Patent Application
20220298231
Incorporation by Reference of Sequence Listing Provided as a Text File A Sequence Listing is provided herewith in a text file, BOULT-037_SEQ_LIST_ST25, created on May 16, 2022 and having a size of 298,454 bytes. The contents of the text file are incorporated herein by reference in its entirety.
The present invention relates to novel molecules that can be employed for the prevention, alleviation, treatment and/or diagnosis of diseases, disorders and abnormalities associated with alpha-synuclein (α-synuclein, A-synuclein, aSynuclein, A-syn, α-syn, aSyn, a-syn) aggregates including, but not limited to, Lewy bodies and/or Lewy neurites, such as Parkinson's disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease. The invention relates to alpha-synuclein binding molecules, in particular to alpha-synuclein antibodies or an antigen-binding fragment thereof or a derivative thereof and uses thereof. The present molecules can also be used for determining a predisposition to such a disorder, disease or abnormality, monitoring residual disorder, disease or abnormality, or predicting the responsiveness of a patient who is suffering from such a disorder, disease or abnormality to the treatment with a certain medicament.
Many degenerative diseases are associated with extracellular or intracellular deposits of amyloid or amyloid-like proteins that contribute to the pathogenesis as well as to the progression of the disease. The best characterized amyloid protein that forms extracellular aggregates is amyloid beta (Aβ).
Amyloid-like proteins that form mainly intracellular aggregates, include, but are not limited to alpha-synuclein, tau, and huntingtin (htt). Diseases involving alpha-synuclein aggregates are generally listed as synucleinopathies (or a-synucleinopathies) and these include, but are not limited to, Parkinson's disease (PD). Synucleinopathies include Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), diffuse Lewy body disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, and Down syndrome. Synucleinopathies with neuronal and glial aggregates of alpha-synuclein include but are not limited to multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy). Other diseases that may have alpha-synuclein-immunoreactive lesions include traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick type C1 disease, frontotemporal dementia with Parkinsonism linked to chromosome 17), motor neuron disease, Huntington's disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Creutzfeldt-Jakob disease, ataxia telangiectasia, Meige's syndrome, subacute sclerosing panencephalitis, Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder (Jellinger, Mov Disord 2003, 18 Suppl. 6, S2-12; Galvin et al., JAMA Neurology 2001, 58 (2), 186-190; Kovari et al., Acta Neuropathol. 2007, 114(3), 295-8; Saito et al., J Neuropathol Exp Neurol. 2004, 63(4), 323-328; McKee et al., Brain, 2013, 136 (Pt 1), 43-64; Puschmann et al., Parkinsonism Relat Disord 2012, 1851, S24-S27; Usenovic et al., J Neurosci. 2012, 32(12), 4240-4246; Winder-Rhodes et al., Mov Disord. 2012, 27(2), 312-315; Ferman et al., J Int Neuropsychol Soc. 2002, 8(7), 907-914; Smith et al., J Pathol. 2014; 232:509-521, Lippa et al., Ann Neurol. 1999 March; 45(3):353-7; Schmitz et al., Mol Neurobiol. Aug. 22, 2018; Charles et al., Neurosci Lett. Jul. 28, 2000; 289(1):29-32; Wilhelmsen et al., Arch Neurol. 2004 March; 61(3):398-406; Yamaguchi et al., J Neuropathol Exp Neurol. 2004, 80th annual meeting, vol. 63; Askanas et al., J Neuropathol Exp Neurol. 2000 July; 59(7):592-8).
Alpha-synuclein is a 140 amino acid long, cytosolic protein abundantly and predominantly expressed in the CNS and localized in pre-synaptic terminals (Burré J., J Parkinsons Dis. 2015; 5(4):699-713). Alpha-synuclein is a natively unfolded protein but adopts secondary structure of mostly helical nature upon association with lipid vesicles or membranes (Iwai et al., Biochemistry 1995, 34(32), 10139-10145). The physiological function of alpha-synuclein still remains elusive. Because of the association of alpha-synuclein to synaptic vesicles and its presynaptic localization it is suggested that it regulates synaptic activity and plasticity, neurotransmitter release, dopamine production and metabolism, vesicle trafficking, synaptic vesicle pool maintenance and chaperone-like activity (Cabin et al., J Neurosci. 2002; 22:8797-8807; Chandra et al., Cell. 2005; 123:383-396).
The sequence of alpha-synuclein can be divided into three main domains: 1) the N-terminal region comprising of residues 1-60, which contains 11-mer amphipathic imperfect repeat residues with highly conserved hexamer (KTKEGV). This region has been implicated in regulating alpha-synuclein association to lipid membranes and its internalization; 2) the hydrophobic Non-Amyloid beta Component (NAC) domain spanning residues 61-95; which is essential for alpha-synuclein fibrillization; and 3) the C-terminal region spanning residues 96-140 which is highly acidic and proline-rich, has no distinct structural propensity.
Alpha-synuclein has been shown to undergo several post translational modifications, including truncations, phosphorylation, ubiquitination, sumoylation, oxidation, nitration, acetylation, glycation, glycosylation, and/or transglutaminase covalent cross linking (Fujiwara et al., Nat Cell Biol 2002, 4(2), 160-164; Hasegawa et al., J Biol Chem 2002, 277(50), 49071-49076; Li et al., Proc Natl Acad Sci USA 2005, 102(6), 2162-2167; Oueslati et al., Prog Brain Res 2010, 183, 115-145; Schmid et al., J Biol Chem 2009, 284(19), 13128-13142; Dorval et al., J Biol Chem. 2006, 281(15):9919-24; Ruzafa et al., PlosOne 2017 12(5):e0178576; Ischiropoulos et al., Ann N Y Acad Sci. 2003, 991, 93-100; Munch et al., J Chem Neuroanat. 2000; 20:253-257; Marotta et al., Chembiochem. 2012; 13:2665-2670). The majority of these modifications involve residues within the C-terminal region.
Several phosphorylation sites have been detected in the carboxyl-terminal region on Tyr-125, -133, and -136, and on Ser-129 (Negro et al., FASEB J 2002, 16(2), 210-212). Extensive and selective phosphorylation of alpha-synuclein at Ser-129 is evident in synucleinopathy lesions, including Lewy bodies (Fujiwara et al., Nat Cell Biol 2002, 4(2); 160-164). Other post-translational modifications in the carboxyl-terminal, including glycosylation on Ser-129 (McLean et al., Neurosci Lett 2002, 323(3), 219-223) and nitration on Tyr-125, -133, and -136 (Takahashi et al., Brain Res 2002, 938 (1-2), 73-80), may affect aggregation of alpha-synuclein. Truncation of the carboxyl-terminal region by proteolysis has been reported to play a role in alpha-synuclein fibrillogenesis in various neurodegenerative diseases (Rochet et al., Biochemistry 2000, 39(35), 10619-10626). Full-length as well as partially truncated and insoluble aggregates of alpha-synuclein have been detected in highly purified Lewy bodies (Crowther et al., FEBS Lett 1998, 436(3), 309-312).
Abnormal protein aggregation is a common feature in aging brain and in several neurodegenerative diseases, even though a clear role in the disease process remains to be defined. In in vitro models, alpha-synuclein readily assembles into filaments resembling those isolated from brain of patients with Lewy Body dementia and familial PD (Crowther et al., FEBS Lett 1998, 436(3), 309-312). Alpha-synuclein and its mutated forms (e.g. A53T and A30P) have a random coil conformation and do not form significant secondary structures in aqueous solution at low concentrations; however, at higher concentrations they are prone to self-aggregate, producing amyloid fibrils (Wood et al., J Biol Chem 1999, 274(28), 19509-19512). Several differences in the aggregation behavior of the PD-linked mutants and the wild-type protein have been documented. Monomeric alpha-synuclein aggregates in vitro form stable fibrils via a metastable oligomeric (i.e., protofibril) state (Volles et al., Biochemistry 2002, 41(14), 4595-4602).
Parkinson's disease (PD) is the most common neurodegenerative motor disorder. PD is mainly an idiopathic disease, although in at least 5% of the PD patients the pathology is linked to mutations in one or several specific genes. Several point mutations have been described in the alpha-synuclein gene (A30P, E46K, H50Q, G51 D, A53T) which cause familial PD with autosomal dominant inheritance. Furthermore, duplications and triplications of the alpha-synuclein gene have been described in patients that developed PD underlining the role of alpha-synuclein in PD pathogenesis (Lesage et al., Hum. Mol. Genet., 2009, 18, R48-59). The pathogenesis of PD remains elusive, however, growing evidence suggests a role for the pathogenic folding of the alpha-synuclein protein that leads to the formation of amyloid-like fibrils. Indeed, the hallmarks of PD are the presence of intracellular alpha-synuclein aggregate structures called Lewy Bodies in the nigral neurons, as well as the death of dopaminergic neurons in the substantia nigra and elsewhere. Alpha-synuclein is a natively unfolded presynaptic protein that can misfold and aggregate into larger oligomeric and fibrillar forms which are linked to the pathogenesis of PD. Studies have implicated small soluble oligomeric and protofibrillar forms of alpha-synuclein as the most neurotoxic species (Lashuel et al., J. Mol. Biol., 2002, 322, 1089-102), however the precise role of alpha-synuclein in the neuronal cell toxicity remains to be clarified (review: Cookson, Annu. Rev. Biochem., 2005, 74, 29-52).
Recent evidence from cellular and animal models suggests that pathological and/or aggregated alpha-synuclein can spread from one neuron to another. Once inside the new cell alpha-synuclein aggregates act as seeds, recruiting endogenous alpha-synuclein and advancing protein aggregation (Luk et al., Science. 2012, 338(6109):949-5; Tran et al., Cell Rep. 2014, 7(6):2054-65). Moreover, the transynaptic spreading of pathological and/or aggregated alpha-synuclein could explain the progressive advancing of Lewy pathology through defined anatomical connected brain areas in PD that was first described by Braak and colleagues (Braak et al., Neurobiol. Aging. 2003; 24:197-211).
Consequently, the cell-to-cell spreading of pathological and/or aggregated alpha-synuclein renders immunotherapy as a compelling target for new therapeutic approaches aiming to alleviate, treat, retard or halt the progression of PD and other synucleinopathies. Antibodies described herein inhibit and/or delay seeded and/or spontaneous alpha-synuclein aggregation, and this functional feature would allow them to bind to alpha-synuclein seeds in the extracellular space to either neutralize the seeds and consequently delay or inhibit the propagation of alpha-synuclein aggregates or facilitate the clearance of these spreading species. The development of such therapies for PD and other synucleinopathies would addresses an unmet medical need since currently only symptomatic treatments are available.
The diagnosis of Parkinson's disease is largely clinical and depends on the presence of a specific set of symptoms and signs (the initial core feature being bradykinesia, rigidity, rest tremor and postural instability), a slowly progressive course, and a response to drug treatment. The final confirmation of the diagnosis is made by post-mortem neuropathological analysis. Strategies are being developed to apply recent advances of the cause of Parkinson's disease to the development of biochemical biomarkers as well as imaging biomarkers (Schapira, Curr Opin Neurol 2013; 26(4):395-400). Such biomarkers that have been investigated in different body fluids (cerebrospinal fluid (CSF), plasma, saliva) include alpha-synuclein levels but also DJ-1, Tau and Abeta, as well as neurofilaments proteins, interleukins, osteopontin and hypocrontin (Schapira, Curr Opin Neurol 2013; 26(4):395-400), but so far none of these biomarkers alone or in combination can be used as a determinant diagnostic test. Antibodies for diagnostic application that selectively recognize and bind to certain pathological structures of alpha-synuclein would have the potential to be used as biomarkers with high sensitivity and specificity. To our knowledge no approved biomarker for monitoring pathological alpha-synuclein levels is currently on the market or available for clinical trials despite a crucial needs for Parkinson's disease research and drug development (Eberling et al., J Parkinsons Dis. 2013; 3(4):565-7).
WO2017/207,739 provides antibodies that specifically bind human alpha-synuclein with a high affinity and reduces alpha-synuclein spreading in vivo.
It is an object of the present invention to provide alpha-synuclein binding molecules that can be employed to treat, alleviate and/or prevent a disease, disorder or abnormality associated with alpha-synuclein aggregates, such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In another aspect, it is an object of the present invention to provide molecules that can be employed to diagnose, monitor disease progression of, and/or monitor drug activity against, a disease, disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies, Lewy neurites and/or glial cytoplasmic inclusions, such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
The invention generally relates to an alpha-synuclein binding molecule, which inhibits and/or delays seeded and/or spontaneous alpha-synuclein aggregation.
