduce Parkinson's disease-like motor impairment after chronic treatment in rat AAV model. The effect of chronic treatment with GM37 or PBS in AAV-human-α-synuclein rats on motor asymmetry is evaluated in cylinder test. Each rat is tested for paw use by monitoring for 5 minutes. The percentage of right paw use (ipsilateral to injection) and left paw use (contralateral + right paw) is calculated for each animal compared to GFP-PBS rats (as shown on y-axis) *, **p<0.05 and 0.01. PBS-treated rats still have significant asymmetry in paw use, while antibody GM37-treated animals no longer have significant impairment (Example 9). [Figure 20-1]Figures 20A-20C show that chronic treatment with antibody GM37 can reduce pathological α-synuclein phosphorylation induced by injection of pathological α-synuclein fibril seeds into the mouse striatum. Figure 20A shows a schematic diagram showing the relative treatment times for seed injection and cell counting. Antibody GM37 was administered one day before injection of recombinant α-synuclein fibril seeds into the dorsal striatum of mice, then once a week for 6 weeks. The dosing regimen was either 15 mg / kg intravenously (iv) or 30 mg / kg intraperitoneally (ip). Figure 20B shows the exposure levels of GM37 in plasma based on injection site and dose. Once a week, samples were taken before injection of a new antibody dose. Figure 20C shows the exposure levels of GM37 (csf) based on dose and injection site at the end of the study. Figure 20D compares the number of cells with phosphorylated α-synuclein positive inclusions counted per six sections in the substantia nigra after treatment with GM37 or PBS control. Phosphorylated α-synuclein inclusions were significantly reduced intracellularly in mice treated with both 15 mg / kg intravenous (iv) and 30 mg / kg intraperitoneal (ip) GM37 compared to PBS treated mice (Example 10). [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above. [Figure 21] FIG. 1 shows an alignment of human alpha (SEQ ID NO: 10), beta (SEQ ID NO: 37) and gamma (SEQ ID NO: 38) synuclein proteins. Amino acid residues that differ from alpha-synuclein are highlighted. Gaps are indicated by dots. SwissProt numbers are shown in brackets. [Figure 22] Alignment of alpha-synuclein orthologues (cynomolgus monkey, SEQ ID NO: 39; rat, SEQ ID NO: 40; mouse, SEQ ID NO: 41) is shown. Amino acid residues that differ from human alpha-synuclein (SEQ ID NO: 10) are highlighted. SwissProt numbers are shown in brackets. [Diagram 23]Transient expression of GM37, designated GM37 wild type (wt), and three GM37 mutants, designated GM37 var 1, 2, and 3, is shown. Asterisks indicate that data are determined after protein A purification and neutralization. † indicates that data are calculated from the yield obtained after protein A and neutralization relative to the scale of the expression culture (0.4 L). [Figure 24] Competitive ELISA measuring binding of four antibodies GM37 wt, GM37 var 1, GM37 var 2 and GM37 var 3 to human α-synuclein. Plates coated with α-synuclein are used to detect the amount of antibody remaining after pre-incubation in solutions of each antibody (0.3 μg / ml) with increasing concentrations of α-synuclein (0-1000 nM). All four antibodies show similar binding to α-synuclein. [Diagram 25] A table comparing the binding rate kinetic parameters of GM37wt and mutants 1-3 to immobilized recombinant human α-synuclein. Binding was measured using SPR and binding rates were determined using a 1:1 binding algorithm (BIAcore® T200). [Figure 26] The effect of α-synuclein antibodies on phosphorylated α-synuclein levels in mouse primary neurons treated with pathological α-synuclein fibril seeds is compared. Primary neurons were treated with seeds (10 ng) in the presence or absence of the four GM37, GM37 var 1, GM37 var 2 and GM37 var 3 (2 μg). Neurons were fixed and stained after 3 weeks and analyzed by Cellomics ARRAYSCAN™ for α-synuclein phosphoserine 129 positive spots. Cells treated with seeds alone or seeds and an isotype control antibody (B12) show significantly increased levels of phosphorylation. Cells treated with GM37wt and the three mutants can inhibit phosphorylation of α-synuclein, all of which show the same level of phosphorylation as unseeded cells. Data are shown as mean ± SD determined from 7 images per well in 5 wells. N=2. [Figure 27] The temperature-dependent aggregation of wt GM37, var1, var2 and var3 are compared. Samples of each antibody were subjected to a steady increase in temperature over time and the level of aggregation was measured simultaneously by multi-angle light scattering (Prometheus NT.48, NanoTemper Technologies). The temperature of onset of aggregation is similar for GM37 and GM37-mutants, but the lowest level of aggregation is observed for GM37-Var2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] definition As used herein, the term "alpha-synuclein" means Synonymous with "intracellular protein" and alpha-synuclein protein isoforms (e.g., P37840, 1-3) in UniProt. -synuclein amino acid numbering is shown below with respect to SEQ ID NO: 10. and methionine (M) is amino acid residue 1: [ka]

[0029] The present invention relates to a method for the preparation of a compound capable of specifically binding to α-synuclein, particularly human α-synuclein. In particular, the antibodies and fragments thereof are It exhibits the ability to specifically bind to an epitope within 112-117 of human α-synuclein.

[0030] The term "antibody" (Ab) in the context of the present invention refers to an antibody that binds to an epitope of a molecule (an "antigen"). In certain embodiments of the present invention, According to the hypothesis, a natural antibody is a fragment of an immunoglobulin molecule. Contains a tetramer consisting of at least two heavy (H) chains and at least two light (L) chains Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and three regions ( The heavy chain is composed of a heavy chain constant region consisting of CH1, CH2 and CH3. IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (IgA1 and and IgA2 subtypes), of any isotype, including IgM and IgE. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The heavy and light chain variable regions are composed of the κ and λ chains. , typically involved in antigen recognition, while the heavy and light chain constant regions are involved in the expression of various cells of the immune system. (e.g., effector cells) and the first component of the classical complement system (C1q), It can mediate the binding of immunoglobulins to tissues or host factors. The VH and VL regions are "Complementarity" is interspersed with regions of more conserved sequence called framework regions (FR). Each VH and VL can be further subdivided into regions of hypervariability called "target region" or "target determining region." From the non-terminus to the carboxy-terminus, the order is: FR1-CDR1-FR2-CDR2-FR It consists of three CDR regions and four FR regions arranged as 3-CDR3-FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. Some are "isolated" so that they exist in a different physical environment than they might exist in nature. or modified to differ from natural antibodies in amino acid sequence and their is an antigen-binding fragment of

[0031] The term "epitope" means an antigenic determinant capable of specific binding to an antibody. A pitope is usually a surface group of a molecule, such as an amino acid or sugar side chain. uping) and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and linear epitopes are characterized by the fact that binding to the former, but not the latter, occurs during denaturing and solubility Epitopes are distinct from other epitopes in that they are constantly lost in the presence of a medium. The amino acid residues that are effectively blocked by the specific antigen-binding peptide (in other words, the amino acid residues are amino acid residues and bonds directly involved in binding, such as those within the footprint of the peptide The term "112-117 epitope" may include other amino acid residues that are not directly involved in the synthesis of the peptide. The term consists of at least 4 of the 6 amino acid residues of human α-synuclein, 112–117. The 112-117 epitope is a region of human α-synuclein that contains - Any of residues 1 to 111 of synuclein (including any of residues 106 to 111) ) also ligated to any of the residues 118 to 140 of human α-synuclein (residues 118 to 120 As used herein, the term "antibody" refers to an antibody that binds to the 112-117 epitope. It binds to at least four of the six amino acid residues of human α-synuclein. If the antibody is capable of specifically binding to lein, it is said to be a "112-117 epitope." It is said that the antibody is capable of specifically binding to an epitope within the IgG antibody.

[0032] As used herein, the term "antigen-binding fragment of an antibody" refers to an antigen-binding fragment of an antibody that is an A fragment, portion or region of an antibody capable of specifically binding to a top ) or domain (regardless of how it is produced (e.g., by cleavage, recombinantly, synthetically, etc.), and thus "antigen-binding" The term "antigen-binding fragment of an antibody" refers to, for example, an "antigen-binding fragment of an antibody" that is an "epitope-binding fragment of an antibody." "epitope binding" is intended to be the same as "epitope binding" It is intended that an antigen-binding fragment may comprise one, two, or more of the CDR regions of such an antibody. may contain three, four, five or all six and specifically bind to such epitopes It is possible, however, that specificity for such an epitope differs from that of such an antibody. Preferably, however, the antigen-binding fragment is an antigen-binding fragment that exhibits specificity, affinity or selectivity. would contain all six of the CDR regions of such an antibody. The fragment may be part of a single polypeptide chain (e.g., scFv) or may be a fragment of a single polypeptide chain. or may comprise an amino terminus and a carboxyl terminus (e.g., bispecific antibodies, F ab fragment, Fab 2 Two or more polypeptides each having a The fragments of an antibody that exhibit antigen-binding capacity may be part of or include a dock chain. The antibody may be obtained, for example, by protease cleavage of an intact antibody. The two regions of the v fragment, VL and VH, are naturally encoded by separate genes. or polynucleotides encoding such gene sequences (e.g., The VL and VH regions combine to form a single monovalent antigen-binding molecule. Protein chains (known as single-chain Fvs (scFvs); see, e.g., Bird et al. al., (1988) Science 242:423-426; and Huston et al.(1988)Proc.Natl.Acad.Sci.(USA)8 5:5879-5883) The linker may be linked using recombinant methods. Alternatively, a single polypeptide Flexible phosphorylation is too short to allow the VL and VH regions of the chain to bind together. Bispecific antibodies can be produced by using a carrier (e.g., less than about 9 residues). bispecific antibody, diabody, or similar molecule. (two such polypeptide chains bind together to form a bivalent antigen-binding molecule) (For a description of bispecific antibodies, see, for example, PNAS USA 90(14), 6444-8 (1993). Antigen binding agents included within the scope of the present invention. Examples of recombinant fragments include (i) Fab' or Fab fragments, VL, VH, A monovalent fragment consisting of the CL and CH1 regions, or a fragment of the antibody of International Publication No. 200705978 (ii) F(ab')2 fragments, monovalent antibodies, It contains two Fab fragments linked by a disulfide bridge in the range region. (iii) a Fd fragment consisting essentially of a VH and a CH1 domain; (iv) an Fv fragment consisting essentially of a VL and a VH domain; (v) an Fv fragment consisting essentially of a VH domain Essentially, domain antibodies (Holt et al.; Trends Biotechnol. ol.2003 Nov;2i(ll):484-90), also known as dAb fragments (Ward et al., Nature 341, 544-546 (1989)); (vi) camelid or nanobody (Revets et al. ;Expert Opin Biol Ther.2005 Jan;5_(l):l (II-24) and (vii) isolated complementarity determining regions (CDRs). In addition, the two regions of the Fv fragment, VL and VH, are encoded by separate genes. However, they are expressed as a single tag in which the VL and VH regions combine to form a monovalent molecule. Protein chains (known as single chain antibodies or single chain Fv (scFv), e.g., Bi rd et al., Science 242, 423-426 (1988) and Hu ston et al., PNAS USA 85, 5879-5883 (1988) The recombinant method is further enhanced by synthetic linkers that allow them to be produced as These and other useful antibody fragments in the context of the present invention can be conjugated using The term antibody, unless otherwise specified, refers to a polypeptide that is a polypeptide that is a polypeptide of interest. Antibody-like polypeptides, such as humanized antibodies and humanized antibodies, as well as polypeptides that are subject to enzymatic cleavage, peptide synthesis, The antigen (antigen-binding domain) may be provided by any known technique, including synthesis, and recombinant techniques. It is also understood that the present invention includes antibody fragments that retain the ability to specifically bind to the target antibody (fragment). The antibodies generated can be of any isotype. As used herein, "isotype" refers to the immunoglobulin that is encoded by heavy chain constant region genes. The antibody or antibody that is the IgG1, IgG2, IgG3, or IgG4 antibody is referred to as the IgG class. Antibody fragments may be obtained using conventional techniques known to those skilled in the art; Suitable fragments that can be synthesized are readily available for use in the same manner as intact antibodies. can be screened.

[0033] The term "bispecific antibody" refers to two independent antibodies that each target an independent target. These targets are located on different proteins. The epitopes may be present on the same target or different epitopes present on the same target. The monospecific bivalent antibody molecule is formed by compensatory amino acid modifications in the constant region of the HC of the parent monospecific bivalent antibody molecule. The resulting heterodimeric antibody can be generated using two different parent monospecific antibodies. The amino acid alterations in the Fc region have been shown to be consistent over time. Stable bispecific heterodimeric antibodies provide improved stability (Ridgw ay et al.,Protein Engineering 9,617-621( 1996), Gunasekaran et al., JBC 285, 19637-1 (2010), Moore et al., MAbs 3:6 546-557 (201 1), Strop et al., JMB 420, 204-219 (2012), Me tz et al.,Protein Engineering 25:10 571- 580(2012), Labrijn et al., PNAS 110:113,51 45-5150(2013), Spreter Von Kreudenstein e t al., MAbs 5:5 646-654 (2013)). Bispecific antibodies are S The two monospecific scfvs can then be combined to produce a single molecule. The antibodies can then form stable heterodimers to generate a single bispecific molecule. (Mabry et al., PEDS 23:3 115 -127(2010). Bispecific molecules have dual binding capabilities. For example, CNS diseases To deliver therapeutic antibodies across the blood-brain barrier to treat It targets both cell transport and surface receptors.

[0034] GM37, GM-37, GM37 wild type (wt), mab37 and 6004-37 The terms are used interchangeably herein and all refer to the same antibody.

[0035] The term antibody GM37 refers to the heavy chain and CDRs 1-3 as shown in SEQ ID NOs: 1-3. An antibody comprising or consisting of light chain CDR1 to 3 shown in SEQ ID NOs: 4 to 6, or In one embodiment, the antibody GM37 or The antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 and / or a heavy chain variable region of SEQ ID NO: 8. The antibody GM37 may comprise or consist of a light chain variable region of SEQ ID NO:8. A light chain consisting of a variable region and a kappa constant region of SEQ ID NO: 17, together with a variable region of SEQ ID NO: 7. and the constant region of SEQ ID NO:18.

[0036] Deamination of proteins, in this case antibodies, occurs during production and storage, and in vivo. Deamination can also occur naturally, making it difficult to control the quality of the final drug. Deamination can also affect the activity of the molecule in some cases. However, the location of the relevant asparagine can be difficult to predict with certainty. However, in some cases, it may be influenced by the asparagine-glycine motif. Potential deamination motifs for the GM37 antibody are found, but the potential for deamination One site was found to be residue 54 of the heavy chain. Subsequent replacement of the lagin was not straightforward, and three mutants of GM37 (GM37 mutant (v ar) 1, 2 and 3) retain the activity of the original GM37 (GM37 wild type (wt)). I found out that...

[0037] The term GM37 mutant refers to deaminated mutants 1, 2, or 3, Mutant 1 has an N54S substitution compared to the GM37 antibody described herein above. Whereas mutant 2 has an N54Q substitution and mutant 3 has N54H.

[0038] Here, antibody GM37 variants (var) 1, 2 and 3 comprise CDR1 and CDR2 from GM37. and 3) from heavy chains shown in SEQ ID NOs: 1 and 3 and GM37 shown in SEQ ID NOs: 4 to 6 33, except that variant 1 comprises or consists of the light chain CDRs 1 to 3 of SEQ ID NO: 33. 2, variant 2 has CDR2 of SEQ ID NO: 34, and variant 3 has CDR3 of SEQ ID NO: 35. and antibodies or their antigen-binding fragments, which differ in their heavy chain CDR2s such that they have the same CDR2 as the heavy chain CDR2. This is intended to include ment.

[0039] In one embodiment, the antibody GM37 mutant or antigen-binding fragment thereof is heavy chain variable regions of SEQ ID NOs: 30, 31 and 32 for variants 1, 2 and 3, respectively; and the light chain variable region of SEQ ID NO: 8. Antibody GM37 may comprise or consist of the sequence SEQ ID NO:3 together with a light chain consisting of a variable region of SEQ ID NO:8 and a kappa constant region of SEQ ID NO:17 IgG comprising a heavy chain consisting of a variable region of 0, 31 or 32 and a constant region of SEQ ID NO: 18 It may be an antibody.

[0040] The terms GM285, GM-285, mab285 and 6004-285 are used herein. They are used synonymously in the specification and all refer to the same antibody.

[0041] The term antibody GM285 refers to the heavy chain and CDRs 1-3 shown in SEQ ID NOs: 20-22. An antibody comprising or consisting of light chain CDR1 to 3 shown in SEQ ID NOs: 23 to 25, or It is intended to include antigen-binding fragments thereof. In one embodiment, the antibody GM37 or an antigen-binding fragment thereof, comprising the heavy chain variable region of SEQ ID NO: 26 and / or the heavy chain variable region of SEQ ID NO: For example, the antibody GM37 can comprise or consist of the light chain variable region of the sequence SEQ ID NO:2, together with a light chain consisting of a variable region of SEQ ID NO:27 and a kappa constant region of SEQ ID NO:29 The antibody may be an IgG antibody comprising a heavy chain consisting of a variable region of SEQ ID NO:6 and a constant region of SEQ ID NO:28.

[0042] The GM285 antibody is a polypeptide that binds to sequences 112 to 115 (I It specifically binds to an epitope within the LED; SEQ ID NO: 19.

[0043] Unless otherwise specified herein, the numbering of amino acid residues in this region is , IMGT®, International ImMunoGeneTics Information System® Or Kabat, EA, Wu, TT, Perry, HM, Gottes mann, K. S. & Foeller, C. (1991).Sequences of Proteins of Immunological Interest,5th edited by,NIH Publication no.91-3242, U.S. Department of Health and Human Services. (USDepartment of Health and Human Services ces).Chothia, C. & Lesk, AM (1987).Canonical al structures for the hypervariable doma ins of immunogloblins.J.Mol.Biol.196, 901 -917).

[0044] "Anti-α-synuclein antibody" or "α-synuclein antibody" (as the context requires) and are used interchangeably herein) refers to α-cyclodextrin as defined herein above. nuclein or alpha-synuclein fragments, in particular SEQ ID NO: 9 and / or 19 An antibody or antigen-binding fragment thereof that specifically binds to the corresponding sequence of α-synuclein. This is a case in point.

[0045] As used herein, the term "human antibody" ("humAb" or "HuAb"). The uMab antibody (which may be abbreviated as "uMab") is a recombinant antibody that contains variable regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention are intended to include antibodies having a human genomic DNA sequence and constant region. It may include amino acid residues not encoded by the cell lineage immunoglobulin sequence (e.g., By random or site-specific mutagenesis in vitro or during gene rearrangement , or mutations induced by somatic mutations).

[0046] As used herein, "monoclonal antibody" or "monoclonal antibody The term "composition" refers to a preparation of antibody molecules of single molecular composition. A cloned antibody composition displays a single binding specificity and affinity for a particular epitope. In certain embodiments, a monoclonal antibody may be composed of two or more Fab regions, This increases the specificity for two or more targets. The term "monoclonal antibody composition" refers to any particular method of production (e.g., recombinant It is not intended to be limited by the type of gene (e.g., transgenic, hybridoma, etc.) used in the present invention. stomach.

