Agents, uses and methods for treating synucleinopathies

Abstract

The present invention relates to novel monoclonal anti-alpha-synuclein antibodies. These antibodies can be used to treat synucleinopathies, such as Parkinson's disease (including idiopathic and genetic forms of Parkinson's disease), diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.

Description

Field of Invention:

[0001] This invention relates to a novel class of monoclonal antibodies that specifically bind to α-synuclein, and also to methods for using these molecules and their α-synuclein-binding fragments for the treatment and diagnosis of synucleinosis.

[0002] References to sequence lists:

[0003] This application includes one or more sequence lists (in accordance with 37C.FR1.821, etc.), which are disclosed in a computer-readable medium (filename: 0992_ST25.txt, created on June 22, 2016, and 44kB in size), the entire contents of which are incorporated herein by reference. Background of the invention:

[0005] Synucleinopathy, also known as Lewy body disease (LBD), is characterized by the deposition of intracellular protein aggregates of Lewy bodies (LB) and / or Lewy neurites (of which the protein α-synuclein is the main component), visible under a microscope. (Jellinger, Mov) Disord, January 2012; 27(1):8-30; McKeith et al., Neurology (1996) 47:1113-24). Synucleinopathies include Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease) and diffuse Lewy body (DLB) disease (also known as Lewy body dementia (DLB), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease (CAPD), pure autonomic failure (PAF), and multiple system atrophy (MSA; e.g., oligopontocerebellar atrophy, striatum-nigrostriatum degeneration, and Hey-Dr syndrome)). Synucleinopathies often involve degeneration of the dopaminergic nigrostriatum system, resulting in the core motor deficits in Parkinson's disease (rigidity, bradykinesia, resting tremor). However, Lewy bodies and dystrophic Lewy neurites are also widely present in the central, peripheral, and autonomic nervous systems and brain regions, as well as other organs, in connection with non-motor dysfunctions such as dementia and autonomic deficits. In Parkinson's disease and other synucleinopathies, several non-motor signs and symptoms are thought to precede motor symptoms. For example, such early signs include rapid eye movement sleep behavior disorder (RBD) and loss of smell, as well as constipation (Mahowald et al., Neurology (2010) 75:488-489). In older adults, synucleinopathies remain a common cause of motor impairment and cognitive decline (Galasko et al., Archives of Neurology (1994) 51:888-95).

[0006] Alpha-synuclein is a member of a protein family that includes β- and γ-synuclein, as well as synoretin. Alpha-synuclein is expressed in a normal synaptic state and is thought to play a role in regulating synaptic vesicle release, thereby affecting neural communication, plasticity, learning, and memory.

[0007] Several studies have suggested that α-synuclein plays a central role in the pathogenesis of Parkinson's disease (PD). Under pathological conditions, this protein can aggregate to form intracellular insoluble fibrils. For example, synuclein accumulates in Leukopenia leukopenia (Spillantini et al., Nature (1997) 388:839-40; Takeda et al., Journal of Pathology (1998) 152:367-72; Wakabayashi et al., Neuroscience Letters (1997) 239:45-8). Mutations in the α-synuclein gene, as well as diploid and triploid duplications of the gene, co-segregate with rare familial forms of Parkinson's disease (Kruger et al., Nature Genetics (1998) 18:106-8; Polymeropoulos et al., Science (1997) 276:2045-7). An important finding is that α-synuclein is secreted into the extracellular fluid and is present in plasma and cerebrospinal fluid (CSF). Several studies, such as those by Pacheco et al. (2015) and others (Pacheco et al., J Neurochem, March 2015, 132(6):731-4; Conway, Proceedings of the National Academy of Sciences of the United States of America (2000) 97:571-576; Volles et al., J. Biochem. 42:7871-7878, 2003), have shown that extracellular synuclein plays a pathogenic role in the brain. They have demonstrated that extracellular α-synuclein oligomers possess neurotoxicity to the plasma membrane of brain neurons. Another interesting hypothesis based on synuclein secretion data is that prion-like transmission of α-synuclein forms the basis for the progression of Parkinson's disease and other synucleinic disorders (Li et al., 2014, *Nat RevNeurol*, Feb. 2014, 10(2):92-8; Hansen and Li, 2012, *Trends Mol Med*, May 2012, 18(5):248-55). These findings have generated the expectation that extracellular synuclein can be targeted with immunotherapy (Vekiarellis et al., 2011, *Lancet Neurol*, Nov. 2011, 10(11):1015-25).

[0008] Naturally occurring α-synuclein autoantibodies have been shown to exist in PD patients and healthy controls (Smith et al., 2012, PLoS One, 2012, 7(12):e52285; Maetzler et al., 2014, PLoS One, 21 Feb, 2014, 9(2):e88604; Papachroni et al., 2007, J Neurochem, May 2007, 101(3):749-56; and Wolfe et al., 2002, Neurology, 14 May 2002, 58(9):1435-6). Increased levels of α-synuclein autoantibodies compared to healthy controls have been reported in PD patients (Gruden et al., 2011, J Neuroimmunology). Neuroimmunol, April 2011, 233(1-2):221-7; Gruden et al., 2012, Neuroimmunomodulation, 2012, 19(6):334-42; and Yanamandra, 2011, PLoS One, April 25, 2011, 6(4):e18513) or reduced α-synuclein autoantibodies in PD patients (Besong-Agbo et al., 2013, Neurology, January 8, 2013, 80(2):169-75). It was proposed early on after the discovery of autoantibodies that circulating anti-α-synuclein autoantibodies may play a protective role against α-synuclein aggregation (Woulfe et al., 2002, Neurology, May 14, 2002, 58(9):1435-6).

[0009] Overexpression of α-synuclein in transgenic mice to mimic some pathogenic aspects of Lewy body disease. Several different transgenic mouse lines overexpressing α-synuclein have been produced over the past few decades (described in review Koehler et al., 2014, PLoSOne, May 31, 2013, 8(5):e64649; Fleming and Chessele, 2006, Behav Pharmacol, Sep 2006, 17(5-6):383-91; Springer and Kahle, 2006, CurrNeurol NeurosciRep, Sep 2006, 6(5):432-6). Mouse lines with Thy-1 and PDGF-β promoters develop motor disorders and cognitive deficits and have been used to demonstrate the neuroprotective effects of antibodies against α-synuclein in vivo. However, none of the transgenic lines exhibit strong dopaminergic neuronal degeneration, and the motor phenotype is typically driven by expression within motor neurons that do not usually degenerate in Parkinson's disease. Therefore, it remains unclear whether the potentially positive outcomes of disease-modifying therapies are mediated through effects on dopaminergic neurons or other central nervous system neurons.

[0010] A robust finding in transgenic mouse models is that long-term overexpression of human α-synuclein impairs synaptic function. Studies using both in vitro and in vivo systems have shown that overexpression of wild-type (wt) human α-synuclein in the hippocampus impairs synaptic transmission (Nemani et al., 2010, Neuron, Jan 14, 2010, 65(1):66-79; Paumier et al., 2013, PLoS One, Aug 1, 2013, 8(8):e70274). This has been shown in the CA1 region of the hippocampus, where both studies found reduced basal synaptic transmission. The underlying mechanism is hypothesized that intracellular accumulation of α-synuclein leads to the release of dysfunctional synapses. However, recent findings regarding the extracellular space of α-synuclein secreted into the synapse and the toxic effects of α-synuclein oligomers on synaptic function suggest the possibility that extracellular α-synuclein plays a role in synaptic dysfunction, and thus demonstrate the ability of therapeutic antibodies to rescue defects.

[0011] Using viral vectors to overexpress α-synuclein represents an important way to mimic PD in rodents because this approach produces a relatively rapid degeneration of substantia nigra neurons, a characteristic that has not been reproduced in mice or rats through genetic mutation (Kirik and Bjorklund, 2003, Trends Neurosci, July 2003, 26(7):386-92). Furthermore, viral gene delivery revealed the ability of wild-type α-synuclein to induce nigrostriatal disease (Kirik et al., 2002, J Neurosci, April 1, 2002, 22(7):2780-91), a finding consistent with evidence in PD with family forms having duplicative and tripplicative duplicative α-synuclein (Lee and Trojanowski, 2006, Neuron, October 5, 2006, 52(1):33-8). In one study, a pool of goat antibodies targeting the N-terminus of α-synuclein has been shown to combat dopaminergic cell death and mitigate behavioral deficits in an AAV-α-synuclein-based rat model of Parkinson's disease (Shahaduzzaman et al., 2015, PLoS One, February 6, 2015, 10(2):e0116841).

[0012] Recent studies have shown that prion-like transmission of α-synuclein pathology leads to both α-synuclein pathogenesis and dopaminergic cell death (Luk et al., 2012, Science, 16 Nov 2012, 338(6109):949-53). This model has been used to show that α-synuclein antibodies can alleviate this pathology (Tran et al., 2014, Cell Reports, 26 June 2014, 7(6):2054-65). In this model, antibody treatment reduces the accumulation of phosphorylated α-synuclein in several brain regions, including dopaminergic neurons in the substantia nigra, and reduces the development of motor deficits.

[0013] Besides mutations, alternative splicing of the α-synuclein gene and post-translational modifications of the protein, such as phosphorylation, ubiquitination, nitration, and truncation, can produce the following protein forms of α-synuclein, which have an increased ability to form aggregated and / or toxic forms of α-synuclein (Beyer and Ariza, Molecular Neurobiology, April 2013, 47(2):509-24). However, the precise pathological types of α-synuclein remain unknown. Various types of misfolded / aggregated / secreted forms, ranging from oligomers to fibrils, and different post-translational modifications have been associated with toxicity, but there is no consensus on which is most important (if there is indeed only a single toxic type).

[0014] Overall, the accumulation of α-synuclein with similar morphological and neurological alterations in different animal models, including humans, mice, and flies, suggests that this molecule plays a central role in the pathogenesis of Lewy body disease.

[0015] Several different α-synuclein antibodies have been shown to have therapeutic effects in preclinical animal models. Antibodies targeting epitopes containing α-synuclein residues 91–99 and those targeting epitopes containing α-synuclein residues 118–126 have both been shown to affect motor and cognitive deficits in transgenic mice (Games et al., 2014, J Neurosci, July 9, 2014, 34(28):9441–54). The most advanced of these antibodies is a humanized version of the mouse monoclonal antibody 9E4, which targets epitopes containing α-synuclein residues 118–126 and is currently in a phase I clinical trial. C-terminal antibodies targeting epitopes containing α-synuclein residues 120-140 (Bae et al., 2012, J Neurosci, 26 Sep 2012, 32(39):13454-69) have also been shown to influence cell-to-cell pathological propagation in preclinical models. In addition, antibodies targeting conformational types such as α-synuclein oligomers and fibrils have been shown to at least reduce levels of these presumed toxic α-synuclein types (Linstrom). (Vaikath et al., Neurobiology of Disease, Sep 2014, 69:134-43; and Spencer et al., Molecular Therapy, Oct 2014, 22(10):1753-67). These conformational antibodies, such as mab47, which reduce α-synuclein oligomer levels in vivo, also show targeting the C-terminal epitopes of α-synuclein amino acids 121-125 (US 20120308572). Other conformations of fibrils and oligomer-specific antibodies also target the C-terminal sequence (Vaikath et al., Neurobiology of Disease, 2015, 79:81-99).

[0016] Because the toxic forms of α-synuclein are unknown, ideally, therapeutic antibodies should be able to bind to most types of α-synuclein formed through alternative splicing or post-translational modifications (e.g., truncation), as well as oligomeric and fibrillary forms. As discussed above, one problem with antibodies currently being tested as therapeutics in preclinical models is that many target C-terminal epitopes that are not found in some of the major truncated forms of α-synuclein. For example, the essential amino acids for 9E4 binding are asparagine 122 and tyrosine 125 (alanine scan as stated in patent US20140127131), and this means that the antibody cannot bind to α-synuclein truncated at amino acids 119 and 122, some of the major truncated types found in Parkinson's brain tissue (Kellie et al., Sci Rep., 2014, 4:5797). Antibody 274 and antibody mab47 are in the same situation (US 8,632,776). Furthermore, N-terminal antibodies may not bind to some of the major truncated types of α-synuclein lacking the first amino acid, such as α-synuclein truncated to amino acids 5-140. For antibody 9E4, a proposed mechanism of action is to prevent truncation at amino acid 119-122 in the extracellular space, as the antibody will bind to the same region as the protease cleavage of α-synuclein (Games et al., 2014, J Neurosci, July 9, 2014, 34(28):9441-54). Similar mechanisms of action can also be found with antibodies in the vicinity of said site, and therefore many antibodies around this region are expected to have this activity.

[0017] There is some evidence to support the toxic effects of truncated α-synuclein species in animal models. It has been shown that expression of truncated α-synuclein under the tyrosine hydroxylase promoter leads to nigrostriatal pathology, which is uncommon in transgenic α-synuclein models (Tofaris et al., 2006, J Neurosci, April 12, 2006, 26(15):3942-50; Wakamatsu et al., 2006, Neurobiol Aging, April 2008, 29(4):574-85). For example, expression of amino acids 1-130 of human α-synuclein with the A53T mutation results in embryonic loss of dopaminergic neurons in the substantia nigra pars compacta, but expression of the full-length protein does not (Wakamatsu et al., 2006, Neurobiol Aging, April 2008, 29(4):574-85). Expression of the 120-amino acid α-synuclein molecule under the promoter of calcium / calmodulin-dependent protein kinase IIα (CaMKII-α) is associated with α-synuclein aggregation and progressive deficits in cortico-hippocampal memory tests, including the Barnes maze and new object recognition (Hall et al., 2015, Exp Neurol, February 2015, 264:8-13). Furthermore, co-expression of C-terminal truncated α-synuclein in the rat AAV model enhanced the pathology induced by full-length α-synuclein (Ulusoy et al., 2010, Eur J Neurosci, August 2010, 32(3):409-22).

[0018] In this invention, antibodies (such as "GM37" and "GM285", described in the examples) were produced that can bind to toxic α-synuclein fragments 1-119 / 122 and neutralize this truncated form of α-synuclein. The antibodies of this invention (such as GM37 and GM285) are capable of binding to other oligomeric forms of α-synuclein and altering its uptake by other CNS resident cells in a manner that reduces disease transmission. Furthermore, the antibodies of this invention (such as GM37 and 285) have surprisingly been found to be superior to prior art antibodies such as 9E4 in binding to different types of α-synuclein in the human brain, and to exhibit surprisingly excellent effects in clearing extracellular α-synuclein and normalizing impaired synaptic transmission induced by the presence of abnormal α-synuclein in vivo. To further illustrate their therapeutic capabilities, the antibodies of this invention (such as GM37 and 285) were able to prevent the onset of motor performance-related diseases in a rat model of Parkinson's disease. Finally, antibodies GM37 and GM285 were able to inhibit the seeding and phosphorylation of endogenous α-synuclein aggregates induced by extracellular addition of recombinant pathological α-synuclein seeds in primary mouse neurons. Antibodies such as GM37 and GM285 also inhibited the in vivo seeding of α-synuclein pathology into dopaminergic neurons using a mouse model of Parkinson's disease, further supporting the therapeutic potential of these antibodies in preventing cell-to-cell propagation of the pathology. In conclusion, these data strongly support the use of these novel antibodies (such as GM37 and GM285) as new therapeutic agents that can alter the disease by inhibiting the mechanisms by which the pathology spreads among patients with neuronal Parkinson's disease.

[0019] In another aspect of the invention, three amino acid variants of the GM37 antibody are provided. All of these variants have similar functional outcomes to the parent antibody (GM37) but with improved manufacturability. These variants reduce the risk of post-translational modifications within the binding domain of the GM37 antibody and provide improvements in antibody production. This is advantageous because large-scale clinical or commercial manufacturing of antibodies is complex and expensive, and providing homogeneous products for pharmaceutical drugs is particularly critical for immunoglobulins and proteins. Invention Overview:

[0021] This invention relates to novel monoclonal antibodies and antigen-binding fragments thereof, which are capable of specifically binding to epitopes within amino acids 112-117 (SEQ ID NO:9 (ILEDMP)) of α-synuclein. Epitopes bound by the antibodies of the present invention or their antibody-binding fragments (such as exemplary antibodies “GM37” or “GM285”) are referred to herein as “112-117 epitopes”. The antibodies of the present invention specifically bind to epitopes within these 112-117 epitopes and can (according to one embodiment) compete with antibodies GM37 or GM285 in binding to epitopes within amino acids 112-117. For example, the antibodies of the present invention or their antigen-binding fragments thereof can competitively bind to epitopes within amino acids 112-117 of human α-synuclein, the antibody having a heavy chain consisting of a variable domain of SEQ ID NO:7 and a light chain consisting of a variable domain of SEQ ID NO:8. This competitive binding inhibition can be determined using assays and methods well known in the art, such as unlabeled binding assays like surface plasmon resonance (SPR). For example, human α-synuclein is immobilized on a surface and incubated with or without a reference antibody 'GM37' before incubation with the antibody or binding fragment to be tested. Alternatively, a pairwise mapping method can be used, wherein the reference antibody 'GM37' is immobilized on the surface, the human α-synuclein antigen binds to the immobilized antibody, and then the ability of a second antibody to simultaneously bind to human α-synuclein is tested (see '...'). 'Test Manual', GE Healthcare LifeSciences, 29-0194-00AA 05 / 2012; its contents are incorporated herein by reference.

[0022] More specifically, the GM285 antibody binds to epitopes within residues 112-117 of the α-synuclein, which include residues 112-115 of the α-synuclein (ILED; SEQ ID NO:19).

[0023] In one embodiment, the present invention relates to the monoclonal antibody GM37, its variants (e.g., GM37 variant 1, GM37 variant 2 and GM37 variant 3), or GM285.

[0024] Specifically, the present invention provides a monoclonal antibody GM37, variants thereof (e.g., GM37 variant 1, GM37 variant 2, and GM37 variant 3), or GM285, and comprising an antibody and its derivatives having a sufficient number (e.g., 1, 2, or 3) of light chain CDRs and a sufficient number (e.g., 1, 2, or 3) of heavy chain CDRs to form a binding site capable of specifically binding to a human synuclein protein. Preferably, as defined below, such an antibody will have three light chain CDRs and three heavy chain CDRs. The amino acid residues in this region are numbered according to... International Immunogenetic Information (theinternationalImMunoGeneTics information ) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS and Foeller, C. (1991), Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, C. and Lesk, AM (1987), Canonical structures For The Hypervariable domains of Immunoglobulins, J.Mol.Biol. 196, 901-917.

[0025] In one embodiment, the monoclonal antibody or its antigen-binding fragment has a synuclein antigen-binding fragment, which includes or comprises the following:

[0026] (a) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1; and / or

[0027] (b) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO:2; and / or

[0028] (c) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3; and / or

[0029] (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:4; and / or

[0030] (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and / or

[0031] (f) The light chain CDR3 having the amino acid sequence of SEQ ID NO:6;

[0032] This synuclein antigen-binding fragment can specifically bind to human α-synuclein.

[0033] In one embodiment, the monoclonal antibody or its antigen-binding fragment has a synuclein antigen-binding fragment, which includes or comprises the following:

[0034] (a) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1;

[0035] (b) Heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 33, 34 or 35;

[0036] (c) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3;

[0037] (d) Light chain CDR1 having the amino acid sequence of SEQ ID NO:4;

[0038] (e) The light chain CDR2 having the amino acid sequence of SEQ ID NO:5; and

[0039] (f) The light chain CDR3 having the amino acid sequence of SEQ ID NO:6;

[0040] This synuclein antigen-binding fragment can specifically bind to human α-synuclein.

[0041] In another embodiment, the monoclonal antibody or its antigen-binding fragment has a synuclein antigen-binding fragment, which includes or consists of the following:

[0042] (a) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:20; and / or

[0043] (b) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO:21; and / or

[0044] (c) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO:22; and / or

[0045] (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO:23; and / or

[0046] (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO:24; and / or

[0047] (f) Light chain CDR3 having the amino acid sequence of SEQ ID NO:25.

[0048] This synuclein antigen-binding fragment can specifically bind to human α-synuclein.

[0049] In one embodiment, the monoclonal antibody or antigen-binding fragment possesses a synuclein antigen-binding fragment that comprises (in its CDR, its variable domain, its backbone residues, or its constant domain) an amino acid sequence that differs from that of naturally occurring anti-α-synuclein antibodies, and that amino acid sequence (relative to such naturally occurring anti-α-synuclein antibodies) exhibits:

[0050] (i) Different affinities (KD) for α-synuclein;

[0051] (ii) They differ in their ability to inhibit the protease truncation of α-synuclein;

[0052] (iii) The ability to reverse damage to basal synaptic transmission differs in F28-snca transgenic mice;

[0053] (iv) The ability to reduce α-synuclein levels in the mouse hippocampus varies, as measured by in vivo microdialysis; and / or

[0054] (v) The ability to restore motor function in rat models of Parkinson's disease varies when administered long-term;

[0055] (vi) Differences in the ability to prevent α-synuclein seeding (e.g., accumulation of insoluble phosphorylated α-synuclein in vitro and / or in mouse models of Parkinson's disease); and / or

[0056] (vii) The ability to bind truncated α-synuclein differs in the human brain.

[0057] The antibodies and their antigen-binding fragments of the present invention can be used in methods for treating, diagnosing, or imaging the following synucleinic diseases, such as Parkinson's disease (PD, including idiopathic and hereditary forms of Parkinson's disease), diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Gaucher disease (GD), combined Alzheimer's and Parkinson's disease (CAPD), pure autonomic failure, and multiple system atrophy.

[0058] Brief description of the attached figures

[0059] Figure 1The immunization protocols used to generate hybridomas are shown. The table below lists the immunogens and differences in mouse strains used to identify GM37 and GM285. Different HCo17-Balb / c and HCo12 / Balb / c mice were immunized independently (these mice are described below). Hybridomas expressing GM37 were identified from mice immunized with full-length α-synuclein containing amino acid 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). Hybridomas expressing the antibody GM285 were derived from an immunization protocol in which HCo12-Balb / c mice were immunized with full-length monomeric α-synuclein amino acid 1-140 and then boosted with full-length fibril α-synuclein (Example 1).

[0060] Figure 2 (inset AC) shows the screening for GM37 that binds to α-synuclein, α-synuclein homologues, and orthologs.

[0061] A) Use a wash-free ELISA (FMAT) to bind antibody GM37 to α-synuclein.