In one embodiment, the invention relates to an alpha-synuclein binding molecule, which
Accordingly, the invention relates in its broadest aspect to binding molecules, in particular antibodies or antigen-binding fragments thereof, which bind alpha-synuclein. In a preferred embodiment of the invention, the binding molecules, in particular antibodies or antigen-binding fragments thereof, inhibit and/or delay the aggregation of seeded and/or spontaneous alpha-synuclein aggregation and are capable of recognizing and binding to pathological and/or aggregated alpha-synuclein, particularly human alpha-synuclein, in vitro and/or in vivo. Alpha-synuclein is a soluble protein that has the propensity to spontaneously aggregate and form soluble oligomers or soluble/insoluble protofibrils or mature fibrils or detergent-insoluble aggregates under certain conditions. Seeded alpha-synuclein aggregation is the aggregation accelerated by pathological alpha-synuclein, so called “seeds”.
The alpha-synuclein binding molecules of the invention, in particular antibodies or antigen-binding fragments thereof, block cell-to-cell spreading and/or delay and/or inhibit the aggregation of alpha-synuclein protein or fragments thereof. Thus, an alpha-synuclein binding molecule within the present invention inhibits and/or delays seeded and/or spontaneous alpha-synuclein aggregation; and is capable of recognizing and binding to pathological and/or aggregated alpha-synuclein, particularly human alpha-synuclein, in vitro and in vivo. An alpha-synuclein binding molecule within the present invention inhibits and/or delays seeded and/or spontaneous alpha-synuclein aggregation; and is capable of recognizing and binding to pathological and/or aggregated alpha-synuclein, particularly human alpha-synuclein, in vitro or in vivo.
It is preferred within the invention that the alpha-synuclein binding molecule, in particular the antibody or antigen-binding fragment thereof, additionally has one or more, preferably two or more, more preferably 3 or more, more preferably 4 or more, even more preferably all of the functional features (i) to (vi):
It is preferred within the invention that the alpha-synuclein binding molecule, in particular the antibody or antigen-binding fragment thereof, additionally has one or more, preferably two or more, more preferably 3 or more, more preferably 4 or more, even more preferably all of the functional features (i) to (vi):
In particular alpha-synuclein binding molecules of the invention, in particular antibodies or antigen-binding fragments thereof, inhibit and/or delay aggregation of alpha-synuclein protein or fragments thereof.
In one embodiment, alpha-synuclein binding molecules of the invention, in particular antibodies or antigen-binding fragments thereof, inhibit the formation of alpha-synuclein aggregates, including but not limited to, Lewy Bodies, Lewy Neurites, and/or glial cytoplasmic inclusions.
The alpha-synuclein binding molecules, especially antibodies or antigen-binding fragments thereof, of the invention may selectively bind aggregated alpha-synuclein and/or pathological alpha-synuclein in preference to non-aggregated alpha-synuclein and/or non-pathological alpha-synuclein (such as monomeric alpha-synuclein).
In some embodiments of the invention, the antibody is a monoclonal antibody. In some embodiments, the antibody is a murine, murinized, human, humanized, or chimeric antibody.
In some embodiments of the invention, the antibody, or antigen-binding fragment or derivative thereof having a binding characteristic of an antibody described herein, is an antibody having the variable regions VH and/or VL of the amino acid sequences, respectively, set forth in SEQ ID NO: 10 and SEQ ID NO: 14; SEQ ID NO: 20 and SEQ ID NO: 24; SEQ ID NO: 30 and SEQ ID NO: 34; SEQ ID NO: 40 and SEQ ID NO: 44; SEQ ID NO: 50 and SEQ ID NO: 54; SEQ ID NO: 60 and SEQ ID NO: 64; SEQ ID NO: 70 and SEQ ID NO: 74; SEQ ID NO: 30 and SEQ ID NO: 84; SEQ ID NO: 90 and SEQ ID NO: 94; SEQ ID NO: 100 and SEQ ID NO: 104; SEQ ID NO: 110 and SEQ ID NO: 114; SEQ ID NO: 280 and SEQ ID NO: 284; SEQ ID NO: 290 and SEQ ID NO: 194; SEQ ID NO: 140 and SEQ ID NO: 144; SEQ ID NO: 150 and SEQ ID NO: 154; SEQ ID NO: 160 and SEQ ID NO: 164; SEQ ID NO: 170 and SEQ ID NO: 174; SEQ ID NO: 180 and SEQ ID NO: 184; SEQ ID NO: 190 and SEQ ID NO: 194; SEQ ID NO: 200 and SEQ ID NO: 204; SEQ ID NO: 210 and SEQ ID NO: 214; SEQ ID NO: 220 and SEQ ID NO: 224; SEQ ID NO: 230 and SEQ ID NO: 234; SEQ ID NO: 240 and SEQ ID NO: 244; SEQ ID NO: 250 and SEQ ID NO: 254; SEQ ID NO: 260 and SEQ ID NO: 264; SEQ ID NO: 270 and SEQ ID NO: 274; SEQ ID NO: 300 and SEQ ID NO: 304; SEQ ID NO: 310 and SEQ ID NO: 314; SEQ ID NO: 320 and SEQ ID NO: 324; SEQ ID NO: 330 and SEQ ID NO: 334; SEQ ID NO: 340 and SEQ ID NO: 344; SEQ ID NO: 350 and SEQ ID NO: 354; SEQ ID NO: 360 and SEQ ID NO: 364; SEQ ID NO: 370 and SEQ ID NO: 374; SEQ ID NO: 380 and SEQ ID NO: 384; SEQ ID NO: 390 and SEQ ID NO: 394; SEQ ID NO: 400 and SEQ ID NO: 404; SEQ ID NO: 410 and SEQ ID NO: 414; SEQ ID NO: 420 and SEQ ID NO: 424; SEQ ID NO: 430 and SEQ ID NO: 434; SEQ ID NO: 440 and SEQ ID NO: 414; SEQ ID NO: 450 and SEQ ID NO: 424; SEQ ID NO: 460 and SEQ ID NO: 464; SEQ ID NO: 470 and SEQ ID NO: 474; SEQ ID NO: 480 and SEQ ID NO: 484; SEQ ID NO: 490 and SEQ ID NO: 494; SEQ ID NO: 500 and SEQ ID NO: 504; SEQ ID NO: 510 and SEQ ID NO: 514; SEQ ID NO: 520 and SEQ ID NO: 524; SEQ ID NO: 530 and SEQ ID NO: 534; SEQ ID NO: 540 and SEQ ID NO: 544; SEQ ID NO: 550 and SEQ ID NO: 554; SEQ ID NO: 560 and SEQ ID NO: 564; SEQ ID NO: 570 and SEQ ID NO: 574; SEQ ID NO: 580 and SEQ ID NO: 584; SEQ ID NO: 590 and SEQ ID NO: 474; SEQ ID NO: 600 and SEQ ID NO: 554; SEQ ID NO: 610 and SEQ ID NO: 614; SEQ ID NO: 610 and SEQ ID NO: 624; SEQ ID NO: 610 and SEQ ID NO: 634; SEQ ID NO: 610 and SEQ ID NO: 644; SEQ ID NO: 620 and SEQ ID NO: 614; SEQ ID NO: 620 and SEQ ID NO: 624; SEQ ID NO: 620 and SEQ ID NO: 634; SEQ ID NO: 620 and SEQ ID NO: 644; SEQ ID NO: 630 and SEQ ID NO: 614; SEQ ID NO: 630 and SEQ ID NO: 624; SEQ ID NO: 630 and SEQ ID NO: 634; SEQ ID NO: 630 and SEQ ID NO: 644; SEQ ID NO: 640 and SEQ ID NO: 614; SEQ ID NO: 640 and SEQ ID NO: 624; SEQ ID NO: 640 and SEQ ID NO: 634; SEQ ID NO: 640 and SEQ ID NO: 644; SEQ ID NO: 650 and SEQ ID NO: 614; SEQ ID NO: 650 and SEQ ID NO: 624; SEQ ID NO: 650 and SEQ ID NO: 634; SEQ ID NO: 650 and SEQ ID NO: 644; SEQ ID NO: 660 and SEQ ID NO: 614; SEQ ID NO: 670 and SEQ ID NO: 614; SEQ ID NO: 680 and SEQ ID NO: 614; SEQ ID NO: 690 and SEQ ID NO: 614; SEQ ID NO: 690 and SEQ ID NO: 624; SEQ ID NO: 700 and SEQ ID NO: 614; SEQ ID NO: 700 and SEQ ID NO: 624; SEQ ID NO: 710 and SEQ ID NO: 614; SEQ ID NO: 710 and SEQ ID NO: 624; SEQ ID NO: 720 and SEQ ID NO: 614; SEQ ID NO: 720 and SEQ ID NO: 624.
The invention therefore also provides an alpha-synuclein binding antibody having the variable regions VH and/or VL of the amino acid sequences, respectively, set forth in SEQ ID NO: 10 and SEQ ID NO: 14; SEQ ID NO: 20 and SEQ ID NO: 24; SEQ ID NO: 30 and SEQ ID NO: 34; SEQ ID NO: 40 and SEQ ID NO: 44; SEQ ID NO: 50 and SEQ ID NO: 54; SEQ ID NO: 60 and SEQ ID NO: 64; SEQ ID NO: 70 and SEQ ID NO: 74; SEQ ID NO: 30 and SEQ ID NO: 84; SEQ ID NO: 90 and SEQ ID NO: 94; SEQ ID NO: 100 and SEQ ID NO: 104; SEQ ID NO: 110 and SEQ ID NO: 114; SEQ ID NO: 280 and SEQ ID NO: 284; SEQ ID NO: 290 and SEQ ID NO: 194; SEQ ID NO: 140 and SEQ ID NO: 144; SEQ ID NO: 150 and SEQ ID NO: 154; SEQ ID NO: 160 and SEQ ID NO: 164; SEQ ID NO: 170 and SEQ ID NO: 174; SEQ ID NO: 180 and SEQ ID NO: 184; SEQ ID NO: 190 and SEQ ID NO: 194; SEQ ID NO: 200 and SEQ ID NO: 204; SEQ ID NO: 210 and SEQ ID NO: 214; SEQ ID NO: 220 and SEQ ID NO: 224; SEQ ID NO: 230 and SEQ ID NO: 234; SEQ ID NO: 240 and SEQ ID NO: 244; SEQ ID NO: 250 and SEQ ID NO: 254; SEQ ID NO: 260 and SEQ ID NO: 264; SEQ ID NO: 270 and SEQ ID NO: 274 SEQ ID NO: 300 and SEQ ID NO: 304; SEQ ID NO: 310 and SEQ ID NO: 314; SEQ ID NO: 320 and SEQ ID NO: 324; SEQ ID NO: 330 and SEQ ID NO: 334; SEQ ID NO: 340 and SEQ ID NO: 344; SEQ ID NO: 350 and SEQ ID NO: 354; SEQ ID NO: 360 and SEQ ID NO: 364; SEQ ID NO: 370 and SEQ ID NO: 374; SEQ ID NO: 380 and SEQ ID NO: 384; SEQ ID NO: 390 and SEQ ID NO: 394; SEQ ID NO: 400 and SEQ ID NO: 404; SEQ ID NO: 410 and SEQ ID NO: 414; SEQ ID NO: 420 and SEQ ID NO: 424; SEQ ID NO: 430 and SEQ ID NO: 434; SEQ ID NO: 440 and SEQ ID NO: 414; SEQ ID NO: 450 and SEQ ID NO: 424; SEQ ID NO: 460 and SEQ ID NO: 464; SEQ ID NO: 470 and SEQ ID NO: 474; SEQ ID NO: 480 and SEQ ID NO: 484; SEQ ID NO: 490 and SEQ ID NO: 494; SEQ ID NO: 500 and SEQ ID NO: 504; SEQ ID NO: 510 and SEQ ID NO: 514; SEQ ID NO: 520 and SEQ ID NO: 524; SEQ ID NO: 530 and SEQ ID NO: 534; SEQ ID NO: 540 and SEQ ID NO: 544; SEQ ID NO: 550 and SEQ ID NO: 554; SEQ ID NO: 560 and SEQ ID NO: 564; SEQ ID NO: 570 and SEQ ID NO: 574; SEQ ID NO: 580 and SEQ ID NO: 584; SEQ ID NO: 590 and SEQ ID NO: 474; SEQ ID NO: 600 and SEQ ID NO: 554; SEQ ID NO: 610 and SEQ ID NO: 614; SEQ ID NO: 610 and SEQ ID NO: 624; SEQ ID NO: 610 and SEQ ID NO: 634; SEQ ID NO: 610 and SEQ ID NO: 644; SEQ ID NO: 620 and SEQ ID NO: 614; SEQ ID NO: 620 and SEQ ID NO: 624; SEQ ID NO: 620 and SEQ ID NO: 634; SEQ ID NO: 620 and SEQ ID NO: 644; SEQ ID NO: 630 and SEQ ID NO: 614; SEQ ID NO: 630 and SEQ ID NO: 624; SEQ ID NO: 630 and SEQ ID NO: 634; SEQ ID NO: 630 and SEQ ID NO: 644; SEQ ID NO: 640 and SEQ ID NO: 614; SEQ ID NO: 640 and SEQ ID NO: 624; SEQ ID NO: 640 and SEQ ID NO: 634; SEQ ID NO: 640 and SEQ ID NO: 644; SEQ ID NO: 650 and SEQ ID NO: 614; SEQ ID NO: 650 and SEQ ID NO: 624; SEQ ID NO: 650 and SEQ ID NO: 634; SEQ ID NO: 650 and SEQ ID NO: 644; SEQ ID NO: 660 and SEQ ID NO: 614; SEQ ID NO: 670 and SEQ ID NO: 614; SEQ ID NO: 680 and SEQ ID NO: 614; SEQ ID NO: 690 and SEQ ID NO: 614; SEQ ID NO: 690 and SEQ ID NO: 624; SEQ ID NO: 700 and SEQ ID NO: 614; SEQ ID NO: 700 and SEQ ID NO: 624; SEQ ID NO: 710 and SEQ ID NO: 614; SEQ ID NO: 710 and SEQ ID NO: 624; SEQ ID NO: 720 and SEQ ID NO: 614; SEQ ID NO: 720 and SEQ ID NO: 624.