[0047] The term "humanized" refers to a gene derived from a non-human species, generally prepared using recombinant techniques. The antigen-binding sites from immunoglobulins and the structures and / or structures of human immunoglobulins or a molecule having the remaining immunoglobulin structure based on the sequence. Complete non-human antibody variable regions fused to human constant regions, or appropriate humanized versions of the human variable regions. Only the complementarity determining regions (CDRs) of such variable regions grafted onto the framework regions The framework residues of such humanized molecules may include any of the wild-type (e.g., They may be fully human, or the sequences may have served as a basis for humanization. It may be modified to contain one or more amino acid substitutions not found in human antibodies. This reduces or eliminates the possibility that the constant regions of the molecule act as immunogens in human individuals. However, the possibility of an immune response to foreign variable regions remains (LoBug lio,AFet al.(1989)“Mouse / Human Chimeri c Monoclonal Antibody In Man:Kinetics An d Immune Response”,Proc.Natl.Acad.Sci.(U Another approach provides constant regions of human origin. Not only that, but we have also modified the variable regions to reshape them as closely as possible to the human form. The variable regions of both the heavy and light chains bind to the corresponding antigen. Each antibody has three complementarity determining regions (CDRs) that determine its binding ability and respond differently to different antigens. The complementarity determining regions (CDRs) of a gene are relatively conserved in a given species and have a structure that is similar to that of the CDR scaffold. It is known that the ribonuclease is flanked by four framework regions (FRs) that are thought to provide When a non-human antibody is prepared against a specific antigen, the variable region is derived from the non-human antibody. The CDRs of the target human antibody are then grafted onto the FRs present in the target human antibody to form a "reshaped" antibody. The application of this technique to various antibodies can be described in detail in Sato, Kei, et al. t al.(1993)Cancer Res 53:851-856.Riechma nn, L. et al. (1988) “Reshaping Human Antibodies” dies for Therapy”,Nature 332:323-327;Ver hoeyen, M. et al. (1988) “Reshaping Human An tibodies:Grafting An Antilysozyme Activi ty”,Science 239:1534-1536;Kettleborough, CA et al. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting:The Importance Of Framework Residues On Loo p Conformation”,Protein Engineering 4:77 3-3783;Maeda,H.et al.(1991)“Construction Of Reshaped Human Antibodies With HIV-N eutralizing Activity”,Human Antibodies H ybridoma 2:124-134;Gorman,S.D.et al.(199 1)“Reshaping A Therapeutic CD4 Antibody” ,Proc.Natl.Acad.Sci.(U.S.A.)88:4181-4185 ;Tempest,P.R.et al.(1991)“Reshaping A Hu man Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection i n vivo”,Bio / Technology 9:266-271;Co,M.S. et al.(1991)“Humanized Antibodies For An tiviral Therapy”,Proc.Natl.Acad.Sci.(U.S .A.)88:2869-2873;Carter,P.et al.(1992)“H umanization Of An Anti-p185her2 Antibody For Human Cancer Therapy”,Proc.Natl.Aca d.Sci.(U.S.A.)89:4285-4289;およびCo,M.S.et al.(1992)“Chimeric And Humanized Antibod ies With Specificity For The CD33 Antige This has been reported by Dr. In embodiments, a humanized antibody contains all of the CDR sequences (e.g., all of the sequences derived from a murine antibody). In another embodiment, the humanized mouse antibody comprises all six CDRs. The antibody may have one or more CDRs (one, two, three, four, , 5, 6), which are derived from one or more CDRs from the original antibody. " Also referred to as one or more CDRs. The ability to humanize antigens is well known (e.g., U.S. Patent Nos. 5,225,539; 5,530,101; Specification No. 85,089; Specification No. 5,859,205; Specification No. 6,407,213 (See detailed description; Id. No. 6,881,557).

[0048] As used herein, an antibody or antigen-binding fragment thereof refers to an antibody that binds to another epitope. More frequently, more rapidly, for a longer period, and / or If a molecule reacts or associates with another molecule (i.e., an epitope) with a higher affinity or avidity, In one embodiment, an antibody of the invention is said to "specifically bind" to a region of a target polypeptide (a polypeptide that is a polypeptide of interest). or an antigen-binding fragment thereof that binds to its target (human α-synuclein) rather than to another molecule at least 10 times stronger; preferably at least 50 times stronger, more preferably at least Preferably, the antibody, or antigen-binding fragment thereof, binds to the physiological Thus, the 11th residue of human α-synuclein binds to the 11th residue of human α-synuclein under physiological conditions, e.g., in vivo. "Specifically binds" to an epitope within 2-117 (ILEDMP (SEQ ID NO: 9)) The antibody capable of isolating human α-synthesis cells with such specificity and / or under such conditions. An antibody capable of binding to an epitope within residues 112-117 of klein or an antibody thereof Suitable methods for determining such binding are within the skill of the art. Methods are known and exemplary methods are described in the accompanying Examples. When used in the context of binding of an antibody to a given antigen, the term "binding" typically refers to the binding of an antibody to a given antigen. The antigen was used as the ligand and the antibody as the analyte in a BIAcore® 3000 or by surface plasmon resonance (SPR) techniques on either the T200 or T200 instruments. Approximately 10% of the decision was made -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less KD refers to binding with an affinity corresponding to a given antigen or closely related antigens. At least as strong as its affinity for binding to non-specific antigens (e.g., BSA, casein) 10 times lower, e.g. at least 100 times lower, e.g. at least 1,000 times lower, e.g. For example, at least 10,000-fold lower, e.g., at least 100,000-fold lower KD. The amount at which the affinity is lower depends on the KD of the antibody. Therefore, if the KD of an antibody is very low (i.e., the antibody is very specific), the antibody will bind to the antigen. The amount at which the affinity for the antibody is at least 10,000-fold lower than the affinity for a nonspecific antigen. It could be.

[0049] As used herein, the term "kd" (sec-1 or 1 / sec) refers to a specific The term "koff" refers to the dissociation rate constant of an antibody-antigen interaction. Said value is also called the koff value.

[0050] As used herein, the term "ka" (M-1 x sec-1 or 1 / M sec) The term refers to the binding rate constant of a particular antibody-antigen interaction.

[0051] As used herein, the term "KD" (M) refers to the molecular weight of a particular antibody-antigen interaction. This refers to the dissociation equilibrium constant of an effect, and is obtained by dividing kd by ka.

[0052] As used herein, the term "KA" (M-1 or 1 / M) refers to a specific This refers to the equilibrium constant of the antibody-antigen interaction, and is obtained by dividing ka by kd. do.

[0053] In one embodiment, the present invention provides a method for producing a composition comprising the steps of: i. 0.5-10 nM of α-synuclein, such as 1-5 nM or 1-2 nM Binding affinity (KD); ii. the ability to inhibit protease cleavage of α-synuclein fibrils; iii. Impairment of basal synaptic transmission in F28-snca transgenic mice Ability to improve; iv. α-synuclein in the mouse hippocampus as measured by in vivo microdialysis Ability to reduce levels; v. When administered chronically, it restores motor function in a rat model of Parkinson's disease. Ability to restore vi. Alpha-Synuclein Seeding (in vitro and / or Parkinson's their ability to prevent the accumulation of insoluble phosphorylated α-synuclein in mouse models of glaucoma; and / or vii. Ability to bind truncated α-synuclein in human brain The present invention relates to an antibody or antigen-binding fragment thereof which exhibits one or more of the following:

[0054] The binding affinity (KD) for α-synuclein was determined using the method described in, for example, Example 2. This can be determined using methods well known in the art.

[0055] The term "ability to inhibit protease cleavage of α-synuclein fibrils" refers to the ability of primary dermal Calpain-dependent expression of human α-synuclein fragment 1-119-122 in stromal neurons 1-induced formation (see Example 5).

[0056] "Improvement of impaired basal synaptic transmission in F28-snca transgenic mice" The term "ability to induce a neuronal response" refers to the slope of the fEPSP evoked by the neuronal response, as measured, for example, electrophysiologically. As shown by the results of including the ability to improve the decline in synaptic transmission and plasticity in the CA1 region (see Example 6) reference).

[0057] "Levels of α-synuclein in the mouse hippocampus as measured by in vivo microdialysis" The term "ability to reduce the amount of vasopressin in the hippocampus" refers to the ability to reduce the amount of vasopressin in the hippocampus as measured using in vivo microdialysis. Human α-synuclein levels in freely moving F28-snca transgenic mice These include the ability to reduce the risk of heart failure (see Example 7).

[0058] "Restores motor function in a rat model of Parkinson's disease when administered chronically" The term "ability to induce Parkinson's disease in rats using recombinant adeno-associated virus vectors" refers to This includes the ability to reduce or eliminate motor asymmetry in rAAV models ( See Example 8).

[0059] In some antibodies, only a small portion of the CDRs, called SDRs, are required for binding. A subset of the essential CDR residues are required to retain binding in the humanized antibody. CDR residues that do not contact the relevant epitope and are not in the SDRs are selected based on previous studies. (e.g. residues H60-H65 in CDR H2 are often not required) From the Kabat CDR region outside the hothia hypervariable loop (Kabat e t al. (1992) SEQUENCES OF PROTEINS OF IMMU NOLOGICAL INTEREST,National Institutes o f Health Publication No.91-3242;Chothia, C. et al. (1987) “Canonical Structures For The Hypervariable domains Of Immunoglobu lins”, J. Mol. Biol. 196:901-917), molecular modeling and / or by experiments, or by Gonzales, NR et al. 2004) “SDR Grafting Of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity”,Mol.Immu Nol. 41:863-872. - such a position where there is no CDR residue or the entire donor CDR is omitted In humanized antibodies, the amino acid occupying this position is identical to that in the recipient antibody sequence (Kaba The amino acids in the CDRs included may occupy corresponding positions (according to the corresponding numbering). The number of such substitutions of the receptor for the donor amino acid reflects the balance of competing considerations. Such substitutions reduce the number of mouse amino acids in the humanized antibody, resulting in , which is potentially advantageous in reducing potential immunogenicity. However, substitutions may also be made to improve affinity. It is preferable to avoid a significant decrease in affinity. The position of substitution within R and the substituting amino acid may also be selected empirically.

[0060] A single amino acid modification of a CDR residue can result in loss of functional binding (R udikoff,S.etc.(1982)“Single Amino Acid S ubstitution Altering Antigen-Binding Spe cificity”,Proc.Natl.Acad.Sci.(USA)79(6): 1979-1983) provided a means to systematically identify alternative functional CDR sequences. In one preferred method for obtaining such mutant CDRs, The polynucleotide that corresponds to the target gene is mutated (e.g., by random mutation, site-specific methods (e.g., polymerase chain reaction with primers encoding the mutated locus) The CDRs are then generated with substituted amino acid residues by enzyme-mediated amplification. The identity of relevant residues in the (functional) CDR sequences is compared with the identity of the replaced (non-functional) mutant CDR sequences. BLOSUM62.iij substitutions for that substitution by comparing with the identity of the columns A score can be assigned to each of the alignments. The BLOSUM system ranks the alignments by their reliability. Provides a matrix of amino acid substitutions created by analyzing a database of columns (Eddy,SR(2004)“Where Did The BLOSUM6 2 Alignment Score Matrix Come From?”,Nat ure Biotech.22(8):1035-1036;Henikoff,JG (1992) “Amino acid substitution matrices from protein blocks”,Proc.Natl.Acad.Sci (USA)89:10915-10919;Karlin,S.et al.(199 0)“Methods For Assessing The Statistical Significance Of Molecular Sequence Feat ures By Using General Scoring Schemes”,P roc.Natl.Acad.Sci.(USA)87:2264-2268;Alts chul, SF (1991) “Amino Acid Substitution” Matrices From An Information Theoretic P erspective”, J. Mol. Biol. 219, 555-565. Currently, the most advanced The BLOSUM database at the end is the BLOSUM62 database (BLOSUM62. Table 1 shows the BLOSUM62.iij substitution scores (higher scores indicate lower substitutions). The more conservative the substitution, the more likely it is that the substitution will not affect function. The resulting antigen-binding fragment containing the CDRs is unable to bind to α-synuclein. For example, the BLOSUM62.iij substitution score is considered to be insufficiently conservative. Then, new replacement candidates with higher replacement scores are selected and generated. For example, if the original residue is glutamate (E) and the non-functional replacement residue is histidine (H), If so, the BLOSUM62.iij substitution score is 0, and the more conservative change (aspartate such as to partate, asparagine, glutamine, or lysine are preferred.

[0061] [Table 1]

[0062] Thus, the present invention provides the use of random mutagenesis to identify improved CDRs. In the context of the present invention, conservative substitutions are those found in one or more of the following three tables: The amino acid sequence may be defined by substitutions within the range of amino acid types reflected in .

[0063] [Table 2]

[0064] [Table 3]

[0065] [Table 4]

[0066] A more conservative substitution grouping would be Leucine-Isoleucine, Phenylalanine-Tyrosine, Lysine-Arginine, Arginine Nin-valine, and asparagine-glutamine.

[0067] Further groups of amino acids are described, for example, in Creighton (1984) Proteins :Structure and Molecular Properties(2d E d.1993), the original published by WH Freeman and Company The formula can be formulated using the above principles.

[0068] Phage display technology is an alternative to increasing (or decreasing) CDR affinity. This technique, called affinity maturation, can be used instead of mutations or "CDR warping." "King walking" is used, and re-selection is performed by selecting the target antigen. or an antigenic antigen-binding fragment thereof, compared to the initial or parent antibody. In this case, an antibody having a CDR that binds to the antigen with higher (or lower) affinity is selected. Identify (see, e.g., Glaser et al. (1992) J. Immunology 149:3903). Mutagenesis of entire codons rather than single nucleotides is Generate a semi-randomized repertoire of amino acid mutations from a pool of mutant clones A library of mutant clones can be constructed, each of which contains a single CDR in a single CDR. Mutations that vary by amino acid modification and represent each possible amino acid substitution for each CDR residue Mutants that have increased (or decreased) binding affinity for an antigen include The immobilized mutants can be screened by contacting them with labeled antigen. Any screening method known in the art can be used to screen for increased or decreased antibody responses to the antigen. The method can be used to identify mutant antibodies with different affinities (e.g., ELISA) Wu et al.1998,Proc.Natl.Acad.Sci.(USA )95:6037;Yelton et al., 1995, J. Immunology 155:1994). CDR walking, which randomizes the light chain, can be used. Possible (Schier et al., 1996, J. Mol. Bio. 263:55 See 1.

[0069] Methods for achieving such affinity maturation are described, for example, in: Krause, J. C. et al. al.(2011) “An Insertion Mutation That Di storts Antibody Binding Site Architectur e Enhances Function Of A Human Antibody” ,MBio.2(1)pii:e00345-10.doi:10.1128 / mBio .00345-10;Kuan,CTet al.(2010)“Affinity -Matured Anti-Glycoprotein NMB Recombina nt Immunotoxins Targeting Malignant Glio mas And Melanomas”,Int.J.Cancer 10.1002 / ijc.25645;Hackel, BJet al. (2010) “Stabil ity And CDR Composition Biases Enrich Bi nder Functionality Landscapes”, J.Mol.Bio l.401(1):84-96;Montgomery,DLet al.(200 9) “Affinity Maturation and Characterization ion Of A Human Monoclonal Antibody Again st HIV-1 gp41”,MAbs 1(5):462-474;Gustchi Na, E. et al. (2009) “Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library A nd Directed Against The Internal Trimeri c Coiled-Coil Of Gp41 Yields A Set Of Fa bs With Improved HIV-1 Neutralization Po tency And Breadth”, Virology 393(1):112-1 19; Finally, W. J. et al. (2009) “Affinity Maturat ion Of A Humanized Rat Antibody For Anti -RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plast icity Both Inside And Outside The Comple mentarity-Determining Regions”, J. Mol. Bio l. 388(3):541-558; Bostrom, J. et al. (2009) “ Improving Antibody Binding Affinity And Specificity For Therapeutic Development” , Methods Mol. Biol. 525:353-376; Steidl, S. e t al. (2008) “In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification”,Mol.Immunol.46(1):1 35-144; and Barderas, R. et al. (2008) “Affini ty Maturation Of Antibodies Assisted By In Silico Modeling”,Proc.Natl.Acad.Sci.( USA) 105(26):9029-9034.

[0070] Thus, the sequences of CDR variants of the included antibodies or antigen-binding fragments thereof The columns are sorted by substitution; for example, four amino acid residues substituted, three amino acid residues substituted, two or one amino acid residue from the parent antibody, GM37, GM37 va r may be different from the sequence of CDRs 1 to 3, or 285. According to one embodiment of the present invention, Amino acids in the CDR regions are replaced with conservative substitutions as defined in the three tables above. It is further envisioned that the

[0071] As used herein, "treatment" or "treating" refers to The term "treating" refers to improving the progression or severity of a disease or disorder, to retard, alleviate, or inhibit the development of one or more of such diseases or disorders; This means improving, delaying, alleviating, or suppressing a number of symptoms or side effects. For purposes of clarity, "treatment" or "treating" refers to obtaining beneficial or desired clinical results. The present invention further refers to a method for the treatment of a disease comprising administering to a subject a therapeutically effective amount of ... Not detected, but partial or total, detectable or undetectable Regardless of the cause, symptoms are reduced, the extent of the disability or illness is reduced, or the condition remains stable (i.e., not worsening). (not including) a disease or disorder condition, delay or slowing of the progression of a disease or disorder condition, This includes improvement or alleviation of the disorder state, and remission of the disease or disorder.

[0072] "Effective amount" as applied to an antibody or antigen-binding fragment thereof of the present invention is intended to to achieve a desired biological effect or desired therapeutic outcome (including but not limited to a clinical outcome). "Therapeutically effective amount" refers to an amount sufficient, at dosages and for periods of time necessary, to The term "antibody" as applied to an antibody or antigen-binding fragment thereof of the present invention means a disorder or improve, alleviate, or stabilize the progression of a disease state or the symptoms of a disorder or disease or an antibody or antigen-binding fragment thereof sufficient to inhibit, retard, reduce, or delay In one embodiment, the method of the present invention is intended to represent the amount of other chemical fragments. The present invention provides for the administration of an antibody, or an antigen-binding fragment thereof, in combination with a compound. In such cases, an "effective amount" is the amount of the combination sufficient to cause the intended biological effect. be.

[0073] A therapeutically effective amount of an anti-α-synuclein antibody or antigen-binding fragment thereof of the present invention. The method includes determining the disease state, age, sex, and weight of the individual, and whether anti-α-synuclein antibodies are present in the individual. A therapeutically effective amount may vary depending on factors such as the ability of the compound to elicit a desired response in a subject. In addition, any toxic or adverse effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The quantity.

[0074] As indicated above, the present invention relates in particular to a method for the preparation of a nucleic acid sequence encoding amino acids 112-11 of human α-synuclein. 7 (SEQ ID NO: 9 (ILEDMP)) In one embodiment, the antibody is a clonal antibody that binds 112-113 of alpha-synuclein. Able to compete with antibody GM37 for binding to an epitope within 17 amino acids .

[0075] GM37, its mutants GM37 var 1-3 and GM285 are exemplified. The antibodies of the present invention, as well as α-synuclein binding fragments thereof, It binds to a toxic α-synuclein fragment consisting of residues 1–119 / 122 of the α-synuclein complex (e.g. For example, they bind to α-synuclein fragments extracellularly, thereby allowing them to be taken up by cells. It is possible to neutralize its toxicity by preventing it from being absorbed into the blood. - a peptide capable of binding to an epitope within amino acids 112-117 of synuclein The antibodies are more efficient than previous antibodies such as antibody 9E4 in binding to toxic α-synuclein species in the human brain. It is superior to conventional antibodies in removing extracellular α-synuclein and inhibiting α-synuclein in vivo. The present invention has an excellent effect in normalizing synaptic transmission disorders induced by insulin. Antibodies to ameliorate the manifestation of the relevant motor phenotype in a rat model of Parkinson's disease It is also possible.

[0076] The antibodies of the present invention are preferably human or humanized antibodies.

[0077] The present invention provides a method for reducing α-synuclein aggregate formation in a patient, the method comprising: administering to a patient in need of such treatment a therapeutically effective amount of an antibody of the present invention. A method is also provided.

[0078] Additionally, the antibody may be formulated with a pharma- ceutically acceptable carrier, diluent and / or stabilizer. The antibodies of the present invention may be used in therapy. In particular, the antibodies of the present invention may be used in the treatment of parkinsonism. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV), combined Alzheimer's disease Synucleinopathies such as Parkinson's disease, pure autonomic failure, and multiple system atrophy It can be used to treat inflammatory bowel disorders.

[0079] The treatment contemplated by the present invention may be long-term, with the patient receiving treatment for at least 2 weeks. For example, treatment may be for at least one month, six months, a year or longer.

[0080] The antibodies of the present invention, and antigen-binding fragments thereof, can be expressed in a variety of cell lines, including human cell lines, non-human cell lines, and the like. mammalian cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-2 1 cell line, mouse cell line (myeloma cell line, etc.), fibrosarcoma cell line, PER.C6 cell line, It can be produced in HKB-11 cell line, CAP cell line and HuH-7 human cell line ( Dumont et al., 2015, Cri t Rev Biotechnol.Sep 18:1-13.).

[0081] The antibodies of the present invention can be used in the methods described, for example, in Kohler et al., Nature 256, 495 (1975) It may be a clonal antibody or may be produced by recombinant DNA methods. Clonal antibodies can also be used, for example, as described in Clackson et al., Nature 35 2, 624-628 (1991) and Marks et al., J. MoI. Biol. phage antibodies using the techniques described in J. Immunol. 222, 581-597 (1991). The monoclonal antibodies may be isolated from a library. The monoclonal antibodies may be obtained from any suitable source. Thus, for example, a monoclonal antibody can be used to target, for example, a cell expressing an antigen on its surface. or from mice immunized with the antigen of interest in the form of a nucleic acid encoding the antigen of interest. The hybridoma is prepared from the mouse splenic B lymphocyte cells obtained. The local antibodies can also be obtained from immunized humans or non-human mammals (rats, rabbits, dogs, These antibodies are obtained from hybridomas derived from antibody-expressing cells of various mammals (such as horses, goats, and primates).

[0082] In one embodiment, the antibody of the present invention is a human antibody. Monoclonal antibodies are produced by transgenic mice that carry parts of the human immune system rather than the mouse system. Such transgenic and The transchromosomal mice are referred to herein as HuMAb mice and KM mice, respectively. This includes mice.