[0062] B) Using SPR (Fortebio), the binding of antibody GM37 was specific for α-synuclein (α figure) and did not bind to other related synuclein family proteins, β-synuclein (β figure), and γ-synuclein (γ figure). Using SPR (Fortebio Octetred), GM37 showed similar binding to α-synuclein from cynomolgus monkeys (cynomolgus monkey figure) and mice (mouse figure) (Example 1).

[0063] C) Using SPR (Fortebio Octetred), the binding of antibody GM285 is specific to α-synuclein and does not bind to other related synuclein family proteins, β-synuclein, and γ-synuclein. Measured using SPR (Fortebio Octetred), GM285 showed similar binding to α-synuclein from cynomolgus monkeys (cynomolgus monkey inset) and mice (mouse inset) (Example 1).

[0064] Figure 3 (Small image AC) shows the real-time binding affinity of GM37.

[0065] A) such as through SPR ( The binding of antibody GM37 to α-synuclein, as determined by 3000 (x-axis) and measured in RU (relative units), is shown on the y-axis (x-axis). Goat anti-human IgG was immobilized on a CM5 chip. GM37 was captured on the goat anti-human IgG-immobilized chip, and the binding of human α-synuclein at concentrations (3.125, 6.25, 12.5, 25, 50, 100 nM) to the surface was tested. The sensor surface was regenerated between each cycle.

[0066] B) Convert the signals from different concentrations of binding into binding curves.

[0067] C) The binding constant of antibody GM37 was calculated (expressed as hlgG1-6004-037-C106S) (Example 2).

[0068] Figure 4 (Small image AC) shows the real-time binding affinity of the GM285.

[0069] A) such as through SPR ( The binding of antibody GM285 to α-synuclein, as determined by 3000 (x-axis) and measured by RU, is shown (y-axis). Goat anti-human IgG was immobilized on a CM5 chip. GM285 was captured on the goat anti-human IgG immobilized chip, and the binding of human α-synuclein at a series of concentrations (3.125, 6.25, 12.5, 25, 50, 100 nM) to the surface was tested. The sensor surface was regenerated between each cycle.

[0070] B) Convert signals from binding at different concentrations into binding curves.

[0071] C) The binding constant of antibody GM285 (denoted as hlgG1-6004-285) was calculated (Example 2).

[0072] Figure 5 (Inset AC) shows the real-time binding affinity of the contrast antibody 9E4.

[0073] A) such as through SPR ( The binding of 9E4 to α-synuclein (y-axis) as determined by RU (3000) shows the change over time (x-axis). Goat anti-human IgG was immobilized on a CM5 chip. 9E4 was captured on the chip by binding to goat anti-human IgG, which was immobilized on the chip. The binding of a series of human α-synuclein concentrations (3.125, 6.25, 12.5, 25, 50, 100 nM) to the surface was tested. The sensor surface was regenerated between each cycle.

[0074] B) Convert signals from binding at different concentrations into binding curves.

[0075] C) The binding constant of antibody 9E4 was calculated. (Example 2).

[0076] Figure 6 The amino acid sequence of alpha-synuclein is shown. Major truncated sites in human brain tissue identified by mass spectrometry (arrows) (Kellie JF, Kellie JF, 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, July 23, 2014, 4:5797, doi:10.1038 / srep05797).

[0077] Figure 7 (Inset AB) shows the epitope mapping of antibodies GM37 and GM285. ELISA data show the relative levels of antibody binding to a continuous polypeptide (20-mer) from amino acid sequence 95-132 of α-synuclein (other unbound polypeptides are not shown).

[0078] A) The GM37 epitope requires the polypeptide sequence ILEDMP (SEQ ID NO:9) to bind completely.

[0079] B) GM285 requires the peptide ILED (SEQ ID NO:19) to bind completely. (Example 3).

[0080] Figure 8 (Small figure AB) shows a schematic diagram of the truncated form of α-synuclein.

[0081] A) The binding epitopes of GM37 / 285 (ILEDMP; SEQ ID NO:9) and 9E4 (NEAYE; SEQ ID NO:36) are shown in bold on the amino acid sequence of the α-synuclein (SEQ ID No:10). Arrows indicate... Figure 6 The C-terminal truncation site.

[0082] B) The major truncated forms of α-synuclein have been identified from human brain material. Sizes based on amino acid count are shown on the right. Full-length α-synuclein is 140 amino acids. Because epitope subtraction is possible, GM37, its variants 1-3, and GM285 should bind the full-length and fragments 1-119 / 122 and 1-135. Antibody 9E4 will bind only the full-length and fragment 1-135. The specificity of the smaller C-terminal fragment remaining after truncation is not shown.

[0083] Figure 9 The images show antibodies GM37 and GM285, immunoprecipitates, and full-length and truncated α-synuclein from the human brain. A coarse homogenate of human DLB brain was incubated with test antibodies (beads (no antibody, B12-human IgG1 control antibody that does not bind α-synuclein, GM-37, GM37 variant 2, GM-285, and mouse (m)9E4), and the immunodepleted supernatant and immunoprecipitated material were separated on SDS-PAGE. Immunoblotting showed bands representing full-length and different truncated forms of α-synuclein from the supernatant that were depleted and immunoprecipitated (IP) along with the antibodies. It can be seen that the GM37, GM37v2, and GM285 antibodies depleted the major α-synuclein species in the supernatant, and IP showed these species, truncated 1-135, 1-119 / 122 species, and full-length α-synuclein. 9E4 did not affect the 1-119 / 122 species, but only IP showed the full-length and 1-135 (Example 4).

[0084] Figure 10 A schematic diagram showing the proteolytic activity of α-synuclein protofibrils cleaved by calpain at amino acid 119 / 122. α-synuclein protofibrils (PFF) were added to cultures with (PFF+) or without (PFF) test antibodies. The presence of GM-37 / 285 inhibited the formation of truncated α-synuclein in cells and secreted into cell culture media.

[0085] Figure 11AGM37 inhibited the formation of truncated bands (12 kDa) in both PFF-treated primary mouse cortical cultures and cell lysates. Proteins were separated by SDS-PAGE and subjected to Western blotting to detect different types of α-synuclein. Two monomeric α-synuclein bands were detected at 12 kDa and 14 kDa in cells treated with only PFF or the control antibody (B12), representing truncated and full-length α-synuclein, respectively. Only a weak 12 kDa band was observed in the presence of GM-37, indicating that most lysis was prevented. This effect was also reflected in the cell culture medium. The relative level of accumulation was also inhibited by the presence of GM-37, as reflected in the reduced relative intensity of the 14 kDa band. Alternatively, there may be a reduced number of cellularly uptaken 14 kDa bands. (Example 5)

[0086] Figure 11B The dose-dependent inhibition of α-synuclein fibrillary proteolysis by antibodies GM37, GM37 variant 2, and GM285 was demonstrated. At low antibody concentrations (0, 1 μg / ml), a band representing full-length (FL) α-synuclein and a band representing C-terminal truncated (CT) α-synuclein were observed in cell lysates from primary mouse cortical cultures (indicated by arrows). Increasing antibody concentrations to 1 μg / ml, 5 μg / ml, and 10 μg / ml resulted in reduced α-synuclein fibrillary proteolysis in cells. This phenomenon was observed with antibodies GM37, GM37v2, and GM285. Control samples were treated with human IgG1 antibody B12, which does not recognize α-synuclein. Controls without antibody addition (No ab) and cells without α-synuclein fibrillation addition (No α-synuclein) were also included. Compared to the B12 or “antibody-free” control, the total amount of α-synuclein was also reduced in samples treated with 37, 37v2, and 285, indicating that all three antibodies reduced α-synuclein accumulation in cells in a concentration-dependent manner. Actin bands at the top of the gel show equal sample loading (Example 5).

[0087] Figure 12 shows the effects of GM37 and GM285 on the seeding of α-synuclein aggregation and α-synuclein phosphorylation in mouse primary cortical neurons.

[0088] 12A) An example of an image of primary neurons (appearing as spots or dots in the cell) stained with phosphorylated α-synuclein when cells are seeded using pure or coarse seeds containing 1 ng of α-synuclein.

[0089] 12B) Immunoblot of proteins from primary cortical neurons isolated in soluble and insoluble fractions. The blot was stained with human α-synuclein-specific antibodies (4B12 / Ha-syn), phosphoserine-129-α-synuclein-specific antibodies (ab51253 / pS-a-Syn), and mouse α-synuclein-specific antibodies (D37A2 / Ma-syn), and the blot showed that the addition of coarse seeds to primary neurons led to the accumulation of endogenous mouse α-synuclein and phosphorylated α-synuclein, as well as the accumulation of higher molecular weight polymers of α-synuclein, in the insoluble fraction.

[0090] 12C) GM37, GM37 variant 2, and GM285 inhibited the appearance of phosphorylated α-synuclein, the amount of which was quantified by Cellomics ARRAYSCAN™ automated microscopy as the number of α-synuclein phosphoserine 129 positive spots in cells. GM37, GM37v2, and GM285 reduced the amount of phosphorylated α-synuclein spots in cells in a dose-dependent manner.

[0091] 12D) Western blots of primary cortical neuronal homogenates stained with the highest antibody dose (133 nM) and targeting actin, human α-synuclein, phosphorylated α-synuclein, and mouse α-synuclein showed that antibodies 37, 37v2, and 285 inhibited the truncation of crude α-synuclein seeds taken up by cells in the insoluble fraction. All antibodies also inhibited the accumulation of phosphorylated, endogenous mouse, and higher molecular weight multimers of phosphorylated mouse α-synuclein in the insoluble fraction. Actin bands at the top of the gel indicate equal sample loading (Example 6).

[0092] Figure 13 Basic synaptic transmission was observed in the hippocampus of age-matched, F28-snca transgenic control mice in the CA1 region of the Schaffer collateral. Field excitatory postsynaptic potentials (fEPSPs) were induced by a single stimulus to the Schaffer collateral, and basic synaptic transmission was assessed by measuring the slope of the fEPSP as a function of stimulus intensity. Short-term synaptic plasticity was assessed by inducing double-pulse facilitation. Different stimulus intensities of 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μA were applied sequentially in increasing order, with each intensity repeated 2 to 3 times. Compared to age-matched control mice, basic synaptic transmission was significantly impaired in F28-snca transgenic mice overexpressing wild-type α-synuclein (Example 7).

[0093] Figure 14This study demonstrates the effect of a single systemic dose of human 9E4 (15 mg / kg, intraperitoneal) on basal synaptic transmission impairment in the CA1 region of the Scheffer collateral in the hippocampus of F28-snca transgenic mice. Field excitatory postsynaptic potentials (fEPSPs) were induced by a single stimulus applied to the Scheffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of stimulus intensity. Acute treatment with h9E4 induced a significant reversal of basal synaptic transmission impairment in F28-snca transgenic mice (Tg-snca + h9E4 vs. Tg-snca + PBS, p = 0.002). However, the reversal by h9E4 was only localized, as evidenced by significantly lower basal synaptic transmission compared to littermates treated with PBS (p = 0.007) (Example 7).

[0094] Figure 15 This study demonstrates the effect of systemic administration of a single dose of human GM37 (15 mg / kg, intraperitoneal) or an isotype control antibody (B12) on basal synaptic transmission impairment in the CA1 region of the Scheffer collateral in the hippocampus of F28-snca transgenic mice. Field excitatory postsynaptic potentials (fEPSPs) were induced by a single stimulus applied to the Scheffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of stimulus intensity. Acute treatment with GM37 induced complete reversal of basal synaptic transmission impairment in F28-snca transgenic mice (Tg-snca + GM37 vs. Tg-snca + B12, p = 0.004) (Example 7).

[0095] Figure 16 This study demonstrates the effect of a single systemic dose of human GM285 (15 mg / kg, intraperitoneal) on basal synaptic transmission impairment in the CA1 region of the Scheffer collateral in the hippocampus of F28-snca transgenic mice. Field excitatory postsynaptic potentials (fEPSPs) were induced by a single stimulus applied to the Scheffer collateral, and basal synaptic transmission was assessed by measuring the fEPSP slope as a function of stimulus intensity. Acute treatment with GM285 induced complete reversal of basal synaptic transmission impairment in F28-snca transgenic mice (Tg-snca + GM285 vs. Tg-snca + PBS, p = 0.001) (Example 7).

[0096] Figure 17(Figures A and B) show the effect of systemic administration (15 mg / kg, intraperitoneal) of human 9E4, GM37, or the isotype control antibody (anti-HEL) on the level of human α-synuclein in the interstitial fluid (ISF) of the hippocampus in freely moving F28-snca transgenic mice. The mean of 2–3 baseline values ​​(4–6 h) prior to antibody treatment for each animal was used as the baseline and set as 100%. Differences were analyzed using two-way ANOVA with repeated measures. The baseline level of human α-synuclein in the hippocampus was 8.1 ± 1.1 ng / ml (mean ± SEM, n = 25, not corrected for recovery by in vitro dialysis probes). Compared to the two control antibodies (human 9E4 and the control isotype), administration of GM37 induced a significant reduction in human α-synuclein in the hippocampus of F28 mice. Anti-HEL time points showing significant differences in α-synuclein levels between animals treated with GM37 or the control antibody are indicated by asterisks. (Example 8)

[0097] Figure 18 A schematic diagram showing the antibody treatment timeline (downward arrow) illustrates viral injection and behavioral assessment in a rat AAV human α-synuclein model. Figure 19 The example shown is (Example 9).

[0098] Figure 19 The results showed that long-term treatment with antibody GM37 reduced motor deficits in a rat AAV model of Parkinson's disease. The effect of long-term treatment with GM37 or PBS on motor asymmetry in AAV-human α-synuclein rats was assessed using a cylinder test. Forelimb use in each rat was tested by monitoring for 5 minutes. The percentage of right forelimb use (ipsilateral to injection) and left forelimb use (contralateral to right forelimb +) was calculated for each animal (as shown on the y-axis), compared to GFP-PBS rats, *p<0.05 and **p<0.01. Rats treated with PBS still showed significant asymmetry in paw use, while animals treated with antibody GM37 no longer showed significant deficits (Example 9).

[0099] Figures 20A-20C Long-term treatment with antibody GM37 showed that it could reduce pathological α-synuclein phosphorylation induced by injecting pathological α-synuclein filament seeds into the mouse striatum. Figure 20A A schematic diagram illustrating the effects of seed injection and cell counting relative to treatment time is shown. Antibody GM37 was administered one day prior to seeding recombinant α-synuclein filaments into the dorsal striatum of mice, followed by weekly administration for six weeks. The administration regimen was 15 mg / kg intravenously or 30 mg / kg intraperitoneally. Figure 20BThe levels of GM37 exposure in plasma are shown, depending on the injection site and dose. Weekly samples are taken before each new antibody dose is administered. Figure 20C The study showed the exposure levels of GM37 in cerebrospinal fluid at the end of the study, based on dose and injection site. Figure 20D The number of cells with phosphorylated α-synuclein-positive inclusions in the substantia nigra, counted according to each sixth segment, was compared after treatment with GM37 or a PBS control. Mice treated with PBS showed a significantly reduced number of phosphorylated α-synuclein inclusions in cells compared to mice treated with intravenous injection of 15 mg / kg GM37 and intraperitoneal injection of 30 mg / kg GM37 (Example 10).

[0100] Figure 21 This image shows a comparison of human α (SEQ ID NO:10), β (SEQ ID NO:37), and γ (SEQ ID NO:38) synuclein proteins. Amino acid residues that differ from α-synuclein are highlighted. Gaps are indicated by dots. The numbers in parentheses are SwissProt numbers.

[0101] Figure 22 The image shows the alignment of orthogonal homologs of α-synuclein (cynomolgus monkey, SEQ ID NO:39; rat, SEQ ID NO:40; mouse, SEQ ID NO:41). Amino acid residues different from human α-synuclein (SEQ ID NO:10) are highlighted. The SwissProt number is shown in parentheses.

[0102] Figure 23 This shows the transient expression of GM37 (named GM37 wild-type (wt)) and three GM37 variants (named GM37 variant 1, 2, and 3). Asterisks indicate data determined after protein A purification and neutralization. The data represent yields calculated from protein A and the yield obtained after neutralization, in relation to the size of the expression culture (0.4 L).

[0103] Figure 24 This demonstrates a competitive ELISA for measuring the binding of four antibodies—GM37wt, GM37 variant 1, GM37 variant 2, and GM37 variant 3—to human α-synuclein. Plates coated with α-synuclein were used to detect the amount of antibody remaining after pre-incubation with increasing concentrations of α-synuclein (0–1000 nM) in solutions of each antibody (0.3 μg / ml). All four antibodies showed similar binding to α-synuclein.

[0104] Figure 25 A table is shown comparing the binding kinetics of GM37wt and its variants 1–3 with immobilized recombinant human α-synuclein. This binding was measured using SPR and a 1:1 binding algorithm was employed. T200) determines the binding ratio.

[0105] Figure 26 The effects of α-synuclein antibodies on phosphorylated α-synuclein levels in mouse primary neurons treated with pathological α-synuclein filament seeds were compared. Primary neurons were treated with seeds (10 ng) in or without GM37, GM37 variant 1, GM37 variant 2, and GM37 variant 3 (2 μg). After 3 weeks, neurons were fixed and stained, and α-synuclein phosphorylated serine 129 positive spots were analyzed by Cellomics ARRAYSCAN™. ​​Cells treated with seeds alone or with seeds plus an isotype control antibody (B12) showed significantly increased phosphorylation levels. Cells treated with GM37wt and the three variants were able to inhibit α-synuclein phosphorylation, all showing the same level of phosphorylation as cells that did not receive seeds. Data are presented as mean ± SD, determined from 7 images per well in 5 wells. N = 2.

[0106] Figure 27 Temperature-dependent aggregation of GM37wt, variant 1, variant 2, and variant 3 was compared. Samples of each antibody were subjected to steadily increasing temperatures over time, and aggregation levels were simultaneously determined by multi-angle light scattering (Prometheus NT.48, NanoTemper Technologies). The GM37 and GM37 variants showed similar initiation temperatures for aggregation; however, GM37-variant 2 exhibited the lowest level of aggregation.

[0107] Detailed description of the invention

[0108] definition

[0109] As used herein, the term "α-synuclein" is synonymous with "α-synuclein protein" and refers to any α-synuclein protein isoform (e.g., identified as P37840, 1-3 in UniProt). For SEQ ID NO:10 shown below, amino acid numbers are assigned to α-synuclein, where methionine (M) is amino acid residue 1:

[0110] SEQ ID NO:10:

[0111]

[0112] This invention relates to antibodies and antibody fragments capable of specifically binding to α-synuclein (and particularly to human α-synuclein). In particular, antibodies and fragments thereof exhibit the ability to specifically bind to epitopes within 112-117 of human α-synuclein.

[0113] In the context of this invention, the term "antibody (Ab)" refers to an immunoglobulin molecule, or, according to some embodiments of the invention, a fragment of an immunoglobulin molecule capable of specifically binding to an epitope of a molecule ("antigen"). Naturally occurring antibodies typically comprise tetramers, which generally consist of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, the heavy chain constant domain typically consisting of three domains (CH1, CH2, and CH3). The heavy chains can have any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE. Each light chain consists of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). The light chains include κ and λ chains. Heavy and light chain variable domains are typically responsible for antigen recognition, while heavy and light chain constant domains mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. VH and VL regions can be further subdivided into hypervariable regions called "complementarity-determining regions," interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL consists of three CDR domains and four FR domains, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Heavy and light chain variable domains contain binding domains that interact with the antigen. Of particular interest are antibodies and their antigen-binding fragments that have been "isolated" to exist in a different physical environment than they can in nature, or that have been modified to differ in amino acid sequence from naturally occurring antibodies.

[0114] The term "epitope" refers to an antigenic determinant that specifically binds to an antibody. Epitopes typically consist of surface groups such as amino acid or sugar side chain molecules and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and linear epitopes is that conformational epitopes often lose binding to the former rather than the latter in the presence of denaturing solvents. Epitopes can contain amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, such as amino acid residues effectively blocked by specific antigen-binding peptides (in other words, the amino acid residues are within the influence range of the specific antigen-binding peptide). The term "112-117 epitope" refers to a region of human α-synuclein containing at least four of the six amino acid residues of human α-synuclein (112-117), excluding any residues of human α-synuclein from 1-111 (including any residues from 106-111) and from 118-140 (including residues from 118-120). As used herein, an antibody is considered to specifically bind to an epitope within the “112-117 epitope” if it can specifically bind to human α-synuclein by binding to at least four of the six amino acid residues of the 112-117 epitope.

[0115] As used herein, the term "antigen-binding fragment of an antibody" means a fragment, portion, region, or domain of an antibody capable of specifically binding to an epitope (regardless of how it is generated (e.g., via cleavage, recombination, synthesis, etc.)), and therefore the term "antigen binding" is intended to be synonymous with "epitope binding," such that, for example, "antigen-binding fragment of an antibody" and "epitope-binding fragment of an antibody" have the same meaning. An antigen-binding fragment may contain 1, 2, 3, 4, 5, or all six CDR domains of such an antibody, and despite being capable of specifically binding to such an epitope, may exhibit specificity, affinity, or selectivity for such epitopes that are different from those of this antibody. However, preferably, the antigen-binding fragment contains all six CDR domains of this antibody. An antigen-binding fragment of an antibody may be part of or comprise a single polypeptide chain (e.g., scFv), or may be part of or comprise two or more polypeptide chains (each having an amino terminus and a carboxyl terminus, e.g., a biantibody, a Fab fragment, a Fab2 fragment, etc.). Antibody fragments exhibiting antigen-binding ability can be obtained, for example, by protease cleavage of the intact antibody. More preferably, although the two domains VL and VH of the Fv fragment are naturally encoded by a single gene or a polynucleotide encoding such a gene sequence (e.g., encoding cDNA), these two domains can be linked by a flexible linker using a recombination method, which allows the two domains to become a single protein chain in which the VL and VH regions associate to form a monovalent antigen-binding molecule (called a single-stranded Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. (USA)) 85:5879-5883). Alternatively, by employing a flexible linker that is too short (e.g., less than about 9 residues) to associate the VL and VH regions of a single polypeptide chain together, bispecific antibodies, biantibodies, or similar molecules (where two such polypeptide chains associate together to form a bivalent antigen-binding molecule) can be formed (for a description of biantibodies, see, for example, PNASUSA 90(14), 6444-8(1993)).Examples of antigen-binding fragments included in this invention include (i) Fab' or Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO2007059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in a hinge domain; (iii) Fd fragments consisting essentially of VH and CH1 domains; (iv) Fv fragments consisting essentially of VL and VH domains; (v) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), which consist essentially of VH domains and are also referred to as domain antibodies (Holt et al., Trends Biotechnol. 2003 Nov; 2i(ll):484-90); (vi) camel or nanobodies (Revets et al., Expert Opinion on Biotherapy). Opin Biol Ther., January 2005; 5_(l):l ll-24) and (vii) the isolated complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by isolated genes, they can be linked using a recombination approach via a synthetic adapter that allows them to become a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain antibody or single-chain Fv (scFv); see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). These and other useful antibody fragments in this invention are further discussed here. It should also be understood that, unless otherwise specified, the term antibody also includes antibody-like polypeptides, such as chimeric and humanized antibodies obtained by any known technique (e.g., enzymatic cleavage, peptide synthesis, and recombinant techniques), as well as antibody fragments (antigen-binding fragments) that retain the ability to bind specifically to antigens. Antibodies thus produced can be of any isotype. As used herein, "isotype" refers to a class of immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4) encoded by a heavy chain constant domain gene. Such antibody fragments are obtained using conventional techniques known to those skilled in the art; suitable fragments capable of binding to desired epitopes can be easily screened for practicality in the same manner as for intact antibodies.