In some embodiments, the antibody comprises:
These alpha-synuclein binding antibodies may constitute separate aspects of the invention.
In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid encodes an antibody, or an antigen-binding fragment or derivative thereof, described herein. In some embodiments, a host cell is provided, wherein the host cell comprises an isolated nucleic acid that encodes an antibody, or an antigen-binding fragment or derivative thereof, described herein. In some embodiments, a method of producing an antibody, or an antigen-binding fragment or derivative thereof, is provided, comprising culturing the host cell under conditions suitable for producing the antibody, or the antigen-binding fragment or the derivative thereof.
In some embodiments, an immunoconjugate is provided, wherein the immunoconjugate comprises an isolated antibody, antigen-binding fragment or derivative thereof, described herein and a therapeutic agent. In some embodiments, a labeled antibody, antigen-binding fragment or derivative thereof, is provided, comprising an antibody antigen-binding fragment or derivative thereof, described herein and a detectable label.
In some embodiments, a pharmaceutical composition is provided, comprising an isolated antibody, antigen-binding fragment or derivative thereof, described herein and a pharmaceutically acceptable carrier and/or excipient.
As used herein, the term “isolated” means that the chemical compound, e.g. the nucleic acid or antibody, may have been separated and/or recovered from its natural environment. Within the present invention, the chemical compound is preferably chemically synthesized, or synthesized in a cellular system different from the cell from which it naturally originates, and is thus “isolated” from its naturally associated components. The chemical compound may be isolated from its natural environment by e.g. purification or produced by means of a technical process (including but not limited to e.g. gene synthesis, polymerase chain reaction (PCR), vector purification and protein (antibody) purification). Such chemical compound may be, in particular, a nucleic acid, DNA-, RNA-, or cDNA-sequence, or a peptide, antibody or protein.
The present invention is not limited to an isolated antibody in accordance with the above definition, but also relates to an antibody as such irrespective of its origin.
The same applies to peptides, nucleic acids, DNA, RNA and/or cDNA sequences provided by the present invention, which are encompassed in isolated form, as defined above, or in any other form.
In some embodiments, a method of preventing, alleviating and/or treating a disease, disorder or abnormality associated with alpha-synuclein aggregates or pathological alpha-synuclein, such as Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), Diffuse Lewy Body Disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder, is provided. According to one embodiment, the methods of the invention comprise administering an effective concentration or an effective amount of a binding molecule, particularly an antibody, or an antigen-binding fragment or derivative thereof, of the invention binding alpha-synuclein (e.g., a full-length antibody or an alpha-synuclein binding fragment or derivative of an antibody) as described herein to a subject in need thereof.
In some embodiments, a method of retaining motor capabilities or improving motor deficits of a subject suffering from a synucleopathy, including reducing bradykinesia, rigidity, resting tremor or postural instability is provided, comprising administering an antibody, or an antigen-binding fragment or derivative thereof, described herein or a pharmaceutical composition comprising an antibody, or antigen-binding fragment or derivative thereof, described herein to a subject in need thereof.
In some embodiments, a method of retaining or increasing cognitive capacity of a subject suffering from a synucleopathy is provided, comprising administering an antibody, or antigen-binding fragment or derivative thereof, described herein or a pharmaceutical composition comprising an antibody, or antigen-binding fragment or derivative thereof, described herein to a subject in need thereof.
In some embodiments, an isolated antibody, or an antigen-binding fragment or derivative thereof, described herein is provided for use as a medicament. In some embodiments, an isolated antibody, or an antigen-binding fragment or derivative thereof, described herein is provided for use in alleviating, preventing and/or treating a synucleinopathy in a subject. In some embodiments, use of an antibody, or an antigen-binding fragment or derivative thereof, described herein is provided for manufacture of a medicament for preventing, alleviating and/or treating a disease, a disorder and/or abnormality associated with alpha-synuclein aggregates.
In some embodiments, the disease, disorder and/or abnormality associated with alpha-synuclein aggregate is a synucleinopathy. In some embodiments, the synucleinopathy is Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), Diffuse Lewy Body Disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.
More particularly, the synucleinopathy is selected from Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), and Diffuse Lewy Body Disease.
In some embodiments, a method of detecting alpha-synuclein aggregates including, but not limited to, Lewy bodies, Lewy neurites and/or glial cytoplasmic inclusions, is provided, comprising contacting a sample with an antibody, or antigen-binding fragment or derivative thereof, described herein and detecting the presence of aggregates using methods known in the art. In some embodiments, the sample is a brain sample, a cerebrospinal fluid sample, or a blood sample.
In some embodiments, a method for evaluating an alpha-synuclein binding molecule for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation is provided, the method comprising the steps of: bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and determining the time to reach half-maximum signal of the detectable dye, particularly the signal of fluorescent dye, relative to the seeded aggregation in the absence of binding molecule, wherein an increase in time to reach half-maximum signal of the detectable dye in the presence of binding molecule relative to the seeded aggregation in the absence of binding molecule indicates that the alpha-synuclein binding molecule is capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation.
In further embodiments, a method for selecting/screening an alpha-synuclein binding molecule capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation is provided, the method comprising the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and selecting the alpha-synuclein binding molecule as being able to inhibit and/or delay seeded and/or spontaneous alpha-synuclein aggregation based on the signal of the detectable dye, in particular the fluorescent dye, determined in the absence and presence of the alpha-synuclein binding molecule.
In some embodiments, the method of evaluating or selecting an alpha-synuclein binding molecule capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation is provided, wherein the detectable dye is thioflavin (ThT), which binds to the beta-sheet structure of the aggregated protein.
In some embodiments, the method of evaluating or selecting an alpha-synuclein binding molecule capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation is provided, wherein the alpha-synuclein monomeric protein is covalently linked to the detectable dye, in particular the fluorescent dye, and/or wherein the signal of the detectable dye, in particular the fluorescent dye, is quenching of signal/fluorescence emission upon formation of the protein aggregates. Other detection methods are also envisaged within the scope of the present invention, including, for example, fluorescence resonance energy transfer (FRET) assays or the like. Dyes, in particular fluorescent dyes, are known to the person skilled in the art. Examples include for example green fluorescent protein, yellow fluorescent protein and the like.
In some embodiments, an alpha-synuclein binding molecule is evaluated as capable of inhibiting and/or delaying seeded and/or spontaneous alpha-synuclein aggregation or is selected, respectively, if in step d) of the invention the seeded and/or spontaneous alpha-synuclein aggregation is inhibited and/or delayed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300% in the presence of the alpha-synuclein binding molecule as compared to in the absence of the alpha-synuclein binding molecule. Alternatively, an alpha-synuclein binding molecule may be evaluated as capable of inhibiting and/or delaying seeded and/or spontaneous alpha-synuclein aggregation if the alpha-synuclein binding molecule causes an at least 10 percent increase in aggregation half-time (τ1/2 values) of seeded aggregation relative to the seeded aggregation in the absence of binding molecule.
In some embodiments, a method for determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting seeded alpha-synuclein aggregation comprises the steps of:
In the method for determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting seeded alpha-synuclein aggregation, the composition comprising the alpha-synuclein binding molecule and a transduction reagent is pre-mixed prior to incubation with cells containing and/or expressing a monomeric alpha-synuclein reporter protein. In some embodiments, a method for determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting cellular uptake of pathological and/or aggregated alpha-synuclein comprises the steps of:
In some embodiments of the invention, the alpha-synuclein binding molecule for the method of determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting the seeded alpha-synuclein aggregation comprises preferably an alpha-synuclein antibody or an antigen-binding fragment or derivative thereof, more preferably an antibody or an antigen-binding fragment or derivative thereof of the invention.
In some embodiments of the invention, the transduction reagents under (i) and (ii) of the method of determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting the seeded alpha-synuclein aggregation can be the same or different, preferably the transduction reagents are different, more preferably the transduction reagent under (i) comprises Ab-DeliverIN™ and the transduction reagent under (ii) comprises Lipofectamine™ 2000.
In some embodiments of the invention, the step (iii) of the method of determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting the seeded alpha-synuclein aggregation comprises immunohistochemistry, microscopy, biochemical or flow cytometry detection methods, preferably immunohistochemistry, more preferably immunohistochemistry wherein by measuring fluorescence of the fluorescently labelled alpha-synuclein as expressed by said cells.
In some embodiments of the invention, a method for determining or evaluating an alpha-synuclein binding molecule for the capability of delaying and/or inhibiting the seeded alpha-synuclein aggregation comprises the steps of:
Accordingly, in the context of the present invention, the term “transduction reagent” (or “transfection reagent”) as used herein refers mainly to a formulation that is capable of forming non-covalent complexes with a molecule of interest to be transported intracellularly. Example of transduction reagent includes but is not limited to Ab-DeliverIN™, Lipofectamine™ 2000, Xfect™ Transfection Reagent, ViaFect™ Transfection Reagent, Polyethylenimine (PEI) cellular transfection reagent or FuGENE™.
In some embodiments, an alpha-synuclein binding molecule is evaluated as capable of delaying and/or inhibiting seeded alpha-synuclein aggregation using the methods of the present invention if the seeded alpha-synuclein aggregation is delayed and/or inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300% in the presence of the alpha-synuclein binding molecule as compared to in the absence of the alpha-synuclein binding molecule to be evaluated. Alternatively, an alpha-synuclein binding molecule may be evaluated as capable of delaying and/or inhibiting seeded alpha-synuclein aggregation if the alpha-synuclein binding molecule causes an at least 10% reduction of the level of aggregated alpha-synuclein relative to the level of aggregated alpha-synuclein in the absence of binding molecule.
Within the scope of the present invention, alpha-synuclein may have the sequence of SEQ ID NO: 1. Alpha-synuclein aggregates are multimeric beta-sheet rich assemblies of alpha-synuclein monomers that can form either soluble oligomers or soluble/insoluble protofibrils or mature fibrils which coalesce into intracellular deposits detected as a range of Lewy pathologies in Parkinson's disease and other synucleinopathies. Alpha-synuclein under physiological conditions does not adopt an ordered tertiary structure, rather it is classified as a natively unfolded protein which can exist as a mixture of dynamic and flexible structural conformations. Misfolded alpha-synuclein can form multimeric intermediate oligomeric structures which eventually assemble into highly-ordered fibrillar aggregates.
The term “aggregated alpha-synuclein” as used herein, refers to insoluble or soluble oligomeric and/or polymeric structures composed of alpha-synuclein misfolded monomers and/or multimers and/or assemblies of monomers.
Pathological alpha-synuclein is misfolded or aggregated or post-translationally modified alpha-synuclein that is the main component of Lewy pathologies; Lewy pathologies can be detected as having the following morphologies: Lewy bodies, Lewy neurites, premature Lewy bodies or pale bodies, perikaryal deposits with diffuse, granular, punctate or pleomorphic patterns. Moreover, pathological alpha-synuclein is the major component of intracellular fibrillary inclusions detected in oligodendrocytes also referred to as glial cytoplasmic inclusions and in neuronal somata, axons and nuclei (referred to as neuronal cytoplasmic inclusions) that are the histological hallmarks of multiple system atrophy. Pathological alpha-synuclein in Lewy pathologies often displays substantial increase in post-translational modifications such as phosphorylation, ubiquitination, nitration, and truncation.
Seeds are multimeric beta-sheet rich structures which are composed of alpha-synuclein could be also (i.e. in addition to alpha-synuclein) composed of other amyloidogenic proteins (e.g. Tau, Amyloid β) which can accelerate the aggregation kinetics of alpha-synuclein by elongating the growing multimer and/or by acting as templates for the nucleation of monomers on the seed surface.
Spontaneous aggregation of alpha-synuclein is the aggregation process that progresses without the addition of seeds. Alpha-synuclein is a soluble protein that has the propensity to spontaneously aggregate and form soluble oligomers or soluble/insoluble protofibrils or mature fibrils or detergent-insoluble aggregates under certain conditions.