[0083] HuMAb mice are engineered with targeted mutations that inactivate the endogenous μ and κ chain loci. In addition, unrearranged human heavy chain variable and constant (μ and Y) and light chain variable and A minilocus of human immunoglobulin genes encoding constant (kappa) chain immunoglobulin sequences. (minilocus) (Lonberg, N. et al., Nature 3 68, 856-859 (1994). Thus, mice are The mice showed reduced expression of κ in response to immunization with the introduced human heavy and light chain transgenes. However, they undergo class switching and somatic mutation to form high-affinity human IgG, κ monoclonal antibodies. To generate monoclonal antibodies (Lonberg, N. et al. (1994), supra; onberg, N., Handbook of Experimental Pharm. Acology 113, 49-101 (1994), Lonberg, N. and H. uszar,D.,Intern.Rev.Immunol.Vol.13 65-93 (1995) and Harding, F. and Lonberg, N., Ann.N. Y. Acad. Sci 764 536-546 (1995). The MAb mice were produced according to Taylor, L. et al., Nucleic Acids Research 20,6287-6295(1992), Chen,J.et a l., International Immunology 5,647-656(19 93), Tuaillon et al., J. Immunol. 152, 2912-2 920 (1994), Taylor, L. et al., International Immunology 6,579-591(1994), Fishwild,D.et al.,Nature Biotechnology 14,845-851(199 6) are described in detail in U.S. Patent No. 5,545,806 and U.S. Patent No. 569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,63 No. 3,425, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,877, 397, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,31 No. 8, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,770,429 Specification, U.S. Patent No. 5,545,807 Specification, International Publication No. 98 / 24884 Pamphlet Lett., WO 94 / 25585 Pamphlet, WO 93 / 1227 Pan Fret, WO 92 / 22645 Pamphlet, WO 92 / 03918 See also WO 01 / 09187.

[0084] HCo7, HCo12, HCo17 and HCo20 mice express their endogenous light chains ( JKD disruption in the κ gene (Chen et al., EMBO J. 12, 811 6, pp. 1111-1115 (1993)), C in their endogenous heavy chain genes MD destruction (as described in Example 1 of WO 01 / 14424) ), and the KCo5 human kappa light chain transgene (Fishwild et al., Natl. J. Clin. Soc. 2014). It is described in Ure Biotechnology 14, 845-851 (1996). In addition, the HCo7 mouse carries the HCo7 human heavy chain transgene ( As described in the specification of Patent No. 5,770,429, The HCo12 human heavy chain transgene (WO 01 / 14424) The HCo17 mice have a human heavy chain transgene (as described in Example 2) and Gene (as described in Example 2 of WO 01 / 09187) and the HCo20 mouse carries a HCo20 human heavy chain transgene. background homozygous for disruption of the endogenous mouse heavy chain and kappa light chain loci The cells express human immunoglobulin heavy chain and kappa light chain transgenes in the host.

[0085] In the KM mouse strain, the endogenous mouse kappa light chain gene is expressed as Homozygously disrupted as described in BO J. 12, 811-820 (1993) The endogenous mouse heavy chain gene is The mouse strain was homozygously disrupted as described in Example 1. hwild et al.,Nature Biotechnology 14,845 -851 (1996), harboring a human kappa light chain transgene, KCo5. This mouse strain was developed as described in WO 02 / 43478. Similarly, a human heavy chain transchromosome consisting of chromosome 14 fragment hCF(SC20) was also HCo12-Balb / c, HCo17-Balb / c and HCo20-B The alb / c mouse was used as described in WO 09 / 097006. As shown, HCo12, HCo17 and HCo20 are converted to KCo5[J / K](Balb). It can be generated by crossing.

[0086] In the KM mouse strain, the endogenous mouse kappa light chain gene is expressed as Homozygously disrupted as described in BO J. 12, 811-820 (1993) The endogenous mouse heavy chain gene is The mouse strain was homozygously disrupted as described in Example 1. hwild et al.,Nature Biotechnology 14,845 -851 (1996), harboring a human kappa light chain transgene, KCo5. This mouse strain was developed as described in WO 02 / 43478. Thus, a human heavy chain transfectant composed of chromosome 14 antigen-binding fragment hCF (SC20) was used. It also has chromosomes.

[0087] Spleen cells from these transgenic mice were cultured according to well-known techniques. The method can be used to generate hybridomas secreting monoclonal antibodies of the present invention. Human monoclonal or polyclonal antibodies, or antibodies of the invention derived from other species The present invention also provides another method for producing a transgenic vector for the relevant immunoglobulin heavy and light chain sequences. The generation of a non-human mammal or plant and the production of an antibody in a recoverable form. In connection with transgenic production in mammals, antibodies can be produced by inducing It can be produced in and recovered from the milk of goats, cows, or other mammals. No. 5,827,690, U.S. Pat. No. 5,756,687, U.S. Pat. See U.S. Pat. Nos. 5,750,172 and 5,741,957. I want to.

[0088] The antibodies of the present invention can be of any isotype. The choice of isotype is typically determined by the Specifically, the desired effector function, such as ADCC induction, is guided. The types are IgG1, IgG2, IgG3, and IgG4. Optionally, either α-synuclein or λ can be used. The class can be switched by known methods. For example, the class of the antibody of the present invention, which was originally IgM, The antibody may be class switched to an IgG antibody of the present invention. Further, class switching techniques include , converting one IgG subclass to another, e.g., from IgG1 to IgG2 Thus, the effector functions of the antibodies of the invention can be used to treat a variety of therapeutic For use, for example, isotypes of IgG1, IgG2, IgG3 or IgG4 antibodies may be used. In one embodiment, the antibody of the present invention is an IgG1 antibody. Antibodies are isotypes, such as IgG1 and κ. Antibodies have amino acid sequences that are different from other isotypes. If a protein has the highest homology to that isotype, it is said to be of a particular isotype. do.

[0089] In one embodiment, the antibody of the invention is a full-length antibody, preferably an IgG antibody, in particular In another embodiment, the antibody of the present invention is an antibody fragment or or a single chain antibody.

[0090] Antibodies and antigen-binding fragments thereof can be prepared, for example, by subjecting the antibodies to antigen-binding fragmentation using conventional techniques. and the antigen-binding fragments are obtained in the same manner as described herein for whole antibodies. For example, F(ab')2 antigen-binding fragments can be screened for usefulness. The resulting F(ab') fragment can be generated by treating the antibody with pepsin. The 2 antigen-binding fragments are treated to reduce disulfide bridges to form Fab' antigens. Fab antigen-binding fragments can be produced by binding IgG antibodies to papain. Fab' antigen-binding fragments are obtained by treating IgG antibodies with pepsi F(ab') antigen-binding fragments can also be obtained by thioether or thiol digestion. or disulfide bonds, Fab' antigen-binding fragments can be made by isolating the disulfide bonds in the hinge region of F(ab')2. Fab'-antigen is an antibody-antigen binding fragment obtained by cleaving the bond. The binding fragment may be prepared by isolating the F(ab')2 antigen-binding fragment with dithiothreitol or the like. Antibody antigen-binding fragments can also be obtained by treating the antibody with a reducing agent such as . Such antigen-binding fragments may be produced by expression of a nucleic acid encoding such a fragment in a cell. (e.g., Evans et al., J. Immunol. Meth. 184, 12 For example, a portion of the F(ab')2 antigen-binding fragment may be The chimeric gene encoding such a truncated antibody antigen-binding fragment molecule is To achieve this, the DNA sequence encoding the CH1 region and hinge region of the H chain is inserted, followed by a translation termination sequence. It may include a stop codon.

[0091] In one embodiment, the anti-α-synuclein antibody is a monovalent antibody, preferably a hinge region antibody. No. WO 2007059782 (incorporated by reference in its entirety) having a deletion of (which is incorporated herein by reference). Thus, in one embodiment, In the present invention, the antibody is a monovalent antibody, and the anti-α-synuclein antibody comprises: i) a light receptor of the monovalent antibody; providing a nucleic acid construct encoding a chain, said construct being capable of binding to a selected antigen-specific Nucleotide sequences encoding the VL regions of specific anti-α-synuclein antibodies and the constant C domains of Ig and a nucleotide sequence encoding the VL region of the selected antigen-specific antibody. The nucleotide sequence encoding the Ig CL region and the nucleotide sequence encoding the Ig CL region The peptide sequences are operably linked together and, in the case of the IgG1 subtype, code for the CL region. The nucleotide sequence that binds to the IgG in the presence of polyclonal human IgG or in animals or humans When administered to the human, it is disulfide-coupled with other peptides containing the same amino acid sequence of the CL region. The CL region was modified to not contain any amino acids capable of forming peptide or covalent bonds. ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody; wherein the construct comprises a nucleotide sequence encoding a VH region of a selected antigen-specific antibody. and a nucleotide sequence encoding the constant CH region of human Ig, The nucleotide sequence encoding the H region includes a region corresponding to the hinge region and an Ig subunit. As required by the polyclonal type, other regions of the CH domain, such as the CH3 domain, When administered to animals in the presence of native human IgG or to humans, the CH region of human Ig is identical to that of human Ig. With other peptides containing the amino acid sequence of The antibody has been modified to exclude amino acid residues involved in the formation of non-covalent heavy chain bonds. the nucleotide sequence encoding the VH region of a selected antigen-specific antibody and the said nucleotide sequences encoding the CH region of cDNA clones of p53 and p53 of human genomic DNA; (iii) providing a cellular expression system for producing said monovalent antibody; and (iv) (i) and (ii) by co-expressing the nucleic acid constructs in a cell of the cell expression system. and producing said monovalent antibody.

[0092] Similarly, in one embodiment, the anti-alpha-synuclein antibody (i) a variable region or an antigen-binding portion of an antibody of the invention described herein; and (ii) a CH region of an immunoglobulin or a region thereof that includes the CH2 and CH3 regions wherein the CH region or a region thereof is a region corresponding to a hinge region. and, if the immunoglobulin is not of the IgG4 subtype, the CH region, such as the CH3 region The other regions form disulfides with the same CH regions in the presence of polyclonal human IgG. or form other covalent or stable non-covalent bonds with the same CH region. It has been modified so as not to contain amino acid residues capable of forming inter-heavy chain bonds.

[0093] In a further embodiment, the heavy chain of the monovalent anti-α-synuclein antibody comprises an amino acid sequence comprising the entire hinge region It has been modified to appear deleted.

[0094] In another further embodiment, the sequence of the monovalent antibody is such that it is N-linked glycosylated. It has been modified so as not to contain the receptor site for

[0095] The present invention relates to anti-α-synuclein binding regions (e.g., anti-α-synuclein monoclonal antibodies). Bivalent or polyvalent antibodies targeting two or more epitopes (the α-synuclein binding region of the antibody) Also included are "bispecific antibodies" that are part of a bivalent bispecific scaffold (e.g., the second epitope is Bispecific antibodies have been shown to provide improved transcytosis across biological barriers, such as the blood-brain barrier. (These may contain epitopes of active transport receptors, so that they may demonstrate cytotoxicity.) In a further embodiment, the monovalent Fab of the anti-synuclein antibody binds different proteins. It may be coupled to additional targeting Fab or scfv to generate bispecific antibodies. Bispecific antibodies have dual functions, e.g., therapeutic mechanisms conferred by anti-synuclein binding regions. and bind to receptor molecules to facilitate transport across biological barriers such as the blood-brain barrier. The vesicles may have a transport function that can be combined with the vesicles.

[0096] The anti-α-synuclein antibodies, and antigen-binding fragments thereof, of the present invention may also be used as single chain antibodies. A single chain antibody is a peptide that combines the heavy and light chain Fv regions. The present invention relates to a method for producing an anti-α-synuclein antibody comprising the steps of: , in a single peptide chain (typically about 10, 12, 15 or more amino acids to provide single-chain Fvs (scFvs) linked with a flexible peptide linker (1000 bp, ... Methods for producing such antibodies are described, for example, in U.S. Pat. No. 4,946,778, P luckthun in The Pharmacology of Monoclon al Antibodies,vol.113,Rosenburg and Moor e eds. Springer-Verlag, New York, pp.269-31 5 (1994), Bird et al., Science 242, 423-426 ( 1988), Huston et al., PNAS USA 85, 5879-588 3(1988) and McCafferty et al., Nature 348, 5 52-554 (1990). Single chain antibodies contain only a single VH and VL. is used, it is monovalent, and when two VH and VL are used it is bivalent, Or it may be multivalent when more than two VH and VL are used.

[0097] The anti-α-synuclein antibodies and antigen-binding fragments thereof described herein can be used in any Any suitable number of modified amino acids may be included and / or combined with such conjugate substituents. The compatibility in this regard is generally the same as that of the underivatized parent anti-α-synuclein antibody. Reduces α-synuclein selectivity and / or anti-α-synuclein specificity associated with the body The inclusion of one or more modified amino acids is determined by the ability to substantially retain the amino acid sequence. e.g., increasing the serum half-life of the polypeptide, reducing polypeptide antigenicity, or It may be advantageous to increase the storage stability of the polypeptide. During gene production, co-translationally or post-translationally post-translationally modified (e.g., in mammalian cells) N-linked glycosylation at the NXS / T motif upon expression in the Non-limiting examples of modified amino acids include glycosylated amino acids, sulfuric acid, and the like. Oxidized amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, Acetylated amino acids, acylated amino acids, PEGylated amino acids, biotinylated amino acids, cal Examples of amino acid modifications include carboxylated amino acids, phosphorylated amino acids, etc. There are ample references throughout the literature that serve as useful pointers. er(1998)Protein Protocols On CD-Rom,Huma Modified amino acids can be found, for example, in the glycosylation of Amino acids with PEGylation, farnesylation, acetylation, biosynthesis, amino acids conjugated to lipid moieties, or conjugated to organic derivatizing agents The amino acid may be selected from substituted or conjugated amino acids.

[0098] Anti-α-synuclein antibodies may also be used in combination with polyclonal antibodies, e.g., to increase their serum half-life. Exemplary polymers and their coupling to peptides are shown in Table 1. Methods for coupling to amides are described, for example, in U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,179,337, U.S. Pat. No. 4,495,285 and U.S. Pat. Further exemplary polymers include poly(ethylene glycol) copolymers, poly(ethylene glycol) copolymers, and poly(ethylene glycol) copolymers. Oxyethylated polyols and polyethylene glycols (PEG) (e.g., about 1,0 00 to about 40,000, for example, about 2,000 to about 20,000, for example, about 3,000 to PEG having a molecular weight of 12,000 g / mol.

[0099] The antibodies of the invention may further be used in diagnostic methods or as diagnostic imaging ligands. .

[0100] In one embodiment, the anti-alpha-synuclein comprises one or more radiolabeled amino acids. Radiolabeled anti-α-synuclein antibodies are provided for both diagnostic and therapeutic purposes. (Conjugation to radiolabeled molecules is another possible feature.) Non-limiting examples of ferroelectrics include, but are not limited to, bismuth ( 213 Bi), Carbon ( 11 C , 13 C. 14 C), Chromium ( 51 Cr), Cobalt ( 57 Co, 60 Co), copper( 64 Cu), Dysprosium ( 165 Dy), Erbium ( 169Er), fluorine ( 18 F) ,gadolinium( 153 Gd, 159 Gd), Gallium ( 68 Ga, 67 Ga), germanium Niu( 68 Ge), Gold ( 198 Au), Holmium ( 166 Ho), hydrogen ( 3 H), I Indium ( 111 In, 112 In, 113 In, 115 In), iodine ( 121 I, 1 23 I, 125 I, 131 I), Iridium ( 192 Ir), iron ( 59 Fe), Crypto hmm( 81m Kr), Lanthanum ( 140 La), lutetium ( 177 Lu), manganese ( 5 4 Mn), Molybdenum ( 99 Mo), Nitrogen ( 13 N, 15 N), oxygen ( 15 O), Paraji Umm ( 103 Pd), Phosphorus ( 32 P), potassium ( 42 K), praseodymium ( 142 Pr ),promethium( 149 Pm), rhenium ( 186 Re, 188 Re), Rhodium ( 1 05 Rh), Rubidium ( 81 Rb, 82 Rb), ruthenium ( 82 Ru, 97 Ru), samarium( 153 Sm), Scandium ( 47 Sc), Selenium (75 Se), sodium M( 24 Na), Strontium ( 85 Sr, 89 Sr, 92 Sr), Sulfur ( 35 S), technetium( 99 Tc), thallium ( 201 Tl), Tin ( 113 Sn, 117 Sn) ,xenon( 133 Xe), Ytterbium ( 169 Yb, 175 Yb, 177 Yb), yttrium( 90 Y) and zinc ( 65 Zn) Radiolabeled amino acids and Methods for preparing peptides and related peptide derivatives are known in the art. For example, Junghans et al., Cancer Chemotherapy nd Biotherapy 655-686(2nd edition, Chafne r and Longo, eds., Lippincott Raven (1996)) Also, U.S. Patent No. 4,681,581 and U.S. Patent No. 4,735,210 No. 5,101,827, No. 5,102,990 ( U.S. Reissue Patent No. 35,500, U.S. Patent No. 5,648,471 and See, for example, U.S. Patent Nos. 5,697,902 and 5,697,902, in which a radioisotope is can be combined by the T method (Lindegren, S. et al. (1998) "Ch loramine-T In High-Specific-Activity Rad ioiodination Of Antibodies Using N-Succi nimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate”, Nucl.Med.Biol.25(7):659 -665;Kurth, M. et al. (1993) “Site-Specific Conjugation Of A Radioiodinated Phenethy lamine Derivative To A Monoclonal Antibo dy Results In Increased Radioactivity Lo calization In Tumor”, J.Med.Chem.36(9):12 55-1261;Rea,DWet al.(1990)“Site-specif ically radioiodinated antibody for target ting tumors”,Cancer Res.50(3 Suppl):857s -861s).

[0101] The present invention relates to a fluorescent label (such as a rare earth chelate (e.g., europium chelate)), a fluorescent Fluorescein-type labels (e.g., fluorescein, fluorescein isothiocyanate, 5- Carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinyl amine fluorescein), rhodamine-type labels (e.g., ALEXA FLUOR® 568 (Invitrogen), TAMRA® or dansyl chloride); VIVOTAG 680 XL FLUOROCHROME(TM)(Perkin E lmer), phycoerythrin;umbelliferone, Lissamine;cyanine; Eicoerythrin, Texas Red, BODIPY FL-SE (registered trademark) (Invitrogen) using a fluorochrome or its analogs, all of which are suitable for optical detection. Also provided are anti-α-synuclein antibodies and antigen-binding fragments thereof that are detectably labeled for use in Chemiluminescent labels may be used (e.g., luminol, luciferase, luciferase, etc.). Such diagnosis and detection also relates to the diagnostic compounds of the present invention. The product can be produced by the addition of various enzymes, including but not limited to, horseradish peroxidase. , alkaline phosphatase, β-galactosidase, or acetylcholinesterase Detectable substances include enzymes, including, but not limited to, streptavidin / vitamin B, This is achieved by binding to prosthetic group complexes such as avidin and avidin / biotin. obtain.

[0102] Chemiluminescent labels may be used (e.g., luminol, luciferase, luciferase, Such diagnosis and detection can also be achieved by using the diagnostic molecules of the present invention, Various enzymes, including but not limited to, horseradish peroxidase, alginate, Contains potassium phosphatase, β-galactosidase, or acetylcholinesterase Detectable substances include enzymes or, but are not limited to, streptavidin / biotin. and by binding to prosthetic group complexes such as avidin / biotin. Paramagnetic labels may also be used, preferably those used for positron emission tomography (PET) or monolayer imaging. It is detected using single photon emission computed tomography (SPECT). The sex markers include, but are not limited to, aluminum (Al), barium (Ba), calcium (Ca), and ruthenium (Ru). Ca, Cerium (Ce), Dysprosium (Dy), Erbium (Er), Eu (Eu), Gd (Gd), Ho (Ho), Iridium (Ir), Lithium (Li), Magnesium (Mg), Manganese (Mn), Molybdenum (M), Neo Nd (Nd), Os (Os), Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), Thoronium (Sr), Terbium (Tb), Thulium (Tm), Tin (Sn), Titanium (Ti), tungsten (W), and zirconium (Zi), especially Co +2 , C.R. + 2 , Cr +3 , Cu +2 , Fe +2 , Fe +3 , Ga +3 , Mn +3 , Ni +2 , Ti + 3 , V +3 , and V +4 paramagnetic ions, using various positron emission tomography techniques These include positron emitting metals, and compounds that contain non-radioactive paramagnetic metal ions.

[0103] Thus, in one embodiment, the anti-α-synuclein antibodies of the invention are fluorescently labeled, The labeled antibody may be labeled with a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzyme label. and detecting or measuring the presence or amount of said alpha-synuclein in the brain of a subject. This method can be used to detect the presence of an anti-α-synuclein antibody bound to the α-synuclein. The method may include detecting or measuring in vivo imaging of the α-synuclein-bound protein. The method may include ex vivo imaging of the anti-α-synuclein antibody.

[0104] In a further aspect, the present invention provides one or more of the antibodies or antigen-binding fragments thereof of the present invention. The present invention relates to an expression vector encoding one or more polypeptide chains. The targets can be used for the recombinant production of antibodies and antigen-binding fragments of the invention.