[0116] The term "bispecific antibody" refers to an antibody containing two independent antigen-binding fragments, each targeting a separate target. These targets can be epitopes located on different proteins or different epitopes on the same target. Bispecific antibody molecules can be prepared using compensating amino acid changes in the constant domain of the HC domain of a parental monospecific bivalent antibody molecule. The resulting heterodimeric antibody contains a Fab domain formed from two different parental monospecific antibodies. Amino acid changes in the Fc domain lead to increased stability of the time-dependent bispecific heterodimeric antibody. (Ridgway et al., Protein Engineering 9, 617-621 (1996); Gunasekaran et al., JBC 285, 19637-1 (2010); Moore et al., MAbs 3:6 546-557 (2011); Strop et al., JMB 420, 204-219 (2012); Metz et al., Protein Engineering 25:10 571-580 (2012); Labrijn et al., PNAS 110:113, 5145-5150 (2013); Spreter Von Kreudenstein et al., MAbs 5:5 646-654 (2013)). Bispecific antibodies can also comprise molecules generated using ScFv fusions. Two monospecific ScFvs are then independently linked to an Fc domain capable of forming a stable heterodimer to produce a single bispecific molecule (Mabry et al., PEDS 23:3 115-127 (2010)). Bispecific molecules possess dual binding capabilities. For example, they can be used to deliver therapeutic antibodies across the blood-brain barrier to treat CNS diseases while simultaneously targeting both the therapeutic target and transcellular surface receptors.

[0117] The terms GM37, GM-37, GM37 wild-type (wt), mab37, and 6004-37 are used interchangeably in this document and all refer to the same antibody.

[0118] The term antibody GM37 is intended to include antibodies or antigen-binding fragments thereof that comprise, or consist of, the heavy chain CDR1-3 given in SEQ ID No:1-3 and the light chain CDR1-3 given in SEQ ID No:4-6. In one embodiment, antibody GM37 or its antigen-binding fragment may comprise, or consist of, the heavy chain variable domain of SEQ ID NO:7 and / or the light chain variable domain of SEQ ID NO:8. For example, the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of the variable domain of SEQ ID NO:7 and the constant domain of SEQ ID NO:18 together, and a light chain consisting of the variable domain of SEQ ID NO:8 and the κ constant domain of SEQ ID NO:17.

[0119] Deamination of proteins (in these cases, antibodies) can occur spontaneously during manufacturing and storage, and also in vivo, making the quality of the final pharmaceutical product difficult to control. In some cases, deamination can also affect the molecule's activity. Deamination occurs at asparagine residues, but the position of the relevant asparagine residue can be difficult to predict definitively, although it can be influenced by the asparagine-glycine motif in some cases. Several possible deamination motifs have been found in the GM37 antibody; however, one possible deamination site has been found at residue 54 of the heavy chain. Subsequent substitution of asparagine with another amino acid is not direct, but three variants of GM37 (GM37 variants (var) 1, 2, and 3) have been found to retain the activity of the original GM37 (GM37 wild-type (wt)).

[0120] The term GM37 variant refers to variant 1, 2 or 3 that has been deamination, wherein, compared to the GM37 antibody described herein, variant 1 has an N54S substitution, variant 2 has an N54Q substitution and variant 3 has an N54H substitution.

[0121] Therefore, antibody GM37 variants (var) 1, 2 and 3 are intended to include an antibody or an antigen-binding fragment thereof comprising, or consisting of, the heavy chains CDR1 and 3 of GM37 as given in SEQ ID No:1 and 3, and the light chains CDR1-3 of GM37 as given in SEQ ID No:4-6, but their heavy chain CDR2s are different, with variant 1 having the CDR2 of SEQ ID NO:33, variant 2 having the CDR2 of SEQ ID NO:34 and variant 3 having the CDR2 of SEQ ID NO:35.

[0122] In one embodiment, an antibody GM37 variant or its antigen-binding fragment may comprise, or consist of, the heavy chain variable domains of SEQ ID NO:30, 31, and 32 (for variants 1, 2, and 3, respectively) and / or the light chain variable domain of SEQ ID NO:8. The antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:30, 31, or 32 and a constant domain of SEQ ID NO:18, and a light chain consisting of a variable domain of SEQ ID NO:8 and a κ constant domain of SEQ ID NO:17.

[0123] The terms GM285, GM-285, mab285, and 6004-285 are used interchangeably in this document and all refer to the same antibody.

[0124] The term antibody GM285 is intended to include an antibody or an antigen-binding fragment thereof comprising, or consisting of, the heavy chain CDR1-3 given in SEQ ID No:20-22 and the light chain CDR1-3 given in SEQ ID No:23-25. In one embodiment, antibody GM37 or an antigen-binding fragment thereof may comprise, or consist of, the heavy chain variable domain of SEQ ID NO:26 and / or the light chain variable domain of SEQ ID NO:27. For example, the antibody GM37 may be an IgG antibody comprising a heavy chain consisting of a variable domain of SEQ ID NO:26 and a constant domain of SEQ ID NO:28, and a light chain consisting of a variable domain of SEQ ID NO:27 and a κ constant domain of SEQ ID NO:29.

[0125] The GM285 antibody specifically binds to epitopes in sequence 112-115 (ILED; SEQ ID NO:19) of human α-synuclein (SEQ ID NO:10).

[0126] Unless otherwise stated herein, the amino acid residues in this region are numbered according to International Immunogenetic Information the international ImMunoGeneTics information ) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS and Foeller, C. (1991), Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, C. and Lesk, AM (1987), Canonical structures for the hypervariable domains of immunoglobulins, J.Mol.Biol. 196, 901-917.

[0127] As defined above, an “anti-α-synuclein antibody” or “α-synuclein antibody” (which may be used interchangeably herein, depending on the written context) is an antibody or antigen-binding fragment thereof that specifically binds to α-synuclein or α-synuclein fragments, particularly the α-synuclein sequences corresponding to SEQ ID No. 9 and / or 19.

[0128] As used herein, the term "human antibody" (which may be abbreviated as "humAb" or "HuMab") is intended to include antibodies having variable and constant domains derived from human immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or during gene rearrangement or in vivo by somatic mutations).

[0129] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to preparations of antibody molecules consisting of a single molecule. Conventional monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. In some embodiments, a monoclonal antibody may consist of more than one Fab domain, thereby increasing specificity for more than one target. The terms "monoclonal antibody" or "monoclonal antibody composition" are not intended to be limited to any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

[0130] The term "humanized" refers to molecules typically prepared using recombinant techniques that have an antigen-binding site derived from an immunoglobulin from a non-human species and a residual immunoglobulin structure based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete non-human antibody variable domain fused to a human constant domain or a complementarity-determining region (CDR) of such a variable domain consisting only of an appropriate human framework region grafted onto a human variable domain. The framework residues of such humanized molecules may be wild-type (e.g., fully human) or they may be modified to include substitutions of one or more amino acids not found in human antibodies whose sequences already serve as the basis for humanization. Humanization reduces or eliminates the possibility that the constant domain of the molecule might act as an immunogen in human individuals, but the possibility of an immune response to the exogenous variable domain remains (LoBuglio, AF et al. (1989), "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics and Immune Response," Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. (USA)) 86:4220-4224). Another approach focuses not only on providing human-derived constant domains but also on modifying variable domains to make them as close as possible to human form. It is known that the variable domains of both the heavy and light chains contain three complementarity-determining regions (CDRs) that respond differently to the antigen in question and determine binding capacity, flanked by four framework regions (FRs) that are relatively conserved in a given species and presumably provide the scaffold for the CDRs. When preparing nonhuman antibodies against a specific antigen, the variable domain can be "modified" or "humanized" by grafting a CDR derived from the nonhuman antibody onto the FR present in the human antibody to be modified. This has been demonstrated by Sato, K. et al. (1993), Cancer Research 53:851-856; Riechmann, L. et al. (1988), "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988), "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534-1536; and Kettleborough, CA.=E.G. et al. (1991) "Humanization of a Mouse Monoclonal Antibody By CDR-Grafting: The Importance of Framework Residues On Loop Conformation", Protein Engineering 4:773-3783; Maeda, H. et al. (1991) "Construction of Reshaped Human Antibodies With HIV-Neutralizing Activity", Human Antibodies Hybridoma 2:124-134; Gorman, SD et al. (1991) "Reshaping A "Therapeutic CD4 Antibody", Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. (USA)) 88:4181-4185; Tempest, PR et al. (1991) "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo", Bio / Technology 9:266-271; Co, MS et al. (1991) "Humanized Antibodies For Antiviral Therapy", Proceedings of the National Academy of Sciences 88:2869-2873; Carter, P. et al. (1992) "Humanization of An Anti-p185her2 Antibody for Human Cancer Therapy". For Human Cancer Therapy, Proceedings of the National Academy of Sciences of the United States of America 89:4285-4289; and Science, MSThe application of this method in various antibodies was reported in "Chimeric and Humanized Antibodies With Specificity For The CD33 Antigen" by et al. (1992), J. Immunol. 148:1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs that are altered relative to the original antibody (one, two, three, four, five, or six), which are also referred to as one or more CDRs "derived" from one or more CDRs from the original antibody. The ability to humanize antigens is well known (see, for example, U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

[0131] As used herein, an antibody or antigen-binding fragment thereof is described as "specifically" binding to a region (i.e., an epitope) of another molecule if it reacts or associates with an epitope more frequently, more rapidly, for a longer duration, and / or with greater affinity or affinity compared to other epitopes. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention binds to its target (human α-synuclein) at least 10 times more strongly than another molecule; preferably at least 50 times stronger and more preferably at least 100 times stronger. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, antibodies capable of "specifically binding" to epitopes within residues 112-117 (ILEDMP (SEQ ID No: 9)) of human α-synuclein comprise antibodies or antigen-binding fragments thereof capable of binding to epitopes within residues 112-117 of human α-synuclein with such specificity and / or under such conditions. Methods suitable for determining such binding are known to those skilled in the art, and exemplary methods are described in the appended examples. As used herein, the term "binding" in the context of antibody binding to a predetermined antigen generally refers to the binding of an antibody to a predetermined antigen when the antigen is used as a ligand and the antibody as an analyte. When measured in instruments such as the 3000 or T200 using techniques such as surface plasmon resonance (SPR), the affinity corresponds to approximately 10. -7 M or smaller (such as about 10) -8 M or smaller, such as about 10 -9The antibody binds to a KD (M or smaller) and binds to a predetermined antigen with an affinity corresponding to a KD at least ten times lower than the antibody's affinity for nonspecific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. This lower affinity can be at least 100 times lower, for example at least 1,000 times lower, for example at least 10,000 times lower, or for example at least 100,000 times lower. The amount of lower affinity depends on the antibody's KD, such that when the antibody's KD is very low (i.e., the antibody is highly specific), the amount by which the affinity for the antigen is lower than the affinity for the nonspecific antigen can be at least 10,000 times.

[0132] As used herein, the term "kd" (sec⁻¹ or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as the koff value.

[0133] As used herein, the term "ka" (M-1x sec-1 or 1 / Msec) refers to the association rate constant of a particular antibody-antigen interaction.

[0134] As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing kd by ka.

[0135] As used herein, the term "KA" (M-1 or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing ka by kd.

[0136] In one embodiment, the present invention relates to an antibody or an antigen-binding fragment thereof that exhibits one or more of the following properties:

[0137] i. Binding affinity (KD) for α-synuclein, which is between 0.5-10 nM, for example 1-5 nM or 1-2 nM;

[0138] ii. The ability to inhibit protease truncation of α-synuclein protoplasts;

[0139] iii. The ability to reverse basal synaptic conduction impairment in F28-snca transgenic mice;

[0140] iv. The ability to reduce the level of α-synuclein in the mouse hippocampus, as measured by in vivo microdialysis;

[0141] v. When administered long-term, it can restore motor function in a rat model of Parkinson's disease.

[0142] vi. The ability to prevent α-synuclein seeding (e.g., the accumulation of insoluble phosphorylated α-synuclein in vitro and / or in mouse models of Parkinson's disease); and / or

[0143] vii. The ability to bind truncated α-synuclein in the human brain.

[0144] The binding affinity (KD) of α-synuclein can be determined using methods well known in the art (e.g., as described in Example 2).

[0145] The term “the ability to inhibit protease truncation of α-synuclein fibrils” includes the ability to inhibit calpain-1-induced formation of fragments 1-119-122 of human α-synuclein within primary cortical neurons (see Example 5).

[0146] The term “ability to reverse basal synaptic transmission impairment in F28-snca transgenic mice” includes the ability to reverse synaptic transmission impairment and plasticity in the CA1 region of the hippocampus of F28-snca transgenic mice, as shown by, for example, the induced fEPSP slope measured by electrophysiological means (see Example 6).

[0147] The term “ability to reduce α-synuclein levels in mouse hippocampus by in vivo microdialysis” includes the ability to reduce human α-synuclein levels in awake, freely moving F28-snca transgenic mice in the hippocampus, as measured by in vivo microdialysis (see Example 7).

[0148] The term “the ability to restore motor function in a rat model of Parkinson’s disease when administered over a long period of time” includes the ability to reduce or eliminate motor asymmetry in a rat model of Parkinson’s disease using a recombinant adeno-associated virus vector (rAAV) (see Example 8).

[0149] In some antibodies, only a subset of the CDRs (i.e., the subset of CDR residues required for binding, called SDRs) need to remain bound in the humanized antibody. The identification of CDR residues that do not contact the relevant epitope and are not in the SDR can be based on previous studies (e.g., residues H60-H65 in CDR H2 are generally not required), through molecular modeling and / or empirical methods, or as described in Gonzales, NR et al., (2004) "SDR Grafting Of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity", Molecular Immunology 41:863-872, from the Kabat CDR region located outside the Chothia hypervariable loop (see, Kabat et al., (1992) Sequences of Proteins of Immunological Interest, National Institutes of Health). Health), Publication No. 91-3242; Chothia, C. et al., (1987) "Canonical Structures For The Hypervariable Regions Of Immunoglobulins", Journal of Molecular Biology (J.Mol.Biol.) 196:901-917. In such humanized antibodies, at positions where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid occupying that position can be an amino acid occupying the corresponding position (by Kabat number) in the receptor antibody sequence. The number of such substitutions of the receptor for the donor amino acid in the CDR to be incorporated reflects a balance of competitive considerations. Such substitutions are potentially advantageous in reducing the number of mouse amino acids in the humanized antibody and thus in reducing potential immunogenicity. However, substitutions can also cause changes in affinity, and a significant reduction in affinity is preferably avoided. The substitution positions within the CDR and the amino acids to be substituted can also be selected empirically.

[0150] The fact that a single amino acid alteration of a CDR residue can result in the loss of functional binding (Rudikoff, S. et al. (1982), “Single Amino Acid Substitution Altering Antigen-binding Specificity,” Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. (USA)) 79(6): 1979–1983) provides a means for the systematic identification of alternative functional CDR sequences. In a preferred method for obtaining such variant CDRs, polynucleotide mutagenesis encoding the CDR (e.g., via random mutagenesis or by site-directed methods (e.g., polymerase chain-mediated amplification using primers encoding the mutant locus)) is performed to produce a CDR with the substituted amino acid residue. The substitution score of the BLOSUM62.iij can be identified by comparing the identity of the relevant residue in the original (functional) CDR sequence with the identity of the substituted (non-functional) variant CDR sequence.The BLOSUM system provides amino acid substitution matrices created by analyzing reliable alignments from sequence databases (Eddy, SR (2004) "Where Did The BLOSUM62 Alignment Score Matrix Come From?", Nature Biotech. 22(8):1035-1036; Henikoff, JG (1992) "Amino acid substitution matrices from protein blocks", Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. (USA)) 89:10915-10919; Karlin, S. et al. (1990) "Methods For Assessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes), Proceedings of the National Academy of Sciences of the United States of America 87:2264-2268; Altschul, SF (1991) "Amino Acid Substitution Matrices From An Information Theoretic Perspective". "Perspective", Journal of Molecular Biology (J.Mol.Biol.) 219, 555-565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the substitution scores of BLOSUM62.iij (higher scores indicate more conserved substitutions, and therefore the substitution is less likely to affect function). For example, if the antigen-binding fragment containing the resulting CDR cannot bind to α-synuclein, the BLOSUM62.iij substitution score is considered insufficiently conserved, and new candidate substitutions with higher substitution scores are selected and generated. Thus, for example, if the original residue is glutamic acid (E) and the nonfunctional substitution residue is histidine (H), the BLOSUM62.iij substitution score will be 0, and more conserved variations (such as to aspartic acid, asparagine, glutamine, or lysine) are preferred.

[0151]

[0152] This invention therefore considers the use of random mutagenesis for identifying improved CDRs. In the context of this invention, conservative substitutions can be defined by substitutions within amino acid classes reflected in one or more of the following three tables:

[0153] Classes of conserved substituted amino acid residues:

[0154]

[0155]

[0156] Category of alternative conserved amino acid residue substitutions:

[0157]

[0158] Alternative physical and functional classification of amino acid residues:

[0159]

[0160] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0161] Additional amino acid groups can also be formulated using principles described, for example, in Creighton (1984) Proteins: Structure and Molecular Properties (2nd edition, 1993), WH Freeman and Company.

[0162] Phage display technology can alternatively be used to increase (or decrease) CDR affinity. This technique, known as affinity ripening, employs mutagenesis or “CDR walking,” and reselection uses the target antigen or its antigenic antigen-binding fragment to identify antibodies with CDRs that bind to the antigen with higher (or lower) affinity compared to the initial or parent antibody (see, for example, Glaser et al. (1992), Journal of Immunology 149:3903). Mutagenesis of the entire codon, rather than individual nucleotides, produces a semi-random library of amino acid mutations. Libraries can be constructed consisting of a set of variant clones, each differing by a single amino acid change in a single CDR, and these clones contain variants representing every possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) affinity for the antigen can be screened by contacting immobilized mutants with labeled antigens. Mutant antibodies with increased or decreased affinity for antigens can be identified using any screening method known in the art (e.g., ELISA) (see Wu et al., 1998, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. (USA)) 95:6037; Yelton et al., 1995, Journal of Immunology (J. Immunology) 155:1994). CDR walking of randomized light chains may also be possible (see Schier et al., 1996, Journal of Molecular Biology (J. Mol. Bio.) 263:551).

[0163] Methods for achieving such affinity maturation are described in the following, for example: Krause, JC et al. (2011) "An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of A Human Antibody", MBio.2(1)pii:e00345-10.doi:10.1128 / mBio.00345-10; Kuan, CT et al. (2010) "Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas and..." Melanomas), International Journal of Cancer (Int.J.Cancer) 10.1002 / ijc.25645; Hackel, BJ et al. (2010) "Stability and CDR Composition Biases Enrich Binder Functionality Landscapes", J.Mol.Biol. 401(1):84-96; Montgomery, DL et al. (2009) "Affinity Maturation and Characterization of a Human Monoclonal Antibody Against HIV-1gp41", MAbs 1(5):462-474; Gustchina, E.(2009) et al. "Affinity Maturation By Targeted Diversification Of The CDR-H2Loop Of A Monoclonal Fab Derived From A Synthetic" describes a set of Fabs with improved HIV-1 neutralizing titers and amplitudes through directional diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic primary human antibody library and affinity maturation targeting the coiled helix of the internal trimer of Gp41. Human Antibody Library and Directed Against the Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency and Breadth), Virology 393(1):112-119; Finlay, WJ et al. (2009) "Affinity Maturation of a Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside and Outside the Complementarity-Determining Region" Regions), Molecular Biology (J.Mol.Biol.) 388(3):541-558; Bostrom, J. et al. (2009) "Improving Antibody Binding Affinity And Specificity For Therapeutic Development", Methods Molecular Biology (Methods Mol.Biol.) 525:353-376; Steidl, S. et al. (2008) "In Vitro Affinity Maturation of Human GM-CSF Antibodies By Targeted CDR Diversification". "CDR-Diversification", Molecular Immunology (Mol. Immunol.) 46(1):135-144; and Barderas, R. et al. (2008) "Affinity Maturation of Antibodies Assisted By In Silico Modeling", Proceedings of the National Academy of Sciences (Proc. Natl. Acad.)Sci. (USA)) 105(26):9029-9034. .

[0164] Therefore, the sequence of the CDR variant of the included antibody or its antigen-binding fragment can differ from the sequence of the CDR of the parent antibody, GM37, GM37 variants 1-3, or 285 by substitution; for example, substitution of 4 amino acid residues, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue. According to embodiments of the invention, it is further contemplated that the amino acids in the CDR region can be substituted with conservative substitutions, as defined in the three tables above.

[0165] As used herein, the term "treatment" means improving, slowing, reducing, or reversing the progression or severity of a disease or disorder, or improving, slowing, reducing, or reversing one or more symptoms or side effects of such disease or disorder. For the purposes of this invention, "treatment" further means a method for obtaining a beneficial or desired clinical outcome, wherein "beneficial or desired clinical outcome" includes, but is not limited to, relief of symptoms, reduction of the severity of a disorder or disease, stabilization (i.e., no worsening) of a disease or disorder, delay or slowing of the progression of a disease or disorder, improvement or reduction of a disease or disorder, and relief of a disease or disorder, whether partial or complete, detectable or undetectable.