Lewy bodies are abnormal aggregates of protein that develop inside nerve cells in Parkinson's disease (PD), Lewy body dementia and other synucleinopathies. Lewy bodies appear as spherical masses that displace other cell components. Morphologically, Lewy bodies can be classified as being brainstem or cortical type. Classic brainstem Lewy bodies are eosinophilic cytoplasmic inclusions consisting of a dense core surrounded by a halo of 5-10-nm-wide radiating fibrils, the primary structural component of which is alpha-synuclein; cortical Lewy bodies differ by lacking a halo. The presence of Lewy bodies is a hallmark of Parkinson's disease.
Lewy neurites are abnormal neuronal processes in diseased neurons, containing granular material, abnormal alpha-synuclein filaments similar to those found in Lewy bodies, dot-like, varicose structures and axonal spheroids. Like Lewy bodies, Lewy neurites are a feature of a-synucleinopathies such as dementia with Lewy bodies, Parkinson's disease, and multiple system atrophy.
Glial cytoplasmic inclusions (also referred to as Papp-Lantos inclusions) consist of insoluble alpha-synuclein filamentous aggregates detected in oligodendrocytes in the white matter of multiple system atrophy brains. Alpha-synuclein aggregates in neuronal somata, axons and nuclei, referred to as neuronal cytoplasmic inclusions, are characteristic cytopathological features of multiple system atrophy. The detection of glial cytoplasmic inclusions is considered a hallmark for the neuropathological diagnosis of multiple system atrophy.
An alpha-synuclein binding molecule is a molecule that binds to the pathological and/or aggregated alpha-synuclein protein, such as an alpha-synuclein antibody or fragment thereof, at a specific recognition site, or epitope. Antigen-binding molecules of the invention bind to an epitope within the amino acid sequence of SEQ ID NO: 1. The epitope may be a linear epitope or a non-linear epitope. Preferably antigen-binding molecules of the invention bind to an epitope within amino acids residues 1-15 (SEQ ID NO: 121), 10-24 (SEQ ID NO: 122), 28-42 (SEQ ID NO: 124), 36-40 (SEQ ID NO: 2), 37-51 (SEQ ID NO: 125), 51-57 (SEQ ID NO: 3), 51-58 (SEQ ID NO: 136), 65-74 (SEQ ID NO: 4), 65-81 (SEQ ID NO: 5), 81-120 (SEQ ID NO: 137), 82-96 (SEQ ID NO: 130), 91-105 (SEQ ID NO: 131), 93-95 (GFV), 100-114 (SEQ ID NO: 132), 109-123 (SEQ ID NO: 133), 118-132 (SEQ ID NO: 134), 124-131 (SEQ ID NO: 7), 127-140 (SEQ ID NO: 135), 128-135 (SEQ ID NO: 8) or 131-140 (SEQ ID NO: 9) of human alpha-synuclein of SEQ ID NO: 1. More preferably, antigen-binding molecules of the invention bind to an epitope within amino acids residues 124-131 (SEQ ID NO: 7), 128-135 (SEQ ID NO: 8) or 131-140 (SEQ ID NO: 9) of human alpha-synuclein of SEQ ID NO: 1. Even more preferably, antigen-binding molecules of the invention may bind to an epitope comprising amino acids 126 and 127 of human alpha-synuclein of SEQ ID NO: 1 as critical residues for binding. In another embodiment, antigen-binding molecules of the invention bind to a non-linear epitope within amino acids residues of human alpha-synuclein of SEQ ID NO: 1.
Other alpha-synuclein binding molecules may also include multivalent molecules, multispecific molecules (e.g., diabodies or biparatopic antibodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly created molecules. Illustrative antigen-binding molecules useful in the present invention include antibody-like molecules. An antibody-like molecule is a molecule that can exhibit functions by binding to a target molecule (See, e.g., Current Opinion in Biotechnology 2006, 17:653-658; Current Opinion in Biotechnology 2007, 18:1-10; Current Opinion in Structural Biology 1997, 7:463-469; Protein Science 2006, 15:14-27), and includes, for example, DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229), Adnectin (WO 2002/032925) and fynomers (WO 2013/135588).
An “antigen binding molecule,” as used herein, is any molecule that can specifically or selectively bind to an antigen. A binding molecule may include or be an antibody or a fragment thereof. An alpha-synuclein binding molecule is a molecule that binds to the alpha-synuclein protein, such as an alpha-synuclein antibody or fragment thereof, at a specific recognition site, epitope.
The terms “alpha-synuclein antibody”, “anti-alpha-synuclein antibody” and “an antibody that binds to pathological and/or aggregated alpha-synuclein” or simply “antibody” as used herein refer to an antibody that is capable of binding pathological alpha-synuclein and/or aggregated alpha-synuclein, including, but not limited to, Lewy bodies, Lewy Neurites or glial cytoplasmic inclusions with sufficient affinity such that the antibody is useful as a therapeutic and/or diagnostic agent in targeting alpha-synuclein. In one embodiment, the extent of binding of an alpha-synuclein antibody of the invention to an unrelated, non-alpha-synuclein protein is less than about 10% of the binding of the antibody to alpha-synuclein as measured, e.g., by a radioimmunoassay (RIA).
In general, the term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully-human antibodies and antibody fragments so long as they exhibit the desired antigen-binding activity. Antibodies within the present invention may also be chimeric antibodies (especially mouse VH and VL regions fused with human constant domains), recombinant antibodies, antigen-binding fragments of recombinant antibodies, humanized antibodies or antibodies displayed upon the surface of a phage or displayed upon the surface of a chimeric antigen receptor (CAR) T-cell.
An “antigen-binding fragment” of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
The term “monoclonal antibody” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modified “monoclonal” indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495.
Accordingly, in context of the present invention, the term “antibody” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., “antigen-binding fragment thereof”). Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules. The term also relates to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fv, Fab′, Fab′-SH, F(ab′)2. The term “antibody” also comprises but is not limited to fully-human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
Humanized antibodies are modified antibodies that are also referred to as reshaped human antibodies. A humanized antibody is constructed by transferring the CDRs of an antibody derived from an immunized animal to the complementarity determining regions of a human antibody. Conventional genetic recombination techniques for such purposes are known (see European Patent Application Publication No. EP 239400; International Publication No. WO 96/02576; Sato K. et al., Cancer Research 1993, 53: 851-856; International Publication No. WO 99/51743).
The term “CDR” as employed herein relates to “complementary determining region”, which is well known in the art. The CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1, CDR2 and CDR3 in each V domain. VH-CDR, or CDR-H depicts a CDR region of a variable heavy chain and VL-CDR or CDR-L relates to a CDR region of a variable light chain. VH means the variable heavy chain and VL means the variable light chain. The CDR regions of an Ig-derived region may be determined as described in Kabat “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991); Chothia J., Mol. Biol. 196 (1987), 901-917 or Chothia, Nature 342 (1989), 877-883.
An “Fc” region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
A “Fab′ fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′) 2 molecule.
A “F(ab′)2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′)2 fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains.
The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
Accordingly, in the context of this invention, antibody molecules or antigen-binding fragments thereof are provided, which are humanized and can successfully be employed in pharmaceutical compositions.
An “antibody that binds to an epitope” within a defined region of a protein is an antibody that requires the presence of one or more of the amino acids within that region for binding to the protein.
In certain embodiments, an “antibody that binds to an epitope” within a defined region of a protein is identified by mutation analysis, in which amino acids of the protein are mutated, and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 20% of the binding to unaltered protein. In some embodiments, an “antibody that binds to an epitope” within a defined region of a protein is identified by mutation analysis, in which amino acids of the protein are mutated, and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding to unaltered protein. In certain embodiments, binding of the antibody is determined by FACS, WB or by a suitable binding assay such as ELISA.
The term “binding to” as used in the context of the present invention defines a binding (interaction) of at least two “antigen-interaction-sites” with each other. The term “antigen-interaction-site” defines, in accordance with the present invention, a motif of a polypeptide, i.e., a part of the antibody or antigen-binding fragment of the present invention, which shows the capacity of specific interaction with a specific antigen or a specific group of antigens of alpha-synuclein. Said binding/interaction is also understood to define a “specific recognition”. The term “specifically recognizing” means in accordance with this invention that the antibody is capable of specifically interacting with and/or binding to at least two amino acids of alpha-synuclein as defined herein (also known as “critical residues”), in particular interacting with/binding to at least two amino acids within residues 1-15 (SEQ ID NO: 121), 10-24 (SEQ ID NO: 122), 28-42 (SEQ ID NO: 124), 36-40 (SEQ ID NO: 2), 37-51 (SEQ ID NO: 125), 51-57 (SEQ ID NO: 3), 51-58 (SEQ ID NO: 136), 65-74 (SEQ ID NO: 4), 65-81 (SEQ ID NO: 5), 81-120 (SEQ ID NO: 137), 82-96 (SEQ ID NO: 130), 91-105 (SEQ ID NO: 131), 93-95 (GFV), 100-114 (SEQ ID NO: 132), 109-123 (SEQ ID NO: 133), 118-132 (SEQ ID NO: 134), 124-131 (SEQ ID NO: 7), 127-140 (SEQ ID NO: 135), 128-135 (SEQ ID NO: 8) or 131-140 (SEQ ID NO: 9) of human alpha-synuclein of SEQ ID NO: 1. The residues may form a linear or a non-linear epitope. Preferably, antigen-binding molecule of the invention bind to an epitope within amino acids residues 124-131 (SEQ ID NO: 7), 128-135 (SEQ ID NO: 8) or 131-140 (SEQ ID NO: 9) of human alpha-synuclein of SEQ ID NO: 1. Even more preferably, antigen-binding molecules of the invention may bind to an epitope comprising amino acids 126 and 127 of human alpha-synuclein of SEQ ID NO: 1 as critical residues for binding. The antigen binding molecules of the invention may also bind to a non-linear epitope within amino acids residues of human alpha-synuclein of SEQ ID NO: 1.
Cross-reactivity of antigen-binding molecules, in particular a panel of antibodies or antigen-binding fragments thereof under investigation may be tested, for example, by assessing binding of said panel of antibodies or antigen-binding fragments thereof under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. Only those constructs (i.e. antibodies, antigen-binding fragments thereof and the like) that bind to the certain structure of alpha-synuclein as defined herein, e.g., a specific epitope or (poly)peptide/protein of alpha-synuclein as defined herein but do not or do not essentially bind to any of the other epitope or (poly)peptides of the same alpha-synuclein, are considered specific for the epitope or (poly)peptide/protein of interest and selected for further studies in accordance with the method provided herein. These methods may comprise, inter alia, binding studies, blocking and competition studies with structurally and/or functionally closely related molecules. These binding studies also comprise FACS analysis, surface plasmon resonance (SPR, e.g. with BIACORE™), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or by radiolabeled ligand binding assays.
Accordingly, specificity can be determined experimentally by methods known in the art and methods as described herein. Such methods comprise, but are not limited to Western Blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
It may be understood by a person skilled in the art that the epitopes may be comprised in the alpha-synuclein protein, but may also be comprised in a degradation product thereof or may be a chemically synthesized peptide. The amino acid positions are only indicated to demonstrate the position of the corresponding amino acid sequence in the sequence of the alpha-synuclein protein. The invention encompasses all peptides comprising the epitope. The peptide may be a part of a polypeptide of more than 100 amino acids in length or may be a small peptide of less than 100, preferably less than 50, more preferably less than 25 amino acids, even more preferably less than 18 amino acids. The amino acids of such peptide may be natural amino acids or nonnatural amino acids (e.g., beta-amino acids, gamma-amino acids, D-amino acids) or a combination thereof. Further, the present invention may encompass the respective retro-inverso peptides of the epitopes. The peptide may be unbound or bound. It may be bound, e.g., to a small molecule (e.g., a drug or a fluorophor), to a high-molecular weight polymer (e.g., polyethylene glycol (PEG), polyethylene imine (PEI), hydroxypropylmethacrylate (HPMA), etc.) or to a protein, a fatty acid, a sugar moiety or may be inserted in a membrane. In order to test whether an antibody in question and the antibody of the present invention recognize the same or similar epitope, many assays are known in the art, some of which (e.g. “alanine scanning mutagenesis”) is described in below in example.
Whether an antibody recognizes the same epitope as or an epitope overlapping with an epitope that is recognized by another antibody as provided herein can be confirmed by competition between the two antibodies against the epitope. Competition between the antibodies can be evaluated by competitive binding assays using means such as enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer method (FRET), and fluorometric microvolume assay technology (FMAT®). The amount of antibodies bound to an antigen indirectly correlate with the binding ability of candidate competitor antibodies (test antibodies) that competitively bind to the same or overlapping epitope. In other words, as the amount of or the affinity of test antibodies against the same or overlapping epitope increases, the amount of antibodies bound to the antigen decreases, and the amount of test antibodies bound to the antigen increases. Specifically, the appropriately labeled antibodies and test antibodies are simultaneously added to the antigens, and then the bound antibodies are detected using the label. The amount of the antibodies bound to the antigen can be easily determined by labeling the antibodies in advance. This label is not particularly limited, and the labeling method is selected according to the assay technique used. Specific examples of the labeling method include fluorescent labeling, radiolabeling, and enzyme labeling.