[0105] Expression vectors in the context of the present invention include chromosomal vectors, non-chromosomal vectors, and synthetic vectors. Any suitable DNA, including synthetic nucleic acid vectors (nucleic acid sequences that include a suitable set of expression control elements). or RNA vectors. Examples of such vectors include derivatives of SV40. , bacterial plasmids, phage DNA, baculovirus, yeast plasmids, plasmids and Vectors derived from combinations of phage DNA and viral nucleic acids (RNA or DNA) In one embodiment, the anti-α-synuclein antibody-encoding nucleic acid is an For example, a linear expression element ( For example, Sykes and Johnston, Nat Biotech 12, 35 5-59 (1997), a compacted nucleic acid vector ter (see, e.g., U.S. Pat. No. 6,077,835 and / or International Publication No. WO 00 / 7 (as described in Brochure No. 0087), pBR322, pUC 19 / 18 or pUC 118 / 119, the "midge" minimal size nucleic acid vector. Any plasmid vector (e.g., Schakowski et al., MoI Th Naked DNA or in RNA vectors or CaPO 4 Precipitated Nucleic Acid Vector Constructs, Such as Precipitated Constructs (See, e.g., WO 00 / 46147, Benvenisty and Reshef, PNAS USA 83,9551-55(1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 2,603(1981 ) as described in ). Such nucleic acid vectors and methods of use thereof are well known in the art (see, e.g., U.S. Pat. No. 5,589,466 and and U.S. Patent No. 5,973,972).

[0106] In one embodiment, the vector comprises an anti-α-synuclein antibody or its derivative in a bacterial cell. Examples of such vectors are suitable for expressing antigen-binding fragments of the Blu eScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989) , expression vectors such as pET vectors (Novagen, Madison, WI), etc. Examples include:

[0107] The expression vector may additionally or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Examples of such enzymes include constitutive or induced enzymes such as α-factor, alcohol oxidase, and PGH. Examples of vectors that contain inducible promoters include those described in F. Ausubel et al., ed. .Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), Grant et al., Methods in Enzymol 153,516-544(1987), Mattanovich,D. et al.Methods Mol.Biol.824,329-358(2012) , Celik,E.et al.Biotechnol.Adv.30(5),1108 -1118(2012), Li,P.et al.Appl.Biochem.Biot echnol.142(2),105-124(2007), Boeer,E.et a l.Appl.Microbiol.Biotechnol.77(3),513-52 3(2007), van der Vaart, JMMethods Mol.Bi ol.178,359-366(2002), and Holliger, P.Method ds Mol. Biol. 178, 349-357 (2002)).

[0108] In the expression vector of the present invention, the anti-α-synuclein antibody-encoding nucleic acid can be any suitable It may contain or be associated with promoters, enhancers, and other expression enhancing elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter). promoter / enhancer and RSV, SV40, SL3-3, MMTV, and HI V LTR promoter), an effective poly(A) termination sequence, in E. coli an origin of replication for the plasmid product, an antibiotic resistance gene as a selectable marker, and and / or a convenient cloning site (e.g., a polylinker). It may also include inducible promoters as opposed to constitutive promoters such as MV IE (those skilled in the art will appreciate that However, it is important to note that such terms are in fact descriptive of the degree of gene expression under particular conditions. (Will recognize that.)

[0109] The antibodies of the present invention, and antigen-binding fragments thereof, can be expressed in a variety of cell lines, including human cell lines, non-human cell lines, and the like. mammalian cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-2 1 cell line, mouse cell line (myeloma cell line, etc.), fibrosarcoma cell line, PER.C6 cell line, It can be produced in HKB-11 cell line, CAP cell line and HuH-7 human cell line ( Dumont et al., 2015, Cri t Rev Biotechnol.Sep 18:1-13.).

[0110] In yet another aspect, the present invention relates to an antibody of the invention as defined herein or an anti-body thereof. The original binding region or the transfectant that produces the bispecific molecule of the invention as defined herein. Recombinant eukaryotic or prokaryotic host cells, such as transfectomas. Examples of host cells include yeast, bacteria, and mammalian cells (CHO or HEK). For example, in one embodiment, the present invention provides an anti-α-cytosine kinase inhibitor of the present invention. A cell genomic DNA comprising a sequence coding for the expression of a nucleic acid antibody or an antigen-binding fragment thereof. In another embodiment, the present invention provides a cell comprising a nucleic acid stably integrated into the genome. , a plasmid, a cos, comprising a sequence coding for the expression of the anti-α-synuclein antibody of the present invention. providing cells containing unfused nucleic acid, such as a nucleic acid sequence encoding a nucleic acid vector, ... Provide.

[0111] In a further aspect, the present invention provides a method for producing an anti-α-synuclein antibody of the present invention. The method comprises the steps of: a) producing a hybridoma or host cell of the invention as described herein above; a) culturing the host cells; and b) purifying the antibody of the invention from the culture medium. do.

[0112] In one embodiment, the present invention relates to a preparation, as such terms are used herein. As used herein, it includes anti-alpha-synuclein antibodies as defined herein, and is intended to include alpha-synuclein antibodies as defined herein. or substantially alter the anti-α-synuclein functionality of the preparation. The present invention relates to a preparation that is substantially free of naturally occurring antibodies that do not The preparation does not include naturally occurring serum or purified derivatives of such serum, and is not intended to include anti-α-synthetic antibodies. Mixing the anti-α-synuclein antibody with another antibody that does not alter the functionality of the anti-α-synuclein antibody in the preparation wherein such functionality includes (i) the binding affinity (KD) of the anti-α-synuclein antibody to α-synuclein; (ii) an anti-α-synuclein that inhibits protease cleavage of α-synuclein fibrils; Antibody capabilities; (iii) Impairment of basal synaptic transmission in F28-snca transgenic mice the ability of anti-α-synuclein antibodies to ameliorate harm; (iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis the ability of anti-alpha-synuclein antibodies to reduce levels of (v) When administered chronically, it improves motor function in a rat model of Parkinson's disease. Ability of anti-α-synuclein antibodies to reverse (vi) α-synuclein seeding (in vitro and / or in Parkinson's disease) their ability to prevent certain metabolic disorders, such as the accumulation of insoluble phosphorylated alpha-synuclein in mouse models of Alzheimer's disease; and / or (vii) the ability to bind to truncated α-synuclein in human brain is selected from the group consisting of:

[0113] The present invention is directed in particular to the structure of the amino acid sequence of a natural anti-α-synuclein antibody compared to the structure of the natural anti-α-synuclein antibody. Changes (in either the CDRs, variable regions, framework residues and / or constant regions) and (b) a preparation of such an anti-α-synuclein antibody, which, due to said conformational change, The anti-α-synuclein monoclonal antibody is capable of detecting the α-synuclein protein expressed by the natural anti-α-synuclein antibody. Significantly altered functionality compared to the functionality expected (i.e., difference in functionality of >20%, 40% % difference, 60% difference, 80% difference, 100% difference, 150% difference, greater than 2-fold difference, greater than 4-fold difference, greater than 5-fold difference, or greater than 10-fold difference); Such functionality is (i) Binding affinity of anti-α-synuclein monoclonal antibodies to α-synuclein (KD); (ii) an anti-α-synuclein that inhibits protease cleavage of α-synuclein fibrils; the capacity of monoclonal antibodies; (iii) Impairment of basal synaptic transmission in F28-snca transgenic mice the ability of anti-α-synuclein monoclonal antibodies to ameliorate harm; (iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis and / or the ability of the anti-alpha-synuclein monoclonal antibody to reduce the level of (v) When administered chronically, it improves motor function in a rat model of Parkinson's disease. the ability of anti-α-synuclein monoclonal antibodies to restore (vi) α-synuclein seeding (in vitro and / or in Parkinson's disease) their ability to prevent certain metabolic disorders, such as the accumulation of insoluble phosphorylated alpha-synuclein in mouse models of Alzheimer's disease; and / or (vii) the ability to bind to truncated α-synuclein in human brain In particular, such altered functionality is the result of structural changes and therefore They cannot be separated.

[0114] The term "substantially free" of naturally occurring antibodies refers to the Substantially altering the alpha-synuclein binding properties of a naturally occurring antibody or preparation such as The complete inclusion of such naturally occurring antibodies at a certain concentration in such preparations is not Absence refers to the absence of an antibody that has no naturally occurring counterpart or is not associated with it in nature. A substance is said to be "isolated" if it is separated or purified from other components.

[0115] The term "naturally occurring antibodies" as it relates to such preparations refers to antibodies produced in live human Antibodies (natural antibodies) that are induced in mice or other animals as a natural consequence of the functioning of their immune systems This refers to the autoantibodies that occur during the course of treatment.

[0116] Thus, the preparations of the invention comprise anti-α-synuclein antibodies and antibodies specific to α-synuclein. Thus, additional antibodies intentionally added capable of binding to epitopes not retained This does not exclude, and in fact expressly includes, such preparations which contain The invention is particularly directed to the preparation being used in the treatment of Parkinson's disease (including idiopathic and hereditary Parkinson's disease). , Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson's Disease, Pure Autonomic Failure, and its experimental results showing improved efficacy in treating synucleinopathies such as multiple system atrophy. The present invention includes embodiments.

[0117] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) an anti-alpha-synuclein antibody or antigen-binding fragment thereof as defined herein; or preparations, as such terms are defined herein. A preparation comprising an anti-alpha-synuclein antibody or an antigen-binding fragment thereof, and (ii) a pharma- ceutically acceptable carrier; The present invention relates to a pharmaceutical composition comprising:

[0118] The pharmaceutical composition is described in Remington: The Science and Practice ce of Pharmacy, 22nd Edition, Gennaro, Ed., Disclosed by Mack Publishing Co., Easton, PA, 2013 In accordance with conventional techniques, such as those described in, for example, US Pat. No. 6,399,411, the disclosure of which is incorporated herein by reference in its entirety, a pharma- ceutically acceptable carrier or diluent and It may be formulated with any other known adjuvants and excipients.

[0119] Pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients are The pharmaceutical composition should be suitable for the selected compound of the invention and the selected method of administration. The suitability of the carriers and other ingredients of the composition may be important in determining whether the selected compound or pharmaceutical composition of the present invention is suitable for use in the preparation of pharmaceutical compositions. The determination is based on the lack of significant adverse effects on the desired biological properties of the product (e.g. , modest effect on epitope binding (relative inhibition of 10% or less, 5% or less (e.g., relative inhibition of

[0120] The pharmaceutical compositions of the invention may contain diluents, fillers, salts, buffers, detergents (e.g., Tween- non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or tannins), (protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or pharmaceutical compositions The combination may also contain other materials suitable for inclusion in the formulation. Diluents may affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, lysozyme, etc. In addition, the pharmaceutical compositions or The formulation may also include other carriers, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. The materials include proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers. mer (e.g., latex functionalized sep cellulose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and Large, slowly metabolized, highly soluble lipids, such as lipid aggregates (e.g., oil droplets or liposomes), It may also include molecules.

[0121] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention will vary depending on the particular patient, composition, and dosage form. and the method of administration to obtain an amount of active ingredient effective to achieve the desired therapeutic response. The dosage level selected will depend on the activity of the particular composition of the invention being used, or The amide, route of administration, timing of administration, excretion rate of the specific compound used, duration of treatment, Other drugs, compounds and / or materials used in combination with the particular composition to be used; The age, sex, weight, medical condition, general health and past medical history of the patient to be treated, and The dosage will depend on a variety of pharmacokinetic factors, including similar factors well known in the therapeutic art.

[0122] The pharmaceutical compositions may be administered parenterally, topically, orally or intravenously for prophylactic and / or therapeutic treatment. Administration may be by any suitable route and method, including nasal means. In some embodiments, the pharmaceutical compositions of the present invention are administered parenterally. The phrases "orally administered" and "parenterally administered" refer to enteral and and administration methods other than topical administration, including epidermal, intravenous, intramuscular, intraarterial, intrathecal, intravesical, ocular, Intrafosal, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid In vivo and in vivo injections and infusions include intraspinal, intracranial, intrathoracic, epidural and intrasternal injections and infusions. Further suitable routes of administering the compounds of the invention in vitro are well known in the art. The various pharmaceutical compositions are known and may be selected by one of skill in the art. In one embodiment, the pharmaceutical composition is administered intravenously. It is administered by intravenous or subcutaneous injection or infusion.

[0123] Pharmaceutically acceptable carriers include any suitable carrier that is physiologically compatible with the compounds of the present invention. Suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption agents Retarders and the like.

[0124] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, aqueous Saline, phosphate buffered saline, ethanol, dextrose, polyol (glycerol glycol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil , carboxymethylcellulose colloidal solution, tragacanth gum and injectable organic acids esters (such as ethyl oleate), and / or various buffers. Entities are well known in the pharmaceutical art.

[0125] Pharmaceutically acceptable carriers can be sterile aqueous solutions or solutions for the extemporaneous preparation of sterile injectable solutions or dispersions. Such vehicles and formulations for pharma- ceutically active substances include pharmaceutical preparations, dispersions and sterile powders. The use of such agents is well known in the art. A conventional media or agent may be used in combination with the active compound. Except where not combined, their use in the pharmaceutical compositions of the present invention is contemplated.

[0126] Proper fluidity can be achieved by, for example, the use of coating materials such as lecithin. In some cases, this can be maintained by maintenance of the required particle size and by the use of surfactants.

[0127] The pharmaceutical composition of the present invention may contain a pharma- ceutical acceptable antioxidant, for example (1) a water-soluble antioxidant. Agents (ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, (2) Oil-soluble antioxidants (ascorbyl palmitate, butyl palmitate, etc.) Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), Lecithin , propyl gallate, α-tocopherol, etc.); and (3) metal chelating agents (citric acid, Acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. obtain.

[0128] The pharmaceutical composition of the present invention contains sugars, polyalcohols (mannitol, sorbitol, etc.) in the composition. The solution may also contain isotonic agents such as isotonic acid, for example glycerol, glycerol, or sodium chloride.

[0129] The pharmaceutical compositions of the present invention contain preservatives, which may increase the shelf life or effectiveness of the pharmaceutical composition. agents, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, etc., appropriate for the selected route of administration. The compounds of the present invention can be administered by implants, transdermal patches, and the delivery of compounds from immediate release, such as controlled release formulations, including microencapsulated delivery systems. Such carriers can be prepared with protective carriers. Such carriers include gelatin, glycol monostearate, etc. Lyceryl, Glyceryl Distearate, Biodegradable, Biocompatible Polymer (Ethylene Bis-Acetate) polyanhydrides, polyglycolic acids, collagens, polyorthoesters, and polylactides. Acids, etc.) alone or in combination with waxes or other materials known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art. For example, Sustained and Controlled Release Drug De livery Systems, JR Robinson, ed., Marcel D See Ekker, Inc., New York, 1978.

[0130] In one embodiment, the compounds of the invention are formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration can be formulated as sterile injectable solutions or dispersions. The present invention includes sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of pharmaceutical active substances. The use of such media and agents for the treatment of cancer is well known in the art. Unless the body or agent is incompatible with the active compound, it may be used in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into the compositions.

[0131] Injectable pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be a solution, microemulsion, liposome, or high-drug The carrier may be, for example, water, ethanol, poly(ethylene glycol), or other ordered structures suitable for the concentration. ols (such as glycerol, propylene glycol, and polyethylene glycol), and Suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic acid esters ( The solvent or dispersion medium may be aqueous or non-aqueous, and may contain a solvent such as ethyl oleate. The proper fluidity can be achieved, for example, by the use of a coating such as lecithin, in the case of a dispersion. This can be maintained by maintaining the required particle size and by the use of surfactants. The composition may contain isotonic agents, such as sugars, polyalcohols (glycerol, mannitol, sol It may be preferable to include sodium chloride, sodium phosphate, etc., in the injectable composition. Prolonged absorption can be achieved by using substances that delay absorption of the antibody, such as monostearic acid salts and gelatin. Sterile injectable solutions can be prepared by including in the composition, for example, Incorporating the required amount of active compound into a suitable solvent containing one or a combination of such ingredients. , optionally followed by sterile microfiltration. Generally, the dispersion is in a sterile vehicle containing a suitable dispersion medium and the required other ingredients, e.g., from those enumerated above. It is prepared by incorporating the active compound. Sterile powder for the preparation of a sterile injectable solution In the case of the above, an example of a preparation method is the addition of the active ingredients and any optional additives from previously sterile-filtered solutions thereof. The process is vacuum drying and freeze drying (lyophilization) to obtain a powder of the desired component.

[0132] Sterile injectable solutions can be prepared in a suitable solvent containing one or a combination of ingredients as enumerated above. The desired amount of active compound is incorporated into the formulation, if necessary by subsequent sterile microfiltration. Generally, the dispersion can be prepared by mixing a basic dispersion medium and other required components from those listed above. Sterile injections are prepared by incorporating the active compound into a sterile vehicle containing the ingredients of: In the case of sterile powders for the preparation of solutions for use, an example of a preparation method is the preparation of previously sterile-filtered solutions of those powders. The liquid is vacuum dried and frozen to obtain a powder of the active ingredient and any additional desired ingredients. It is lye (freeze-dried).

[0133] The dosing regimen in the above methods of treatment and uses described herein is preferably selected from the group consisting of: For example, a single bolus may be administered to provide a desired response (e.g., a therapeutic response). The dose may be administered in one dose, several divided doses may be administered over time, or the dose may be administered in multiple doses. The dosage may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. The products may be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a unitary dose for the subject being treated. Each unit refers to a suitable physically discrete unit capable of producing the desired therapeutic effect in association with the required pharmaceutical carrier. The unit dosage forms of the present invention contain a predetermined amount of active compound calculated to produce (a) the unique properties of the active compound and the specific therapeutic effect achieved; and (b) the individual Combination of such active compounds for the treatment of sensitivity in This disclosure is subject to and directly dependent on the limitations inherent in the technology field.

[0134] The effective dosage and administration regimen of the anti-α-synuclein antibody will depend on the disease or condition being treated. Depending on the dosage, it may be determined by one of skill in the art. The dosage is about 0.0001 to about 100 mg / kg, more usually about 0.01 to about 5 mg / kg. The dosage may range from 1 mg / kg body weight to 10 mg / kg body weight. The dosage may be in the range of 1 to 10 mg / kg body weight. For example, about 0.1 to about 10 mg / kg / body weight, about 0.1 to about 5 mg / kg / body weight, about 0. 1 to about 2 mg / kg / body weight, about 0.1 to about 1 mg / kg / body weight, for example about 0.15 mg / kg / body weight, approx. 0.2mg / kg / body weight, approx. 0.5mg / kg / body weight, approx. 1mg / kg / Body weight, about 1.5 mg / kg / body weight, about 2 mg / kg / body weight, about 5 mg / kg / body weight, and Approximately 10 mg / kg / body weight can be mentioned.

[0135] A physician of ordinary skill in the art can administer an effective amount of the pharmaceutical composition required. For example, a physician can easily determine and prescribe the amount of an anti-alpha- - The dose of the synuclein antibody is reduced to a level lower than that required to obtain the desired therapeutic effect. Dosage may be started at 10 mg / kg and gradually increased until the desired effect is achieved. A suitable daily dose of the composition is that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Administration can be by, for example, Administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous. The daily doses are administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. The compound of the present invention may be administered in one, three, four, five, six or more divided doses. The compound can be administered alone, but may also be administered as a pharmaceutical composition as described above. It is preferred to administer the substance.

[0136] The labeled antibodies of the present invention can be used for diagnostic purposes to detect, diagnose, or monitor a disease or disorder. The present invention can be used in, but is not limited to, Parkinson's disease, idiopathic Parkinson's disease , familial Parkinson's disease, diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease LBV, Combined Alzheimer's and Parkinson's Disease, Pure Autonomic Neuropathy Detecting or diagnosing neurodegenerative or cognitive diseases or disorders, including myelopathy or multiple system atrophy (a) providing a fragment of α-synuclein using one or more antibodies that specifically bind to the fragment; determining the presence of alpha-synuclein species and fragments in a cell or tissue sample from a subject; (b) measuring the level of the antigen against a control level, e.g., a level in a normal tissue sample; whereby an increase in the measured level of the antigen compared to a control level of the antigen indicates disease and indicating the disease or disorder or indicating the severity of the disease or disorder.

[0137] The antibodies of the present invention can be assayed by immunohistochemistry using techniques well known in the art. α-synuclein monomers, oligomers, fibrillar forms or fragments of α-synuclein Other antibody-based assays useful for detecting proteins can be used to measure the amount of a protein that is present in the antibody fragment. The methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and Mesoscale Discovery Platform Examples of suitable immunoassay methods include immunoassays based on the ELISA platform (MSD). Any suitable antibody label may be used in such kits and methods, and is known in the art. The labels include enzyme labels (such as alkaline phosphatase and glucose oxidase). ); Radioisotope labeling (iodine ( 125 I, 131 I), Carbon ( 14 C), sulfur ( 35 S ), Tritium ( 3 H), Indium ( 121 In), and technetium ( 99m Tc )); and luminescent labels (such as luminol and luciferase); and fluorescent labels ( fluorescein and rhodamine.