[0166] When applied to the antibodies or antigen-binding fragments thereof of the present invention, "effective amount" means an amount sufficient at the desired dose and for the desired duration to achieve the expected biological effect or desired therapeutic outcome (including, but not limited to, clinical outcome). When applied to the antibodies or antigen-binding fragments thereof of the present invention, the phrase "therapeutic effective amount" is intended to represent an amount of antibody or antigen-binding fragment thereof sufficient to improve, alleviate, stabilize, reverse, slow, reduce, or delay the progression of an impairment or disease state, or the progression of symptoms of such impairment or disease. In embodiments, the method of the present invention provides administration of an antibody or antigen-binding fragment thereof in combination with other compounds. In such cases, "effective amount" is the amount of the combination sufficient to cause the desired biological effect.

[0167] The therapeutically effective amount of the anti-α-synuclein antibody or its antigen-binding fragment of the present invention can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the anti-α-synuclein antibody to elicit a desired response in the individual. The therapeutically effective amount is also the amount by which the beneficial therapeutic effect of the antibody or antibody fraction outweighs any of its toxic or adverse effects.

[0168] As noted above, the present invention particularly relates to monoclonal antibodies that specifically bind to epitopes within amino acids 112-117 of human α-synuclein (SEQ ID NO:9 (ILEDMP)). In one embodiment, the antibody competes with antibody GM37 for binding to epitopes within amino acids 112-117 of α-synuclein.

[0169] The antibodies of the present invention (exemplified by GM37, its variants GM37 variants 1-3 and GM285 and their α-synuclein-binding fragments) are capable of binding to and neutralizing the toxicity of α-synuclein fragments composed of residues 1-119 / 122 of α-synuclein (e.g., by extracellular binding to the α-synuclein fragment, thereby preventing its uptake by cells). Surprisingly, the antibodies of the present invention, capable of binding to epitopes within amino acids 112-117 of α-synuclein, are superior to prior art antibodies (e.g., antibody 9E4) in terms of the types of toxic α-synuclein bound to the human brain, and exhibit excellent effects in clearing extracellular α-synuclein and normalizing impaired synaptic transmission induced by α-synuclein in vivo. The antibodies of the present invention are also capable of reducing the occurrence of related motor phenotypes in a rat model of Parkinson's disease.

[0170] The antibody of the present invention is preferably a human or humanized antibody.

[0171] The present invention also provides a method for reducing the formation of α-synuclein aggregates in patients, the method comprising administering a therapeutically effective amount of the antibody of the present invention to a patient in need of such treatment.

[0172] Furthermore, the antibody can be contained in the composition together with a pharmaceutically acceptable carrier, diluent, and / or stabilizer. The antibodies of the present invention can be used in treatment. In particular, the antibodies of the present invention can be used to treat synucleinopathies such as Parkinson's disease (including idiopathic and hereditary forms of Parkinson's disease), Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.

[0173] The treatment envisioned in this invention can be long-term and patients can receive treatment for at least 2 weeks (e.g., for at least 1 month, 6 months, 1 year or longer).

[0174] The antigen-binding fragments of the antibodies of the present invention can be generated in various cell lines, such as human cell lines, mammalian non-human cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, mouse cell lines (e.g., myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep 18: 1-13, the contents of which are incorporated herein by reference).

[0175] The antibodies of this invention can be, for example, monoclonal antibodies produced by a hybridoma method, first described by Kohler et al., Nature 256, 495 (1975), or monoclonal antibodies produced by a recombinant DNA method. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., Journal of Molecular Biology 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B lymphocytes, which are immunized with an antigen of interest or a nucleic acid encoding the antigen of interest, for example, in a cellular form expressing the antigen on their surface. Monoclonal antibodies can also be obtained from hybridomas of antibody-expressing cells derived from immune humans or from non-human mammals (such as rats, rabbits, dogs, sheep, goats, primates, etc.).

[0176] In one embodiment, the antibody of the present invention is a human antibody. Human monoclonal antibodies against α-synuclein can be generated using transgenic or transchromosomal mice carrying a portion of the human immune system rather than the mouse system. Such transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively.

[0177] HuMAb mice contain a human immunoglobulin microlocus along with a directed mutation that encodes non-rearranged human heavy chain variable and constant (μ and Y) and light chain variable and constant (κ) immunoglobulin sequences. This directed mutation inactivates the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Therefore, mice exhibit reduced expression of mouse IgM or Igκ, and in response to immunity, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations to produce high-affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), see above; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113, 49-101 (1994); Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995); Harding, F. and Lonberg, N., Ann. NYAcad. Sci. 764 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992); Chen, J. et al., International Immunology 5, 647-656 (1993); Tuaillon et al., Journal of Immunology 152, 2912-2920 (1994); Taylor, L. et al., International Immunology 6, 579-591 (1994); Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187.

[0178] Hco7, HCo12, HCo17, and HCo20 mice exhibit JKD disruption in their endogenous light chain (κ) gene (as described in Chen et al., EMBO J. 12, 811-820 (1993)), CMD disruption in their endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), and KCo5 human κ light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). In addition, HCo7 mice carried the HCo7 human heavy chain transgene (as described in US 5,770,429), HCo12 mice carried the HCo12 human heavy chain transgene (as described in Example 2 of WO 01 / 14424), HCo17 mice carried the HCo17 human heavy chain transgene (as described in Example 2 of WO 01 / 09187), and HCo20 mice carried the HCo20 human heavy chain transgene. The resulting mice expressed human immunoglobulin heavy chain and κ light chain transgenes against a background of homozygosity disruption of the endogenous mouse heavy chain and κ light chain loci.

[0179] In the KM mouse strain, the endogenous mouse κ light chain gene has been homozygously disrupted, as described in Chen et al., EMBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene has been homozygously disrupted, as described in Example 1 of WO 01 / 09187. This mouse strain carries the human κ light chain transgene KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosomal consisting of chromosome 14 segment hCF (SC20), as described in WO02 / 43478. HCo12-Balb / c, HCo17-Balb / c, and HCo20-Balb / c mice can be generated by crossing HCo12, HCo17, and HCo20 with KCo5[00J / K](Balb), as described in WO09 / 097006.

[0180] In the KM mouse strain, the endogenous mouse κ light chain gene has been homozygous disrupted, as described in Chen et al., EMBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene has also been homozygous disrupted, as described in Example 1 of WO 01 / 09187. This mouse strain carries the human κ light chain transgene KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosomal consisting of the chromosome 14 antigen-binding fragment hCF(SC20), as described in WO 02 / 43478.

[0181] Spleen cells from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies using well-known techniques. The human monoclonal or polyclonal antibodies of the present invention, or antibodies derived from other species, can also be produced by transgenic processes to generate another non-human mammal or plant that is transgenic for the heavy and light chain sequences of the immunoglobulins of interest, and from which antibodies are produced in a recyclable form. In conjunction with transgenic production in mammals, antibodies can be produced and recovered from the milk of goats, cows, or other mammals. See, for example, US 5,827,690, US 5,756,687, US 5,750,172, and US 5,741,957.

[0182] The antibodies of the present invention can have any isotype. The choice of isotype is typically guided by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant domain κ or λ can be used. If desired, the class of the anti-α-synuclein antibodies of the present invention can be converted using known methods. For example, an antibody of the present invention initially being IgM can be class-converted to an IgG antibody of the present invention. Furthermore, class-conversion techniques can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention can be changed by isotype switching to, for example, IgG1, IgG2, IgG3, or IgG4 antibodies for various therapeutic uses. In one embodiment, the antibody of the present invention is an IgG1 antibody, such as IgG1,κ. An antibody is described as a particular isotype if its amino acid sequence has the highest homology with one isotype relative to other isotypes.

[0183] In one embodiment, the antibody of the present invention is a full-length antibody, preferably an IgG antibody, particularly an IgG1,κ antibody. In another embodiment, the antibody of the present invention is an antibody fragment or a single-chain antibody.

[0184] Antibodies and their antigen-binding fragments can be obtained, for example, by antigen-binding fragmentation using conventional techniques, and the antigen-binding fragments are screened for practicality in the same manner as described herein for whole antibodies. For example, F(ab')2 antigen-binding fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 antigen-binding fragments can be treated to reduce disulfide bonds, thereby generating Fab' antigen-binding fragments. Fab antigen-binding fragments can be obtained by treating IgG antibodies with papain; Fab' antigen-binding fragments can be obtained by digesting IgG antibodies with pepsin. F(ab') antigen-binding fragments can also be generated via thioether bonds or disulfide bonds by binding Fab' as described below. Fab' antigen-binding fragments are antibody antigen-binding fragments obtained by cleaving the disulfide bonds of the hinge domain of F(ab')2. Fab'-antigen-binding fragments can be obtained by treating F(ab')2 antigen-binding fragments with a reducing agent (such as dithiothreitol). Antibody-antigen binding fragments can also be generated by expressing nucleic acids encoding such fragments in recombinant cells (see, for example, Evans et al., Journal of Immunological Methods, 184, 123-38 (1995)). For instance, a chimeric gene encoding a portion of the F(ab')2 antigen-binding fragment could include a DNA sequence encoding the CH1 region and hinge domain of the H strand, followed by a translation stop codon to produce such a truncated antibody-antigen binding fragment molecule.

[0185] In one embodiment, the anti-α-synuclein antibody is a monovalent antibody, preferably a monovalent antibody with a missing hinge domain, as described in WO 2007059782 (which is incorporated herein by reference in its entirety). Thus, in one embodiment, the antibody is a monovalent antibody, wherein the anti-α-synuclein antibody is constructed by a method comprising: i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding a VL region encoding a selected antigen-specific anti-α-synuclein antibody and a nucleotide sequence encoding a constant CL region encoding Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of Ig are operably linked together, and wherein, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that, in the presence of polyclonal human IgG or when administered to animals or humans, the CL region does not contain any amino acids capable of forming disulfide or covalent bonds with other peptides containing the same amino acid sequence of the CL region; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising: The construct comprises a nucleotide sequence encoding a VH region of a selected antigen-specific antibody and a nucleotide sequence encoding a constant CH region of human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that, in the presence of polyclonal human IgG or when administered to an animal, the regions corresponding to the hinge domain and other regions of the CH region (such as the CH3 region, as required by the Ig subtype) do not contain any amino acid residues that participate in forming disulfide bonds or covalent or stable non-covalent heavy chain bonds with other peptides containing the same amino acid sequence of the CH region of human Ig, wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operatively linked together; iii) providing a cell expression system for generating the monovalent antibody; iv) generating the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in the cells of the cell expression system of (iii).

[0186] Similarly, in one embodiment, the anti-α-synuclein antibody is a monovalent antibody comprising:

[0187] (i) the variable domain or antigen-binding portion of the antibody of the present invention described herein, and

[0188] (ii) The CH domain of an immunoglobulin or a domain containing CH2 and CH3 domains, wherein the CH domain or its domains have been modified such that the domain corresponding to the hinge domain and (if the immunoglobulin is not IgG4 subtype) other domains of the CH domain (such as the CH3 domain) do not contain any amino acid residues that can form a disulfide bond with the same CH domain or, in the presence of polyclonal human IgG, form other covalent or stable non-covalent heavy chain bonds with the same CH domain.

[0189] In another embodiment, the heavy chain of the monovalent α-synuclein antibody has been modified so that the entire hinge domain is missing.

[0190] In another embodiment, the sequence of the monovalent antibody has been modified so that it does not contain any receptor sites for N-linked glycosylation.

[0191] The present invention also includes "bispecific antibodies" wherein the anti-α-synuclein binding domain (e.g., the α-synuclein binding domain of an anti-α-synuclein monoclonal antibody) is part of a bivalent or multivalent bispecific scaffold targeting more than one epitope (e.g., the second epitope may include an epitope of an active transport receptor, thus enabling the bispecific antibody to exhibit improved endocytic transport across biological barriers (e.g., the blood-brain barrier)). Therefore, in another embodiment, the monovalent Fab of the anti-synuclein antibody may be linked to another Fab or scfv targeting a different protein to generate a bispecific antibody. The bispecific antibody can have dual functions, such as therapeutic functions conferred by the anti-synuclein binding domain and transport functions that can bind to receptor molecules to enhance translocation across biological barriers (e.g., the blood-brain barrier).

[0192] The anti-α-synuclein antibody and its antigen-binding fragment of the present invention also include a single-chain antibody. A single-chain antibody is a peptide in which the heavy chain and light chain Fv regions are linked. In one embodiment, the present invention provides a single-chain Fv (scFv) wherein the heavy chain and light chain in the Fv of the anti-α-synuclein antibody of the present invention are linked into a single peptide chain by flexible peptide linkers (typically about 10, 12, 15 or more amino acid residues). Methods for producing such antibodies are described, for example, in US 4,946,778; Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994); Bird et al., Science 242, 423–426 (1988); Huston et al., PNAS USA 85, 5879–5883 (1988); and McCafferty et al., Nature 348, 552–554 (1990). Single-chain antibodies can be monovalent if only a single VH and VL are used; they can be bivalent if two VH and VL are used; or they can be polyvalent if more than two VH and VL are used.

[0193] Anti-α-synuclein antibodies and their antigen-binding fragments described herein can be modified by including any suitable number of modified amino acids and / or associating with such conjugated substituents. In this context, suitability is generally determined by the ability to at least substantially preserve the α-synuclein selectivity and / or α-synuclein specificity associated with the underived parental anti-α-synuclein antibody. Introducing one or more modified amino acids can be advantageous in, for example, increasing the peptide's serum half-life, decreasing its antigenicity, or increasing its storage stability. Modification of one or more amino acids can occur, for example, concurrently with or after translation during recombinant production (e.g., N-linked glycosylation at the NXS / T motif during expression in mammalian cells), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprene-modified (e.g., farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, polyethylene glycol-modified amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. Sufficient references are available in the literature to guide those skilled in the art in modifying amino acids. Exemplary practices can be found in Walker (1998), *Protein Protocols On CD-ROM*, Humana Press, Totowa, New Jersey. Modified amino acids may, for example, be selected from glycosylated amino acids, polyethylene glycol-modified amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids linked to lipid moieties, or amino acids linked to organic derivatives.

[0194] Anti-α-synuclein antibodies can also be chemically modified by covalently binding to polymers to, for example, increase their cycling half-life. Exemplary polymers and methods of attaching them to peptides are illustrated, for example, in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Other illustrative polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., molecular weights between about 1,000 and about 40,000, such as between about 2,000 and about 20,000, for example, PEG of about 3,000-12,000 g / mol).

[0195] The antibodies of the present invention can be further used in diagnostic methods or as diagnostic imaging ligands.

[0196] In one embodiment, an anti-α-synuclein antibody comprising one or more radiolabeled amino acids is provided. Radiolabeled anti-α-synuclein antibodies can be used for both diagnostic and therapeutic purposes (binding to a radiolabeled molecule is another possible feature). Non-limiting examples of such labels 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 169 Er), fluorine ( 18 F), Gadolinium 153 Gd, 159 Gd), gallium ( 68 Ga、 67 Ga), germanium ( 68 Ge), gold ( 198 Au, Holmium 166 Ho), hydrogen ( 3 H), Indium 111 In、 112 In、 113 In、 115 In), iodine 121 I, 123 I, 125 I, 131 I), Iridium 192 Ir), iron ( 59 Fe), Krypton 81m Kr), Lanthanum 140 La), Lutetium 177 Lu), manganese 54 Mn), molybdenum 99 Mo), nitrogen ( 13 N、 15 N), oxygen ( 15 O), palladium ( 103 Pd), phosphorus ( 32 P), potassium ( 42 K), Praseodymium ( 142 Pr), Promethium ( 149 Pm), rhenium ( 186 Re、 188 Re), rhodium 105 Rh), rubidium ( 81 Rb、 82 Rb), Ruthenium ( 82 Ru、 97 Ru, samarium 153 Sm), Scandium (47 Sc), selenium ( 75 Se), sodium 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 ( 65Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902). For example, radioisotopes can be bound using the chloramine-T method (Lindegren, S. et al. (1998) "Chloramine-T In High-Specific-Activity Radioiodination Of Antibodies Using N-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate", Nuclear Medicine and Biology (Nucl. Med. Biol.) 25(7):659-665; Kurth, M. et al. (1993) "Site-Specific Conjugation Of A Radioiodinated Phenethylamine Derivative To A Monoclonal Antibody Results In Increased Radioactivity Localization In "Tumor", Journal of Medicinal Chemistry (J.Med.Chem.) 36(9):1255-1261; Rea, DW et al. (1990) "Site-specifically radioiodinated antibody for targeting tumors", Cancer Res. 50(3 Supplement):857s-861s.

[0197] The present invention also provides anti-α-synuclein antibodies and their antigen-binding fragments that are detectably labeled using the following: fluorescent labels (such as rare earth chelates (e.g., europium chelates)), fluorescein-type labels (e.g., fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), and rhodamine-type labels (e.g., ALEXA). 568 (Invitrogen) Or dansyl chloride), VIVOTA G680XL FLUOROCHROME TM (Perkin Elmer), phycoerythrin; umbelliferone, erythramine; anthocyanins; phycoerythrin, Texas red, BODIPY (Ingenie Biotech) or its analogues, all of which are suitable for optical detection. Chemiluminescent labeling (e.g., luminol, luciferase, luciferin, and jellyfish protein) can be employed. Such diagnosis and detection can also be accomplished by coupling the diagnostic molecules of the present invention to detectable substances (including, but not limited to, various enzymes, including but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase) or to prosthetic complexes (such as, but not limited to, streptavidin / biotin and avidin / biotin).

[0198] Chemiluminescent labeling (e.g., luminol, luciferase, luciferin, and jellyfish protein) can be employed. This diagnosis and detection can also be accomplished by coupling the diagnostic molecules of the present invention to detectable substances (including, but not limited to, various enzymes, including but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase) or to prosthetic complexes (such as, but not limited to, streptavidin / biotin and avidin / biotin). Paramagnetic labeling can also be employed, and detection is preferably performed using positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Such paramagnetic markings include, but are not limited to, those containing aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), and zirconium (Zi), and especially Co. +2 CR +2 Cr +3 Cu +2 Fe +2 Fe +3 Ga +3 Mn+3 Ni +2 Ti +3 V +3 and V +4 Compounds of paramagnetic ions, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions.

[0199] Therefore, in one embodiment, the anti-α-synuclein antibody of the present invention can be labeled with fluorescent labeling, chemiluminescent labeling, paramagnetic labeling, radioisotope labeling, or enzyme labeling. The labeled antibody can be used to detect or measure the presence or amount of the α-synuclein in the brain of a subject. This method may include in vivo imaging of detecting or measuring the anti-α-synuclein antibody bound to the α-synuclein, and may also include ex vivo imaging of the anti-α-synuclein antibody bound to the anti-α-synuclein.

[0200] In another aspect, the present invention relates to expression vectors encoding one or more polypeptide chains of the antibody or antigen-binding fragment thereof of the present invention. Such expression vectors can be used for recombinant generation of the antibody and antigen-binding fragment of the present invention.

[0201] In the context of this invention, the expression vector can be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, bacteriophage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding an anti-α-synuclein antibody is contained in a naked DNA or RNA vector, including, for example, linear expression elements (as described in, for example, Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), compact nucleic acid vectors (as described in, for example, US 6,077,835 and / or WO 00 / 70087), plasmid vectors (such as pBR322, pUC 19 / 18, or pUC118 / 119), “midge” minimal-size nucleic acid vectors (as described in, for example, Schakowski et al., Molecular Therapeutics 3, 793-800 (2001)), or as a precipitation-type nucleic acid vector construct, such as a CaPO4 precipitation construct (as described in, for example, WO 00 / 46147; Benvenisty and Reshef, PNASUSA 83, 9551-55 (1986); Wigler et al., Cell 14, 725 (1978); and Coraro and Pearson, Somatic Cell Genetics 2, 603 (1981). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0202] In one embodiment, the vector is suitable for expressing anti-α-synuclein antibodies or antigen-binding fragments thereof in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), pET vector (Novagen, Madison, Wisconsin), etc.

[0203] The expression vector can also be, or alternatively, a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, those containing constitutive or inducible promoters (such as α-factors, alcohol oxidases, and PGH) (Reviewed in: F. Ausubel et al., eds., 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 in Molecular Biology). Mol. Biol.) 824, 329-358 (2012); Celik, E. et al. Advances in Biotechnology (Biotechnol. Adv.) 30(5), 1108-1118 (2012); Li, P. et al. Applied Biochemistry and Biotechnology (Appl. Biochem. Biotechnol.) 142(2), 105-124 (2007); Bohr, E. et al. Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.) 77(3), 513-523 (2007); Van der Vaart, JM. Molecular Biology Methods 178, 359-366 (2002) and Holliger, P. Molecular Biology Methods 178, 349-357 (2002).

[0204] In the expression vector of the present invention, the nucleic acid encoding an anti-α-synuclein antibody may contain or be associated with any suitable promoter, enhancer, and other elements that facilitate expression. Examples of such elements include strongly expressive promoters (e.g., the human CMV IE promoter / enhancer along with RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), efficient poly(A) termination sequences, origins of replication for plasmid production in *E. coli*, antibiotic resistance genes as selective markers, and / or convenient cloning sites (e.g., multi-linker). The nucleic acid may also contain inducible promoters as opposed to constitutive promoters (such as CMV IE) (those skilled in the art will recognize that such terms are actually descriptive of the degree of gene expression under certain conditions).

[0205] The antibodies or antigen-binding fragments of the present invention can be generated in various cell lines, such as human cell lines, mammalian non-human cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, mouse cell lines (e.g., myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep 18: 1-13, the contents of which are incorporated herein by reference).

[0206] In even further aspects, the present invention relates to recombinant eukaryotic or prokaryotic host cells (such as transfected tumors) that produce antibodies of the present invention as defined herein or their antigen-binding domains, or bispecific molecules of the present invention as defined herein. Examples of host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells). For example, in one embodiment, the present invention provides cells comprising nucleic acids stably integrated into the cell genome containing an expression sequence encoding the anti-α-synuclein antibody of the present invention or its antigen-binding fragment. In another embodiment, the present invention provides cells comprising non-integrated nucleic acids (such as plasmids, phage particles, phage particles, or linear expression elements) containing an expression sequence encoding the anti-α-synuclein antibody of the present invention.

[0207] In another aspect, the present invention relates to a method for generating the anti-α-synuclein antibody of the present invention, the method comprising the steps of: a) culturing hybridoma or host cells of the present invention as described above, and b) purifying the antibody of the present invention from the culture medium.