Herein, the “antibody that binds to the overlapping epitope” or “antibody that binds to the same epitope” refers to a test antibody that can reduce the amount of binding of the labeled antibody by at least 50% at a concentration that is usually 100 times higher, preferably 80 times higher, more preferably 50 times higher, even more preferably 30 times higher, and still more preferably 10 times higher than a concentration of the non-labeled antibody at which binding of the non-labeled antibody reduces the amount of binding of the labeled antibody by 50% (1050). The epitope recognized by the antibody can be analyzed by methods known to those skilled in the art, and for example, it can be performed by Western blotting and such.
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, the antibody comprises:
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 12; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; (c) VH-CDR3 comprising the amino acid sequence YSY; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 26; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 33; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 37.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 45; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 47.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 52; (c) VH-CDR3 comprising the amino acid sequence YSF; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 55; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 56; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 62; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 65; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 67.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; (c) VH-CDR3 comprising the amino acid sequence YSY; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 75; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 77.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 33; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 91; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 92; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 93; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 95; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 96; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 97.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 101; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 102; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 103; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 111; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 113; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 115; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 117.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 281; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 282; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 283; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 285; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 286; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 287.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 192; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 193; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 195; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 96; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 197.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 141; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 142; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 143; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 145; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 151; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 152; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 153; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 161; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 162; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 163.
In some embodiments, an alpha-synuclein antibody comprises at least four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 161; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 162; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 163; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 165; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 167.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 171; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 172; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 173; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 175; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 176; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 177.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 181; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 182; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 183; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 187.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 201; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 202; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 153; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 206; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 211; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 212; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 213; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 215; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 216; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 217.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 222; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 223; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 225; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 96; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 227.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 231; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 232; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 233; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 235; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 236; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 237.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 242; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 243; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 225; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 96; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 247.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 252; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 253; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 255; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 256; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 257.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 261; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 262; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 263; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 265; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 176; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 267.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 271; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 272; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 273; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 275; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 276; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 277.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 301; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 302; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 303; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 307.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 311; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 312; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 313; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 315; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 67.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 321; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 322; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 323; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 325; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 326; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 327.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 151; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 332; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 333; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 335; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 336; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 341; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 342; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 343; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 345; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 346; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 347.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 351; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 352; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 353; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 355; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 356; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 357.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 361; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 362; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 363; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 365; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 367.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 371; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 372; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 373; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 345; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 376; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 347.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 351; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 382; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 383; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 385; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 386; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 387.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 351; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 382; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 393; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 395; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 356; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 357.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 351; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 382; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 393; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 405; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 356; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 357.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 411; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 412; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 413; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 421; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 422; (c) VH-CDR3 comprising the amino acid sequence GNY; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 425; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 426; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 427.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 431; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 432; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 433; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 435; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 436; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 437.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 151; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 442; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 443; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 461; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 462; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 463; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 465; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 467.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 141; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 472; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 473; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 475; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 476; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 477.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 481; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 482; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 483; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 165; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 487.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 141; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 492; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 493; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 495; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 496; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 497.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 151; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 502; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 503; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 336; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 311; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 512; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 513; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 515; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 516; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 517.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 521; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 522; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 463; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 525; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 467.
In some embodiments, an alpha-synuclein antibody comprises at least one, two or three CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 371; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 532; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 533.
In some embodiments, an alpha-synuclein antibody comprises at least four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 371; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 532; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 533; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 345; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 376; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 537.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 341; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 542; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 543; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 345; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 376; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 347.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 551; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 552; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 553; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 555; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 557.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 551; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 552; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 563; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 565; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 557.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 571; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 202; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 573; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 581; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 582; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 583; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 585; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 586; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 587.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 612; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 615; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 612; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 625; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 612; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 663; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 615; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 612; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 673; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 615; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 612; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 683; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 615; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, three, four, five, or six CDRs selected from (a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 612; (c) VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 683; (d) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 625; (e) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, or three CDRs selected from (a) VH-CDR1 comprising the amino acid sequence selected from SEQ ID NO: 11, 21, 31, 41, 61, 91, 101, 111,141, 151, 161, 171, 181, 201, 211, 231, 261, 271, 281, 301, 311, 321, 341, 351, 361, 371, 411, 421, 431, 461, 481, 521, 551, 571 and 581, (b) VH-CDR2 comprising the amino acid sequence selected from SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 92, 102, 112, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 302, 312, 322, 332, 342, 352, 362, 372, 382, 412, 422, 432, 442, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 582 and 612, (c) VH-CDR3 comprising the amino acid sequence selected from SEQ ID NO: 13, YSY, 33, 43, YSF, 93, 103, 113, 143, 153, 163, 173, 183, 193, 213, 223, 233, 243, 253, 263, 273, 283, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393, 413, GNY, 433, 443, 463, 473, 483, 493, 503, 513, 533, 543, 553, 563, 573, 583, 663, 673 and 683.
In some embodiments, an alpha-synuclein antibody comprises at least one, two, or three CDRs selected from (a) VL-CDR1 comprising the amino acid sequence selected from SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 145, 165, 175, 195, 215, 225, 235, 255, 265, 275, 285, 315, 325, 335, 345, 355, 365, 385, 395, 405, 425, 435, 465, 475, 495, 515, 525, 555, 565, 585, 615 and 625, (b) VL-CDR2 comprising the amino acid sequence selected from SEQ ID NO: 16, 26, 36, 46, 56, 76, 96, 106, 176, 206, 216, 236, 256, 276, 286, 326, 336, 346, 356, 376, 386, 426, 436, 476, 496, 516 and 586, (c) VL-CDR3 comprising the amino acid sequence selected from SEQ ID NO: 17, 27, 37, 47, 67, 77, 87, 97, 107, 117, 167, 177, 187, 197, 217, 227, 237, 247, 257, 267, 277, 287, 307, 327, 347, 357, 367, 387, 427, 437, 467, 477, 487, 497, 517, 537, 557 and 587.
In another embodiment, the alpha-synuclein antibody comprises a heavy chain variable domain (VH) selected from SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 90, 100, 110, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710 and 720 including post-translational modifications of that sequence.
In a particular embodiment, the heavy chain variable domain (VH) comprises at least one, two, or three CDRs selected from (a) VH-CDR1 comprising the amino acid sequence selected from SEQ ID NO: 11, 21, 31, 41, 61, 91, 101, 111, 141, 151, 161, 171, 181, 201, 211, 231, 261 271, 281, 301, 311, 321, 341, 351, 361, 371, 411, 421, 431, 461, 481, 521, 551, 571 and 581, (b) VH-CDR2 comprising the amino acid sequence selected from SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 92, 102, 112, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 302, 312, 322, 332, 342, 352, 362, 372, 382, 412, 422, 432, 442, 462, 472, 482, 492, 502, 512, 522, 532, 542, 552, 582 and 612, (c) VH-CDR3 comprising the amino acid sequence selected from SEQ ID NO: 13, YSY, 33, 43, YSF, 93, 103, 113, 143, 153, 163, 173, 183, 193, 213, 223, 233, 243, 253, 263, 273, 283, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393, 413, GNY, 433, 443, 463, 473, 483, 493, 503, 513, 533, 543, 553, 563, 573, 583, 663, 673 and 683.
In another embodiment, the alpha-synuclein antibody comprises a light chain variable domain (VL) selected from SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 144, 154, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, 274, 284, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, 404, 414, 424, 434, 464, 474, 484, 494, 504, 514, 524, 544, 554, 564, 574, 584, 614, 624, 634 and 644 including post-translational modifications of that sequence.
In a particular embodiment, the light chain variable domain (VL) comprises at least one, two, or three CDRs selected from (a) VL-CDR1 comprising the amino acid sequence selected from SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 145, 165, 175, 195, 215, 225, 235, 255, 265, 275, 285, 315, 325, 335, 345, 355, 365, 385, 395, 405, 425, 435, 465, 475, 495, 515, 525, 555, 565, 585, 615 and 625 (b) VL-CDR2 comprising the amino acid sequence selected from SEQ ID NO: 16, 26, 36, 46, 56, 76, 96, 106, 176, 206, 216, 236, 256, 276, 286, 326, 336, 346, 356, 376, 386, 426, 436, 476, 496, 516 and 586, (c) VL-CDR3 comprising the amino acid sequence selected from SEQ ID NO: 17, 27, 37, 47, 67, 77, 87, 97, 107, 117, 167, 177, 187, 197, 217, 227, 237, 247, 257, 267, 277, 287, 307, 327, 347, 357, 367, 387, 427, 437, 467, 477, 487, 497, 517, 537, 557 and 587.
In some embodiments, the invention relates to an antibody selected from ACI-7067-1101C8-Ab2, ACI-7067-1102G3-Ab1, ACI-7067-1106A8-Ab2, ACI-7067-1107G5-Ab2, ACI-7067-1108H1-Ab1, ACI-7067-1111B12-Ab2, ACI-7067-1112H8-Ab2, ACI-7067-1108B11-Ab2, ACI-7067-1113D10-Ab1, ACI-7067-1116F2-Ab1, ACI-7067-1206E5-Ab1, ACI-7079-2501B11-Ab3, ACI-7079-2501D10-Ab1, ACI-7079-2501G2-Ab2, ACI-7079-2503C6-Ab1, ACI-7079-2504A6-Ab1, ACI-7079-2506E2-Ab2, ACI-7079-2506F3-Ab1, ACI-7079-2507B3-Ab1, ACI-7079-2511B3-Ab3, ACI-7079-2601B6-Ab1, ACI-7079-2602G4-Ab4, ACI-7079-2603C1-Ab3, ACI-7079-2603F3-Ab1, ACI-7079-2605B3-Ab2, ACI-7079-2606A6-Ab2, ACI-7079-2509E5-Ab2, ACI-7087-4119E10-Ab2, ACI-7087-4125E6-Ab1, ACI-7088-4301 D5-Ab2, ACI-7088-4301E12-Ab2, ACI-7088-4301H3-Ab2, ACI-7088-4303A1-Ab1, ACI-7088-4303A3-Ab1, ACI-7088-4303B6-Ab2, ACI-7088-4303H6-Ab1, ACI-7088-4305H7-Ab1, ACI-7088-4317A4-Ab1, ACI-7089-4409F1-Ab1, ACI-7089-4415G5-Ab1, ACI-7089-4417G6-Ab1, ACI-7089-4418C5-Ab1, ACI-7089-4418F6-Ab1, ACI-8033-5A12-Ab1, ACI-8033-25A3-Ab1, ACI-8033-1G10-Ab1, ACI-8033-19A2-Ab1, ACI-8033-8C10-Ab1, ACI-8033-7A2-Ab1, ACI-8033-1A12-Ab1, ACI-8033-4F3-Ab1, ACI-8033-17F5-Ab1, ACI-8033-18C11-Ab1, ACI-8033-18D12-Ab1, ACI-8033-1F8-Ab1, ACI-8033-22E5-Ab1, ACI-8033-27D8-Ab1, ACI-8033-21C8-Ab1, hACI-7067-1101C8-Ab2_H1L1, hACI-7067-1101C8-Ab2_H1 L2, hACI-7067-1101C8-Ab2_H1 L3, hACI-7067-1101C8-Ab2_H1 L4, hACI-7067-1101C8-Ab2_H2L1, hACI-7067-1101C8-Ab2_H2L2, hACI-7067-1101C8-Ab2_H2L3, hACI-7067-1101C8-Ab2_H2L4, hACI-7067-1101C8-Ab2_H3L1, hACI-7067-1101C8-Ab2_H3L2, hACI-7067-1101C8-Ab2_H3L3, hACI-7067-1101C8-Ab2_H3L4, hACI-7067-1101C8-Ab2_H4L1, hACI-7067-1101C8-Ab2_H4L2, hACI-7067-1101C8-Ab2_H4L3, hACI-7067-1101C8-Ab2_H4L4, hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H5L2, hACI-7067-1101C8-Ab2_H5L3, hACI-7067-1101C8-Ab2_H5L4, hACI-7067-1101C8-Ab2_H6L1, hACI-7067-1101C8-Ab2_H7L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2, hACI-7067-1101C8-Ab2_H10L1, hACI-7067-1101C8-Ab2_H10L2, hACI-7067-1101C8-Ab2_H11L1, hACI-7067-1101C8-Ab2_H11L2, hACI-7067-1101C8-Ab2_H12L1 and hACI-7067-1101C8-Ab2_H12L2. In certain preferred embodiments, the antibody may be selected from hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2, hACI-7067-1101C8-Ab2_H10L1, hACI-7067-1101C8-Ab2_H10L2, hACI-7067-1101C8-Ab2_H11L1, hACI-7067-1101C8-Ab2_H11L2, hACI-7067-1101C8-Ab2_H12L1 and hACI-7067-1101C8-Ab2_H12L2. As demonstrated herein, these humanized antibodies display advantageous affinity to alpha synuclein, expression levels and sequence identity to the human acceptor framework. They all delay seeded aggregation. In certain preferred embodiments, the antibody may be selected from hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2 and hACI-7067-1101C8-Ab2_H10L1. As demonstrated herein, these humanized antibodies display improved affinity against the aggregated form of alpha synuclein compared to the chimeric antibody cACI-7067-1101C8-Ab2. In certain preferred embodiments, the antibody may be selected from hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2, hACI-7067-1101C8-Ab2_H10L1 and hACI-7067-1101C8-Ab2_H10L2. As demonstrated herein, these humanized antibodies display efficacy in delaying alpha synuclein aggregation compared to the chimeric antibody cACI-7067-1101C8-Ab2.