[0138] Presence of labeled anti-α-synuclein antibodies or their α-synuclein-binding fragments can be detected in vivo for diagnostic purposes. In one embodiment, diagnosis is achieved by administering to a subject a) an effective amount of administering such a labeled molecule to a subject; and b) waiting for a predetermined time interval after administration. In this way, the labeled molecules are concentrated at sites of Aβ deposition (if any) and the unbound labeled molecules are concentrated at sites of Aβ deposition (if any). c) allowing the background to be removed until it reaches background level; and d) detecting the level of the labeled molecule above background levels. Indicating that a subject is suffering from a disease or disorder or indicating the severity of a disease or disorder As shown, the method includes detecting the labeled molecule in the subject. The molecule may be an imaging molecule suitable for detection using a particular imaging system known to those skilled in the art. The background level is the amount of labeled antibody detected relative to the amount of antibody present in a particular image. and comparing the results to standard values ​​previously determined for the aging system. The method and method that can be used in the diagnostic method of the present invention The imaging and systems include, but are not limited to, computed tomography (CT), positron Whole-body scans such as PET (positron emission tomography), magnetic resonance imaging (MRI), and ultrasonography Examples include ultrasound testing.

[0139] In a further aspect, the present invention relates to an antibody of the present invention or an antibody thereof for use in medicine. Regarding the original binding fragment.

[0140] In a further aspect, the present invention provides a method for treating, diagnosing or imaging a synucleinopathy. The present invention also relates to an antibody or antigen-binding fragment thereof for use in detecting a patient infected with a HIV-1 virus.

[0141] In one embodiment, the monoclonal antibody, or antigen-binding fragment thereof, Parkinson's disease, idiopathic Parkinson's disease, familial Parkinson's disease, diffuse Lewy body disease (D LBD), Lewy body variant Alzheimer's disease (LBV), combined Alzheimer's disease and for use in treating Parkinson's disease, pure autonomic failure, or multiple system atrophy. This is for the purpose.

[0142] In a further aspect, the present invention provides a method for treating, diagnosing or imaging a synucleinopathy. Use of an antibody of the invention, or an antigen-binding fragment thereof, in the manufacture of a medicament for treating Regarding use.

[0143] In a further aspect, the present invention provides an effective dosage of an antibody of the present invention, or an antigen-binding fragment thereof. Treatment of Parkinson's disease or other synucleinopathies, comprising administering fragments thereof , relating to diagnostics or imaging.

[0144] Preferably, in the uses and methods of these aspects of the invention, the treatment is chronic, Preferably, the incubation period is at least 2 weeks, e.g., at least 1 month, 6 months, 1 year or more. It covers more than that.

[0145] In a further aspect, the present invention relates to an antibody, or an antigen-binding fragment thereof, of the present invention. A kit comprising:

[0146] [Table 5]

[0147] EMBODIMENTS OF THE PRESENT DISCLOSURE As will be apparent from the text and examples, the present invention further relates to the following embodiments: do.

[0148] 1. Amino acids 112 to 117 in α-synuclein (SEQ ID NO: 9 (ILEDMP) ) a monoclonal antibody capable of specifically binding to an epitope within the Combined fragments.

[0149] 2. A molecule according to embodiment 1, which competes with antibody GM37 for binding to said epitope. A monoclonal antibody, or an antigen-binding fragment thereof.

[0150] 3. GM37, GM37 mutant 1, GM37 mutant 2 or GM37 mutant 3. A monoclonal antibody, or an antigen-binding fragment thereof, according to embodiment 1.

[0151] 4. Amino acids 112 to 115 in alpha-synuclein (SEQ ID NO: 19 (ILED)) A monoclonal antibody capable of specifically binding to an epitope in a Combined fragment.

[0152] 5. The monoclonal antibody according to embodiment 1 or 4, which is GM285, or Antigen-binding fragment.

[0153] 6. The method of any preceding embodiment, wherein the antibody comprises or consists of an intact antibody. A clonal antibody, or an antigen-binding fragment thereof.

[0154] 7. Fv fragments (e.g., single chain Fv and disulfide-linked Fv), Fab-like Fv Fragments (e.g., Fab fragments, Fab' fragments and F(ab) 2 Hula fragments) and domain antibodies (e.g. single V H Variable region or V L Variable region) The preceding embodiment comprises or consists of an antigen-binding fragment selected from the group consisting of: 2. The monoclonal antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 11.

[0155] 8. Monoclonal antibodies are classified into subtypes IgG1, IgG2, IgG3 and IgG4. The monoclonal antibody of any one of the preceding embodiments is selected from the group consisting of: A null antibody, or an antigen-binding fragment thereof.

[0156] 9. The antibody or antigen-binding fragment has the following characteristics: (i) 0.5 to 10 nM of α-synuclein, such as 1 to 5 nM or 1 to 2 nM The binding affinity (K D ); (ii) the ability to inhibit protease cleavage of α-synuclein fibrils; (iii) Impairment of basal synaptic transmission in F28-snca transgenic mice the ability to remedy harm; (iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis Ability to reduce levels of; (v) When administered chronically, it restores motor function in a rat model of Parkinson's disease. Ability to repay; (vi) α-synuclein seeding (in vitro and / or in Parkinson's disease) their ability to prevent the accumulation of insoluble phosphorylated alpha-synuclein in mouse models of Alzheimer's disease; and / or (vii) the ability to bind to truncated α-synuclein in human brain 5. The monoclonal antibody of any one of the preceding embodiments, exhibiting one or more of: or an antigen-binding fragment thereof.

[0157] 10. The monoclonal antibody of any one of the preceding embodiments, which is a human or humanized antibody. A monoclonal antibody, or an antigen-binding fragment thereof.

[0158] 11. The following CDR: a) GFTFSSYAMT (SEQ ID NO: 1) or 4 or less amino acid differences, or 3 or more or less than two amino acid differences, or less than one amino acid difference amino acid sequence; b) AIRS(N / S / Q / H)GDRTD YADSVKG (SEQ ID NOs: 2, 33, 3 4, 35) or four or fewer amino acid differences, or three or fewer amino acid differences, or two or fewer or an amino acid sequence having no more than one amino acid difference; or c) AKNWAPFDS (SEQ ID NO: 3) or 4 or less amino acid differences, or 3 or less or 2 or less amino acid differences, or 1 or less amino acid differences Acid sequence The monoclonal antibody or any one of embodiments 1 to 3 and 6 to 10, comprising a heavy chain variable region comprising A monoclonal antibody, or an antigen-binding fragment thereof, as described herein.

[0159] 12. SEQ ID NO: 1 and 3 and SEQ ID NO: 2 and one of 33, 34 or 35 12. The monoclonal antibody of embodiment 11, comprising a heavy chain variable region comprising one CDR; and an antigen-binding fragment thereof.

[0160] 13. [ka] 12. The method of claim 11, comprising or consisting of a heavy chain variable region selected from the group consisting of: The monoclonal antibody or antigen-binding fragment thereof.

[0161] 14. The following CDR: a) ASQSVSSSYLA (SEQ ID NO: 4) or four or fewer amino acid differences, or or less than two amino acid differences, or less than one amino acid difference Amino acid sequence; b) GASSRAT (SEQ ID NO: 5) or 4 or less amino acid differences, or 3 or less amino acid differences Amino acid difference, or no more than two amino acid differences, or no more than one amino acid difference Array; or c) QQYGSSPWT (SEQ ID NO: 6) or 4 or less amino acid differences, or 3 or less or 2 or less amino acid differences, or 1 or less amino acid differences Acid sequence The monoclonal antibody or any one of embodiments 1 to 3 and 6 to 13, comprising a light chain variable region comprising A monoclonal antibody, or an antigen-binding fragment thereof, as described herein.

[0162] 15. The antibody of embodiment 14, comprising a light chain variable region comprising the CDRs of SEQ ID NOs: 4, 5 and 6. The monoclonal antibody or antigen-binding fragment thereof.

[0163] 16. Amino acid sequence: [ka] 15. The antibody or any of embodiments 14 and 15, comprising a light chain variable region comprising or consisting of: Antigen-binding fragment.

[0164] 17. Amino acid sequence: [ka] 15. The monoclonal antibody of embodiment 14, comprising a light chain comprising or consisting of is an antigen-binding fragment thereof.

[0165] 18. A light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:8 and a sequence comprising or consisting of any of the amino acids represented by numbers 7, 33, 34 or 35 The monoclonal antibody or the monoclonal antibody according to any one of embodiments 1 to 3 and 6 to 17, is an antigen-binding fragment thereof.

[0166] 19. Increased stability to prevent aggregation or unfolding as shown in Figure 27 It has increased thermal stability, such as 2% to 5% at temperatures above 65°C compared to GM37 wt. 10% more stable, and 2% to 8% more stable at temperatures above 65°C compared to GM37 wt. or 2%-5% more stable at temperatures above 65°C compared to GM37 wt. a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain comprising the amino acid sequence of SEQ ID NO: 34 3 and 4, comprising a heavy chain variable region comprising or consisting of the amino acids shown. 19. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 6 to 18.

[0167] 20. The following CDR: a) AASGFTFSRFTMT (SEQ ID NO: 20) or no more than four amino acid differences; and has 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences an amino acid sequence having b) AISGSGGGTS YADSVKG (SEQ ID NO: 21) or four or fewer amino acids or 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less an amino acid sequence having an amino acid difference; or c) AKNWAPFDY (SEQ ID NO: 22) or 4 or fewer amino acid differences, or 3 or more or less than two amino acid differences, or less than one amino acid difference Amino acid sequence A monoclonal antibody or a monoclonal antibody according to any one of embodiments 1 to 10, comprising a heavy chain variable region comprising A monoclonal antibody, or an antigen-binding fragment thereof.

[0168] 21. The heavy chain variable region of embodiment 2 comprises the CDRs of SEQ ID NOs: 20, 21 and 22. 1. A monoclonal antibody or antigen-binding fragment thereof according to claim 0.

[0169] 22. Amino acid sequence [ka] 21. The monoclonal antibody of embodiment 20, comprising a heavy chain variable region comprising or consisting of: An antibody or an antigen-binding fragment thereof.

[0170] 23. The following CDR: d) RASQSVSRSYLA (SEQ ID NO: 23) or no more than four amino acid differences; or Have 3 or less amino acid differences, or 2 or less amino acid differences, or 1 or less amino acid differences the amino acid sequence; e) GASSRAT (SEQ ID NO: 24) or 4 or less amino acid differences, or 3 or less Amino acid difference, or no more than two amino acid differences, or no more than one amino acid difference acid sequence; or f) QQYGSSPWT (SEQ ID NO: 25) or 4 or fewer amino acid differences, or 3 or more or less than two amino acid differences, or less than one amino acid difference Amino acid sequence The monoclonal antibody or the antibody of embodiments 1 to 10 and 20 to 2, comprising a light chain variable region comprising 2. A monoclonal antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 11.

[0171] 24. Embodiment 2, comprising a light chain variable region comprising the CDRs of SEQ ID NOs: 23, 24 and 25. 3. A monoclonal antibody or an antigen-binding fragment thereof according to claim 3.

[0172] twenty five. [ka] 25. The method of claim 24, comprising a light chain variable region comprising or consisting of the amino acid sequence of An antibody or an antigen-binding fragment thereof.

[0173] 26. A light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 27 and a sequence A heavy chain variable region comprising or consisting of any of the amino acids shown in sequence number 26. The monoclonal antibody or antigen-binding fragment thereof of any one of the preceding embodiments. Fragment.

[0174] 27. The monoclonal antibody of any one of the preceding embodiments, comprising an Fc region. or an antigen-binding fragment thereof.

[0175] 28. The method of any preceding embodiment, further comprising: A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

[0176] 29. The moiety for increasing in vivo half-life is polyethylene glycol (PEG), Selected from the group consisting of human serum albumin, glycosylation groups, fatty acids and dextran. 29. The monoclonal antibody or antigen-binding fragment thereof described in embodiment 28.

[0177] 30. Any of the preceding embodiments, wherein the antibody polypeptide further comprises a detectable moiety. 1. A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

[0178] 31. The detectable moiety may be a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label or 31. The monoclonal antibody or antigen-binding fragment thereof of embodiment 30, which is an enzyme label. segment.

[0179] 32. The method of embodiment 30 or 31, wherein the detectable moiety comprises or consists of a radioisotope. 32. The monoclonal antibody or antigen-binding fragment thereof according to claim 31.

[0180] 33. A radioisotope is 99m Tc, 111 In, 67 Ga, 68 Ga, 72 As, 8 9 Zr, 123 I and 201 The moiety according to embodiment 32 is selected from the group consisting of Tl. Monoclonal antibodies or antigen-binding fragments thereof.

[0181] 34. The method of embodiment 30, wherein the detectable moiety comprises or consists of a paramagnetic isotope. A monoclonal antibody or antigen-binding fragment thereof as described herein.

[0182] 35. Paramagnetic isotopes are 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe 35. The monoclonal antibody or its antigen-binding domain according to embodiment 34, selected from the group consisting of Combined fragment.

[0183] 36. The detectable moiety is a marker for SPECT, PET, MRI, optical or ultrasound imaging. 36. The method according to any one of embodiments 30 to 35, which is detectable by imaging techniques such as fluoroscopy. The monoclonal antibody or antigen-binding fragment thereof.

[0184] 37. The detectable moiety indirectly binds to an antibody or its antigen-binding fragment via a binding moiety. The monoclonal antibody according to any one of embodiments 30 to 36, and an antigen-binding fragment thereof.

[0185] 38. The bond is 1,4,7,10-tetraazacyclododecane-1,4,7,10 , Derivatives of diethylenetriamine tetraacetate (DOTA), deferoxamine (DFO), diethylenetriamine pentaacetate (DFO) S-2-(4-isothiocyanatobenzyl)-1,4,7-diacetate (DTPA) derivative Derivatives of triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,8,1 Derivatives of 1-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA) 38. The monoclonal antibody or antigen-binding fragment thereof according to embodiment 37, selected from the group consisting of: segment.

[0186] 39. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments. or an isolated nucleic acid molecule encoding a component polypeptide chain thereof.

[0187] 40. The nucleic acid molecule of embodiment 39, wherein the molecule is a cDNA molecule.

[0188] 41. The nucleic acid according to embodiment 30 or 31, encoding an antibody heavy chain or a variable region thereof acid molecule.

[0189] 42. Any one of embodiments 39 to 41, encoding an antibody light chain or its variable region. A nucleic acid molecule according to claim 1.

[0190] 43. A vector comprising a nucleic acid molecule according to any one of embodiments 39 to 42.

[0191] 44. The nucleic acid molecule according to any one of embodiments 39 to 42 or embodiment 43. A recombinant host cell comprising the vector.

[0192] 45. A method for producing an antibody or antigen-binding fragment according to any one of embodiments 1 to 27. a method for generating an antibody or antigen-binding fragment thereof comprising: 45. A method comprising culturing a host cell according to embodiment 44 under conditions allowing the host cell to grow in the culture medium.

[0193] 46. ​​The monoclonal antibody or antigen-binding fragment according to any one of embodiments 1 to 35. A pharmaceutical composition comprising the fragment and a pharma- ceutically acceptable carrier.

[0194] 47. A monoclonal antibody or a method thereof according to any one of embodiments 1 to 35 for use in medicine. An antigen-binding fragment of.

[0195] 48. For use in treating, diagnosing or imaging synucleinopathies, A monoclonal antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 35.

[0196] 49. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher's disease, Diffuse Lewy Body Disease (DLBD), Lewy Body Variant Alzheimer's Disease (LBV), Combined Alzheimer's and Parkinson's Disease, Pure Autonomic Failure and Multiple System Atrophy The monoclonal antibody or its derivatives according to embodiment 48 for use in treating a disease. Antigen-binding fragment.

[0197] 50. In the manufacture of drugs for treating, diagnosing or imaging synucleinopathies The monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35. use.

[0198] 51. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher's disease, Diffuse Lewy Body Disease (DLBD), Lewy Body Variant Alzheimer's Disease (LBV), Combined Alzheimer's and Parkinson's Disease, Pure Autonomic Failure and Multiple System Atrophy The use of a monoclonal antibody or a medicament for treating a disease, comprising the steps of: uses of antigen-binding fragments thereof.

[0199] 52. A method for treating, diagnosing, or imaging a synucleinopathy in a subject. 47. The method of claim 46, further comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 46. method.

[0200] 53. Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher's disease, Diffuse Lewy Body Disease (DLBD), Lewy Body Variant Alzheimer's Disease (LBV), Combined Alzheimer's and Parkinson's Disease, Pure Autonomic Failure and Multiple System Atrophy 49. The antibody or antigen-binding fragment thereof for use according to embodiment 48 for treating a disease. or the use according to embodiment 50, or the method according to embodiment 52.

[0201] 54. The antibody for use according to embodiment 52 or 53, wherein the treatment is long-term, or or an antigen-binding fragment thereof; or a use; or a method.

[0202] 55. Long-term treatment is for at least 2 weeks, e.g., at least 1 month, 6 months, 1 year or more of the antibody or antigen binding thereof for use according to embodiment 52. fragment; or use; or method.

[0203] 56. For use according to any one of embodiments 47 to 55, wherein the subject is a human being. An antibody, or an antigen-binding fragment thereof; or a use; or a method.

[0204] 57. A kit comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35.

[0205] 58. The kit according to embodiment 57, for use in medical treatment.

[0206] 59. A method for detecting or measuring the presence or amount of alpha-synuclein in the brain or body fluids of a subject. The monoclonal antibody or its derivatives according to any one of embodiments 30 to 35 for use in determining whether a Antigen-binding fragment.

[0207] 60. The detection or measurement is performed using the anti-synuclein bound to the alpha-synuclein. The monoclonal antibody or its derivatives according to embodiment 59, including in vivo imaging of the antibody. An antigen-binding fragment of.

[0208] 61. The detection or measurement is performed using an anti-synuclein antibody bound to the alpha-synuclein. The monoclonal antibody according to any one of embodiments 30 to 35, including ex vivo imaging of the antibody. or an antigen-binding fragment thereof. EXAMPLES

[0209] Example 1: Antibody Screening 1. Immunogen and Ligand Production The following proteins were obtained or produced for use as immunogens as shown in FIG. Mice were immunized with three immunogens: full-length recombinant human α-synuclein fibrils; Human α-synuclein recombinant protein (Rpeptide, Bogart, Georgia) and human α-synucleic acid containing amino acids 1 to 119 In order to generate fibrils from full-length α-synuclein, Rpe ptide (Bogart, Georgia) freeze-dried product (catalog number S-10 01-2) was used. This was diluted with 20 mM Tris at a protein concentration of 1 mg / ml. and 300 mM NaCl buffer. The cells were incubated at 37°C in a Vortemp 56 shaker incubator (Labnet Incubator). Each wafer was measured at 200 rpm at In a 96-well plate containing ceramic beads with a diameter of 70 μm in the well, the protein solution The mixture was incubated in 170 μl aliquots, and after fibril formation, thioflavin T was added. The fluorescence in one of the wells was measured. Nuclein was dissolved in water to obtain a concentration of 1 mg / ml.

[0210] Recombinant α-synuclein containing amino acids 1 to 119 was produced using the following construct: A synthetic gene encoding a six amino acid histidine tag followed by factor Xa cleavage was prepared. The site and sequence encoding human α-synuclein amino acids 1-119: [ka] was synthesized by Genscript and inserted into the pET24a(+) expression vector (Novag) The plasmid pGamma was cloned into the NdeI-XhoI sites of pGamma-2.

[0211] The expression vector was transformed into E. coli BL21 and purchased from Novagen. Overnight Express self-guiding system (User protocol) l TB383 rev.H1005) and single colonies were picked for expression. The scale was 500 ml final culture volume. The cells were centrifuged at 6000 g for 10 min. The cells were then harvested by isolation and subsequently purified with BugBuster protein extraction reagent (User Profile). After dissolution, the samples were centrifuged. The supernatant was used for further purification.

[0212] His-tagged proteins were lysed in 20 mM sodium phosphate pH 7.5, 1 M NaCl A 5 ml HisTrap column (GE Healthcare) equilibrated in (A-buffer) was After sample application and washing with A-buffer, the proteins were purified on Elute to a gradient of 0.25 M imidazole in A-buffer over 20 column volumes. Fractions of 5 ml were collected and analyzed by SDS-PAGE. Fractions containing the target protein were pooled, concentrated and purified with 10 mM Tris pH 7.4, 300 mM NaCl The results were applied to a S200 (26 / 60) size exclusion column (GE Healthcare). Again, fractions were screened according to the presence of a band of the predicted size on SDS-PAGE. Pooled.