[0208] In one embodiment, the present invention relates to a formulation, as used herein, comprising an anti-α-synuclein antibody as defined herein, and substantially free of naturally occurring antibodies that do not bind to α-synuclein or substantially alter the anti-α-synuclein function of the formulation. Thus, such a formulation does not include naturally occurring serum or purified derivatives of such serum, and comprises a mixture of an anti-α-synuclein antibody and another antibody that does not alter the function of the anti-α-synuclein antibody in the formulation; wherein such function is selected from the group consisting of:

[0209] (i) The binding affinity (KD) of the anti-α-synuclein antibody to α-synuclein;

[0210] (ii) The ability of this anti-α-synuclein antibody to inhibit the protease truncation of α-synuclein fibrils;

[0211] (iii) The ability of this anti-α-synuclein antibody to reverse damage to basal synaptic transmission in F28-snca transgenic mice;

[0212] (iv) The ability of this anti-α-synuclein antibody to reduce α-synuclein levels in the mouse hippocampus, as measured by in vivo microdialysis; and

[0213] (v) The ability of this anti-α-synuclein antibody to restore motor function when administered long-term in a rat model of Parkinson's disease;

[0214] (vi) The ability to prevent α-synuclein seeding (e.g., the accumulation of insoluble phosphorylated α-synuclein in vitro and / or in mouse models of Parkinson's disease); and / or

[0215] (vii) The ability to bind truncated α-synuclein in the human brain.

[0216] This invention specifically relates to a formulation of an anti-α-synuclein antibody having a structural alteration in its amino acid sequence (in any of its CDRs, variable domains, framework residues, and / or constant domains) relative to the structure of naturally occurring anti-α-synuclein antibodies, wherein the structural alteration results in a significant change in the functionality exhibited by the anti-α-synuclein antibody monoclonal antibody relative to naturally occurring anti-α-synuclein antibodies (i.e., a difference of more than 20%, more than 40%, more than 60%, more than 80%, more than 100%, more than 150%, more than 2-fold, more than 4-fold, more than 5-fold, or more than 10-fold in terms of functionality); wherein such functionality is:

[0217] (i) The binding affinity (KD) of the anti-α-synuclein monoclonal antibody to α-synuclein;

[0218] (ii) The ability of this anti-α-synuclein monoclonal antibody to inhibit the protease truncation of α-synuclein fibrils;

[0219] (iii) The ability of this anti-α-synuclein monoclonal antibody to reverse damage to basal synaptic transmission in F28-snca transgenic mice;

[0220] (iv) The ability of this anti-α-synuclein monoclonal antibody to reduce α-synuclein levels in the mouse hippocampus, as measured by in vivo microdialysis; and / or

[0221] (v) In a rat model of Parkinson's disease, the ability of anti-α-synuclein monoclonal antibodies to restore motor function when administered long-term;

[0222] (vi) The ability to prevent α-synuclein seeding (e.g., the accumulation of insoluble phosphorylated α-synuclein in vitro and / or in mouse models of Parkinson's disease); and / or

[0223] (vii) The ability to bind truncated α-synuclein in the human brain.

[0224] In particular, such functional changes are a result of structural changes and are therefore inseparable from them.

[0225] The term "substantially free" of naturally occurring antibodies means that such naturally occurring antibodies are completely absent in such formulations, or that the concentration of such naturally occurring antibodies contained in such formulations does not materially affect the α-synuclein binding properties of these formulations. Antibodies are described as "isolated" if they do not have a naturally occurring counterpart or have been isolated or purified from the components that naturally accompany them.

[0226] When referring to such preparations, the term "naturally occurring antibody (naturally present antibody)" means an antibody (including naturally occurring autoantibodies) that is generated in the body of a living human or other animal as a natural result of the functioning of the immune system.

[0227] Therefore, the formulations of the present invention do not exclude and indeed explicitly include such formulations containing anti-α-synuclein antibodies and intentionally added additional antibodies capable of binding to epitopes not present in α-synuclein. Such formulations particularly include embodiments in which the formulation exhibits enhanced efficacy in treating the following synucleinogenic diseases, such as Parkinson's disease (including congenital and hereditary forms of Parkinson's disease), Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.

[0228] In even further aspects, the present invention relates to pharmaceutical compositions comprising:

[0229] (i) Anti-α-synuclein antibodies or antigen-binding fragments thereof (both as defined herein), or formulations containing such anti-α-synuclein antibodies or antigen-binding fragments thereof (as defined herein), and

[0230] (ii) Pharmaceutically acceptable carriers.

[0231] Pharmaceutical compositions may be formulated using pharmaceutically acceptable carriers or diluents, together with any other known adjuvants and excipients, according to conventional techniques disclosed in Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Gennaro, Mack Publishing Co., Easton, Pennsylvania, 2013.

[0232] Pharmaceutically acceptable carriers or diluents, along with any other known adjuvants and excipients, should be suitable for the selected compounds and the selected route of administration of the present invention. The suitability of the carriers and other components of the pharmaceutical composition is determined based on the lack of a significant adverse effect on the desired biological properties of the selected compounds or pharmaceutical compositions of the present invention with respect to epitope binding (e.g., less than a substantial effect (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0233] The pharmaceutical compositions of the present invention may further comprise diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition. The selected diluents do not affect the bioactivity of the composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextran solution, and Hank's solution. Furthermore, the pharmaceutical composition or formulation may also comprise other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc. The composition may also comprise large, slowly metabolized macromolecules such as proteins, polysaccharides (like chitosan), polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized cross-linked agarose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0234] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and route of administration. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention or its amide, route of administration, time of administration, excretion rate of the specific compound used, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, disease condition, general health status, and medical history of the patient being treated, and similar factors well known in the medical field.

[0235] The pharmaceutical composition may be administered by any suitable route and manner, including parenteral, topical, oral, or intranasal means for prophylactic and / or therapeutic treatment. In one embodiment, the pharmaceutical composition of the invention is administered parenterally. As used herein, the phrase "parenteral administration" means a route of administration other than enteral and topical administration, typically by injection, and includes intradermal, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion. Other suitable routes for administering the compounds of the invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art. In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0236] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delay agents, as well as analogs that are physiologically compatible with the compounds of the present invention.

[0237] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextran, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethyl cellulose colloidal solutions, tragacanth gum, and injectable organic esters (such as ethyl oleate) and / or various buffers. Other carriers are well known in the pharmaceutical industry.

[0238] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injections or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art. The use of any conventional media or reagent in the pharmaceutical compositions of the present invention is contemplated, except where such media or reagents are incompatible with the active compound.

[0239] Appropriate flowability can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size (in the case of dispersions), and by using surfactants.

[0240] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0241] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyols (such as mannitol, sorbitol, glycerol) or sodium chloride.

[0242] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, or buffers, which can enhance the shelf life or efficacy of the pharmaceutical composition. The compounds of the present invention can be prepared with carriers that protect the compounds from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers (such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) (alone or with waxes), or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0243] In one embodiment, the compounds of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injections or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art. The use of any conventional media or reagents in the pharmaceutical compositions of the present invention is contemplated, except where such media or reagents are incompatible with the active compound. Additional active compounds may also be incorporated into the composition.

[0244] Injectable pharmaceutical compositions must generally be sterile and stable under the conditions of production and storage. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate flowability can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size (in the case of dispersions), and by using surfactants. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as glycerol, mannitol, sorbitol), or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay antibody absorption (such as monostearate and gelatin) in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of ingredients, such as those listed above, into a suitable solvent, followed by sterilization and microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a base dispersion medium and other desired components, such as those listed above. Examples of preparation methods for sterile powders used to prepare sterile injectable solutions include vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient and any other desired components from a previously sterile filtered solution.

[0245] Sterile injectable solutions can be prepared by incorporating the desired amount of an active compound with one or a combination of the ingredients listed above into a suitable solvent, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a base dispersion medium and other desired ingredients from those listed above. Examples of methods for preparing sterile powders for sterile injectable solutions include vacuum drying and freeze-drying (lyophilization), which produce powders of the active ingredient along with any additional desired ingredients from their previously sterile filtered solution.

[0246] Dosing regimens in the above-described treatment methods and uses are adjusted to provide the best expected response (e.g., therapeutic response). For example, a single large dose (bolus) may be administered, several fractionated doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and dosage uniformity, parenteral compositions may be formulated in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as a single dose to a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect associated with the desired drug carrier. The description of unit dosage forms of the present invention is subject to and directly depends on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the field of compounding such active compounds to obtain sensitive treatment in an individual.

[0247] The effective dose and dosing regimen of anti-α-synuclein antibodies depend on the disease or condition to be treated and can be determined by a person skilled in the art. On any given day the dose is administered, the dose ranges from about 0.0001 to about 100 mg / kg of host body weight, and more typically from about 0.01 to about 5 mg / kg of host body weight. For example, the dose may be 1 mg / kg of body weight or 10 mg / kg of body weight or in the range of 1-10 mg / kg of body weight. Thus, exemplary doses include: from about 0.1 to about 10 mg / kg / body weight, from about 0.1 to about 5 mg / kg / body weight, from about 0.1 to about 2 mg / kg / body weight, from about 0.1 to about 1 mg / kg / body weight, such as about 0.15 mg / kg / body weight, about 0.2 mg / kg / body weight, about 0.5 mg / kg / body weight, about 1 mg / kg / body weight, about 1.5 mg / kg / body weight, about 2 mg / kg / body weight, about 5 mg / kg / body weight, or about 10 mg / kg / body weight.

[0248] Physicians with ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician may begin administering the anti-α-synuclein antibody used in the pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the suitable daily dose of the composition of the present invention will be the minimum amount of compound that effectively produces a therapeutic effect. Such an effective dose will generally depend on the factors described above. Administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous. If desired, the effective daily dose of the pharmaceutical composition may be administered throughout the day in two, three, four, five, six, or more sub-dose portions at appropriate time intervals, optionally in unit dosage form. Although the compounds of the present invention may be administered alone, it is preferred to administer the compound as a pharmaceutical composition as described above.

[0249] The labeled antibodies of this invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases or disorders. This invention provides for the detection or diagnosis of neurodegenerative or cognitive diseases or disorders, including but not limited to Parkinson's disease, idiopathic Parkinson's disease, common Parkinson's disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy. The detection or diagnosis comprises: (a) determining the type and fragment of α-synuclein in a subject's cell or tissue sample using one or more antibodies that specifically bind to α-synuclein; and (b) comparing the level of the antigen to a control level (e.g., the level in a normal tissue sample), whereby an increase in the measured level of the antigen compared to the control level indicates a disease or disorder, or indicates the severity of the disease or disorder.

[0250] Using immunohistochemical methods well-known in the art, the antibodies of the present invention can be used to determine α-synuclein monomers, α-synuclein oligomers, fibrillary forms, or fragments in biological samples. Other antibody-based methods for detecting proteins include immunoassays (such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA)) and mesoscale discovery platform (MSD) assays. Suitable antibody labeling can be used in such kits and methods, and labeling known in the art includes enzyme labeling, such as alkaline phosphatase and glucose oxidase; and radioisotope labeling, such as iodine (…). 125 I, 131 I), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), Indium 121 In) and technetium ( 99m Tc); and luminescent markers, such as luminol and luciferase; and fluorescent markers, such as luciferin and rhodamine.

[0251] The presence of labeled anti-α-synuclein antibodies or their α-synuclein-binding fragments can be detected in vivo for diagnostic purposes. In one embodiment, the diagnosis includes: a) administering an effective amount of such labeled molecule to a subject; b) waiting a time interval after administration to allow the labeled molecule to accumulate at Aβ deposition sites (if present) and to allow unbound labeled molecules to be cleared to background levels; c) determining the background level; and d) detecting the labeled molecule in the subject such that the detection of labeled molecules above the background level indicates that the subject has the disease or disorder, or indicates the severity of the disease or disorder. According to such an embodiment, the labeled molecule is labeled with an imaging portion adapted for detection using a specific imaging system known to those skilled in the art. The background level can be determined by a variety of methods known in the art, including comparing the amount of labeled antibody detected with a standard value previously determined for a specific imaging system. Methods and systems that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole-body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound examinations.

[0252] In another aspect, the present invention relates to antibodies or antigen-binding fragments thereof for use in medicine.

[0253] In another aspect, the present invention relates to antibodies or antigen-binding fragments thereof for use in the treatment, diagnosis or imaging of synucleinosis.

[0254] In one embodiment, a monoclonal antibody or its antigen-binding fragment is used in the treatment of Parkinson's disease, idiopathic Parkinson's disease, common forms of Parkinson's disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.

[0255] In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof of the present invention in the production of medicaments for the treatment, diagnosis or imaging of synucleinosis.

[0256] In another aspect, the present invention relates to the treatment, diagnosis, or imaging of Parkinson's disease or other synucleinic diseases, including administration of an effective dose of the antibody of the present invention or its antigen-binding fragment.

[0257] Preferably, in the uses and methods of those aspects of the invention, the treatment is long-term, and preferably lasts for at least 2 weeks, such as at least 1 month, 6 months, 1 year or longer.

[0258] In another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0259]

[0260]

[0261] Embodiments of the present invention

[0262] As will be apparent from the text and examples, the invention further relates to the following embodiments:

[0263] 1. A monoclonal antibody or its antigen-binding fragment that can specifically bind to an epitope (SEQ ID NO:9(ILEDMP)) within amino acids 112-117 of α-synuclein.

[0264] 2. The monoclonal antibody or its antigen-binding fragment as described in Example 1 competes with antibody GM37 for binding to the epitope.

[0265] 3. The monoclonal antibody or its antigen-binding fragment according to Example 1, which is GM37, GM37 variant 1, GM37 variant 2 or GM37 variant 3.

[0266] 4. A monoclonal antibody or its antigen-binding fragment that can specifically bind to an epitope (SEQ ID NO:19(ILED)) within amino acids 112-115 of α-synuclein.

[0267] 5. The monoclonal antibody or its antigen-binding fragment according to Example 1 or 4, which is GM285.

[0268] 6. The monoclonal antibody or its antigen-binding fragment according to the above embodiments, wherein the antibody comprises or is composed of a complete antibody.

[0269] 7. The monoclonal antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments comprises or is composed of an antigen-binding fragment selected from the group consisting of: Fv fragments (e.g., single-chain Fv and disulfide-bonded Fv), Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments), and domain antibodies (e.g., single-chain Fv and F(ab)2 fragments). H Variable domain or V L (variable field).

[0270] 8. The monoclonal antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the monoclonal antibody is selected from the group consisting of antibodies of IgG1, IgG2, IgG3 and IgG4 subtypes.

[0271] 9. The monoclonal antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the antibody or antigen-binding fragment exhibits one or more of the following properties:

[0272] (i) Binding affinity for α-synuclein (K D The binding affinity is between 0.5 and 10 nM, for example, 1-5 nM or 1-2 nM;

[0273] (ii) The ability to inhibit the protease truncation of α-synuclein protofibrils;

[0274] (iii) The ability to reverse basal synaptic conduction impairment in F28-snca transgenic mice;

[0275] (iv) The ability to reduce the level of α-synuclein in the mouse hippocampus, as measured by in vivo microdialysis;

[0276] (v) The ability to restore motor function in a rat model of Parkinson's disease when administered long-term;

[0277] (vi) The ability to prevent α-synuclein seeding (e.g., accumulation of insoluble phosphorylated α-synuclein in vitro and / or in mouse models of Parkinson's disease); and / or

[0278] (vii) The ability to bind truncated α-synuclein in the human brain.

[0279] 10. The monoclonal antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments is human or humanized.

[0280] 11. A monoclonal antibody or a monoclonal antibody or antigen-binding fragment thereof according to Examples 1-3 and 6-10, comprising a heavy chain variable domain containing the following CDRs:

[0281] a) GFTFSSYAMT (SEQ ID NO:1) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference;

[0282] b) AIRS(N / S / Q / H)GDRTD YADSVKG (SEQ ID No: 2, 33, 34, 35) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference; or

[0283] c) AKNWAPFDS (SEQ ID NO:3) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference. 12. The monoclonal antibody or its antigen-binding fragment according to Example 11, comprising a heavy chain variable domain comprising a CDR of SEQ ID No:1 and 3, and one of SEQ ID No:2 and 33, 34, or 35.

[0284] 13. The monoclonal antibody or its antigen-binding fragment according to Example 11 comprises or consists of a heavy chain variable domain selected from the group consisting of:

[0285] a) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA PGKGLEWVSA IRSNGDRTDYADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO: 7),

[0286] b) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA PGKGLEWVSA IRSSGDRTDYADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO: 30),

[0287] c) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA PGKGLEWVSA IRSQGDRTDYADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO: 31), or

[0288] d) EVQLLESGGG LVQTGGSLRL SCAASGFTFS SYAMTWVRQA PGKGLEWVSA IRSHGDRTDYADSVKGRFTI SRDNSQNTLY LQMNSLRAED TAVYYCAKNW APFDSWGQGT LVTVSS (SEQ ID NO: 32).

[0289] 14. A monoclonal antibody or a monoclonal antibody or antigen-binding fragment thereof according to Examples 1-3 and 6-13, comprising a light chain variable domain containing the following CDRs:

[0290] a) ASQSVSSSYLA (SEQ ID NO:4) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference;

[0291] b) GASSRAT (SEQ ID NO:5) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference; or

[0292] c) QQYGSSPWT (SEQ ID NO:6) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference.

[0293] 15. The monoclonal antibody or its antigen-binding fragment according to Example 14, comprising a light chain variable domain comprising the CDRs of SEQ ID NO: 4, 5 and 6.

[0294] 16. The antibody or antigen-binding fragment thereof according to Example 14 comprises a light chain variable domain, which comprises or is composed of the following amino acid sequence:

[0295] EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK (SEQ ID NO: 8).

[0296] 17. The monoclonal antibody or its antigen-binding fragment according to Example 14 comprises a light chain variable domain, which comprises or is composed of the following amino acid sequence:

[0297] EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK (SEQ ID NO: 8).

[0298] 18. The monoclonal antibody or antigen-binding fragment thereof according to Examples 1-3 and 6-17, comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises or is composed of the amino acid sequence of SEQ ID NO:8, and the heavy chain variable domain comprises or is composed of any of the amino acids given in SEQ ID No:7, 33, 34 or 35.

[0299] 19. The monoclonal antibody or antigen-binding fragment thereof according to Examples 1-3 and 6-18, comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises or is composed of the amino acid sequence of SEQ ID NO:8, and the heavy chain variable domain comprises or is composed of the amino acid given in SEQ ID NO:34, the antibody and its antigen-binding fragment having increased thermal stability, for example as... Figure 27 The increased stability shown in the study demonstrates the prevention of aggregation and spread, exhibiting greater than 2%-10% stability at temperatures above 65°C compared to GM37wt, greater than 2%-8% stability at temperatures above 65°C compared to GM37wt, or greater than 2%-5% stability at temperatures above 65°C compared to GM37wt.

[0300] 20. A monoclonal antibody or a monoclonal antibody or antigen-binding fragment thereof according to Examples 1-10, comprising a heavy chain variable domain containing the following CDRs:

[0301] a) AASGFTFSRFTMT (SEQ ID NO:20) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference;

[0302] b) AISGSGGGTS YADSVKG (SEQ ID NO:21) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference; or

[0303] c) AKNWAPFDY (SEQ ID NO:22) or an amino acid sequence having no more than 4 amino acid differences, or no more than 22 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference.

[0304] 21. The monoclonal antibody or antigen-binding fragment thereof according to Example 20, comprising a heavy chain variable domain comprising the CDRs of SEQ ID NO: 20, 21 and 22.

[0305] 22. The monoclonal antibody or its antigen-binding fragment according to Example 20, comprising a heavy chain variable domain, the heavy chain variable domain comprising or consisting of the following amino acid sequence.

[0306] EVQLLESGGG LVQPGGSLRL SCAASGFTFS RFTMTWVRQA PGKGLEWVSA ISGSGGGTSYADSVKGRLTV SRDNSKNTLY LQMNSLRAED TAVYYCAKNW APFDYWGQGT LVTVSS (SEQ ID NO 26). 23. A monoclonal antibody or the monoclonal antibody or antigen-binding fragment thereof according to Examples 1-10 and 20-22, comprising a light chain variable domain containing the following CDRs:

[0307] d) RASQSVSRSYLA (SEQ ID NO:23) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference;

[0308] e) GASSRAT (SEQ ID NO:24) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference; or

[0309] f) QQYGSSPWT (SEQ ID NO:25) or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference.

[0310] 24. The monoclonal antibody or antigen-binding fragment thereof according to Example 23, comprising a light chain variable domain comprising the CDRs of SEQ ID NO: 23, 24 and 25.

[0311] 25. The antibody or antigen-binding fragment thereof according to Example 24 comprises a light chain variable domain, which comprises or is composed of the following amino acid sequence:

[0312] EIVLTQSPGT LSLSPGERAT LSCRASQSVS RSYLAWYQQK PGQAPRLLIY GASSRATGIPDRFSGSGSGT DFTLTVSRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK (SEQ ID NO: 27).

[0313] 26. The monoclonal antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:27, and the heavy chain variable domain comprising or consisting of any of the amino acids given in SEQ ID NO:26.

[0314] 27. The monoclonal antibody or its antigen-binding fragment according to any one of the foregoing embodiments comprises an Fc region.

[0315] 28. The monoclonal antibody or its antigen-binding fragment according to any one of the foregoing embodiments further comprises a portion for increasing the half-life of the agent in vivo.

[0316] 29. The monoclonal antibody or antigen-binding fragment thereof according to Example 28, wherein the portion used to increase the half-life in vivo is selected from the group consisting of: polyethylene glycol (PEG), human serum albumin, glycosylated groups, fatty acids and dextran.

[0317] 30. The monoclonal antibody or antigen-binding fragment thereof according to any one of the foregoing embodiments, wherein the antibody polypeptide further comprises a detectable portion.

[0318] 31. The monoclonal antibody or its antigen-binding fragment according to Example 30, wherein the detectable portion is fluorescently labeled, chemiluminescently labeled, paramagnetically labeled, radioisotope labeled, or enzyme labeled.

[0319] 32. The monoclonal antibody or antigen-binding fragment thereof according to Example 30 or 31, wherein the detectable portion comprises or is composed of a radioactive isotope.

[0320] 33. The monoclonal antibody or its antigen-binding fragment according to Example 32, wherein the radioactive isotope is selected from the group consisting of the following: 99m Tc, 111 In、 67 Ga、 68 Ga、 72 As、 89 Zr、 123 I and 201 Tl.

[0321] 34. The monoclonal antibody or antigen-binding fragment thereof according to Example 30, wherein the detectable portion comprises or is composed of a paramagnetic isotope.

[0322] 35. The monoclonal antibody or its antigen-binding fragment according to Example 34, wherein the paramagnetic isotope is selected from the group consisting of the following: 157 Gd, 55Mn, 162 Dy、 52 Cr and 56 Fe.