In some embodiments, an antibody binds to the same or similar epitope (totally or partially overlapping epitope) as an antibody selected from ACI-7067-1101C8-Ab2, ACI-7067-1102G3-Ab1, ACI-7067-1106A8-Ab2, ACI-7067-1107G5-Ab2, ACI-7067-1108H1-Ab1, ACI-7067-1111B12-Ab2, ACI-7067-1112H8-Ab2, ACI-7067-1108B11-Ab2, ACI-7067-1113D10-Ab1, ACI-7067-1116F2-Ab1, ACI-7067-1206E5-Ab1, ACI-7079-2501B11-Ab3, ACI-7079-2501D10-Ab1, ACI-7079-2501G2-Ab2, ACI-7079-2503C6-Ab1, ACI-7079-2504A6-Ab1, ACI-7079-2506E2-Ab2, ACI-7079-2506F3-Ab1, ACI-7079-2507B3-Ab1, ACI-7079-2511B3-Ab3, ACI-7079-2601B6-Ab1, ACI-7079-2602G4-Ab4, ACI-7079-2603C1-Ab3, AC1-7079-2603F3-Ab1, ACI-7079-2605B3-Ab2, ACI-7079-2606A6-Ab2, ACI-7079-2509E5-Ab2, AC1-7087-4119E10-Ab2, ACI-7087-4125E6-Ab1, ACI-7088-4301 D5-Ab2, ACI-7088-4301E12-Ab2, ACI-7088-4301H3-Ab2, ACI-7088-4303A1-Ab1, ACI-7088-4303A3-Ab1, ACI-7088-4303B6-Ab2, ACI-7088-4303H6-Ab1, ACI-7088-4305H7-Ab1, ACI-7088-4317A4-Ab1, ACI-7089-4409F1-Ab1, ACI-7089-4415G5-Ab1, ACI-7089-4417G6-Ab1, ACI-7089-441805-Ab1, ACI-7089-4418F6-Ab1, ACI-8033-5A12-Ab1, ACI-8033-25A3-Ab1, ACI-8033-1G10-Ab1, ACI-8033-19A2-Ab1, ACI-8033-8C10-Ab1, ACI-8033-7A2-Ab1, ACI-8033-1A12-Ab1, ACI-8033-4F3-Ab1, ACI-8033-17F5-Ab1, ACI-8033-18011-Ab1, ACI-8033-18D12-Ab1, ACI-8033-1F8-Ab1, ACI-8033-22E5-Ab1, ACI-8033-27D8-Ab1, ACI-8033-2108-Ab1, hACI-7067-1101C8-Ab2_H1 L1, hACI-7067-1101C8-Ab2_H1 L2, hACI-7067-1101C8-Ab2_H1 L3, hACI-7067-1101C8-Ab2_H1 L4, hACI-7067-1101C8-Ab2_H2L1, hACI-7067-1101C8-Ab2_H2L2, hACI-7067-1101C8-Ab2_H2L3, hACI-7067-1101C8-Ab2_H2L4, hACI-7067-1101C8-Ab2_H3L1, hACI-7067-1101C8-Ab2_H3L2, hACI-7067-1101C8-Ab2_H3 L3, hACI-7067-1101C8-Ab2_H3L4, hACI-7067-1101C8-Ab2_H4L1, hACI-7067-1101C8-Ab2_H4L2, hACI-7067-1101C8-Ab2_H4L3, hACI-7067-1101C8-Ab2_H4L4, hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H5L2, hACI-7067-1101C8-Ab2_H5L3, hACI-7067-1101C8-Ab2_H5L4, hACI-7067-1101C8-Ab2_H6L1, hACI-7067-1101C8-Ab2_H7L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2, hACI-7067-1101C8-Ab2_H10L1, hACI-7067-1101C8-Ab2_H10L2, hACI-7067-1101C8-Ab2_H11L1, hACI-7067-1101C8-Ab2_H11L2, hACI-7067-1101C8-Ab2_H12L1 and hACI-7067-1101C8-Ab2_H12L2. In certain preferred embodiments, the antibody binds to the same or similar epitope (totally or partially overlapping epitope) as an antibody selected from hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2, hACI-7067-1101C8-Ab2_H10L1, hACI-7067-1101C8-Ab2_H10L2, hACI-7067-1101C8-Ab2_H11L1, hACI-7067-1101C8-Ab2_H11L2, hACI-7067-1101C8-Ab2_H12L1 and hACI-7067-1101C8-Ab2_H12L2. As demonstrated herein, these humanized antibodies display advantageous affinity to alpha synuclein, expression levels and sequence identity to the human acceptor framework. They all delay seeded aggregation. In certain preferred embodiments, the antibody binds to the same or similar epitope (totally or partially overlapping epitope) as an antibody selected from hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2 and hACI-7067-1101C8-Ab2_H10L1. As demonstrated herein, these humanized antibodies display improved affinity against the aggregated form of alpha synuclein compared to the chimeric antibody cACI-7067-1101C8-Ab2. In certain preferred embodiments, the antibody binds to the same or similar epitope (totally or partially overlapping epitope) as an antibody selected from hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2, hACI-7067-1101C8-Ab2_H10L1 and hACI-7067-1101C8-Ab2_H10L2. As demonstrated herein, these humanized antibodies display efficacy in delaying alpha synuclein aggregation compared to the chimeric antibody cACI-7067-1101C8-Ab2.
In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 2. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 3. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 4. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 5. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence comprising amino acids 93-95 of SEQ ID NO: 1. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 7. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 8. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 9. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 121. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 136. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 130. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 131. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 134. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 135. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 122. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 124. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 125. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 132. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 133. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same or similar epitope comprising the sequence SEQ ID NO: 137. In some embodiments, an isolated antibody is provided wherein the isolated antibody binds to the same or similar non-linear epitope within amino acids residues of human alpha-synuclein of SEQ ID NO: 1. The term “the same or similar epitope” references any antibody provided herein.
Antibodies binding the same epitope as any of the antibodies provided herein are also part of the invention. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 2. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 3. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 4. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 5. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence comprising amino acids 93-95 of SEQ ID NO: 1. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 7. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 8. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 9. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 121. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 136. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 130. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 131. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 134. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 135. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 122. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 124. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 125. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 132. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 133. In some embodiments, an isolated antibody is provided, wherein the isolated antibody binds to the same epitope comprising the sequence SEQ ID NO: 137. In some embodiments, an isolated antibody is provided wherein the isolated antibody binds to the same non-linear epitope within amino acids residues of human alpha-synuclein of SEQ ID NO: 1. The term “the same epitope” references any antibody provided herein.
In accordance with the above, in certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
Amino acids may be grouped according to common side-chain properties:
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a murine Fc region sequence (e.g.: IgG1, IgG2a or IgG2b) comprising an amino acid modification (e.g. substitution) at one or more amino acid positions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions (e.g. an IgG4 isotype including the S228P mutation).
In certain embodiments, the Fc region is mutated to increase its affinity to FcRn at pH 6.0 and consequently extend the antibody half-life. Antibodies with enhanced affinity to FcRn include those with substitution of one or more of Fc region residues 252, 253, 254, 256, 428, 434, including the so called YTE mutation with substitution M252Y/S254T/T256E (Dall' Acqua et al, J Immunol. 169:5171-5180 (2002)) or LS mutation M428L/N434S (Zalevsky et al, Nat Biotechnol. 28(2): 157-159 (2010)).
In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement activation and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes and microglia express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235, 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581) or the so-called “DANG” FC mutant with substitution of residues 265 to alanine and 297 to Glycine. Alternatively, antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235 and 329, so-called “PG-LALA” Fc mutant with substitution of residues 234 and 235 to alanine and 329 to glycine (Lo, M. et al., Journal of Biochemistry, 292, 3900-3908). Other known mutations at position 234, 235 and 321, the so called TM mutant containing mutations L234F/L235E/P331S in the CH2 domain, can be used (Oganesyan et al. Acta Cryst. D64, 700-704. (2008)). Antibodies from the human IgG4 isotype include mutations S228P/L235E to stabilize the hinge and to reduce FgR binding (Schlothauer et al, PEDS, 29 (10):457-466).
Other Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821.
Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acid encoding an alpha-synuclein antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20). In one embodiment, a method of making an anti-alpha-synuclein antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of an alpha-synuclein antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
The present invention also relates to the production of specific antibodies against native polypeptides and recombinant polypeptides of alpha-synuclein. This production is based, for example, on the immunization of animals, like mice. However, also other animals for the production of antibody/antisera are envisaged within the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbit, mice, goats, donkeys and the like. The polynucleotide encoding a correspondingly chosen polypeptide of alpha-synuclein can be subcloned into an appropriate vector, wherein the recombinant polypeptide is to be expressed in an organism being suitable for its expression, for example in bacteria. Thus, the expressed recombinant protein can be injected into a mice and the resulting specific antibody can be, for example, obtained from the mice serum being provided by intra-cardiac blood puncture. Many other strategies are known in the art, such as the use of DNA vaccine strategies which is well-known in the art and encompass liposome-mediated delivery, by gene gun or jet injection and intramuscular or intradermal injection. Thus, antibodies directed against a polypeptide or a protein or an epitope of alpha-synuclein, in particular the epitope of the antibodies provided herein, can be obtained by directly immunizing the animal by directly injecting intramuscularly the vector expressing the desired polypeptide or a protein or an epitope of alpha-synuclein. The amount of obtained specific antibody can be quantified using an ELISA, which is also described herein below. Further methods for the production of antibodies are well known in the art, see, e.g. Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.
Accordingly, antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, DNA encoding the antibody of interest is inserted into an expression vector. Insertion into an expression vector is carried out such that the expression will take place under the control of expression regulatory regions such as enhancers and promoters. Next, host cells are transformed using this expression vector to express the antibodies. Appropriate combinations of the host and expression vector can be used in this step.
Examples of the vectors include M13 series vectors, pUC series vectors, pBR322, pBluescript, and pCR-Script. In addition to these vectors, for example, pGEM-T, pDIRECT, or pT7 can also be used for the purpose of cDNA subcloning and excision.
Particularly, expression vectors are useful for the purpose of producing the antibody. For example, when the host is E. coli such as JM109, DH5α, HB101, or XL1-Blue, the expression vectors indispensably have a promoter that permits efficient expression in E. coli, for example, lacZ promoter (Ward et al., Nature (1989) 341, 544-546; and FASEB J (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoter. Examples of such vectors include the vectors mentioned above as well as pGEX-5X-1 (manufactured by Pharmacia), “QIAexpress system” (manufactured by QIAGEN), pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).
The vectors may contain a signal sequence for polypeptide secretion. In the case of production in the periplasm of E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4397) can be used as the signal sequence for polypeptide secretion. The vectors can be transferred to the host cells using, for example, calcium chloride methods or electroporation methods.
In addition to the E. coli expression vectors, examples of the vectors for producing the antibody of the present invention include mammal-derived expression vectors (e.g., pcDNA3 (manufactured by Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, and pCDM8), insect cell-derived expression vectors (e.g., “Bac-to-BAC baculovirus expression system” (manufactured by GIBCO BRL), and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., “Pichia Expression Kit” (manufactured by Invitrogen Corp.), pNV11, and SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608 and pKTH50).
For the purpose of expression in animal cells such as CHO cells, COS cells, or NIH3T3 cells, the vectors indispensably have a promoter necessary for intracellular expression, for example, SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res (1990) 18, 5322), CAG promoter (Gene (1991) 108, 193), or CMV promoter and, more preferably, have a gene for screening for transformed cells (e.g., a drug resistance gene that can work as a marker by a drug (neomycin, G418, etc.)). Examples of the vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
An exemplary method intended to stably express the gene and increase the number of intracellular gene copies involves transfecting CHO cells deficient in nucleic acid synthesis pathway with vectors having a DHFR gene serving as a complement thereto (e.g., pCHOI) and using methotrexate (MTX) in the gene amplification. An exemplary method intended to transiently express the gene involves using COS cells having a gene which expresses an SV40 T antigen on their chromosomes to transform the cells with vectors having a replication origin of SV40 (pcD, etc.). Also, a replication origin derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), or the like may be used. The expression vectors for increasing the number of gene copies in a host cell system can additionally contain a selection marker such as an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, or a dihydrofolate reductase (dhfr) gene.
The antibodies of the present invention obtained by the methods described above can be isolated from inside host cells or from outside of the cells (the medium, or such), and purified to practically pure and homogeneous antibodies. The antibodies can be separated and purified by methods routinely used for separating and purifying antibodies, and the type of method is not limited. For example, the antibodies can be separated and purified by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting-out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectrofocusing, dialysis, recrystallization, and such.