[0213] To remove the N-terminal tag, purified His-tagged α-synuclein 1–119 was , using Novagen kit (69037-3FRX) at a 1:50 ratio, Xa After overnight incubation, factor Xa was incubated with Rest agarose was used to batchwise remove cleaved α-synuclein 1-11. 9 was purified by permissive HisTrap chromatography as described above. The flow through was purified by -synuclein 1-119 was obtained and concentrated in a Centricon concentrator to approximately 4 The solution was concentrated to 0.100 μg / ml.

[0214] α-Synuclein (Rpeptide) was rehydrated in PBS at 2 mg / ml and Peroxynitirite (100 μL / mg protein) The nitrosylated α-synuclein was then added dropwise to 5 L of Dialyzed in PBS and stored at -20°C.

[0215] Dopamine was used to oxidize α-synuclein. 10 mM in PBS, pH 7.4 An equal volume of 200 μM Dopamine-HCL (Sigma P5244) prepared in and 20 mM of α-synuclein (Rpeptide) in 10 mM PBS, pH 7.4 The resulting 14 uM α-synuclein / 100 uM dose was The mixture was incubated overnight at 37°C. The solution was dialyzed in PBS and stored at -20°C.

[0216] Various native and chimeric forms of synuclein proteins were identified using anti-α-synuclein antibodies. A diverse library was generated for screening. The screening constructs were: These included: human, mouse, rat and cynomolgus monkey α-synuclein; human β-synuclein, human γ-synuclein (Figures 21 and 22) and finally, α-synuclein. α-synuclein derivatives lacking residues 120–140 of nuclein. Two shuffle constructs: A-Syn-AAKK -BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, A-Syn These constructs were synthesized from human α-serotype 1 (BBKK-BAP) and human α-serotype 2 (BBKK-BAP) (SEQ ID NOs: 11 to 14). Nuclein (A), human β-synuclein (B) and chicken α-synuclein (K). Each site of the ligand contained a linear stretch of To facilitate specific biotinylation, a biotin receptor peptide was fused to the C-terminus of the ligand. The gene containing the tide (BAP) tag was cloned. Biotinylation was performed using soluble ELISA. The format allows the conjugation of ligands to the beads used. Construct a mammalian expression vector carrying an in-BAP tag fusion construct (ASynBAP) The ligand was transfected into HE cells using transient transfection (Genmab A / S). The expression was carried out in K293 cells.

[0217] 2.Immunization The antibody HuMab-Synuclein was engineered to be mouse, preventing the expression of a completely mouse antibody. HuMA, a mouse with double knockout of immunoglobulin (Ig) heavy chain and mouse kappa light chain b Immunization of mouse strains HCo17-BALB / c and HCo12-BALB / c mice (human monoclonal antibody; Medarex Inc., San Jose, CA) Various mouse strains were bred with human Ig heavy chain and human Ig κ light chain loci. The mouse was made transgenic by insertion of human VH (heavy chain variable region) and VL ( The number of light chain variable region genes differs.

[0218] Forty-eight mice were immunized intraperitoneally (IP) and intravenously with 20 μg of antigen at 14-day intervals. The mice were then immunized subcutaneously (SC, at the base of the tail) with the same immunogen four times IP and four times SC. A maximum of eight immunizations were performed.

[0219] The first immunization was performed in complete Freund's adjuvant (CFA; Difco Laborat The assay was performed with α-synuclein immunogen in 100 mM NaCl (Olive Trees, Detroit, MI, USA) and the following: Immunizations were performed in incomplete Freund's adjuvant (IFA). Serum antibody titers were sufficient. (sera dilutions of 1 / 50 or less were found in at least two consecutive, biweekly screening In the screening event, the antigen-specific screening assay described herein above When the results were positive in the 100-mL IgG4 antibody test, mice were injected with 100 mL of IgG4 antibody 4 and 3 days before fusion. Two doses of 10 μg of α-synuclein immunogen protein in 1 μL of PBS intravenously (IV) A further boost was administered.

[0220] The immunization protocol is shown in FIG.

[0221] Antibody 37 binds to C-terminal truncations of α-synuclein containing amino acids 1-60 and 1-119. Alternately, from an immunization protocol using human full-length α-synuclein fibrils Ta.

[0222] Antibody 285 used human α-synuclein monomers 1–140 for the first four immunizations. In the absence of antibody titers, immunization was performed using the fibril (ip / s c) or alternatively, immunization was continued with the monomer.

[0223] 3.HuMab Hybridoma Generation HuMAb mice with sufficient antigen-specific antibody titer expression as defined above were sacrificed and The spleen and lymph nodes adjacent to the distal aorta and vena cava were harvested. Fusion of splenocytes and lymph node cells from CEEF was performed essentially according to the manufacturer's instructions. 50 Electrofusion System(Cyto Pulse Scie The fusion was performed by electrofusion using a fusion kit (Inces, Glen Burnie, MD, USA). The fusion cells were cultured in 10% Fetal C in HyQ mADCF-Mab (Perbio). lone I Bovine serum (Perbio), 1 mM sodium pyruvate (C ambrex), 0.5 U / mL penicillin, 0.5 U / mL streptomycin ( Cambrex), 50 μM 2-mercaptoethanol (Invitrogen), 6 0.00 ng / mL interleukin 6 (IL-6) (Strathmann), 1× HA T (Sigma) and 0.5 mg / mL kanamycin (Invitrogen). After 10 days, the supernatant was collected and the cells were seeded in fusion medium containing HyQ mADCF-M. 10% Fetal Clone I Bovine serum in ab, 0.5U / mL Nicillin, 0.5 U / mL streptomycin, 600 ng / mL IL-6 and 1 The hybridoma cultures were washed with harvest medium containing ×proHT (Cambrex). The supernatants were screened by the primary screening assay. These were characterized for binding to four orthologs: human, mouse, and rat and cynomolgus monkey, human α-synuclein, β-synuclein and human γ- synuclein (SEQ ID NOs: 37 to 41), and finally, Their ability to bind to a human α-synuclein derivative lacking residues 120-140 was investigated. The test was carried out.

[0224] Screening of anti-α-synuclein antibodies was performed using an automated liquid dispensing system. ed liquid handling system)(Genmab A / S) The assay was performed using a high-throughput suspension ELISA format. The analysis was performed using two systems: FMA (Applied Biosystems) The 384-well plate was read using the T 8200 and Molecular Dev Using an ImageXpress Velos Cytometer manufactured by ices, A 36-well plate was read.

[0225] In the primary screen, clones were selected from those that bind to eight different ligands. These were characterized by their ability to synthesize a series of four shuffled constructs: A-Syn-A AKK-BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, A- Syn-BBKK-BAP (SEQ ID NOs: 11 to 14), α-synuclein 120 to 140 deletion Loss-BAP, nitrated human α-synuclein-BAP and oxidized human α-synuclein- It contained BAP.

[0226] Briefly, serum or supernatant potentially containing α-synuclein-specific antibodies was collected by In addition to the beads, binding to α-synuclein and / or α-synuclein-derived constructs Binding of the anti-α-synuclein antibody is mediated by the fluorescent conjugate, DyLigh. Detected using t649-conjugated goat anti-human IgG (Fc specific). Anti-α-synuclein antibodies, LB509 and Syn211, were used as positive controls in the screen. To ensure specific detection of α-synuclein antibodies, anti-α-synuclein antibodies were included in the study. Nuclein serum pool negative in 384-well format antibody titer screening Human ChromPure IgG was used as a control in a 1536-well plate. Mat8 - Used in bead-based assays.

[0227] Hybridoma cells from the best primary well were cultured in 40% CloneMedia ( Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (H Each primary culture was inoculated onto a semi-solid medium prepared from 100% maltodextrin (100% maltodextrin, ... For the wells, a Genetix black 6-well plate was seeded. Each well From the clones, 25 subclones were obtained using the ClonePix system (Genetix). The subclones were harvested in collection medium. After 7 days, the supernatants of the subclones were harvested using synuclein. The cells were again screened for rhein-specific human IgG binding, and the human IgG concentration was adjusted to 100 mg / mL at 100°C. The primary sera were measured using a 100-μg / mL ELISA kit (Fortebio, Menlo Park, USA). The best subclones were selected from the wells and treated with 600 ng / mL IL-6, 0.5 U / mL Contains only 0.5 mL of penicillin, 0.5 U / mL of streptomycin, and 1× proHT. The subclones were expanded in an expansion medium containing from one 96-well plate well to one 24-well plate well, The wells were expanded into six 24-well plate wells and six 6-well plate wells. The clones obtained by this process were designated primary clones (PC).

[0228] Further antibody binding studies were performed using Octet 384RED (Fortebio, Menlo). The HuMab antibody solution at 2 μg / ml was used as a sample diluent. The antibody was prepared by dilution in a 20 ml buffer (ForteBio, art. No. 18-5028). Amine-reactive sensors (ForteBio, art. no. 18-0008) were prepared using HuM Prior to coupling to the amine-reactive sensor, HuMab was immobilized on The coupling was carried out at 30° C. and 1000 rpm as follows: Amine-reactive sensors were incubated in PBS Pre-wet followed by EDC / N (per manufacturer's instructions) for 300 seconds. HS (ForteBio.Art.no.18-1033 / 18-1034) activation solution The activated sensor was immobilized with HuMab for 600 seconds.

[0229] 37 in Octet to recombinant human, cynomolgus monkey and mouse α-synuclein and 285, and the lack of binding to human β- or γ-synuclein is shown in Figure 2. can be.

[0230] 4. Sequence analysis of synuclein-specific HuMab variable regions and cloning in expression vectors Training SMART RACE cDNA Amplifica was used according to the manufacturer's instructions. Total RNA was isolated from 0.2–5 × 106 hybridomas using a cloning kit (Clontech). 5'-RACE-complementary DNA (cDNA) was prepared from hybridoma cells and The VH and VL coding regions were amplified by PCR and Igation-independent cloning (Aslanidis, C. and PJde J ong, Nucleic Acids Res 1990;18(20):6069-7 4) p33G1f and p33κ expression vectors (human IgG1 / κ constant region coding region) were For each antibody, 16 16 VL clones and 16 VH clones were sequenced. Clones containing the desired reading frame (ORF) were selected for further study and expression. All combinations of heavy and light chain vectors were transfected with 293fectin. The genes were transiently co-expressed in reestyle™ 293-F cells.

[0231] In the case of GM37, sequencing of the VH region revealed a CDR3 region at position 106. Extra cysteines in the ribonuclease region were identified. Possible misfolding and disulfide bonds To estimate the possibility of a decrease in antibody activity due to the formation of cysteine, It was mutated to

[0232] The comparative antibody 9E4 was synthesized using the VH and VL sequences derived from the hybridoma PTA-8221. (US Patent Application Publication No. 20080175838) (SEQ ID NOs: 42 and 43) It was made based on.

[0233] 5. Antibody Expression / Purification Antibodies were transfected using pTT5 vector and PEIpro( National Research Council of Canada , was produced by transfection in HEK293 6E cells. The heavy and light chains were transfected into HEK293 cells using PEIpro (VWR). 24 hours after transfection, cells were supplemented with TN1 (Sigma). Cells were grown until viability approached 50%, and antibody yields were assayed using easy IgG titer. The supernatant of the culture was filtered through a 0.2 μm dead end filter. The eluate was filtered over a filter and then loaded onto a 5 mL Protein A column (rProtein A FF, Am The mixture was packed in a 0.1 M citric acid-NaOH, pH The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and 12.6 mM NaH 2PO 4 , 140 mM NaCl, pH 7.4 (B. Braun) overnight After dialysis, the sample was sterile filtered on a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE, and concentration was assessed by turbidimetry and absorbance at 280 nm. The purified antibody was aliquoted and stored at -80°C.

[0234] Example 2: Characterization of antibodies using surface plasmon resonance Real-time binding of antibodies to α-synuclein was measured using BIAcore® 30 The capture surface was measured using the first 100 μg / cm2 CM5 chip (BIAcore®). In one flow cell (Fc1) and a second flow cell (Fc2), polyclonal rabbit Mouse Antibody Capture Kit (GE He althcare, Cat. no: BR-1008-38)) The mouse antibody was prepared by ELISA to achieve a ligand level of approximately 500 RU. The sample (ASynBAP) was injected into Fc2 at 30 μl / min at the concentration required for capture. The baseline was allowed to stabilize for 10 min before injection into 1-2. ASynBAP was then Each was tested at 100-3200 nM and 25-3200 RU. The highest concentration in was tested in duplicate. At the end of each cycle, the surface was reconstituted with 10 mM glycine -Regenerate with HCl, pH 1.7 (30 s injection) to release the captured mouse antibodies and analytes. The HBS-EP (GE Healthcare, Cat. No: BR-1001) was removed. -88) was used as the test buffer and sample diluent in all experiments, and the assay The experiments were carried out at 25° C. All samples were kept at 4° C. before collection.

[0235] Capture antibody was immobilized, but α-synuclein antibody was not captured, recorded in Fc1 The response values ​​obtained by the 1:1 or 2:1 binding algorithm were subtracted from the response values ​​in Fc2. The rhythms were analyzed using BIAevaluation software version 4.1.1. The results are shown in Figures 3, 4 and 5, which show that human α-synthetic Binding of antibodies 37, 285 and 9E4 to klein is shown.

[0236] Example 3: Epitope Mapping Epitope mapping of antibodies against α-synuclein was performed using Pepscan (Peps can Zuidersluisweg 2 8243 RC Lelystad,th The study was carried out at the University of Illinois (Netherlands) using a series of overlapping linear peptides. Binding of the antibodies to each of the selected 20-mer peptides was assessed by Pepscan-based ELI. A linear peptide array covering the entire coding sequence of α-synuclein was tested in SA. All peptides containing oxidized methionine or nitrosylated tyrosine were After washing, the peptide array was incubated with the primary antibody solution at 25 °C overnight. Antibody-peroxidase conjugate (SBPC) at a 1 / 1000 dilution for 1 hour at 37 °C. A, cat.nr.2010-05). After washing, peroxy 2,2'-Azino-di-3-ethylbenzothiazoline sulfonate (ABTS) enzyme substrate ) and 2 μl / ml of 3 percent H2O2 were added. After 1 hour, color development was measured. Color development was quantified using a charge-coupled device (CCD)-camera and an image processing system. For data processing, we used a 0-3 The values ​​were obtained from a CCD camera range of 000mAU. The results were quantified and the Peplab data Occasionally, wells contained air bubbles resulting in false positive values ​​and the card had to be manually adjusted. The values ​​caused by air bubbles are scored as 0. Binding data for antibodies 37 and 285 to peptides containing the ED are seen in FIG.

[0237] Example 4: Human brain homogenates of the cingulate cortex from patients with Lewy body dementia Immunoprecipitation of α-synuclein from Human DLB or healthy controls ( * Crude homogenates of cingulate cortex derived from The ability of the antibodies to bind and destroy α-synuclein from mice was analyzed by immunoprecipitation. Frozen samples from human cingulate cortex (Tissue Solutions Ltd, (obtained from Scotland) were cut in a cryostat and 100 mg samples were 1600 μl containing protease inhibitors and phosphatase inhibitors (Roche) Added to cellytic M cell lysis reagent (Sigma C2978). 5000rp 4 × 30 s at 400 rpm using a Precellys bead homogenizer (Bertin tech Brain tissue was homogenized using a 300-mL PBS (Nologies, France) until the sample was completely dissolved. The solution was centrifuged at 3000 × g, and the supernatant was used as a crude homogenate for immunoprecipitation. and used it.

[0238] For immunoprecipitation, 10 μg of antibody was added according to the manufacturer's instructions (Life Technologies, Magnetic Dynabeads Protein G beads were used (Gies, Paisley, UK). Crude brain homogenates were diluted 30-fold in lysis buffer (Sigma). The bound dynabeads were mixed with 500 ul of diluted homogenate and incubated on a rotator. The mixture was incubated at room temperature for 90 minutes with continuous mixing at 4°C. The beads were washed in wash buffer and the bound antigen was eluted using the Dynabeads G protocol, Life Technologies, Paisley UK) Therefore, elution was performed using a non-denaturing elution buffer. Monoclonal anti-human α-synuclein antibody (4B12, Thermo Scientific) The disrupted α-synuclein was visualized by Western blotting using The band patterns representing the various molecular weight forms showed that the 37, 37v2 and 285 antibodies were predominant. Various α-synuclein species, full-length α-synuclein (FL asyn 1–140) and C-terminal truncated species (1–135 and 1–119 / 122) could be immunoprecipitated, whereas Antibody 9E4 is unique in that it is unable to immunoprecipitate the truncated species 1-119 / 122. 7 differs between mutant 2 and 285 antibodies and the comparative antibody 9E4 (Figure 9).

[0239] Example 5: Inhibition of protease cleavage of α-synuclein fibrils by antibodies in cell culture Recombinant α-synuclein monomers and fibrils are synthesized by primary neurons in culture. As shown diagrammatically in FIG. 10, α-synuclein can be introduced into neurons. After incorporation of the ribonuclease, it is found that the major protease-sensitive site at amino acids 119 / 122 At the site of action, it can be processed by intracellular proteases such as calpain I. To investigate the cleavage of α-synuclein by α-aspartase, mouse primary cortical neurons were cultured using E lvang et al.2009(Elvang et al..J Neuroch 2009;110(5):1377-87) and astrocytes were prepared. At DIV3 (3 days in vitro), cells were treated with cytarabine to inhibit cell proliferation. At DIV4 (4 days in vitro), neurons were treated with 0.7 μM final concentration of (Cup Sonicated preformed α-aminobutyric acid (5 min at 50% power in a horn sonicator) Synuclein fibrils (PFFs) were treated alone or together with antibodies at the indicated concentrations. After 24 hours of incubation, the medium was collected and the cells were lysed. The cells were incubated with 4B12 antibody (Figure 11A) (Pierce MA1-90346) and a secondary antibody Mouse antibodies were used in both media and cell lysates: 4B12+anti-mouse After probing with , the blot was stripped and reprobed with anti-human IgG antibody. In the blot in Fig. 2, 14 and 1 were detected in the medium from cells treated with PFF only. A strong band was found at 2 kDa, where 14 kDa corresponds to the full-length α-synuclein. nuclein (FL-asyn), 12 kDa represents the C-terminal cleavage fragments 1–11 9 / 122 (CT-asyn). In addition, it represents SDS-resistant oligomeric species. Most likely, there was a higher molecular weight band. Combined treatment with either ribozyme or ribozyme did not alter this pattern of protein degradation or uptake.

[0240] In the medium from cells treated with fibrils together with 37, the full-length α-synuclein was predominantly There was a small amount of a truncated band (12 kD) and a small amount of a cleaved band (14 kD). In cell lysates from cells treated with fibrils together with This indicates that 37 prevents the cleavage of FL-α-synuclein. The total amount of L-α-synuclein was significantly increased in cells treated with PFFs alone or with the B12 control antibody. Several groups (Games et al., Am J of Pa thol,Vol.182,No.3,March,2013;Ritchie et al,Health,Vol.4,Special Issue,1167-1177, 2012;Mishizen-Eberz,Biochemistry,2005,44 ,7818-7829;Dufty et al,Am J of Pathol,Vo l.170, No.5, May 2007) demonstrated that α-synuclein binds to calpain- It has been shown that fibrillized α-synthetic α-protein can be cleaved by 1. The cleavage site of calpain-1 for klein was found to be in the region 114-122. (Mishizen-Eberz, J of Neurochem, 86, 836-8 47, 2003). In vivo, in transgenic animals and in the human brain, / 122 appears to be the major cleavage product in α-synuclein, and cleavage is It is likely to be located after asparte 119 or asparagine 122. is deamidated to aspartate and has similar cleavage specificity to calpain These results suggest that antibody 37 binds α-synuclein, which is cleaved by another protease. The epitope of antibody 37 is capable of inhibiting the C-terminal cleavage of the enzyme carbapenem. The binding of 37 to α-synuclein is related to calpain-1 because it overlaps with the calpain-1 binding site. These antibodies can directly inhibit binding and cleavage mediated by ribozyme (FIGS. 10 and 11).

[0241] The 285 epitopes overlap 37 epitopes and inhibit protease cleavage. The amino acid sequence of 37v2 is predicted to have three amino acids in one CDR. 37v2 has the same binding as 37, except for the difference in 7. It would be expected that the antibody would inhibit the cleavage of the ribozyme. To investigate whether the effect of PFFs on the function of primary cortical neurons was induced by simultaneous addition of PFFs and antibodies for 24 h. The experiment was set up. The concentration of PFF was kept constant (10 μg / ml), while the control antigens to be tested The concentrations of corpus B12, and antibodies 37, 37v2, and 285 were 10, 5, 1, and 0.1. ug / ml. α-synuclein on Western blot was Detection was performed using an antibody (Abcam) that recognizes an epitope within the region 103-108. As a result, it binds to both FL and C-terminally truncated α-synuclein (Figure 11B). As can be seen in Fig. 11B, GM37, 37v2 and GM285 all bind to proteases. There is a dose-dependent inhibition of enzyme cleavage, with near complete inhibition of cleavage at high concentrations of antibody.