[0323] 36. The monoclonal antibody or antigen-binding fragment thereof according to Examples 30 to 35, wherein the detectable portion can be detected by imaging techniques such as SPECT, PET, MRI, optical or ultrasound imaging.

[0324] 37. A monoclonal antibody or antigen-binding fragment thereof according to any one of Examples 30 to 36, wherein the detectable portion is indirectly linked to the antibody or antigen-binding fragment thereof via a linker portion.

[0325] 38. The monoclonal antibody or antigen-binding fragment thereof according to Example 37, wherein the linker is selected from the group consisting of: derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isocyanothiobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA).

[0326] 39. An isolated nucleic acid molecule or a polypeptide chain thereof, encoding an antibody or an antigen-binding fragment thereof according to any one of the foregoing embodiments.

[0327] 40. The nucleic acid molecule according to Example 39, wherein the molecule is a cDNA molecule.

[0328] 41. The nucleic acid molecule according to Example 30 or 31 encodes an antibody heavy chain or its variable domain.

[0329] 42. A nucleic acid molecule according to any one of Examples 39 to 41, encoding an antibody light chain or its variable domain. 43. A vector comprising a nucleic acid molecule according to any one of Examples 39 to 42.

[0330] 44. A recombinant host cell comprising a nucleic acid molecule according to any one of Examples 39 to 42 or a vector according to Example 43.

[0331] 45. A method for generating an antibody or antigen-binding fragment according to any one of Examples 1 to 27, the method comprising culturing host cells as defined in Example 44 under conditions that allow expression of the encoded antibody or antigen-binding fragment thereof.

[0332] 46. ​​A pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment according to any one of Examples 1 to 35, and a pharmaceutically acceptable carrier.

[0333] 47. The monoclonal antibody or antigen-binding fragment thereof as described in Examples 1-35, for use in medicine.

[0334] 48. The monoclonal antibody or its antigen-binding fragment according to Examples 1-35, for use in the treatment, diagnosis or imaging of synucleinosis.

[0335] 49. The monoclonal antibody or antigen-binding fragment thereof as described in Example 48, for use in the treatment of Parkinson's disease (including idiopathic and hereditary forms of Parkinson's disease), Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.

[0336] 50. Use of the monoclonal antibody or antigen-binding fragment thereof as described in Examples 1-35 in the production of a medicament for the treatment, diagnosis or imaging of synucleinosis.

[0337] 51. Use of the monoclonal antibody or antigen-binding fragment thereof as described in Example 50 in the production of medicaments for the treatment, diagnosis or imaging of Parkinson's disease (including idiopathic and hereditary forms of Parkinson's disease), Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure and multiple system atrophy.

[0338] 52. A method for treating, diagnosing, or imaging synucleinosis in a subject, the method comprising administering to the subject an effective dose of the pharmaceutical composition as described in Example 46.

[0339] 53. The antibody or antigen-binding fragment thereof for use as described in Example 48, or for use as described in Example 50, or for treatment of Parkinson's disease (including idiopathic and hereditary forms of Parkinson's disease), Gaucher disease, diffuse Lewy body disease (DLBD), Lewy body variant (LBV) of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, pure autonomic failure, and multiple system atrophy.

[0340] 54. The antibody or antigen-binding fragment thereof for use as described in Example 52 or 53; or the use; or the method, wherein the treatment is long-term.

[0341] 55. The antibody or antigen-binding fragment thereof for use as described in Example 52; or the use; or the method, wherein the long-term treatment lasts for at least 2 weeks, such as at least 1 month, 6 months, 1 year or longer.

[0342] 56. An antibody or antigen-binding fragment thereof for use according to any one of Examples 47 to 55; or an application; or a method, wherein the subject is a human.

[0343] 57. A kit comprising an antibody or an antigen-binding fragment thereof as described in Examples 1-35.

[0344] 58. The kit according to Example 57 is intended for use in medicine.

[0345] 59. The monoclonal antibody or antigen-binding fragment thereof as described in Examples 30-35, for use in detecting or measuring the presence or amount of the α-synuclein in the brain or body fluids of a subject.

[0346] 60. A monoclonal antibody or antigen-binding fragment thereof as described in Example 59, wherein the detection or measurement comprises in vivo imaging of the anti-synuclein antibody bound to the α-synuclein.

[0347] 61. The monoclonal antibody or antigen-binding fragment thereof as described in Examples 30-35, wherein the detection or measurement comprises in vitro imaging of the anti-synuclein antibody bound to the α-synuclein.

[0348] Example

[0349] Example 1: Antibody Screening

[0350] 1. Immunogen and ligand production

[0351] like Figure 1As shown, the following proteins were obtained or produced for use as immunogens. Mice were immunized using three immunogens: full-length recombinant human α-synuclein fibrils; recombinant human α-synuclein proteins containing amino acids 1-60 (Rpeptide, Bogart, Georgia); and recombinant human α-synuclein proteins containing amino acids 1-119. To obtain fibrils from full-length α-synuclein, lyophilized products from Rpeptide, Bogart, Georgia (catalog number S-1001-2) were used. This product was dissolved at a protein concentration of 1 mg / ml in a buffer of 20 mM Tris and 300 mM NaCl. To prepare fibrils, protein solution was aliquoted into 170 μl portions in 96-well plates containing ceramic beads of 70 μm diameter in each well and incubated at 200 rpm in a Vortemp 56 shaker incubator (Labnet International, Edison, NJ, USA) at 37°C for 7 days. After fibril formation, thiamine T was added, and fluorescence was measured in one of the wells. Recombinant α-synuclein containing amino acids 1-60 was dissolved in water to obtain a concentration of 1 mg / mL.

[0352] The following construct was used to prepare a recombinant α-synuclein containing amino acids 1-119: a histidine tag encoding 6 amino acids, followed by a gene for synthesizing the Xa factor cleavage site and the coding sequence for amino acids 1-119 of human α-synuclein:

[0353]

[0354] The synthetic gene was synthesized by Genscript and cloned into the NdeI-XhoI site of the pET24a(+) expression vector (Novagen).

[0355] The expression vector was transformed into *E. coli* BL21, and single colonies were selected for expression using an overnight automated expression induction system from Novagen (user agreement TB383rev.H1005). The final culture volume was 500 ml. Cells were harvested by centrifugation at 6000 g for 10 min and subsequently lysed using BugBuster protein extraction reagent (user agreement TB245Rev.0304). After lysis, samples were separated by centrifugation, and the supernatant was used for further purification.

[0356] His-labeled proteins were purified using a 5 mL HisTrap column (GE Healthcare), equilibrated in 20 mM sodium phosphate (pH 7.5) and 1 M NaCl (buffer A). After sample loading and washing with buffer A, proteins were eluted with a gradient of 0.25 M imidazole in buffer A, using 20 column volumes. 5 mL fractions were collected and analyzed by SDS-PAGE. Fractions containing the target protein were pooled, concentrated, and applied to an S200 (26 / 60) size ...

[0357] To remove the N-terminal tag, purified his-tagged α-synuclein 1-119 was incubated with factor Xa at a ratio of 1:50 using a Novagen kit (69037-3FRX). After overnight incubation, factor Xa was removed batchwise using Xarrest agarose. As described above, the cleaved α-synuclein 1-119 was finally purified by licensed HisTrap chromatography. The purified α-synuclein 1-119 was obtained from the effluent and concentrated to approximately 400 μg / ml using a Centricon concentrator.

[0358] α-synuclein (Rpeptide) was rehydrated in PBS at 2 mg / ml while peroxynitrite (100 μL / mg protein) was added dropwise with stirring. The nitrosylated α-synuclein was then dialyzed in 5 L PBS and stored at -20°C.

[0359] Dopamine was used to oxidize α-synuclein. Equal volumes of a 200 μM dopamine-HCl (Sigma P5244) solution prepared in 10 mM PBS (pH 7.4) and a 28 μM α-synuclein (Rpeptide) solution in 10 mM PBS (pH 7.4) were combined. The resulting 14 μM α-synuclein / 100 μM dopamine solution was incubated overnight at 37 °C in O / N conditions. The oxidized α-synuclein was then dialyzed in PBS and stored at -20 °C.

[0360] A diverse library of synuclein proteins, including both native and chimeric forms, was generated to screen for antibodies against α-synuclein. The constructs screened included: α-synuclein from humans, mice, rats, and cynomolgus monkeys; human β-synuclein; and human γ-synuclein. Figure 21 and 22The study also included α-synuclein derivatives lacking residues 120-140. Furthermore, a series of four hybrid constructs were generated: A-Syn-AAKK-BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, and A-Syn-BBKK-BAP (SEQ ID No: 11-14). These constructs contained linear segments of human α-synuclein (A), human β-synuclein (B), and chicken α-synuclein (K). The cloned genes contained a biotin receptor peptide (BAP) tag fused to the C-terminus of the ligands to facilitate site-specific biotinylation of each ligand. Biotinylation allowed the ligands to attach to beads used in soluble ELISA. A mammalian expression vector carrying different α-synuclein BAP tag fusion constructs (ASynBAP) was constructed. The ligands were expressed in HEK293 cells using transient transfection (Genmab A / S).

[0361] 2. Immunity

[0362] The antibody HuMab-synuclein is derived from the immunization of HuMAb mouse strains HCo17-BALB / c and HCo12-BALB / c mice, which feature a double knockout of both the mouse immunoglobulin (Ig) heavy chain and the mouse κ light chain, thereby preventing the expression of a purely mouse antibody (human monoclonal antibody; Medarex Inc., San Jose, California, USA). Different mouse strains are transgenic by inserting human Ig heavy chain and human Igκ light chain loci, distinguished by the number of human VH (variable domain of the heavy chain) and VL (variable domain of the light chain) genes.

[0363] Forty-eight mice were immunized at 14-day intervals by alternating intraperitoneal (IP) and subcutaneous (SC) administration of the same immunogen. A maximum of eight immunizations were administered: four IPs and four SCs.

[0364] The mice were first immunized with α-synuclein immunogen in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, Michigan, USA), and subsequent immunizations were performed with incomplete Freund's adjuvant (IFA). When serum titers were found to be adequate (positive for at least two consecutive bi-weekly screening events in an antigen-specific screening assay as described above, with a serum dilution of 1 / 50 or less being detected), the mice were additionally boosted intravenously (IV) twice, four and three days prior to fusion, with 10 μg of α-synuclein immunogen in 100 μL PBS.

[0365] The immunization regimen is shown in Figure 1 .

[0366] Antibody 37 was derived from an immunization regimen using full-length human α-synuclein fibrils, alternating with truncated C-terminal forms of α-synuclein containing amino acids 1-60 and 1-119.

[0367] Antibody 285 was derived from the following immunization protocol, in which human α-synuclein monomer 1-140 was used for the first four immunizations. If no titer was obtained, immunization was continued using fibrils (intraperitoneal / subcutaneous); otherwise, monomers were used.

[0368] 3. HuMab hybridoma production

[0369] HuMAb mice with sufficient antigen-specific titers as defined above were sacrificed, and spleens and lymph nodes were collected from both sides of the abdominal aorta and vena cava. The fusion of spleen and lymph node cells with mouse myeloma cell lines was performed via electrofusion using the CEEF 50 electrofusion system (Cyto Pulse Sciences, Glenburn, MD, USA), essentially following the manufacturer's instructions. The fused cells were seeded in fusion medium containing 10% Fetal Clone I bovine serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U / mL penicillin, 0.5 U / mL streptomycin (Cambrex), 50 μM 2-mercaptoethanol (Ingenieur), 600 ng / mL interleukin-6 (IL-6) (Strathmann), 1x HAT (Sigma), and 0.5 mg / mL kanamycin (Ingenieur). Ten days later, the supernatant was harvested and the cells were restored using harvest medium containing 10% Fetal Clone I bovine serum (Perbio), 0.5 U / mL penicillin, 0.5 U / mL streptomycin, 600 ng / mL IL-6, and 1x proHT (Cambrex). The supernatant of hybridoma cultures was screened by primary screening assays. The supernatant was characterized by binding to eight different ligands. These ligands included four orthogonal homologs: human α-synuclein, β-synuclein, and human γ-synuclein (SEQ ID NO 37-41) from human, mouse, rat, and cynomolgus monkeys, and their ability to bind to human α-synuclein derivatives lacking α-synuclein residues 120-140 was finally tested.

[0370] Anti-α-synuclein antibodies were screened using a high-throughput suspension ELISA format via an automated liquid handling system (Genmab A / S). Plate readings were performed using two systems: an FMAT 8200 from Applied Biosystems was used to read 384-well plates, and an ImageXpress Velos cell analyzer from Molecular Devices was used to read 1536-well plates.

[0371] In the primary screening, clones were characterized by their ability to bind eight different ligands. These included a series of four hybrid constructs: A-Syn-AAKK-BAP, A-Syn-BAAK-BAP, A-Syn-BBAA-BAP, A-Syn-BBKK-BAP (SEQ ID NOs: 11-14), α-synuclein 120-140 deletion-BAP, nitrated human α-synuclein-BAP, and oxidized human α-synuclein-BAP.

[0372] In short, serum or supernatant potentially containing α-synuclein-specific antibodies was added to the beads to allow binding to α-synuclein and / or α-synuclein-derived constructs. Binding to the anti-α-synuclein antibody was detected using a fluorescent conjugate—Fc-specific DyLight649 conjugated goat anti-human IgG. Two known mouse anti-α-synuclein antibodies, LB509 and Syn211, were included in the screening as positive controls. To ensure specific detection of the α-synuclein antibody, a serum pool containing anti-α-synuclein was used as a negative control in the 384-well titer screening, while human ChromPure IgG was used in the 1536-well 8-bead assay.

[0373] Hybrid tumor cells from the optimal primary wells were seeded in a semi-solid medium prepared with 40% Cloning Medium (CloneMedia) (Genetix, Hampshire, UK) and 60% HyQ 2x Complete Medium (Hyclone, Waltham, USA). For each primary well, cells were seeded in the wells of a Genetix Black 6-well plate. Twenty-five subclones were picked from each well using the ClonePix system (Genetix, Ltd.). The subclones were then placed in harvest medium. After seven days, the subclonal supernatants were screened again for synuclein-specific human IgG binding, and human IgG concentrations were measured using Octet (Fortebio, Menlo Park, USA). The optimal subclones were selected from each primary well and amplified in amplification medium containing only 600 ng / mL IL-6, 0.5 U / mL penicillin, 0.5 U / mL streptomycin, and 1x proHT. Subclones are expanded from one 96-well plate to one 24-well plate, then to four 24-well plates, and finally to six 6-well plates. Clones obtained in this way are designated as primary clones (PCs).

[0374] Further antibody binding studies were conducted using Octet 384RED (Fortebio, Menlo Park, USA). A 2 μg / mL HuMab antibody solution was prepared by dilution in sample diluent (Fortebio, Item 18-5028). Amine-reactive sensor protein (Fortebio, Item 18-0008) was used for HuMab immobilization. HuMab was diluted in MES pH 6.0 buffer (18-5027) before coupling to the amine-reactive sensor protein. Coupling was performed at 30°C and 1000 rpm as follows: the amine-reactive sensor protein was pre-wetted in PBS and then activated for 300 seconds with EDC / NHS (Fortebio, Items 18-1033 / 18-1034) activation solution (according to manufacturer's instructions). The activated sensor protein was immobilized with HuMab over a period of 600 seconds.

[0375] In Octet, 37 and 285 bind to recombinant human, cynomolgus monkey, and mouse α-synuclein, but not to human β- or γ-synuclein, as shown in Figure 2.

[0376] 4. Sequence analysis and cloning of the synuclein-specific HuMab variable domain in the expression vector

[0377] From 0.2 to 5 x 10 6Total RNA was prepared from hybridoma cells and 5'-RACE-complementary DNA (cDNA) was prepared from 100 ng of total RNA using the SMARTRACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. The VH and VL coding regions were amplified by PCR and directly cloned into p33G1f and p33κ expression vectors (containing the coding sequence for the human IgG1. / κ constant domain) by ligating independent clones (Aslanidis, C. and PJ de Jong, Nucleic Acids Res 1990; 18(20):6069-74). For each antibody, 16 VL clones and 16 VH clones were sequenced. Clones with the correct open reading frame (ORF) were selected for further study and expression. Vectors for transient co-expression of all combinations of heavy and light chains were used in Freestyle™ 293-F cells using 293fectin.

[0378] In the case of GM37, sequencing of the VH region identified an additional cysteine ​​residue at position 106 of the CDR3 domain. To eliminate the possibility of misfolding due to disulfide bond formation and the potential loss of antibody activity, the cysteine ​​residue at position 106 was mutated to a serine residue.

[0379] The contrast antibody 9E4 was generated based on the VH and VL sequences derived from hybridoma PTA-8221 (US Patent 20080175838) (SEQ ID NO: 42 and 43).

[0380] 5. Antibody expression / purification

[0381] Antibodies were generated in HEK293 6E cells via transfection using the pTT5 vector and PEIpro as a transient transfection reagent (National Research Council of Canada). In summary, heavy and light chains were transfected into HEK293 cells using PEIpro (VWR), and TN1 (Sigma-Aldrich) was added to the cells 24 hours after transfection. Cells were grown until viability approached 50%, and antibody production was measured using a simple IgG titer (Thermo). Culture supernatant was filtered through a 0.2 μm dead-end filter, loaded onto a 5 mL Protein A column (rProtein A FF, Amersham Bioscience), and eluted with 0.1 M citric acid-NaOH (pH 3). The eluent was immediately neutralized with 2M Tris-HCl (pH 9) and dialyzed against 12.6mM NaH₂PO₄, 140mM NaCl, pH 7.4 (B. Braun), O / N. After dialysis, the sample was aseptically filtered through a 0.2μm dead-end filter. Purity was determined by SDS-PAGE, and concentration was measured by turbidimetric assay and absorbance at 280 nm. The purified antibody was aliquoted and stored at -80°C.

[0382] Example 2: Characterizing antibodies using surface plasmon resonance

[0383] use The real-time binding of the antibody to α-synuclein was measured using a CM5 chip. Capture surfaces were prepared in the first flow cells (Fc1) and second flow cells (Fc2) using amine-coupled polyclonal rabbit anti-mouse antibody (partial mouse antibody capture kit, GE Healthcare, catalog number: BR-1008-38). Mouse antibody was captured in Fc2 at the concentration required to achieve a ligand level of approximately 500 RU. Baselines were allowed to stabilize for 10 minutes before injecting the analyte (ASynBAP) at 30 μl / min in Fc1-2. ASynBAP was run at 100–3200 nM and 25–3200 RU, respectively. The highest concentration was repeated once in each titration series. At the end of each cycle, the surface was regenerated with 10 mM glycine-HCl (pH 1.7) (30-second injection) to remove captured mouse antibody and analyte. HBS-EP (GE Healthcare, catalog number: BR-1001-88) was used as the run buffer, and sample dilutions and experiments were run at 25°C in all experiments. All samples were kept at 4°C before being obtained.

[0384] The responses recorded in Fc1 (where the capture antibody has been fixed but no α-synuclein antibody has been captured) are subtracted from the responses recorded in Fc2. Using BIA evaluation software version 4.1.1, the 1:1 or 2:1 binding algorithm is fitted to the dataset. Results showing the binding of antibodies 37, 285, and 9E4 to human α-synuclein can be obtained... Figure 3 , 4 As seen in 5.

[0385] Example 3: Tabletop Plotting

[0386] Antibody mapping for α-synuclein epitopes was performed using an array of overlapping linear peptides on a Pepscan (Pepscan Zuidersluisweg 2 8243RC, Lelystad, The Netherlands). Binding of the antibody to each synthesized 20-meric peptide was detected by a Pepscan-based ELISA. Linear peptide arrays covering the entire coding sequence of α-synuclein, as well as all peptides with oxidized methionine or nitrosotyrosine, were incubated with a primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated for 1 hour at 25°C with a 1 / 1000 diluted antibody-peroxidase conjugate (SBA, cat.nr.2010-05). After washing, the peroxidase substrate 2,2'-azono-di-3-ethylbenzylthiazoline sulfonate (ABTS) and 2 μl / ml of 3% H2O2 were added. Colorimetric assay was performed after 1 hour. Quantitative colorimetric development was performed using a charge-coupled device (CCD) camera and image processing system. For data processing, values ​​ranging from 0 to 3000 mAU were obtained from the CCD camera, similar to a standard 96-well plate ELISA reader. The results were quantified and stored in the Peplab database. Occasionally, a well contained air bubbles that caused false positive values; the card was manually checked, and any values ​​caused by air bubbles were recorded as 0. Binding data for antibodies 37 and 285 with peptides containing the sequences ILEDMP or ILED, respectively, are available in [data missing]. Figure 7 I saw it in the middle.

[0387] Example 4: Immunoprecipitation of α-synuclein from human brain homogenate derived from the cingulate cortex of a Lewy body dementia patient

[0388] The ability of antibodies to bind to and pull down α-synuclein from coarse homogenates of the cingulate cortex from human DLB or healthy controls (marked *) was analyzed by immunoprecipitation. Frozen samples of the human cingulate cortex (available from Tissue Solutions, Scotland) were dissected in a cryostat, and 100 mg of sample was added to 1600 μl of celLytic M cell lysis reagent (Sigma C2978), which contains protease inhibitors and phosphatase inhibitors (Roche). Brain tissue was homogenized using a Precellys bead homogenizer (Bertin Technologies, France) at 4 × 30 seconds, 5000 rpm until the sample was completely dissolved. The solution was centrifuged at 3000 × g, and the supernatant was used as the coarse homogenate for immunoprecipitation.

[0389] For immunoprecipitation, 10 μg of antibody was mixed with Dynabeads protein G beads according to the manufacturer's instructions (LifeTechnologies, Paisley, UK). The crude brain homogenate was diluted 30-fold in lysis buffer (Sigma). The antibody-conjugated dynabeads were mixed with 500 μL of the diluted homogenate and incubated for 90 minutes at room temperature in a vortex mixer. After incubation, the beads were washed in washing buffer according to the manufacturer's instructions (Dynabeads G protocol, LifeTechnologies, Paisley, UK), and the bound antigen was eluted with non-denaturing elution buffer. The immunoprecipitation yield was visualized using Western blotting with the mouse monoclonal anti-human α-synuclein antibody (4B12, Thermo Scientific). The band patterns representing different molecular weight forms of pulled-down α-synuclein differ between antibodies 37, 37v2, and 285 and the contrast antibody 9E4 because antibodies 37, 37v2, and 285 can immunoprecipitate the major α-synuclein species: full-length α-synuclein (FLasyn 1-140) and C-terminal truncated species (1-135 and 1-119 / 122), while antibody 9E4 cannot immunoprecipitate the truncated species 1-119 / 122. Figure 9 .