The chromatographies include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). The chromatographic methods described above can be conducted using liquid-chromatography, for example, HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Columns using protein A include, for example, Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The present invention includes antibodies that are highly purified using these purification methods.
The obtained antibodies can be purified to homogeneity. Separation and purification of the antibodies can be performed using separation and purification methods generally used for protein separation and purification. For example, the antibodies can be separated and purified by appropriately selecting and combining column chromatography such as affinity chromatography, filtration, ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and such, without limitation (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Columns used for affinity chromatography include, for example, protein A columns and protein G columns.
Alpha-synuclein antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art. In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, BIACore®, FACS, immunofluorescence or immunohistochemistry.
In another aspect, competition assays may be used to identify an antibody that competes with any of the antibodies described herein for binding to aggregated or pathological alpha-synuclein. In certain embodiments, such a competing antibody binds to the same or similar epitope (e.g., a linear or a conformational epitope with total or partial overlap) that is bound by an antibody described herein. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).
The invention also provides immunoconjugates comprising an alpha-synuclein antibody provided herein conjugated to one or more therapeutic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), radioactive isotopes (i.e., a radioconjugate), blood brain barrier penetration moieties or detectable labels.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease or disorder or abnormality, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease, disorder or abnormality. In particular embodiments, the binding molecules of the invention are for preventing, slowing down, halting, retaining and/or improving the motor capabilities or motor deficits, cognitive capabilities or cognitive deficits, or behavioral impairments of a subject suffering from a synucleopathy. In further particular embodiments, the binding molecules of the invention are for improving motor capabilities, in particular facial expression, speech, ocular motor dysfunction, tremor at rest, action tremor, increased tone, rapid alternating movement of hands, finger tapping, leg agility, Heel-Shin test, arising from chair, posture, body sway and/or gait; improving cognitive deficits, in particular as measured by MoCA (Montreal Cognitive Assessment) or Addenbrookes Cognitive Examination; and/or improving behavioral impairments, in particular using NPI scale, wherein the synucleopathy is multiple system atrophy (MSA).
In a further embodiment, when the synucleopathy is Parkinson's disease, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease, the binding molecules of the invention are for: (i) improving motor capabilities, in particular activities of daily living (speech, salivation, swallowing, handwriting, cutting food and handling utensils, dressing, hygiene, turning in bed and adjusting bed clothes, falling, freezing when walking, walking, tremor, sensory complaints related to Parkinsonism), motor examination (speech, facial expression, tremor at rest, action or postural tremor of hands, rigidity, finger taps, hand movements, rapid alternating movements of hands, leg agility, arising from chair, posture, gait, postural stability, body bradykinesia and hypokinesia, dyskinesias, clinical fluctuations), symptomatic orthostatis, repeated falls and syncope, and/or transient unexplained loss of consciousness; and/or (ii) improving cognitive deficits; and/or (iii) improving behavioral impairments, in particular behavior and mood (intellectual Impairment, thought disorder, depression, motivation/initiative), delusions, hallucinations, agitation/aggression, depression/dysphoria, anxiety, elation/euphoria, apathy/indifference, irritability/lability, motor disturbance, nighttime behavior, and/or appetite/eating, deficits of attention, executive functions, visuospatial ability, visual hallucination; and/or (iv) improving rapid eye movement (REM) sleep disorders, in particular insomnia, hypersomnolence.
In one embodiment, a pharmaceutical composition is provided comprising the antibody, antigen-binding fragment thereof or derivative thereof, as an active ingredient and a pharmaceutically acceptable carrier and/or excipient. For example, the antibody, antigen-binding fragment thereof or derivative thereof may be combined, as appropriate, with pharmaceutically acceptable carriers or media such as sterilized water or saline solution, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, and binders, for example, and formulated into a pharmaceutical preparation. Examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, cannellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.
The amount of the active ingredient in these preparations can be set as appropriate within the designated range of doses.
In another embodiment, the present disclosure provides a product comprising at least (i) a container (e.g., an injection); (ii) a pharmaceutical composition comprising the antibody, antigen-binding fragment thereof or derivative thereof as an active ingredient within the container; and (iii) a document instructing that the antibody, antigen-binding fragment thereof or derivative thereof be administered according to a desired dosage regimen. Additionally, a label, a syringe, an injection needle, a pharmacologically acceptable medium, an alcohol cotton cloth, plaster, and the like may be additionally packaged, as appropriate, with this product. The container may be a bottle, a glass bottle, or a syringe, for example, and may be made of any of various materials such as glass and plastics. The container contains the pharmaceutical composition, and has an outlet sealed with a rubber stopper, for example. The container is provided with, for example, a label indicating that the pharmaceutical composition is for use in preventing or treating a selected pathological condition. In some cases, this label may describe the embodiment where the antibody, antigen-binding fragment thereof or derivative thereof is used in combination with an additional medicament.
An antibody, immunoconjugate, pharmaceutical composition of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
Antibodies, immunoconjugates, pharmaceutical compositions of the invention may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disease or disorder or abnormality being treated, the particular subject being treated, the clinical condition of the individual patient, the cause of the disease or disorder or abnormality, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody or immunoconjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disease or disorder or abnormality in question. The effective amount of such other agents depends on the amount of antibody or immunoconjugate present in the formulation, the type of disease, or disorder or abnormality or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
It is understood that any of the above formulations or therapeutic methods may be carried out using both an immunoconjugate of the invention and an alpha-synuclein antibody.
In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid encodes an antibody described herein.
In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 18 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 28 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 38 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 48 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 58 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 68 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 78 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 98 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 108 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 118 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 288 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 298 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 148 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 158 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 168 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 178 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 188 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 198 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 208 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 218 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 228 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 238 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 248 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 258 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 268 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 278 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 308 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 318 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 328 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 338 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 348 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 358 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 368 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 378 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 388 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 398 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 408 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 418 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 428 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 438 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 448 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 458 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 468 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 478 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 488 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 498 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 508 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 518 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 528 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 538 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 548 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 558 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 568 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 578 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 588 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 598 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 608 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 618 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 628 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 638 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 648 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 658 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 668 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 678 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 688 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 698 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 708 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 718 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 728 encoding an alpha-synuclein antibody.
In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 19 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 29 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 39 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 49 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 59 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 69 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 79 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 89 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 99 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 109 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 119 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 289 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 199 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 149 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 159 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 169 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 179 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 189 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 209 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 219 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 229 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 239 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 249 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 259 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 269 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 279 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 309 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 319 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 329 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 339 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 349 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 359 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 369 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 379 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 389 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 399 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 409 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 419 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 429 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 439 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 449 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 459 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 469 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 479 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 489 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 499 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 509 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 519 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 529 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 539 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 549 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 559 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 569 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 579 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 589 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 609 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 619 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 629 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 639 encoding an alpha-synuclein antibody. In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid comprises SEQ ID NO: 649 encoding an alpha-synuclein antibody.
In certain embodiments, a binding molecule or an antibody provided herein has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10−8 M or less, e.g. from 10−8 M to 10−13 M, e.g., from 10−9 M to 10−13 M), in particular with respect to binding alpha-synuclein, in particular aggregated alpha-synuclein and/or pathological alpha-synuclein.
In certain embodiments, a binding molecule or an antibody provided herein has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10−8 M or less, e.g. from 10−8 M to 10−13 M, e.g., from 10−9 M to 10−13 M), in particular with respect to binding pathological and/or aggregated alpha-synuclein, including but limited to protofibrils, fibrils, oligomers, Lewy Body, Lewy neurites and/or glial cytoplasmic inclusions.
In one embodiment, KD is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at −10 response units (RU).
In some embodiments, an antibody, particularly an isolated antibody of the invention as described herein that binds human alpha-synuclein is provided, wherein the antibody binds aggregated alpha-synuclein and/or pathological alpha-synuclein with a KD of less than 100 nM, less than 10 nM, less than 1 nM, less than 200 pM, less than 100 pM, or less than 10 pM. Preferably, the antibody of the invention binds aggregated alpha-synuclein and/or pathological alpha-synuclein with a KD of less than 100 nM, less than 10 nM, less than 1 nM, less than 200 pM, less than 100 pM, or less than 10 pM.
The binding molecules, especially antibodies, of the invention may selectively bind aggregated alpha-synuclein and/or pathological alpha-synuclein in preference to non-aggregated alpha-synuclein and/or non-pathological alpha-synuclein (such as monomeric alpha-synuclein). This selectivity may be measured in terms of dissociation (or “off”) rates (kd). Thus, the binding molecules, especially antibodies, of the invention may display slower, preferably significantly slower, dissociation rates (kd) from aggregated alpha-synuclein and/or pathological alpha-synuclein (such as fibrillar alpha-synuclein) compared to non-aggregated alpha-synuclein and/or non-pathological alpha-synuclein (such as monomeric alpha-synuclein). For example, the binding molecules, especially antibodies, of the invention may display at least 10-fold, preferably at least 100-fold, and more preferably at least 1000-fold slower dissociation rates (kd) from aggregated alpha-synuclein and/or pathological alpha-synuclein (such as fibrillar alpha-synuclein) compared to non-aggregated alpha-synuclein and/or non-pathological alpha-synuclein (such as monomeric alpha-synuclein). This selectivity may be measured in terms of relative dissociation constant (KD). Thus, the binding molecules, especially antibodies, of the invention may display lower, preferably significantly lower, dissociation constants (KD) with respect to aggregated alpha-synuclein and/or pathological alpha-synuclein (such as fibrillar alpha-synuclein) compared to non-aggregated alpha-synuclein and/or non-pathological alpha-synuclein (such as monomeric alpha-synuclein). For example, the binding molecules, especially antibodies, of the invention may display at least 10-fold, more preferably at least 20-fold, and more preferably at least 100-fold lower dissociation constants (KD) with respect to aggregated alpha-synuclein and/or pathological alpha-synuclein (such as fibrillar alpha-synuclein) compared to non-aggregated alpha-synuclein and/or non-pathological alpha-synuclein (such as monomeric alpha-synuclein). KD and kd may be measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at −10 response units (RU). Specific methodology is described in the Examples section herein (see “Affinity measurements on alpha-synuclein monomers and alpha-synuclein fibrils by SPR” and “Characterization of ACI-7067-1101C8-Ab2 humanized variants by Surface Plasmon resonance (SPR)”), which may be applied according to the invention as a reference method.
The binding molecules, especially antibodies, of the invention may inhibit and/or delay seeded and/or spontaneous alpha-synuclein aggregation with an IC50 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM or ≤0.1 nM. The IC50 may be obtained by measuring the percentage of de novo alpha-synuclein aggregates formed, relative to conditions in the absence of antibody, as a function of antibody concentration. Dose-response curves may be plotted and IC50 values obtained using Equation 6. See
The binding molecules, especially antibodies, of the invention may inhibit and/or delay seeded and/or spontaneous alpha-synuclein aggregation as quantified by a percent change in the aggregation half-time (T1/2). Suitable methodology for measuring the aggregation half-time is provided herein, see the Examples “Inhibition or delay of seeded alpha-synuclein aggregation”, which description can be applied mutatis mutandis. Antibodies of the invention significantly increase, such as at least a 10% increase in, T112 values, as normalized to aggregation in the absence of antibody.
In some embodiments, an antibody, antigen-binding fragment thereof or derivative thereof, is provided which binds to human alpha-synuclein within an epitope comprised in SEQ ID NO: 1. In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 36-40 (SEQ ID NO: 2). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 1-15 (SEQ ID NO: 121). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 51-57 (SEQ ID NO: 3). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 51-58 (SEQ ID NO: 136). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 65-74 (SEQ ID NO: 4). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 65-81 (SEQ ID NO: 5). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 82-96 (SEQ ID NO: 130). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 91-105 (SEQ ID NO: 131). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 93-95 (GFV) of SEQ ID NO: 1. In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 118-132 (SEQ ID NO: 134). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 124-131 (SEQ ID NO: 7). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 127-140 (SEQ ID NO: 135). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 10-24 (SEQ ID NO: 122). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 128-135 (SEQ ID NO: 8). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 131-140 (SEQ ID NO: 9). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 28-42 (SEQ ID NO: 124). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 37-51 (SEQ ID NO: 125). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 100-114 (SEQ ID NO: 132). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 109-123 (SEQ ID NO: 133). In some particular embodiments, an antibody is provided which binds to human alpha-synuclein within amino acids residues 81-120 (SEQ ID NO: 137). In some embodiments, an antibody is provided which binds to a non-linear epitope within amino acids residues of human alpha-synuclein of SEQ ID NO: 1. More preferably, antigen-binding molecule of the invention bind to an epitope within amino acids residues 124-131 (SEQ ID NO: 7), 128-135 (SEQ ID NO: 8) or 131-140 (SEQ ID NO: 9) of human alpha-synuclein of SEQ ID NO: 1. Even more preferably, antigen-binding molecules of the invention may bind to an epitope comprising amino acids 126 and 127 of human alpha-synuclein of SEQ ID NO: 1 as critical residues for binding.