[0242] Example 6: Antibody-mediated inhibition of seeding of α-synuclein aggregation in cell culture Several studies have demonstrated that exogenous addition of recombinant α-synuclein fibrillar aggregates enhances cell They enter the IL-1 receptor and recruit endogenous α-synuclein, forming an innate immune response similar to LB. It has been shown to be capable of inducing α-synuclein aggregation and phosphorylation in vitro and in vivo. (Volpicelli-Daley et al. 2011, Luk et al. al.2012a, Luk et al.2012b, Recasens et al. 2013, Peelaerts et al. 2015). To examine seeding of endogenous mouse α-synuclein by the α-synuclein-binding protein, we performed the same procedure as above. The prepared mouse primary cortical neurons were plated in a 96-well plate (15,000 cells per well). At day 5 of in vitro culture (DIV), the cells are plated in 50% of the medium. Replace and supplement with cytosine arabinoside (final concentration of 1 uM). On day 6 (DIV6), replace half of the medium with α-synuclein fibrillar material, crude fibril seeds or Replace the medium with glial conditioned medium containing either pure seeds or crude fibril seeds. The monomers were prepared from isolated recombinant human α-synuclein and were then purified using Amino Acids. on Ultra 100.000 cut-off filter (Millipore cat. No. UFC510096) and concentrated to a concentration of 1 mg / ml in PBS, pH 7.4. To prepare crude fibril seeds, the monomer solution was adjusted to (assessed by daily measurements of Thioflavin S), continuous mixing (800 The mixture was incubated in a thermomixer at 37°C with 500 rpm. To minimize the amount of precipitation, a drop of mineral oil was added to cover the solution. The time is 5-7 days, and the pure seeds are obtained by centrifugation of crude fibrillar seeds and purifying them. The aggregated pellet produced from the incubation was resuspended in fresh PBS and sonicated. Antibodies are added once at DIV6 along with the α-synuclein crude seed. Replace half of the medium in the cultures with glial conditioned medium every week to maintain them up to DIV21. Neurons were fixed and α-amino acid S129 (abcam 51253) was added. Phosphorylation of synuclein was detected using rabbit antibodies specific for synuclein phosphorylation, followed by fluorescently labeled anti-rabbit antibodies. Staining for phospho-synuclein and fluorescence were analyzed by automated fluorescence microscopy, Cellomics Quantify using Arrayscan. Nuclei are detected in one channel and valid cells are counted. The phosphorylated α-synuclein spots were located around the nucleus, which represents the cytoplasm of the cells. The spots were detected in a separate channel at a certain ring-forming region. The average number of spots per cell was calculated. An example of cell staining is shown in FIG. 12A. Phosphorylated α-synuclein spots were Crude or pure seeds (1–10 per well) were not seen in treated neurons. Neurons incubated with 1,2-dichlorophenylalanine (ng) induced phosphorylation of α-synuclein. In neurites, phosphorylated synuclein is expressed as spots or puncta. and some of the phospho-synuclein in neurites appears elongated.

[0243] For differentiation studies, cells were harvested in phosphate-buffered saline solution (PBS) and centrifuged. The pellet was resuspended in 1% Triton buffer containing protease inhibitors. The samples were kept on ice for 15 min and then sonicated. The samples were incubated at 4°C for 3 The supernatant was collected and used as the soluble fraction. The pellet was washed once in triton buffer and resuspended in 1% SDS buffer. After turbidity, the samples were sonicated and centrifuged at 100,000×g for 30 min. The supernatant is collected as the insoluble fraction. Protein concentration is measured and the samples are diluted to 4–12%. The proteins were examined on SDS-PAGE gels and blotted onto membranes to detect α-synuclein and phosphorylated α Synuclein (S129P) was detected by 4B12 / 1904 antibody (Thermo sc ientific:MA1-90346-human synuclein), S129P-asyn anti (abcam 51253) and mouse synuclein antibody (Cell Signaling-D3 7A6).

[0244] FIG. 12B shows soluble and soluble IgG from primary neurons with and without crude seeds. As can be seen from FIG. 12B, the addition of seeds , endogenous mouse α-synuclein and p-S129-α-synuclein in the insoluble fraction of cells. This results in the accumulation of multimers of rhein and phosphorylated mouse α-synuclein.

[0245] To test whether antibodies could inhibit seeding, we used α-synuclein Inseed was used at a concentration of 6.6 nM (10 ng / well). and α-synuclein seeds were added together at DIV6 to form a dose response (133 Starting from the highest antibody concentration of 100 nM to 133 pM), neurons were fixed again and the host Cells were stained for hog-synuclein (Abcam 51253) and fluorescence was measured using an automated Quantification was performed using fluorescence microscopy and Cellomics arrayscan. The resulting spots / specks were counted in a Cellomics arrayscan. As can be seen in Figure 12C, antibodies 37, 37v2 and 285 all showed dose-dependent Reduces α-synuclein phosphorylation in neurons, and The two compounds had similar maximum inhibition rates (approximately 70-75%) and EC50 values ​​of approximately 5 nM. Cellular proteins in soluble and insoluble fractions after treatment with antibody at a low concentration (133 nM) Protein fractionation was as shown in Figure 12D, with antibodies 37, 37v2, and 285 all showing Also, cleavage of recombinant crude seeds and accumulation of C-terminal truncated fragments (CT a-syn) Inhibits accumulation of phosphorylated endogenous mouse α-synuclein and aggregated forms in the insoluble fraction The results show that the compound reduced mouse α-synuclein levels.

[0246] Example 7. Acute electrophysiological effects of α-synuclein antibodies in vivo High expression levels of human α-synuclein regulate the mouse α-synuclein promoter The F28-snca transfectant model overexpresses wild-type α-synuclein in mice. in the hippocampus of fecund mice (Westerlund M, et al. Mol Cell Neurosci.2008 Dec;39(4):586-91). 4~6 Age-matched male F28-snca transgenic mice and age-matched control mice Evaluation of synaptic transmission and plasticity in the hippocampal CA1 region during in vivo electroporation The data show that basal synaptic transmission was significantly higher in rats than in age-matched control mice. In comparison, the expression level of α-amyloides in F28-snca transgenic mice was significantly decreased. (Figure 13).

[0247] Four to six month old F28-snca transgenic mice and age-matched counterparts were Male mice (CRO breeding, Taconic Europe A / S) were cultured in a controlled temperature The animals were housed in a single cage under the following conditions: temperature (22±1.5°C) and humidity (55–65%) for 12 hours. The mice were kept on a 12-hour light / dark cycle (lights on at 06:00 h). Water and water were available ad libitum.

[0248] The animals were anesthetized with an intraperitoneal (ip) injection of urethane (1.2 g / kg). The mouse was then placed in a stereotaxic frame and its body temperature was adjusted to 37.5 °C by a heating pad. The skull was then exposed by adjusting the anterior and posterior cingulate cortex. A platinum wire was placed on the frontal bone to act as a reference and the Paxi nos and Franklin (Paxinos and Franklin's e Mouse Brain in Stereotaxic Coordinates The following arrangement is based on the diagram in the book "The Hippocampus: A Neuropsychological Study of the Hippocampus," 4th Edition, 2001. Additional holes were drilled for the insertion of recording and stimulating electrodes: recording, 1.5–1.7 of bregma; mm posterior, 1.0–1.2 mm lateral to the midline, and 1.4–1.7 mm below the brain surface; stimulation, 1.8–2.0 mm posterior to bregma, 1.5–1.7 mm lateral to the midline, and 1 on the brain surface. The animal was kept in the stereotaxic frame throughout the entire recording period. Their level of anesthesia was checked periodically.

[0249] Field potentials (fEPSPs) were recorded in the CA1 by electrical stimulation of the Schaffer collaterals every 30 s. The depth of the recording electrode was adjusted to the time when a negative fEPSP was recorded in response to a monopolar square pulse. The slope of the evoked fEPSP was adjusted until it was recorded at 30–40° from the maximum amplitude of the fEPSP. Measured at 70%.

[0250] Once optimal fEPSPs were evoked, basal synaptic transmission was measured as a function of stimulation intensity and evoked fEPSPs. The relationship between the slope of the SP and the intensity of the stimulation (input-output relationship) was evaluated. 5, 50, 75, 100, 150, 200, 300, 400, and 500 μA, This is applied successively from lowest to highest intensity, and repeated 2-3 times at each intensity. Basal synaptic transmission is in F28-snca transgenic mice compared with age-matched controls It was found that the level of

[0251] Confirmed impairment of basal synaptic transmission in F28-snca transgenic mice We have investigated the ability of GM37 and GM2 to block α-synuclein-mediated effects using 85 and the control h9E4 were tested.

[0252] 3 to 6 hours after administration of a single dose of antibody at a dose of 15 mg / kg (intraperitoneal (ip)) In all experiments, recordings were made later. Basal synaptic transmission was recorded, if possible, in both basal and substantia nigra of each animal. Recordings were made in both hippocampi and recorded as individual experiments.

[0253] Acute treatment with h9E4 reduces basal inflammatory responses in F28-snca transgenic mice induced a significant improvement in the impairment of synaptic transmission (Tg-snca+h9E4 vs. Tg-sn ca+PBS, p=0.002, Figure 14). However, the improvement by h9E4 was not observed in PB As shown by the striking difference from basal synaptic transmission in littermates treated with S ( p=0.007), but only partially.

[0254] Acute treatment with GM37 reduces basal inflammatory responses in F28-snca transgenic mice The results showed that the synaptic transmission was significantly improved by Tg-snca+GM37 vs. Tg-sn Ca+PBS, p=0.004, Figure 15). Transgenic mice treated with GM37 Basal synaptic transmission in mice was significantly lower than that in PBS-treated littermates. 15), indicating complete amelioration of the disorder.

[0255] GM285 also inhibits basal synaptic transmission in F28-snca transgenic mice The GM285-treated transgenic mice induced a significant improvement in the lesions of the GM285-treated mice (Fig. 16). Basal synaptic transmission in mice was significantly lower than that in PBS-treated littermates. This is not significantly different from the transfer, indicating complete amelioration of the disorder.

[0256] Example 8. Microperfusion to assess human α-synuclein in the brain of awake, freely moving animals Analysis Using the push-pull microdialysis method, human α-synuclein in brain interstitial fluid (ISF) was detected. The levels of acetylcholine in mice were evaluated. Mice were kept in a 24-well plate at 10°C under controlled temperature (22 ± 1.5°C) and humidity conditions ( They were housed in single hutches at 55–65% CO2 concentration and had a 12:12 h light / dark cycle (06: The mice were kept in a dark room with the lights on at 00h. Food and water were available ad libitum. The study was performed in the hippocampus of F28-snca transgenic mice (50-54 weeks old). To enable microdialysis in the hippocampus, mice were anesthetized with isoflurane. A cerebral guide cannula (CMA) was stereotactically implanted into the brain, and the Paxinos and Microdialysis probes were positioned within the hippocampus according to the diagram in Franklin 2001. (Probe tip placement: 3.1 mm posterior and 2.8 mm lateral to the bregma, and 1.3 mm to the membrane). Fixation screws and acrylic cement were used to fix the guide cannula. After implantation of the cannula, the mice were surgically treated for 2–3 days before dialysis. Recovered from surgery.

[0257] On the day of the experiment, a 2 mm, 1000 kDa cutoff CMA probe was inserted into the guide cannula. The probe was then inserted into a microdialysis peristaltic pump (MAB20; The microdialysis probe was connected to a 100-μm suction tube (Crobiotech) and operated in push-pull mode. The inlet tube was connected to a peristaltic pump that perfused the probe with artificial cerebrospinal fluid (aCSF; in mM: 1 47NaCl, 2.7KCl, 1.2CaCl 2 , 0.85MgCl 2 ) was connected. To prevent loss of perfusate from the probe by drawing the perfusate out of the tubing, a peristaltic port was used. The pump was further connected to the outlet tube. As the perfusion buffer, 25% bovine albumin fraction V( On the day of use, dilute 0.2% of the IgG1 antibody (Sigma) with artificial CSF and filter through a 0.1 μm membrane. The actual flow rate of the pump was measured without the probe attached. The sample tube was The pump was then weighed before and after sampling over a period of time and the flow rate was calculated. A constant flow rate of 1 μL / min was set. A 120 min sampling schedule was used throughout the duration of the experiment. To avoid interference from tissue damage, the experimental period was limited to 14 to 48 hours after probe implantation. 14–16 hours after the start of the experiment, GM37, human 9E4, or isoform A control antibody (anti-HEL) was injected intraperitoneally (ip) at 15 mg / kg, and six additional Samples (12 hour collections) were collected. The dialysates were stored at -80°C. The concentrations of the peptides were determined by ELISA (Covance ELISA kit).

[0258] The average of 2-3 basal values ​​(4-6 hours) before antibody treatment was taken as the baseline. The data were analyzed to assess statistical relevance. The results were evaluated using two-way analysis of variance (ANOVA) with repeated measures. The basal level of klein was 8.1±1.1 ng / ml (mean±SEM, n=25 (not corrected for in vitro dialysis probe recovery). Administration of GM37 was The F28 mouse 9E4 antibody, human 9E4, and the isotype control (anti-HEL) were compared. The treatment induced a significant decrease in human α-synuclein in the hippocampus of mice (Figure 17).

[0259] Example 9: Chronic effects of α-synuclein antibodies in vivo. Antibody GM37 inhibits α-synuclein in rats. Improves the motor phenotype in a model of Kinson's disease. Targeting of human α-synuclein to dopaminergic neurons in the rat midbrain Highly specific overexpression is achievable using recombinant adeno-associated viral vectors (rAAV). This is associated with a progressive loss of dopaminergic cells in the substantia nigra and movement disorders. .

[0260] Adult female Sprague-Dawley rats (225–250 g) were cultured as previously described. Using the enhancer element from the cytomegalovirus promoter, chicken β- Adeno-associated virus of the AAV2 / 5 serotype containing the actin promoter, followed by human Injection of α-synuclein cDNA and the WPRE element resulted in the expression of human α-synuclein in the substantia nigra (SN). -synuclein was expressed (Xu L, Daly T, Gao C, Flotte T R, Song S, Byrne BJ, Sands MS, Ponder KP(200 1) In this model, human α-synuclein expression was associated with increased dopaminergic neuronal activity. It has been shown that this leads to neurodegeneration in the brain (Maingay M, et al. CNS Spectr.2005 Mar;10(3):235-44). In this model, To test the effect of synuclein therapeutic antibodies, antibody treatment was performed 2-4 days after virus injection. The study was started before and continued until the end of the study (Figure 18). PBS administration was used as a control. GM37 was administered weekly at a dose of 15 mg / kg (IP). The mice were injected twice with the human wild-type α-synuclein gene or green fluorescent protein (GF Viral particles (rAAV2 / 5) containing rAAV2 / 5 were injected into the SN on one side. 0.0 ml / kg subcutaneously (sc) with Hypnorm® and Dormicum® The animals were anesthetized with a combination of acetaminophen (TMS) and placed in a stereotaxic frame. The animal's body temperature was maintained with a heating pad. The temperature was adjusted to 37.5°C by a suction device and the skull was exposed. Following the diagram in (Paxinos & Watson, 1998), the following arrangement is A hole was drilled above the SN: 5.5 mm posterior and 2.0 mm lateral to the bregma. 3 μL of rA A single injection of AV2 / 5-α-syn or rAAV2 / 5-GFP was performed using a stereotactic injector (STI). A Hamilton syringe connected to a endotaxic injector was used. The procedure was performed at a depth of 7.2 mm below the dura mater and with a flow rate of 0.2 μL / min. The needle was left in place for an additional 5 min to allow for diffusion of the extracellular vector. After surgical intervention, the animals were returned to their home cages and placed in a heated environment, where they were anesthetized and The motor asymmetry test in the cylinder test was performed before and after AAV injection. The mice were evaluated 3, 7, and 10 weeks after AAV injection. Data shown are for the left front paw and the right front paw. This corresponds to the proportion of use of the right front paw compared to the total use of both front paws. The behavior of each animal was recorded for a total of 5 min. Contact with the left and right front paws was recorded over the 5 min period. Manual scoring of frequency was performed on the final day of the study, 10 weeks after virus injection. In AAV-syn compared to AAV-GFP-injected rats (p=0.012), There is a significant impairment in the behavior of GM37-treated animals compared to GFP rats (g p = 0.163 and p = 0.407, respectively, for GM37-treated The findings suggest that antibody GM37 may be a promising treatment for chronic myelopathy in this rat model. We show that the motor phenotype of Parkinson's disease can be improved by using .

[0261] Example 10: Chronic effects of alpha-synuclein antibodies in vivo. Antibody GM37 inhibits endogenous mouse alpha-synuclein Inhibits α-synuclein aggregation seeding and phosphorylation. Delivery of recombinant α-synuclein to the dorsal striatum of wild-type mice Injection of more preformed fibrils recruits endogenous mouse α-synuclein and induces its transport in the cortex, hemisphere, and brain. Induces the formation of Ser-129 phosphorylated aggregates within neurons in the gyrus and substantia nigra (Luk et al.2012,Science.2012 Nov 16;338 (6109):949-53). The α-synuclein-specific monoclonal antibody GM37 In vivo formation of phosphorylated α-synuclein inclusions induced by α-synuclein fibrils A total of 45 mice were used to examine whether the use of GABA in mice could reduce the appearance of GM37 at 30 mg / kg intraperitoneally (ip), GM37 at 15 mg / kg intravenously (i One day later, mice were anesthetized and administered either ip (.v.) or vehicle (PBS). 2 ul of recombinant human α-Syn crude seeds (Example 6) prepared as described in ) (total of 2 μg crude seeds per animal) were stereotaxically injected into one brain hemisphere. To inject the inoculum, the skull is opened by drilling a hole, and the inoculum is delivered to the dorsal striatum (the dura mater). A single glass pipette was inserted into the right forebrain so that it was oriented +2.6 mm below the brainstem. (+0.5 mm anterior to bregma, +2.0 mm lateral to midline). After recovery, the mice were sacrificed on day 45. Mice were injected intraperitoneally (ip) or intravenously (iv) once a week for up to 1 min. Groups of 15 mice / group received GM37 15 mg / kg intravenously (iv). , GM37 30 mg / kg was administered intraperitoneally (ip), or PBS (10 ml / kg) was administered intraperitoneally. The doses were administered intraperitoneally (ip) once a week.

[0262] To measure plasma antibody concentrations, cheek blood was collected once a week immediately before the next injection. The plasma was obtained by spinning at 2000 g and incubated at room temperature for 1 h. The CSF samples were incubated for 5 min and then the supernatants were frozen at -20°C. At termination, plasma and CSF samples were taken and frozen at -20°C. The concentration of human IgG was determined using a 100% IgG antibody. 6-1 (M1268) was used for capture and incubating plasma or CSF in the wells. followed by sulfo-TAG goat anti-human (MSD cat no: R32AJ-1 ) was used as the detection antibody. Plates were analyzed by electrochemiluminescence by MSD.

[0263] The antibody levels in plasma are shown in FIG. 20B, which shows the antibody plasma concentration and plasma The antibody levels in the csf are shown in FIG. 20C and in the plasma These results indicate that approximately 0.1% of antibody levels in csf can be measured.

[0264] At 45 days from the time of injection of α-synuclein fibrillar seeds, mice were anesthetized and injected with PBS. were perfused transcardially followed by neutral buffered paraformaldehyde (4%). The brains were removed and incubated overnight in neutral buffered paraformaldehyde for post-fixation. Immunohistochemistry was performed by Neuroscience Associates on 4-thick slices. The experiments were performed on 5 μM serial sections. Using the technique, up to 25 mouse brains were cultured together in blocks of three. Embed, cut 45 μm-thick frozen sections in the coronal plane, and place them in cups containing antigen storage solution. Every sixth section was stained with an antibody against Ser-129 phosphorylated α-synuclein ( Stained with anti-α-synuclein (phospho-S129) antibody [Psyn / 81A] ab184674) By staining, we revealed a Ser129 phosphorylated α-synuclein-reactive structure.

[0265] Images at 10x magnification from 5–7 sections covering the entire substantia nigra from every 6th section were collected. Quantification of pSyn lesions was performed by manually counting immunoreactive positive cells from the images. Counts were performed blinded. Cell counts in the amygdala and substantia nigra were analyzed using one-way ANOV. A, followed by Bonferroni t-test analysis, where the effect of GM37 antibody was compared with PBS compared with treatment.