[0390] Example 5: Antibodies in cell cultures inhibit protease truncation of α-synuclein fibrils.

[0391] Recombinant α-synuclein monomers and fibrils can be taken up by primary neurons in culture medium. For example, in Figure 10The diagram illustrates that after α-synuclein is taken up in neurons, it can be processed by intracellular proteases such as calpain I, which has a major protease-sensitive site at amino acid 119 / 122. To investigate protease truncation of α-synuclein, mouse primary cortical neurons were prepared as described in Elvang et al. (2009) (Elvang et al., Journal of Neurochemistry, 2009, 110(5):1377-87) and treated with cytarabine at DIV3 (3 days in vitro) to inhibit astrocyte growth. At DIV4 (4 days in vitro), neurons were treated alone or with a specified concentration of antibody at a final concentration of 0.7 μM with pre-formed α-synuclein fibrils (PFFs) by sonication (5 minutes, at 50% power in a cup-angle sonicator). After 24 hours of culture, the culture medium was harvested and the cells were lysed. The cells were then treated with 4B12 antibody ( Figure 11A (Pierce Ma1-90346) and a second anti-mouse antibody were used to perform Western blotting on both culture medium and cell lysates. After detection with 4B12+ anti-mouse, the blot was stripped and re-detected with anti-human IgG antibody. The 4B12 blot showed strong bands at 14 and 12 kDa in the culture medium treated with PFF alone, where 14 kDa represents the full-length α-synuclein (FL-ASYN) and 12 kDa represents the C-terminal truncated fragment 1-119 / 122 (CT-asyn). In addition, there were higher molecular weight bands, most likely representing SDS-resistant oligomers. Co-treatment with the isotype control antibody B12 did not alter this pattern of proteolysis or uptake.

[0392] In the culture medium from cells treated with fibrils and 37, full-length α-synuclein (14 kD) was predominantly present, along with a small amount of truncated terminal bands (12 kD). In cell lysates from cells treated with fibrils and 37, only full-length α-synuclein was present, indicating that 37 prevented the cleavage of FL-α-synuclein. Furthermore, the total amount of FL-α-synuclein was reduced compared to cells treated with only PFF or B12 control antibodies. Several groups have shown that α-synuclein can be cleaved by calpain-1 (Games et al., *American Journal of Pathol*, 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., *American Journal of Pathol*, Vol. 170, No. 5, May 2007). The cleavage site of calpain-1 on fibrillary α-synuclein has been found in region 114-122 (Mishizen-Eberz, *Journal of Neurochemistry*, 86, 836-847, 2003). In transgenic animals and the human brain, 1-119 / 122 appears to be the major cleavage product of α-synuclein. Cleavage likely involves aspartic acid 119 or asparagine 122 followed by deamidation to aspartic acid, and then cleavage by calpain or another protease with similar cleavage specificity. These results indicate that antibody 37 can inhibit C-terminal truncation of α-synuclein. The epitope of antibody GM37 overlaps with the binding site of the enzyme calpain-1, therefore, 37 binding to α-synuclein can directly inhibit calpain-1-mediated binding and cleavage. Figure 10 and 11).

[0393] Epitopes 285 and 37 overlap and are also expected to inhibit protease cleavage. The amino acid sequence of 37v2 differs from 37 at only one amino acid in the CDR region and exhibits similar binding; therefore, it is also expected to inhibit protease cleavage in a similar manner to 37. To investigate whether the antibody effect is dose-dependent, a 24-hour assay was established using PFF and antibodies co-applied to primary cortical neurons. The concentration of PFF was stable (10 μg / ml), while the concentrations of the control antibody B12 and antibodies 37, 37v2, and 285 were 10, 5, 1, and 0.1 μg / ml, respectively. α-synuclein on a Western blot was detected using the 1904 / 4B12 antibody (Abcam), which has epitopes in regions 103-108 that bind to both full-length and C-terminally truncated α-synuclein. Figure 11B ). For example from Figure 11B As can be seen, GM37, 37v2 and GM285 have dose-dependent inhibition of protease cleavage, and at high antibody concentrations, they have almost complete inhibition of cleavage.

[0394] Example 6: Antibody-mediated inhibition of α-synuclein aggregate seeding in cell cultures

[0395] Several studies have shown that exogenously added recombinant α-synuclein fibrillary aggregates can enter cells and recruit endogenous α-synuclein, as well as induce α-synuclein aggregation and phosphorylation in vitro and in vivo, similar to LB. (Volpicelli-Daley et al., 2011; Luk et al., 2012a; Luk et al., 2012b; Recasens et al., 2013; Peelaerts et al., 2015). To study the seeding of endogenous mouse α-synuclein by recombinant α-synuclein seeding, mouse primary cortical neurons prepared as described above were seeded in 96-well plates (15,000 cells per well). On day 5 of in vitro culture (DIV), 50% of the medium was altered and supplemented with cytarabine (final concentration 1 uM). At DIV6, using α-synuclein fibrillary material, whether coarse fibrillary seed or pure seed, half of the medium was altered to glial conditioned medium. Crude protofibril seeds were prepared from recombinant human α-synuclein monomers isolated from bacteria. The monomers were filtered through an Amicon Ultra 100,000 cut-off filter (Millipore, catalog number UFC510096) and adjusted to a concentration of 1 mg / ml in PBS (pH 7.4). To prepare crude protofibril seeds, the monomer solution was incubated in a heated stirrer (37°C) with continuous stirring (800 rpm) until template levels were reached (assessed using thiosulfate S via routine measures). To minimize evaporation, droplets of mineral oil were added to cover the solution. The total incubation time was 5–7 days. Pure seeds were prepared from the crude protofibril seeds, which were centrifuged to purify them, and the polymerized particles were resuspended in fresh PBS and sonicated. At DIV6, antibodies were added once along with the crude α-synuclein seeds. Half of the medium in the primary neurons was replaced weekly with glial conditioned medium to maintain them until DIV21. Neurons were immobilized, and phosphorylated α-synuclein amino acid S129-specific rabbit antibody (abcam 51253) was used to stain the phosphorylated synuclein, followed by staining with a fluorescently labeled anti-rabbit antibody. Fluorescence was quantified using an automated fluorescence microscope (Cellomics Arrayscan). Nuclei were detected in one channel to determine the number of effective cells. Phosphorylated α-synuclein spots were detected in another channel within a predefined ring region around the nucleus (thus representing the cytoplasm of the cell). The average number of spots per cell was calculated. An example of cell staining is shown in [illustration missing]. Figure 12A Phosphorylated α-synuclein spots do not appear in untreated neurons. Crude or pure seed-incubated neurons (1-10 ng per well) induce α-synuclein phosphorylation. Figure 12AIn neurites, phosphorylated synuclein appears as spots or dots, and some phosphorylated synuclein appears in an elongated form in neurites.

[0396] For fractionation studies, cells were harvested and centrifuged in phosphate-buffered saline (PBS). The precipitate was resuspended in 1% Triton buffer containing protease inhibitors. Samples were held on ice for 15 minutes, followed by sonication. Samples were centrifuged at 100,000 × g at 4 °C for 30 minutes. The supernatant was collected and labeled as the soluble fraction. The precipitate was washed once in Triton buffer and resuspended in 1% SDS buffer, followed by sonication. Samples were centrifuged again at 100,000 × g for 30 minutes. The supernatant of the insoluble fraction was collected. Protein concentrations were determined and samples were blotted onto membranes on 4%–12% SDS-PAGE gels. α-synuclein and phosphorylated α-synuclein (S129P) were detected using 4B12 / 1904 antibody (Thermo Scientific: MA1-90346 human synuclein), S129P-asyn antibody (Abicon 51253), and mouse synuclein antibody (Cell Signal-D37A6).

[0397] Figure 12B Western blots showing the soluble and insoluble fractions of primary neurons with and without coarse seeds. From Figure 12B It can be seen that the addition of seeds leads to the accumulation of multimers of endogenous mouse α-synuclein, p-S129-α-synuclein, and phosphorylated mouse α-synuclein in the insoluble fraction of the cell.

[0398] To test whether the antibody could inhibit seeding, α-synuclein seed was used at a concentration of 6.6 nM (10 ng / well). Different concentrations of antibody and α-synuclein seed were added together on DIV 6 to obtain a dose response (starting from the highest antibody concentration of 133 nM and decreasing to 133 pM). Neurons were refixed and stained with phosphosynuclein (Ibicon 51253), and fluorescence in the cells was quantified using an automated fluorescence microscope (Cellomics arrayscan). Spots / dots in each well were counted using a Cellonics arrayscan. Figure 12CAs can be seen, antibodies 37, 37v2, and 285 reduced α-synuclein phosphorylation in neurons in a dose-dependent manner, with 37, 37v2, and 285 showing similar maximum inhibition (around 70%-75%) at approximately 5 nM EC50. After treatment with the highest concentration (133 nM), cellular protein fractionation was divided into soluble and insoluble fractions, showing that antibodies 37, 37v2, and 285 inhibited the accumulation of truncated and C-terminal truncated fragments (CT α-syn) of recombinant crude seeds and reduced the accumulation of phosphorylated endogenous mouse α-synuclein and its aggregated form in the insoluble fraction, as shown in... Figure 12D As shown in the image.

[0399] Example 7. Acute electrophysiological effects of α-synuclein antibodies in vivo

[0400] High levels of human α-synuclein were present in the hippocampus of F28-snca transgenic mice, a model of wild-type α-synuclein overexpression under the control of the mouse α-synuclein promoter (Westerlund M et al., Molecular Cell Neurosci, Dec. 2008, 39(4):586-91). Synaptic transmission and plasticity in the CA1 region of the hippocampus were assessed by in vivo electrophysiology in male F28-snca transgenic and age-matched control mice aged 4–6 months. Data showed that basal synaptic transmission was significantly impaired in F28-snca transgenic mice compared to age-matched control mice. Figure 13 ).

[0401] F28-snca transgenic mice aged 4 to 6 months and age-matched control male mice (CRO breeding, Taconic Europe A / S) were individually housed under controlled temperature (22℃ ± 1.5℃) and humidity (55%–65%) conditions and maintained in a 12:12 hour light / dark cycle (lights were turned on at 06:00). Food and water were readily available.

[0402] Animals were anesthetized via intraperitoneal (ip) injection of urethane (1.2 g / kg). Mice were then fixed in a stereotaxic frame, the temperature of which was regulated to 37.5°C using a heating pad, with the skull exposed. Platinum wires were placed in the frontal bone as a reference, and additional holes were drilled for the insertion of recording and stimulation electrodes into the hippocampus. These holes were positioned according to the following coordinates according to the Rat Brain Stereotaxic Atlas (Paxinos and Franklin, Mouse Brain in Stereotaxic Coordinates, 4th Edition, 2001): Recording, 1.5–1.7 mm posterior to the anterior fontanelle, 1.0–1.2 mm lateral to the midline, 1.4–1.7 mm inferior to the cerebral surface; Stimulation, 1.8–2.0 mm posterior to the anterior fontanelle, 1.5–1.7 mm lateral to the midline, 1.5–1.7 mm inferior to the cerebral surface. Animals remained in the stereotaxic frame throughout the recording duration, and their anesthesia level was checked periodically.

[0403] Electrical stimulation via the Scheffer collaterals every 30 seconds evoked field potentials (fEPSPs) in the CA1 region, and the depth of the recording electrodes was adjusted until a negative fEPSP was recorded in response to a unipolar rectangular pulse. The slope of the evoked fEPSP was determined to be between 30% and 70% of the maximum amplitude of the fEPSP.

[0404] Once the optimal fEPSP was induced, basal synaptic transmission was assessed by the relationship between stimulus intensity and the slope of the induced fEPSP (input-output relationship). Different stimulus intensities of 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μA were applied sequentially in increasing order, with each intensity repeated 2 to 3 times. Basal synaptic transmission was found to be significantly impaired in F28-snca transgenic mice compared to age-matched control mice.

[0405] Damage identified by basal synaptic transmission in F28-snca transgenic mice was used to test the ability of GM37, GM285, and the comparative substance h9E4 to block α-synuclein-mediated effects.

[0406] Recordings were performed in all experiments 3 to 6 hours after a single dose of antibody was administered at a dose of 15 mg / kg (ip). If possible, basal synaptic transmission was recorded in both hippocampi of each animal and recorded as a separate experiment.

[0407] Acute treatment with h9E4 induced a significant reversal of basal synaptic transmission impairment in F28-snca transgenic mice (Tg-snca+h9E4 vs. Tg-snca+PBS, p = 0.002). Figure 14However, the reversal via h9E4 was only partial, as indicated by the significant difference in basal synaptic transmission compared to littermates treated with PBS (p = 0.007).

[0408] Acute treatment with GM37 significantly reversed the impairment of basal synaptic transmission induced in F28-snca transgenic mice (Tg-snca+GM37 vs. Tg-snca+PBS, p = 0.004). Figure 15 The basal synaptic transmission in transgenic mice treated with GM37 was not significantly different from that in littermates treated with PBS, indicating complete reversal of the damage. Figure 15 ).

[0409] GM285 also induced a significant reversal of basal synaptic transmission impairment in F28-snca transgenic mice. Figure 16 The basal synaptic transmission in transgenic mice treated with GM285 was not significantly different from that in littermates treated with PBS, indicating complete reversal of the damage.

[0410] Example 8. Microdialysis for evaluating human α-synuclein in the brains of awake, freely moving animals.

[0411] The levels of human α-synuclein in cerebrospinal fluid (ISF) were assessed using a push-pull microdialysis method. Mice were housed individually under controlled temperature (22℃ ± 1.5℃) and humidity (55%–65%) conditions with a 12:12 hour light / dark cycle (lights on at 06:00 h). Food and water were provided free access. The current study was conducted in the hippocampus of F28-snca transgenic mice (50–54 weeks old). To enable microdialysis in the hippocampus, mice were anesthetized with isoflurane and a stereotactic intracerebral guide cannula (CMA) was stereotactically implanted into the brain. The microdialysis probe was positioned in the hippocampus according to a rat stereotactic atlas (theatlas of Paxinos and Franklin 2001) (probe tip coordinates: 3.1 mm posterior to the anterior fontanelle and 2.8 mm lateral to the anterior fontanelle, and 1.3 mm relative to the dura mater). Fixing bolts and acrylic adhesive were used to secure the guide cannula. After cannula implantation, mice were allowed 2-3 days to recover from surgery before dialysis.

[0412] On the day of the experiment, a 2-mm, 1000kDa truncated CMA probe was inserted through a guide cannula. The probe was connected to a two-channel microdialysis peristaltic pump (MAB20; Microbiotech) and operated in push-pull mode. The inlet tube of the microdialysis probe was connected to the peristaltic pump to perfuse the probe with artificial cerebrospinal fluid (aCSF; 147mM NaCl, 2.7mM KCl, 1.2mM CaCl2, 0.85mM MgCl2). The peristaltic pump was also connected to the outlet tube to prevent loss of perfusion fluid from the probe when the fluid was pulled through the tube. As a perfusion buffer, 25% bovine serum albumin fraction V (Sigma) was diluted to 0.2% with artificial CSF on the day of use and filtered through a 0.1-μm filter membrane. The actual flow rate of the pump was determined without the probe connected. The sample tubes were weighed before and after sampling for a given period of time, and the flow rate was calculated. The pump was then set to a constant flow of 1 μL / min. A 120-minute sampling protocol was used throughout the experiment. To avoid interference from tissue damage, the experimental window was set from 14 to 48 hours after probe implantation. 14–16 hours after the start of the experiment, 15 mg / kg of GM37, human 9E4, or an allotype control (anti-HEL) was injected (intraperitoneally), and six additional samples were collected (collected over 12 hours). The dialysate was stored at -80°C. The concentration of human α-synuclein was determined by ELISA (Covance ELISA kit).

[0413] The mean of 2-3 baseline values ​​(4-6 h) prior to antibody treatment in each animal was used as the baseline and set as 100%. Two-way ANOVA with repeated measures was used to assess statistical relevance. The baseline level of human α-synuclein in the hippocampus was 8.1 ± 1.1 ng / ml (mean ± SEM, n = 25, without correction for in vitro dialysis probe recovery). Compared to the two contrasting antibodies (human 9E4 and isotype control (anti-HEL)), administration of GM37 induced a significant reduction in human α-synuclein in the hippocampus of F28 mice. Figure 17 ).

[0414] Example 9: Long-term effects of α-synuclein antibody in vivo. Antibody GM37 improved the motor phenotype in a rat model of Parkinson's disease.

[0415] Targeted expression of human α-synuclein in dopaminergic neurons of the rat midbrain can be achieved using a recombinant adeno-associated virus vector (rAAV) and is associated with progressive loss of dopaminergic cells in the substantia nigra and motor impairment.

[0416] Adult female Sprague-Dawley rats (225–250 g) were used to express human α-synuclein in the substantia nigra (SN) by injection of adeno-associated virus of serotype AAV2 / 5, as previously described. This adeno-associated virus contains a chicken β-actin promoter, an enhancer element with a promoter derived from cytomegalovirus, followed by cDNA of human α-synuclein and WPRE elements (Xu L, Daly T, Gao C, Flotte TR, Song S, Byrne BJ, Sands MS, Ponder KP (2001)). In this model, it has been shown that expression of human α-synuclein leads to neurodegeneration of dopaminergic neurons. (Maingay M et al., Central Nervous System Spectroscopy, March 2005, 10(3):235–44). To test the efficacy of therapeutic antibodies against α-synuclein in this model, antibody treatment was initiated 2 to 4 days prior to viral injection and continued until the end of the study. Figure 18 A control group was administered PBS of the same volume (5 ml / kg:IP). GM37 was administered twice weekly at a dose of 15 mg / kg (IP). Viral particles (rAAV2 / 5) containing the human wild-type α-synuclein or green fluorescent protein (GFP) gene were injected unilaterally into the SN. Animals were then treated with... and The animals were anesthetized with 2.0 ml / kg (subcutaneously) and placed in a stereotactic frame. Their temperature was regulated to 37.5°C using a heating pad, and their skulls were exposed. Based on the rat brain stereotactic atlas (Paxinos and Watson, 1998), a hole was drilled above the right SN at the following coordinates: 5.5 mm posterior to the anterior fontanelle and 2.0 mm lateral to the anterior fontanelle. A single injection of 3 μL of rAAV2 / 5-α-syn or rAAV2 / 5-GFP was administered at a depth of 7.2 mm below the dura mater material, connected to the stereotactic syringe at a flow rate of 0.2 μL / min using a Hamilton syringe. The needle was left in place for an additional 5 minutes to allow diffusion within the SN. Postoperatively, the animals were returned to their cages and placed in a heated environment to allow them to recover from anesthesia. Tests for motor asymmetry in the cylinder test were evaluated before AAV injection and at 3, 7, and 10 weeks after AAV injection. The presented data correspond to the ratio between the use of the right forepaw and the use of both the left and right forepaws. Ten weeks after viral injection, each animal was filmed for a total of 5 minutes in the cylinder, and the number of touches made with the left and right forepaws within those 5 minutes was manually scored on the last day of testing. Significant impairment was observed in AAV-syn at week 10 compared to AAV-GFP injected rats (p = 0.012). A reversal trend was shown for GM37-treated animals, as their performance differed from GFP rats (p = 0.163 and p = 0.407 for GM37, respectively). This finding suggests that antibody GM37 can improve the motor phenotype of Parkinson's disease in this rat model. Figure 18 and 19 ).

[0417] Example 10: Long-term effects of an in vivo α-synuclein antibody. Antibody GM37 inhibits endogenous mouse α-synuclein aggregates and phosphorylated seeding.

[0418] Recombinant protein-based α-synuclein preformed fibrils were injected into the dorsal striatum of wild-type mice to recruit endogenous mouse α-synuclein and induce the formation of Ser-129 phosphorylated aggregates in neurons of the cortex, amygdala, and substantia nigra (Luk et al., 2012, Science, November 16, 2012, 338(6109):949-53). To determine whether the α-synuclein-specific monoclonal antibody GM37 could reduce the formation of phosphorylated α-synuclein inclusions induced by α-synuclein fibrils in vivo, a total of 45 mice were used. Mice were administered 30 mg / kg GM37 (intraperitoneal), 15 mg / kg GM37 (intravenous), or a carrier (intraperitoneal) (PBS). One day later, mice were anesthetized and stereotactically positioned in a hemisphere containing a 2 μL coarse seed of recombinant human α-Syn, prepared as previously described (Example 6) (total 2 μg coarse seed per animal). To inject the coarse seed, the skull was opened by drilling and a single glass pipette (coordinates: anterior fontanelle +0.5 mm, lateral midline +2.0 mm) was inserted into the right forebrain to position the inoculum in the dorsal striatum (subdural +2.6 mm). After recovery, mice received weekly intraperitoneal or intravenous antibody injections until sacrificed at day 45. Groups of 15 mice were administered GM37 via intravenous injection of 15 mg / kg, intraperitoneal injection of 30 mg / kg, or intraperitoneal injection of PBS (10 ml / kg) once weekly.

[0419] To measure antibody concentration in plasma, buccal blood was collected weekly prior to the next injection, i.e., 7 days after the last injection. Plasma was obtained by rotating 2000g, incubated at room temperature for 15 minutes, and then the supernatant was frozen at -20°C. CSF samples were collected at the end of the study and frozen at -20°C. Plasma and CSF samples were analyzed by MSD to determine the concentration of human IgG. Briefly, mouse anti-human IgG (clone MH16-1 (M1268)) was used for capture, plasma or CSF was incubated in wells, followed by sulfo-TAG goat anti-human as the detection antibody (MSD catalog number: R32AJ-1). Electrochemiluminescence analysis of the plates was performed by MSD.

[0420] Antibody levels in plasma are shown to Figure 20B The study showed a dose-dependent increase in plasma antibody concentration and accumulation of plasma antibodies over six weeks. CSF antibody levels were shown in... Figure 20C In addition, it shows that plasma antibody levels of approximately 0.1% can be measured in CSF.

[0421] Forty-five days after α-synuclein fibrillary seeding, mice were anesthetized and perfused cardiacally with PBS, followed by perfusion with neutral-buffered paraformaldehyde (4%). Brains were harvested, fixed in neutral-buffered paraformaldehyde, and cultured overnight. Immunohistochemistry was performed on 45 μm thick serial sections using neuroscience-related methods. Briefly, using... The technique involved embedding up to 25 mouse brains together, dividing them into three sections, and then freezing them in the coronal plane to a thickness of 45 μm. The sections were collected in cups containing antigen preservation solution. Each sixth section was stained with an antibody to form Ser-129 phosphorylated α-synuclein (anti-α-synuclein (phosphorylated S129) antibody [PSYN / 81A]ab184674) to visualize the reactive structure of Ser-129 phosphorylated α-synuclein.

[0422] Quantification of pSyn pathology was performed by manually counting immunoreactive cells derived from 10x magnification images of sections 5–7 covering the entire substantia nigra from each of the sixth sections. Masked counting was conducted. Cell counts in the amygdala and substantia nigra were analyzed by one-way ANOVA followed by a Bonferroni t-test, where the effect of the GM37 antibody was compared to PBS treatment.

[0423] from Figure 20C As can be seen, compared with the PBS control, treatment with antibody GM37 significantly reduced the number of intracellular inclusions in the substantia nigra, regardless of whether intraperitoneal or intravenous treatment was used. These data suggest that antibody GM37 may have a therapeutic effect in PD by blocking the entry of extracellular pathological α-synuclein into neurons, by blocking its propagation between neurons, and / or by promoting clearance from ISF through glial cell uptake. This appearance of inclusions is associated with the loss of dopaminergic neurons and the development of motor disorders in Parkinson's disease in animal models, and treatment with antibody GM37 may have a therapeutic effect on the loss of dopaminergic cells and the development of motor disorders in PD.

[0424] Example 11: Manufacturability of GM37 and GM37 variants

[0425] Anti-α-synuclein antibodies were produced in mammalian cell cultures under conditions simulating those used to manufacture clinical-grade materials for patient use. It is well known that proteins produced in this manner undergo biophysical post-translational modifications that can affect the therapeutic efficacy and stability of the antibody over time. Decades of research have established empirical knowledge that has identified a set of post-translational modifications known to pose a risk to the manufacturability of specific molecules. These post-translational modifications have been shown to be associated with amino acid strings present in the primary sequences of heavy and light chain proteins. Algorithms have been developed that can identify these sequences and determine their potential risks to the manufacturability and manufacturability of therapeutic antibodies.

[0426] Computerized analysis of the primary sequence of antibodies has the potential to reduce the risk of molecules being developed as therapeutics. In particular, detailed analysis of the VH and VL regions can identify unique amino acids considered important for molecular activity but also potentially dangerous for its stability over time. Sequence-specific deamidation has been identified as a potential hazard to protein structure. Protein deamidation can occur on the amide side chains of glutamine or asparagine residues, converting them to carboxylic acid ester groups (Lorenzo et al., PLOS ONE, DOI:10.1371 (December 2015)). For asparagine, non-enzymatic deamidation occurs more rapidly at neutral pH and is therefore considered to pose a higher risk than glutamine. Activity is further affected by subsequent amino acids in the sequence and can occur at rates over days or years. The fate of proteins that have undergone deamidation needs to be experimentally evaluated to determine the effects of the alteration on both its stability and activity.

[0427] We identified the deamidation site within the VH domain of GM37. Amino acid residue 54 is asparagine (N), followed by glycine (G) at position 55. N54 carries a high risk of spontaneous amide formation. To mitigate this risk, we generated a set of three variants, replacing asparagine (N) with serine (S), glutamine (Q), or histidine (H). All three variants were produced in mammalian cell cultures using a transient transfection method (Example 1.5). All three variants exhibited similar expression and purification characteristics to GM37wt. Figure 23 ).

[0428] For each of the eight products, transient transfection was performed using 400 ml of CHOK1SV GS-KO cells that had been cultured in medium for at least 2 weeks. Cells were passaged 24 hours prior to transfection. All transfections were performed via electroporation using GenePulseXcell (Bio-Rad). For each transfection, live cells were resuspended in preheated CD-CHO medium supplemented with 6 mM L-glutamine to a final volume of 2.86 x 10⁻⁶. 7Cells / ml. 40 μg of each of the separately prepared SGV DNAs containing appropriate heavy and light chains was aliquoted into each reaction cuvette (Bio-Rad Laboratories, GenePulser cuvette, 0.4 cm gap, 165-2088), and 700 μl of cell suspension was added. Cells were electroporated at 300 V, 900 μF. Transfected cells were transferred to pre-warmed medium in Erlenmeyer flasks, and the contents of the reaction cuvettes rinsed twice with pre-warmed medium were also transferred to the flasks. Transfected cultures were incubated in a shaking incubator at 36.5 °C, 5% CO2, 85% humidity, and 140 rpm for 6 days. Cell viability was measured at harvest using a Cedex HiRes automated cell counter (Rosche).

[0429] To evaluate the importance of residue 54 in binding to human α-synuclein, we analyzed the binding ability of variants in two different experiments. Using a competitive ELISA format, we assessed the effect of variations on residue 54 on the ability of GM37 to bind α-synuclein in solution. By evaluating the concentrations that inhibited antibody binding to synuclein coated on ELISA plates, we found that all three variants maintained the same binding as GM37wt and bound to α-synuclein with high affinity resulting in IC50s of 1–2 nM. Figure 24 A competition assay was performed at room temperature using each of the following antibodies at a fixed concentration (0.3 μg / ml): GM37 (i.e., GM 37wt), GM37 variant 1, GM37 variant 2, and GM37 variant 3, pre-incubated with human α-synuclein in the range of 0–1000 nM for 60 min. The remaining unbound antibody was captured and measured by electrochemiluminescence (MSD, Gathersburg, MD) on ELISA plates coated with 100 ng / ml recombinant human α-synuclein using an anti-human detection antibody. The IC50 values ​​for the interactions of GM 37wt, GM37 variant 1, GM37 variant 2, and GM37 variant 3 were 1.9 nM, 1.6 nM, 2.1 nM, and 1.4 nM, respectively (e.g., using Prism). (Confirmed).

[0430] Using surface plasmon resonance (SPR), we evaluated the real-time binding kinetics of GM37wt (2 batches) and the three variants (Example 2). Human α-synuclein was captured onto a slide (ligand), and the antibodies were tested as analytes at multiple concentrations. Analysis of binding profiles at multiple antibody concentrations showed that the rates of all four antibodies were identical; similarly, the distance rates measured when the antibodies were removed from the buffer showed no statistically significant differences between the antibodies. Using a 1:1 binding algorithm, all four antibodies had nearly identical binding constants (…). Figure 25 ).

[0431] To assess the impact of changes at N54 on the functional activity of GM37, we analyzed the ability of antibodies to block synuclein seeding activity in primary neuron cultures (Example 6). Seeding levels were determined using antibodies specific to phosphosynuclein. As measured by phosphosynuclein signaling, all three antibodies were able to block seeding ( Figure 26 Furthermore, the level of inhibition was the same for all four antibodies. This cell-based data further confirms the binding data: the binding affinity of human α-synuclein and the inhibition based on seeding in primary cell assays do not require amino acid 54 in the VH domain. Additionally, we found that all three antibodies could be produced using standard expression and purification methods. Interestingly, one of the N54Q variants showed an improvement in production compared to the other variants, which is crucial when antibodies are to be produced on a large scale for commercial purposes. These data support the possibility of mitigating the potential dangers of deamidation by replacing asparagine (N) with other amino acids without concern for potency loss.

[0432] Samples of each antibody (wild-type GM37, variant 1, variant 2, and variant 3) were subjected to temperatures that steadily increased over time, and aggregation levels were simultaneously determined by multi-angle light scattering (Prometheus NT.48, NanoTemper Technologies). The temperatures at which GM37 and the GM37 variants began to aggregate were found to be similar; however, the lowest level of aggregation was observed in GM37-variant 2. Figure 27 ). sequence list <110> H. Lundbeck A / S <120> No new monoclonal anti-α-synuclein antibody <130> 0992 <160> 43 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial <220> <223> GM37 CDR 1 heavy chain <400> 1 Gly Phe Thr Phe Ser Ser Tyr Ala Met Thr 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> GM37 CDR2 heavy chain <400> 2 Ala Ile Arg Ser Asn Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial <220> <223> GM37 CDR3 Heavy Chain <400> 3 Ala Lys Asn Trp Ala Pro Phe Asp Ser 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial <220> <223> GM37 CDR1 Light Chain <400> 4 Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> GM37 CDR 2 Light Chain <400> 5 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 6 <211> 9 <212> PRT <213> artificial <220> <223> GM37 CDR 3 light chain <400> 6 Gln Gln Tyr Gly Ser Ser Pro Trp Thr 1 5 <210> 7 <211> 116 <212> PRT <213> artificial <220> <223> GM37 CDR heavy chain <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Arg Ser Asn Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Ala Pro Phe Asp Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 108 <212> PRT <213> artificial <220> <223> GM 37 light chain <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 6 <212> PRT <213> artificial <220> <223> Epitope 112 - 117 <400> 9 Ile Leu Glu Asp Met Pro 1 5 <210> 10 <211> 140 <212> PRT <213> artificial <220> <223> α - synuclein <400> 10 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140 <210> 11 <211> 165 <212> PRT <213> artificial <220> <223> A‑Syn‑AAKK‑BAP <400> 11 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Asn Ile Ala Ala Ala Thr Gly Leu Val Lys 85 90 95 Lys Asp Gln Leu Ala Lys Gln Asn Glu Glu Gly Phe Leu Gln Glu Gly 100 105 110 Met Val Asn Asn Thr Asp Ile Pro Val Asp Pro Glu Asn Glu Ala Tyr 115 120 125 Glu Met Pro Pro Glu Glu Glu Tyr Gln Asp Tyr Glu Pro Glu Ala Gly 130 135 140 Ser Ala Gly Gly Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys 145 150 155 160 Ile Glu Trp His Glu 165 <210> 12 <211> 165 <212> PRT <213> artificial <220> <223> A‑Syn‑BAAK‑BAP <400> 12 Met Asp Val Phe Met Lys Gly Leu Ser Met Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Ala Lys Gln Asn Glu Glu Gly Phe Leu Gln Glu Gly 100 105 110 Met Val Asn Asn Thr Asp Ile Pro Val Asp Pro Glu Asn Glu Ala Tyr 115 120 125 Glu Met Pro Pro Glu Glu Glu Tyr Gln Asp Tyr Glu Pro Glu Ala Gly 130 135 140 Ser Ala Gly Gly Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys 145 150 155 160 Ile Glu Trp His Glu 165 <210> 13 <211> 162 <212> PRT <213> artificial <220> <223> A‑Syn‑BBAA‑BAP <400> 13 Met Asp Val Phe Met Lys Gly Leu Ser Met Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Arg Glu Gly Val 35 40 45 Val Gln Gly Val Ala Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Ser 50 55 60 His Leu Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala Gly Ser Ala Gly 130 135 140 Gly Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp 145 150 155 160 His Glu <210> 14 <211> 165 <212> PRT <213> artificial <220> <223> A‑Syn‑BBKK‑BAP <400> 14 Met Asp Val Phe Met Lys Gly Leu Ser Met Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Arg Glu Gly Val 35 40 45 Val Gln Gly Val Ala Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Ser 50 55 60 His Leu Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Asn Ile Ala Ala Ala Thr Gly Leu Val Lys 85 90 95 Lys Asp Gln Leu Ala Lys Gln Asn Glu Glu Gly Phe Leu Gln Glu Gly 100 105 110 Met Val Asn Asn Thr Asp Ile Pro Val Asp Pro Glu Asn Glu Ala Tyr 115 120 125 Glu Met Pro Pro Glu Glu Glu Tyr Gln Asp Tyr Glu Pro Glu Ala Gly 130 135 140 Ser Ala Gly Gly Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys 145 150 155 160 Ile Glu Trp His Glu 165 <210> 15 <211> 141 <212> PRT <213> artificial <220> <223> A‑Syn‑120‑140_Del‑BAP <400> 15 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Gly Ser Ala Gly Gly Ser Gly Gly Leu 115 120 125 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 130 135 140 <210> 16 <211> 131 <212> PRT <213> artificial <220> <223> α-synuclein amino acids 1-119 <400> 16 Met Ala His His His His His His Ile Glu Gly Arg Met Asp Val Phe 1 5 10 15 Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val Ala Ala Ala Glu 20 25 30 Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys Thr Lys Glu Gly 35 40 45 Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val Val His Gly Val 50 55 60 Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr Asn Val Gly Gly 65 70 75 80 Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys Thr Val Glu Gly 85 90 95 Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys Lys Asp Gln Leu 100 105 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp Met 115 120 125 Pro Val Asp 130 <210> 17 <211> 106 <212> PRT <213> artificial <220> <223> κ (constant region of LC) <400> 17 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 18 <211> 329 <212> PRT <213> artificial <220> <223> IgG1 (HC constant region) <400> 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 19 <211> 4 <212> PRT <213> Artificial <220> <223> GM285 Position 112-115 <400> 19 Ile Leu Glu Asp 1 <210> 20 <211> 13 <212> PRT <213> Artificial <220> <223> GM285 CDR1 heavy chain <400> 20 Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Thr Met Thr 1 5 10 <210> twenty one <211> 17 <212> PRT <213> Artificial <220> <223> GM285 CDR2 heavy chain <400> twenty one Ala Ile Ser Gly Ser Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> twenty two <211> 9 <212> PRT <213> Artificial <220> <223> GM285 CDR3 heavy chain <400> twenty two Ala Lys Asn Trp Ala Pro Phe Asp Tyr 1 5 <210> twenty three <211> 12 <212> PRT <213> Artificial <220> <223> GM285 CDR1 Light Chain <400> twenty three Arg Ala Ser Gln Ser Val Ser Arg Ser Tyr Leu Ala 1 5 10 <210> twenty four <211> 7 <212> PRT <213> Artificial <220> <223> GM285 CDR2 Light Chain <400> twenty four Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial <220> <223> GM285 CDR3 Light Chain <400> 25 Gln Gln Tyr Gly Ser Ser Pro Trp Thr 1 5 <210> 26 <211> 116 <212> PRT <213> Artificial <220> <223> GM285 VH <400> 26 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Thr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Ala Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 27 <211> 108 <212> PRT <213> artificial <220> <223> GM285 VL <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 329 <212> PRT <213> artificial <220> <223> GM285 IgG1 constant region <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 29 <211> 106 <212> PRT <213> artificial <220> <223> GM285 κ-chain <400> 29 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr �0 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 30 <211> 116 <212> PRT <213> artificial <220> <223> GM37 variant 1 heavy chain <400> 30 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Arg Ser Ser Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Ala Pro Phe Asp Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 31 <211> 116 <212> PRT <213> artificial <220> <22�> GM 37 variant 2 heavy chain <400> 31 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Arg Ser Gln Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Ala Pro Phe Asp Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 32 <211> 116 <212> PRT <213> artificial <220> <223> GM 37 Variant 3 Heavy Chain <400> 32 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Arg Ser His Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Ala Pro Phe Asp Ser Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 33 <211> 17 <212> PRT <213> Artificial <220> <223> GM37 variant 1 heavy chain CDR 2 <400> 33 Ala Ile Arg Ser Ser Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 34 <211> 17 <212> PRT <213> Artificial <220> <223> GM37 variant 2 CDR 2 heavy chain <400> 34 Ala Ile Arg Ser Gln Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 35 <211> 17 <212> PRT <213> Artificial <220> <223> GM37 variant 3 CDR 2 heavy chain <400> 35 Ala Ile Arg Ser His Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 36 <211> 5 <212> PRT <213> Artificial <220> <223> 9E4 combined with epitope <400> 36 Asn Glu Ala Tyr Glu 1 5 <210> 37 <211> 134 <212> PRT <213> Artificial <220> <223> Human β-synuclein <400> 37 Met Asp Val Phe Met Lys Gly Leu Ser Met Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Arg Glu Gly Val 35 40 45 Val Gln Gly Val Ala Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Ser 50 55 60 His Leu Gly Gly Ala Val Phe Ser Gly Ala Gly Asn Ile Ala Ala Ala 65 70 75 80 Thr Gly Leu Val Lys Arg Glu Glu Phe Pro Thr Asp Leu Lys Pro Glu 85 90 95 Glu Val Ala Gln Glu Ala Ala Glu Glu Pro Leu Ile Glu Pro Leu Met 100 105 110 Glu Pro Glu Gly Glu Ser Tyr Glu Asp Pro Pro Gln Glu Glu Tyr Gln 115 120 125 Glu Tyr Glu Pro Glu Ala 130 <210> 38 <211> 127 <212> PRT <213> artificial <220> <223> human γ-synuclein <400> 38 Met Asp Val Phe Lys Lys Gly Phe Ser Ile Ala Lys Glu Gly Val Val 1 5 10 15 Gly Ala Val Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly Val Met Tyr Val Gly Ala Lys Thr Lys Glu Asn Val 35 40 45 Val Gln Ser Val Thr Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Asn 50 55 60 Ala Val Ser Glu Ala Val Val Ser Ser Val Asn Thr Val Ala Thr Lys 65 70 75 80 Thr Val Glu Glu Ala Glu Asn Ile Ala Val Thr Ser Gly Val Val Arg 85 90 95 Lys Glu Asp Leu Arg Pro Ser Ala Pro Gln Gln Glu Gly Glu Ala Ser 100 105 110 Lys Glu Lys Glu Glu Val Ala Glu Glu Ala Gln Ser Gly Gly Asp 115 120 125 <210> 39 <211> 140 <212> PRT <213> artificial <220> <223> Cynomolgus monkey α-synuclein ortholog <400> 39 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Ile Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Gln Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140 <210> 40 <211> 140 <212> PRT <213> artificial <220> <223> Rat α-synuclein ortholog <400> 40 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Thr Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Asn Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Met Gly Lys Gly Glu Glu Gly Tyr Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Ser Ser Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140 <210> 41 <211> 140 <212> PRT <213> artificial <220> <223> Mouse α-synuclein ortholog <409> 41 Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Thr Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Asn Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Met Gly Lys Gly Glu Glu Gly Tyr Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Gly Ser Glu Ala Tyr Glu Met Pro 于亮 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140 <210> 42 <211> 446 <212> PRT <213> artificial <220> <223> 9E4 HC <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ala Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 43 <211> 220 <212> PRT <213> artificial <220> <223> 9E4 LC <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ile Gln Thr Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Lys Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof capable of specifically binding to human α-synuclein, wherein the antibody or antigen-binding fragment thereof comprises: (a) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:1; (b) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:2; (c) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:3; (d) The light chain CDR1, consisting of the amino acid sequence of SEQ ID NO:4; (e) The light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:5; and (f) The light chain CDR3, consisting of the amino acid sequence of SEQ ID NO:

6.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the heavy chain comprises the heavy chain variable domain of SEQ ID NO:7 and the light chain comprises the light chain variable domain of SEQ ID NO:

8.

3. A monoclonal antibody or an antigen-binding fragment thereof capable of specifically binding to human α-synuclein, wherein the antibody or antigen-binding fragment thereof comprises: (a) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:1; (b) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:33; (c) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:3; (d) The light chain CDR1, consisting of the amino acid sequence of SEQ ID NO:4; (e) The light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:5; and (f) The light chain CDR3, consisting of the amino acid sequence of SEQ ID NO:

6.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain comprises the heavy chain variable domain of SEQ ID NO:30 and the light chain comprises the light chain variable domain of SEQ ID NO:

8.

5. A monoclonal antibody or an antigen-binding fragment thereof capable of specifically binding to human α-synuclein, wherein the antibody or antigen-binding fragment thereof comprises: (a) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:1; (b) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:34; (c) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:3; (d) The light chain CDR1, consisting of the amino acid sequence of SEQ ID NO:4; (e) The light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:5; and (f) The light chain CDR3, consisting of the amino acid sequence of SEQ ID NO:

6.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain comprises the heavy chain variable domain of SEQ ID NO:31 and the light chain comprises the light chain variable domain of SEQ ID NO:

8.

7. A monoclonal antibody or an antigen-binding fragment thereof capable of specifically binding to human α-synuclein, wherein the antibody or antigen-binding fragment thereof comprises: (a) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:1; (b) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:35; (c) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:3; (d) The light chain CDR1, consisting of the amino acid sequence of SEQ ID NO:4; (e) The light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:5; and (f) The light chain CDR3, consisting of the amino acid sequence of SEQ ID NO:

6.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain comprises the heavy chain variable domain of SEQ ID NO:32 and the light chain comprises the light chain variable domain of SEQ ID NO:

8.

9. A monoclonal antibody or an antigen-binding fragment thereof capable of specifically binding to human α-synuclein, wherein the antibody or antigen-binding fragment thereof comprises: (a) Heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:20; (b) Heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:21; (c) Heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:22; (d) The light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:23; (e) The light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:24; and (f) The light chain CDR3, consisting of the amino acid sequence of SEQ ID NO:

25.

10. The monoclonal antibody or antigen-binding fragment thereof according to claim 9, wherein the heavy chain comprises the heavy chain variable domain of SEQ ID NO:26 and the light chain comprises the light chain variable domain of SEQ ID NO:

27.

11. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the heavy chain constant domain is composed of SEQ ID NO:18 and the light chain constant domain is composed of the κ constant domain defined in SEQ ID NO:

17.

12. The monoclonal antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the heavy chain constant domain is composed of SEQ ID NO:28 and the light chain constant domain is composed of the κ constant domain defined in SEQ ID NO:

29.

13. A nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-12.

14. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-12 in the preparation of a medicament for treating conucleoprotein diseases.

15. The use according to claim 14, wherein the conucleoprotein disease is multiple system atrophy.

16. A kit comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-12 for use in treatment.

17. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein it is detectably labeled.

18. The monoclonal antibody or antigen-binding fragment thereof according to claim 17, wherein the detectable label is a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzyme label.

19. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 17 or 18 in the preparation of a formulation for detecting or measuring the presence or amount of α-synuclein in the brain of a subject.

20. The use according to claim 19, wherein the detection or measurement comprises in vivo imaging of the antibody or its antigen-binding fragment bound to α-synuclein.

21. The use according to claim 19, wherein the detection or measurement comprises in vitro imaging of the antibody or the antigen-binding fragment thereof bound to α-synuclein.

22. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-12 or 17-18 in the preparation of a medicament for the treatment, diagnosis or imaging of conucleoprotein diseases.

23. The use according to claim 22, wherein the conucleoprotein disease is multiple system atrophy.

24. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 17 or 18 in the preparation of a formulation for detecting or measuring the presence or amount of α-synuclein in the brain or body fluids of a subject.

25. The use according to claim 24, wherein the detection or measurement comprises in vivo imaging of the antibody or its antigen-binding fragment bound to α-synuclein.

26. The use according to claim 24, wherein the detection or measurement comprises in vitro imaging of the antibody or its antigen-binding fragment bound to the α-synuclein.

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