[0266] As can be seen in FIG. 20C, treatment with antibody GM37, either intraperitoneally (i.p.) or intravenously (iv) The number of intracellular inclusions in the substantia nigra compared to PBS controls with either treatment The data show that the antibody GM37 blocks its transmission between neurons and and / or uptake by microglia to promote clearance from the ISF. by blocking the entry of extracellular pathological α-synuclein into neurons. This indicates that the appearance of inclusion bodies may be a therapeutic effect for PD. This is associated with loss of receptors and the development of Parkinson's disease-like movement disorders in animal models. Therefore, treatment with the antibody GM37 suppresses the loss of dopaminergic cells and the development of PD motor deficits. It may have a therapeutic effect against

[0267] Example 11: Manufacturability of GM37 and GM37 mutants Anti-α-synuclein antibodies are used to produce clinical grade material for use in patients. It is produced in mammalian cell culture under conditions that mimic the manufacturing conditions used. The proteins produced may affect the therapeutic efficacy of the antibody as well as its stability over time. It is well known that proteins undergo post-translational modifications that can affect both their biological and biophysical properties. Empirical knowledge confirmed from years of research has enabled the development of specific molecular A series of post-translational modifications known to pose a risk of cytotoxicity were identified. These Post-translational modifications of the heavy and light chains correlate with the amino acid chains present in the primary sequences of the proteins. These sequences have been identified and their potential for therapeutic antibody production and An algorithm was developed that can determine the potential risk to expressivity.

[0268] In silico analysis of the primary sequence of antibodies allows for the identification of molecules for potential development as therapeutic agents. In particular, detailed analysis of the VH and VL regions can be used to derisk the The analysis is important for the activity of the molecule, which may also be a potential risk to its stability over time. Sequence-specific deamidation can identify unique amino acids that are likely to be involved in protein structure. Protein deamidation has been identified as a potential risk for glutamine or aspartate. They occur on the amide side chains of lagin residues and can convert them into carboxylic acid groups (Loren zo et al.PLOSone,DOI:10.1371,Dec.(2015)) Nonenzymatic deamidation at neutral pH occurs more rapidly for asparagine, Therefore, it is considered to be at higher risk than glutamine. This activity is due to the subsequent amino acids in the sequence. This can occur at rates of days or years. The actual product of the protein was analyzed to determine the effect of the changes on both its stability and activity. Therefore, it is necessary to evaluate experimentally.

[0269] The present inventors have identified a site for deamidation within the VH region of GM37. Residue 54 is an asparagine (N) followed by a glycine (G) at position 55. 54 is at high risk of spontaneous deamidation. To reduce this risk, The inventors replaced asparagine (N) with serine (S), glutamine (Q) or histidine A series of three mutants were generated in which the chromosome was replaced with (H). All three mutants were All were produced in mammalian cell culture using transfection methods (Example 1.5). The three mutants showed similar expression and purification characteristics to GM37wt (FIG. 23).

[0270] A minimum of 400 ml transient transfections were performed for each of the eight products. This was performed using CHOK1SV GS-KO cells that had been in culture for 2 weeks. Cells were subcultured 24 hours before transfection. The reaction was performed by electroporation using Gene Pulse XCell (Bio-Rad). For each transfection, 2.86 × 10 viable cells were cultured. 7 Pre-warmed CD-CHO cells supplemented with 6 mM L-glutamine to a concentration of 10 cells / ml. 40 μg of each established antibody containing the appropriate heavy and light chains was resuspended in culture medium. The SGV DNA was then transferred to each cuvette (Bio-Rad, GenePulser cuvette). (0.4 cm gap, 165-2088) and 700 μl of cell suspension was added. The cells were electroporated at 300 V and 900 μF. The transfected cells were Transfer the cuvette to pre-warmed medium in a Lenmeyer flask and rinse twice with pre-warmed medium. The contents of the flask were also transferred to the flask. The transfectant cultures were incubated at 36.5°C, 5% CO in a 20-mL flask. CO 2 The plates were incubated in a shaking incubator at 140 rpm, 85% humidity for 6 days. Cell viability was assessed using a Cedex HiRes automated cell counter (Rosche). was measured at time.

[0271] To assess the importance of residue 54 in binding to human α-synuclein, we analyzed the ability of the mutants to bind in two different experiments. Using the ELISA kit, we show that changes at residue 54 affect α-synuclein in solution. The potential impact of GM37 on the ability of synuclein-coated ELISA To evaluate the concentration of synuclein that can inhibit antibody binding to the A plate, Thus, we conclude that all three mutants maintain the same binding as GM37wt. It was shown to bind to α-synuclein with high affinity resulting in an IC50 of 1-2 nM (Figure 24). Human α-synuclein in the range of 0-1000 nM was assayed at room temperature for 60 minutes. The following antibodies, GM37 (GM37 wt ), GM37 mutant 1, GM37 mutant 2, and GM37 mutant 3, respectively. The competitive assay was performed using a preincubation of 100 μg / ml of 100 μl ... Anti-human detection antibodies were used for electrochemiluminescence (MSD, Gathersburg, MD). The ELISA plate was coated with 100ng / ml recombinant human α-synuclein. The IC50 of the interaction was measured for GM37 wt, GM37 mutant 1, and GM37 1.9 nM, 1.6 nM, and 2.1 nM for mutant 2 and GM37 mutant 3, respectively. M and 1.4 nM (determined using Prism Graphpad®) When this happens.

[0272] Using surface plasmon resonance (SPR), we found that GM37 wt (2 batches) The real-time kinetics of binding of the three mutants was evaluated (Example 2). The lein is captured on a slide (ligand) and the antibodies are tested at multiple concentrations as analytes. Analysis of binding curves in the presence of antibody at multiple concentrations was performed by removing the antibody from the buffer. As with the previous study, the on rates were similar for all four antibodies. The measured off-rates showed statistical differences between the antibodies. Using a 1:1 binding algorithm, all four antibodies gave nearly identical binding. It has a composite constant (Figure 25).

[0273] To evaluate the effect of alterations at N54 on the functional activity of GM37, The authors report the development of an antibody that blocks synuclein seeding activity in primary neuronal cultures. The ability of the seeding level to specifically target phospho-synuclein was analyzed (Example 6). All three antibodies detect the phospho-synuclein signal. When measured with 100% ethanol, seeding was inhibited (Figure 26). The cell-based data showed that the VH region Amino acid 54 in α-synuclein is involved in the binding affinity to human α-synuclein or in primary cells. Further binding data show that β-lactamase is not required for inhibition of seeding in the β-lactamase-based assay. Furthermore, we confirmed that all three of these antibodies were expressed and purified using standard methods. Interestingly, one of the mutants, N54Q, was found to be capable of being produced using the method. One variant showed improved production over other variants, which means that the antibody can be commercially produced on a large scale. These data are very important if the efficacy of By replacing asparagine (N) with another amino acid, the potential risk of deamidation is eliminated. This supports the possibility of reducing the risk of

[0274] The samples of antibodies wt GM37, var1, var2 and var3 were analyzed at different times. The temperature was steadily increased in both cases, and the level of aggregation was monitored using multi-angle light scattering (Prometheus NT.48, NanoTemper Technologies) The onset temperature of aggregation was found to be similar for GM37 and the GM37-mutant. However, the lowest level of aggregation was observed for GM37-Var2 (FIG. 27). The present invention may include the following aspects. [1] A monoclonal antibody capable of specifically binding to human alpha-synuclein, Amino acids 112 to 117 of human α-synuclein (SEQ ID NO: 10) (SEQ ID NO: 9 (ILE An antibody that binds to an epitope in the DMP or an antigen binding thereto Combined fragment. [2] The antibody comprises, for binding to the epitope, a light chain variable region of SEQ ID NO: 8 and a sequence capable of competing with an antibody comprising a heavy chain variable region of No. 7, 30, 31 or 32; The monoclonal antibody of claim 1, or an antigen-binding fragment thereof. [3] Amino acids 112 to 115 of human α-synuclein (SEQ ID NO: 10) (SEQ ID NO: 19 ( The monoclonal antibody according to claim 1, which is capable of specifically binding to an epitope within an ILED. A clonal antibody, or an antigen-binding fragment thereof, that binds to said epitope. [4] The antibody comprises a heavy chain variable region of SEQ ID NO: 26 and a sequence The antibody according to claim 1 or 3, which is capable of competing with an antibody comprising the light chain variable region of sequence number 27. A monoclonal antibody, or an antigen-binding fragment thereof, as described herein. [5] The monoclonal antibody according to any one of claims 1 to 4, comprising or consisting of an intact antibody. A monoclonal antibody, or an antigen-binding fragment thereof. [6] The monoclonal antibody is of the subtype IgG1, IgG2, IgG3 or IgG4 The monoclonal antibody according to any one of claims 1 to 5, An antibody, or an antigen-binding fragment thereof. [7] Fv fragments (e.g., single chain Fv and disulfide-linked Fv), Fab-like fragments Fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments) The group consisting of domain antibodies (e.g., single VH or VL variable regions) Any of claims 1 to 6, comprising or consisting of an antigen-binding fragment selected from A monoclonal antibody, or an antigen-binding fragment thereof, described in any one of claims 1 to 4. [8] The antibody or antigen-binding fragment has the following characteristics: a) 0.5-10 nM of α-synuclein, such as 1-5 nM or 1-2 nM Binding affinity (KD); b) the ability to inhibit protease cleavage of α-synuclein fibrils; c) Improved impairment of basal synaptic transmission in F28-snca transgenic mice The ability to do good; d) Levels of α-synuclein in the mouse hippocampus as measured by in vivo microdialysis. Ability to reduce bells; e) When administered chronically, it restores motor function in a rat model of Parkinson's disease. Ability to; f) α-synuclein seeding (in vitro and / or Parkinson's disease) the ability to prevent the accumulation of insoluble phosphorylated alpha-synuclein in mouse models of / or g) Ability to bind to cleaved α-synuclein in human brain The monoclonal antibody according to any one of claims 1 to 7, which exhibits one or more of the following: or an antigen-binding fragment thereof. [9] The antibody according to any one of claims 1 to 8, which is a human, humanized, recombinant or chimeric antibody. A monoclonal antibody, or an antigen-binding fragment thereof.

[10] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:2; (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO:6 A monoclonal antibody or a monoclonal antibody according to any one of claims 1 to 2 and 5 to 9. Monoclonal antibodies, or fragments thereof.

[11] The antibody of claim 10, comprising a heavy chain variable region of SEQ ID NO: 7 or a light chain variable region of SEQ ID NO: 8. Monoclonal antibodies.

[12] A heavy chain consisting of the variable region of SEQ ID NO: 7 and a light chain consisting of the variable region of SEQ ID NO: 8, The monoclonal antibody of claim 11.

[13] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33; (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO:6 A monoclonal antibody or a monoclonal antibody according to any one of claims 1 to 2 and 5 to 9. Monoclonal antibodies, or fragments thereof.

[14] The antibody according to claim 13, comprising a heavy chain variable region of SEQ ID NO: 30 or a light chain variable region of SEQ ID NO: 8. The monoclonal antibodies described above.

[15] It comprises a heavy chain consisting of the variable region of SEQ ID NO: 30 and a light chain consisting of the variable region of SEQ ID NO: 8. The monoclonal antibody of claim 14.

[16] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 34; (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO:6 A monoclonal antibody or a monoclonal antibody according to any one of claims 1 to 2 and 5 to 9. Monoclonal antibodies, or fragments thereof.

[17] The antibody according to claim 16, comprising a heavy chain variable region of SEQ ID NO: 31 or a light chain variable region of SEQ ID NO: 8. The monoclonal antibodies described above.

[18] 17. The antibody of claim 16, comprising a heavy chain consisting of the variable region of SEQ ID NO: 31 and a variable region of SEQ ID NO: 8. A monoclonal antibody according to claim 1.

[19] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 35; (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO:6 A monoclonal antibody or a monoclonal antibody according to any one of claims 1 to 2 and 5 to 9. Monoclonal antibodies, or fragments thereof.

[20] The antibody according to claim 19, comprising a heavy chain variable region of SEQ ID NO: 32 or a light chain variable region of SEQ ID NO: 8. The monoclonal antibodies described above. [twenty one] 20. The antibody of claim 20, comprising a heavy chain consisting of the variable region of SEQ ID NO: 32 and a variable region of SEQ ID NO: 8. A monoclonal antibody according to claim 1. [twenty two] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20; (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21; (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22; (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 23; (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 24; and (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 25 A monoclonal antibody or a monoclonal antibody according to any one of claims 1, 3 and 5 to 9. Monoclonal antibodies, or fragments thereof. [twenty three] 23. The method of claim 22, comprising the heavy chain variable region of SEQ ID NO: 26 or the light chain variable region of SEQ ID NO: 27. The monoclonal antibody described herein. [twenty four] 2. A heavy chain comprising the variable region of SEQ ID NO: 26 and the variable region of SEQ ID NO: 27. 3. A monoclonal antibody according to claim 3. [twenty five] A preparation comprising the monoclonal antibody according to any one of claims 1 to 24, The preparation is not capable of binding to α-synuclein or the preparation's anti-α- Substantially free of naturally occurring antibodies that do not substantially alter synuclein functionality, Sex, (i) the binding affinity (KD) of said anti-α-synuclein antibody for α-synuclein; (ii) the anti-α-synuclein agent inhibits protease cleavage of α-synuclein fibrils; In-antibody capabilities; (iii) Impairment of basal synaptic transmission in F28-snca transgenic mice the ability of the anti-alpha-synuclein antibody to ameliorate harm; (iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis the ability of the anti-alpha-synuclein antibody to reduce the level of (v) When administered chronically, it improves motor function in a rat model of Parkinson's disease. the ability of the anti-alpha-synuclein antibody to restore (vi) α-synuclein seeding (in vitro and / or in Parkinson's disease) their ability to prevent the accumulation of insoluble phosphorylated α-synuclein in mouse models of Alzheimer's disease; Or (vii) the ability to bind to truncated α-synuclein in human brain A preparation selected from the group consisting of:

[26] A preparation comprising the monoclonal antibody according to any one of claims 1 to 24, The monoclonal antibody has an amino acid sequence similar to that of a natural anti-α-synuclein antibody. a conformational change in the sequence, said conformational change causing said monoclonal antibody to differentiate from said natural anti-α- exhibiting altered functionality compared to the functionality exhibited by a synuclein antibody, said functionality being , (i) the binding affinity of said anti-α-synuclein monoclonal antibody to α-synuclein; Harmony (KD); (ii) the anti-α-synuclein agent inhibits protease cleavage of α-synuclein fibrils; In-monoclonal antibody capabilities; (iii) Impairment of basal synaptic transmission in F28-snca transgenic mice the ability of the anti-alpha-synuclein monoclonal antibody to ameliorate harm; (iv) α-synuclein in mouse hippocampus as measured by in vivo microdialysis the ability of the anti-alpha-synuclein monoclonal antibody to reduce the level of (v) When administered chronically, it improves motor function in a rat model of Parkinson's disease. the ability of the anti-alpha-synuclein monoclonal antibody to restore (vi) α-synuclein seeding (in vitro and / or in Parkinson's disease) their ability to prevent the accumulation of insoluble phosphorylated α-synuclein in mouse models of Alzheimer's disease; Or (vii) the ability to bind to truncated α-synuclein in human brain A preparation selected from the group consisting of:

[27] The monoclonal antibody according to any one of claims 1 to 24 or the monoclonal antibody according to claims 25 to 26. A pharmaceutical composition comprising the preparation according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

[28] A nucleic acid encoding the antibody or fragment thereof according to any one of claims 10 to 24.

[29] A monoclonal antibody according to any one of claims 1 to 24 for use in therapy. or an antigen-binding fragment thereof, or the Preparation.

[30] A compound according to any one of claims 1 to 24 for use in treating a synucleinopathy. The monoclonal antibody according to claim 25 or an antigen-binding fragment thereof or the antibody according to claim 26. The preparation according to any one of claims.

[31] Parkinson's disease, idiopathic and hereditary Parkinson's disease, Gaucher disease (GD), diffuse Diabetic Lewy Body Disease (DLBD), Lewy Body Variant Alzheimer's Disease (LBV), and Combined Treating Alzheimer's and Parkinson's disease, pure autonomic failure or multiple system atrophy The monoclonal antibody of claim 30, or an antigen binding thereof, for use in treating Combined fragment.

[32] A monoclonal antibody according to any one of claims 1 to 24 for use in the manufacture of a medicament. An antibody, or an antigen-binding fragment thereof, or the method according to any one of claims 25 to 26. Its preparation.

[33] Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Gaucher disease (GD) , Diffuse Lewy Body Disease (DLBD), Lewy Body Variant Alzheimer's Disease (LBV), Combined Alzheimer's and Parkinson's disease, pure autonomic failure, or multiple system atrophy 33. The method of claim 32 for use in treating atherosclerosis.

[34] 1. A method of treating Parkinson's disease or other synucleinopathy in a subject, comprising: The monoclonal antibody or its antigen-binding fragment thereof according to any one of claims 1 to 24. or the preparation according to claims 25-26 or the pharmaceutical composition according to claim 27. administering to the subject an effective amount of

[35] 35. The method of claim 34, wherein the treatment is chronic.

[36] 36. The method of claim 35, wherein the long-term treatment lasts for at least two weeks.

[37] 35. The method of claim 34, wherein the subject is a human.

[38] The antibody according to any one of claims 1 to 24, or an antigen thereof, for use in therapy. A binding fragment or a preparation according to any one of claims 25 to 26 or a preparation according to any one of claims 27 to 29. Item 28. A kit comprising the pharmaceutical composition according to item 27.

[39] The monoclonal antibody according to any one of claims 1 to 24, which is detectably labeled. or an antigen-binding fragment thereof.

[40] The detectable label may be a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label or The monoclonal antibody or antigen-binding fragment thereof according to claim 39, wherein the .

[41] The method includes the steps of: detecting or measuring the presence or amount of alpha-synuclein in the brain of a subject; The monoclonal antibody according to any one of claims 39 to 40, or its antigen, for use in a Binding fragments, preparations or pharmaceutical compositions.

[42] The detecting or measuring comprises detecting or measuring the amount of the anti-synuclein antibody bound to the α-synuclein. The monoclonal antibody according to any one of claims 39 to 41, including in vivo imaging. An antibody, antigen-binding fragment thereof, preparation or pharmaceutical composition.

[43] The detection or measurement of the anti-synuclein antibody bound to the alpha-synuclein. or ex vivo imaging of said antigen-binding fragment thereof. The monoclonal antibody, antigen-binding fragment thereof, preparation or is a pharmaceutical composition.

[44] For use in the manufacture of drugs for treating, diagnosing or imaging synucleinopathies The monoclonal antibody or its antigen-binding site according to any one of claims 1 to 24 for use in a method for treating a patient having a pulmonary syndrome. or a preparation according to any one of claims 25 to 26, or 27. The pharmaceutical composition according to claim 27 or the monoclonal antibody according to any one of claims 39 to 40. Use of the antibody or antigen-binding fragment thereof, or preparation or pharmaceutical composition thereof.

[45] The drug is for treating Parkinson's disease (including idiopathic and hereditary Parkinson's disease), Alzheimer's disease (GD), diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson's Disease, Pure Autonomic Failure, and the monoclonal antibody of claim 44 for use in treating multiple system atrophy. Use of the monoclonal antibody, or antigen-binding fragment thereof, or preparation or pharmaceutical composition - Patents.com .

[46] Treating, diagnosing or imaging Parkinson's disease or other synucleinopathies in a subject A method for immunizing a subject with the monoclonal antibody according to any one of claims 1 to 24. or an antigen-binding fragment thereof, or the preparation according to any one of claims 25 to 26. The pharmaceutical composition according to claim 27 or any one of claims 39 to 40. The monoclonal antibody, or antigen-binding fragment thereof, preparation or pharmaceutical composition is administering to said subject an effective amount of

[47] 47. The method of claim 46, wherein the treatment is chronic.

[48] 48. The method of claim 47, wherein the long-term treatment lasts for at least two weeks.

[49] 49. The method of claim 48, wherein the subject is a human.

[50] Detecting or measuring the presence or amount of said alpha-synuclein in the brain or body fluids of the subject. The monoclonal antibody according to any one of claims 39 to 40 for use in An antigen-binding fragment, preparation or pharmaceutical composition of.

[51] The detecting or measuring comprises detecting or measuring the amount of the anti-synuclein antibody bound to the α-synuclein. The monoclonal antibody of claim 50, its antigen-binding fragment, including in vivo imaging. Fragment, preparation or pharmaceutical composition.

[52] The detection or measurement of the anti-synuclein antibody bound to the alpha-synuclein. or ex vivo imaging of said antigen-binding fragment thereof. The monoclonal antibody, antigen-binding fragment thereof, preparation or pharmaceutical composition thereof.

[53] Cell lines, including human cell lines, non-human mammalian cell lines, insect, yeast or bacterial cell lines The antibody or its antigen according to any one of claims 1 to 24, which is produced or manufactured by Combined fragments.

[54] CHO cell lines, HEK cell lines, BHK-21 cell lines, mouse cell lines (myeloma cell lines, etc.) ), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and H 54. The antibody of claim 53, or its antigen binding, produced in uH-7 human cell line. Fragment.

[55] The constant region defined in SEQ ID NO:18 and the kappa constant region defined in SEQ ID NO:17 The monoclonal antibody according to any one of claims 1 to 21, or its antigen-binding fragment. to.

[56] The constant region defined in SEQ ID NO:28 and the kappa constant region defined in SEQ ID NO:29 The monoclonal antibody according to any one of claims 22 to 24, and its antigen-binding fragment. nt.

Claims

[Claim 1] The invention described in the present specification.

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