In some embodiments, an isolated antibody that binds to human alpha-synuclein is provided, wherein the antibody binds extracellular or cytoplasmic alpha-synuclein. In some embodiments an isolated antibody that binds to monomeric or aggregated alpha-synuclein. In some embodiments of the invention, the monomeric, oligomeric or aggregated alpha-synuclein is post-translationally modified, e.g. phosphorylated or nitrosylated. The invention also relates to compositions comprising a binding molecule, particularly an antibody of the invention (including alpha-synuclein antibody fragments and derivatives) as described herein and to therapeutic and diagnostic methods using such compositions in the prevention, diagnosis or treatment of a synucleopathy, wherein an effective amount of the binding molecule is administered to a patient in need thereof.
In certain embodiments, the alpha-synuclein antibodies described herein are useful for detecting the presence of alpha-synuclein in a biological sample. Such methods (specific examples of which are described herein) are typically performed in vitro using an isolated sample. However, they may be performed in vivo in some circumstances, where appropriate. In particular embodiments, the alpha-synuclein antibodies described herein are useful for detecting the presence of aggregated and/or pathological alpha-synuclein, including but not limited to Lewy bodies, Lewy neurites and/or glial cytoplasmic inclusions in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. The biological sample (in all methods reliant upon such detecting) is typically a clinical sample from a mammalian, in particular human, subject. In certain embodiments, a biological sample comprises a cell or tissue, such as cerebrospinal fluid (CSF), a cell or tissue of the brain (e.g., brain cortex or hippocampus), or blood. In some embodiments, a biological sample is cerebrospinal fluid.
In some embodiments, an alpha-synuclein antibody described herein for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of alpha-synuclein in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an alpha-synuclein antibody as described herein under conditions permissive for binding of the alpha-synuclein antibody to alpha-synuclein, and detecting whether a complex is formed between the alpha-synuclein antibody and alpha-synuclein. Such method may be an in vitro and/or in vivo method. Further, the complex formed between the alpha-synuclein antibody and alpha-synuclein in a test biological sample can be compared to the complex formed in a control biological sample (e.g., a biological sample from a healthy subject or subjects). The amount of the complex formed between the alpha-synuclein antibody and alpha-synuclein in a test biological sample can also be quantified and compared to the amount of the complex formed in a control biological sample (e.g., a biological sample from a healthy subject or subjects) or to the average amount of the complex known to be formed in healthy subjects.
In some embodiments, an alpha-synuclein antibody described herein is used to select subjects eligible for therapy, including therapy with an alpha-synuclein antibody, e.g. where alpha-synuclein is a biomarker for selection of patients. For example, in some embodiments, an alpha-synuclein antibody is used to detect whether the subject has a disease, disorder or abnormality associated with alpha-synuclein aggregates including but not limited, Lewy bodies, Lewy neurites and/or Glial cytoplasmic inclusions, or whether the subject is at high risk (or predisposed to) a disease or disorder or abnormality associated with alpha-synuclein aggregates including but not limited, Lewy bodies, Lewy neurites and/or Glial cytoplasmic inclusions.
Exemplary diseases or disorders or abnormality that may be diagnosed using an antibody of the invention include diseases or disorders or abnormalities associated with alpha-synuclein aggregates including, but not limited, Lewy bodies, Lewy neurites and/or Glial cytoplasmic inclusions, that are manifested in a cognitive deficit or behavioral impairment, or motor deficit or impairment such as bradykinesia, rigidity, resting tremor or postural instability. In particular, diseases or disorders or abnormality that may be diagnosed using an antibody, antigen-binding fragment thereof or derivative thereof, of the invention include synucleinopathies such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
Exemplary diseases or disorders or abnormality that may be prevented or treated using an antibody of the invention include diseases, disorders or abnormalities associated with alpha-synuclein aggregates including, but not limited, Lewy bodies, Lewy neurites and/or Glial cytoplasmic inclusions, that are manifested in a cognitive deficit or behavioral impairment, or motor deficit or impairment such as bradykinesia, rigidity, resting tremor or postural instability. In particular, diseases or disorders or abnormality that may be diagnosed using an antibody, antigen-binding fragment thereof or derivative thereof, of the invention include synucleinopathies such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In some embodiments, an immunoconjugate is provided, wherein the immunoconjugate comprises an isolated antibody described herein and a therapeutic agent.
In some embodiments, a labeled antibody is provided, comprising an antibody described herein and a detectable label.
In some embodiments the alpha-synuclein binding molecule of the present invention is linked to a detectable label.
In some embodiments the alpha-synuclein binding molecule is part of an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent.
In some embodiments an alpha-synuclein binding molecule is part of a pharmaceutical composition comprising an alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein specific binding molecule combined with a pharmaceutically acceptable carrier and/or excipient.
In some embodiments an alpha-synuclein binding molecule is part of a diagnostic kit comprising an alpha-synuclein specific binding molecule, or an immunoconjugate wherein the alpha-synuclein specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein specific binding molecule and an alpha-synuclein agonist and cognate molecules, or alternately, antagonists of the same.
In some embodiments an alpha-synuclein binding molecule is used in an immunodiagnostic method for use in the prevention, diagnosis, alleviation of symptoms associated with, or treatment of a disease or disorder or abnormality associated with alpha-synuclein aggregates including, but not limited to, Lewy bodies, Lewy neurites, and/or glial cytoplasmic inclusions.
In some embodiments an alpha-synuclein binding molecule is part of an immunotherapeutic method for the prevention, alleviation of symptoms associated with, or treatment of a synucleinopathy, wherein an effective amount of the alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein binding molecule is administered to a patient in need thereof.
In some embodiments the alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein binding molecule is administered to a patient in need thereof is used to diagnose, prevent, alleviate, delay, inhibit or treat a disease, disorder or abnormality associated with alpha-synuclein aggregates, such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In some embodiments the alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein binding molecule and an alpha-synuclein agonists and cognate molecules, or alternately, antagonists of the same is administered to a patient in need thereof is used in a method for diagnosing or monitoring a disease, disorder or abnormality associated with alpha-synuclein aggregates such as Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), Diffuse Lewy Body Disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.
In some embodiments an alpha-synuclein binding molecule is used in a method for diagnosing presymptomatic disease or disorder or abnormality, or for monitoring disease or disorder or abnormality progression and therapeutic efficacy of a drug, or for predicting responsiveness, or for selecting patients which are likely to respond to the treatment with an alpha-synuclein binding molecule. Said method is preferably performed using a sample of human blood or urine. Most preferably the method involves an ELISA-based or surface adapted assay.
In some embodiments an alpha-synuclein binding molecule is used in a method wherein an alpha-synuclein binding molecule of the present invention is contacted with a sample (e.g., blood, cerebrospinal fluid, or brain tissue) to detect, diagnose or monitor Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In some embodiments an alpha-synuclein binding molecule is used in a method wherein an alpha-synuclein specific binding molecule of the present invention is contacted with a sample (e.g., blood, cerebrospinal fluid, or brain tissue) to detect, diagnose a disease or disorder or abnormality associated with alpha-synuclein aggregates, such as Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), Diffuse Lewy Body Disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.
In some embodiments an alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein binding molecule and an alpha-synuclein agonist and cognate molecules, or alternately, antagonists of the same is administered to a patient in need thereof is used for preventing, alleviating or treating a disease, disorder or abnormality associated with alpha-synuclein aggregates or a synucleinopathy or Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In some embodiments an alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein binding molecule and an alpha-synuclein agonists and cognate molecules, or alternately, antagonists of the same is administered to a patient in need thereof is used for treating a disease or disorder or abnormality associated with alpha-synuclein aggregates, such as Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.
In some embodiments an alpha-synuclein binding molecule, or an immunoconjugate wherein the alpha-synuclein binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising an alpha-synuclein specific binding molecule and an alpha-synuclein agonist and cognate molecules, or alternately, antagonists of the same is administered to a patient in need thereof is used for manufacturing a medicament for treating a disease, disorder or abnormality associated with alpha-synuclein aggregates, or a synucleinopathy or Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In some embodiments, an alpha-synuclein antibody or immunoconjugate for use as a medicament is provided. In some embodiments, an alpha-synuclein antibody or immunoconjugate for use in a method of treatment is provided. In certain embodiments, an anti-alpha-synuclein antibody or immunoconjugate for use in the prevention, diagnosis and/or treatment of a synucleinopathy is provided. In a preferred embodiment of the invention, an alpha-synuclein antibody or immunoconjugate is provided for use in the prevention, diagnosis and/or treatment of a disease, disorder or abnormality associated with alpha-synuclein aggregates, such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In some embodiments, the invention describes the use of an alpha-synuclein antibody or immunoconjugate in the manufacture or preparation of a medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
Antibodies or immunoconjugates of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent.
In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disease or disorders or abnormality described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disease, disorder or abnormality and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or immunoconjugate of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody or immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
The methods of the invention may comprise administering at least one additional therapy, preferably wherein the additional therapy is selected from, but not limited to, neurological drugs, levodopa (e.g. Sinemet®), catechol-O-methyl transferase inhibitors (e.g. entacapone, tolcapone), dopamine agonists, monoamine oxidase B inhibitors (e.g. rasagiline, selegiline) Amantadine, anticholinergic medication, anti-abeta antibodies, anti-Tau antibodies, Tau aggregation inhibitors, beta-amyloid aggregation inhibitors, anti-BACE1 antibodies, and BACE1 inhibitors.
The invention furthermore relates to a method of detecting aggregated and/or pathological alpha-synuclein, including, but not limited to Lewy neurites, Lewy Bodies and/or Glial cytoplasmic inclusions, comprising contacting a sample with the binding molecule of the invention, preferably wherein the sample is a brain sample, a cerebrospinal fluid sample, urine sample or a blood sample.
In some embodiments, the invention encompasses alpha-synuclein binding molecules, particularly antibodies of the invention as described herein that binds aggregated and/or pathological alpha-synuclein and the use of these molecules to diagnose, prevent, alleviate or treat a disease, disorder or abnormality associated with alpha-synuclein aggregates such as Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), or Diffuse Lewy Body Disease.
In another embodiment, a binding molecule, particularly an antibody of the invention as described herein specific for alpha-synuclein is administered to prevent, alleviate or treat a disease, disorder or abnormality associated with alpha-synuclein aggregates selected from Parkinson's Disease, Multiple System Atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), and Diffuse Lewy Body Disease.
In another embodiment, an binding molecules, in particular antibodies or antigen-binding fragments thereof as described herein, binding aggregated and/or pathological alpha-synuclein is contacted with a sample to detect, diagnose or monitor a disease, disorder or abnormality associated with alpha-synuclein aggregates selected from Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with mutations other than alpha-synuclein, pure autonomic failure and Lewy body dysphagia), Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) (“pure” Lewy body dementia), Parkinson's disease dementia (PDD)), Diffuse Lewy Body Disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17, and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial and ALS-dementia complex of Guam), neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia telangiectatica, Meige's syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease as well as other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.
The invention furthermore relates to methods for evaluating an alpha-synuclein binding molecule for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, comprising the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and determining the time to reach half-maximum signal of the detectable dye, particularly the signal of fluorescent dye, relative to the seeded aggregation in the absence of binding molecule. In an alternative or additional embodiment, the method for evaluating an alpha-synuclein binding molecule for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, may comprise the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and determining the time to reach half-maximum signal of the detectable dye, particularly the signal of fluorescent dye, wherein an increase in time to reach half-maximum signal of the detectable dye in the presence of binding molecule relative to the seeded aggregation in the absence of binding molecule indicates that the alpha-synuclein binding molecule is capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation. In a further alternative or additional embodiment, the method for evaluating an alpha-synuclein binding molecule for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, may comprise the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and determining the time to reach half-maximum signal of the detectable dye, particularly the signal of fluorescent dye, and detecting the increase in time to reach half-maximum signal of the detectable dye in the presence of binding molecule relative to the seeded aggregation in the absence of binding molecule, indicating that the alpha-synuclein binding molecule inhibits and/or delays the seeded and/or spontaneous alpha-synuclein aggregation. In a yet further alternative or additional embodiment the method for evaluating an alpha-synuclein binding molecule for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, may comprise the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and measuring the increase in time to reach half-maximum signal of the detectable dye in the presence of the alpha-synuclein binding molecule relative to the seeded aggregation in the absence of binding molecule, as an indication of the binding molecule having capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation.
The invention furthermore relates to a method for screening an alpha-synuclein binding molecule capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, comprising the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and selecting the alpha-synuclein binding molecule as being able to inhibit and/or delay seeded and/or spontaneous alpha-synuclein aggregation based on the signal of the detectable dye, in particular the fluorescent dye, determined in the absence and presence of the alpha-synuclein binding molecule.
The screening or evaluation methods provided herein may further comprise a step of providing alpha-synuclein binding molecules to be screened/evaluated. The binding molecules may for example be provided in form of a library, in particular an antibody library. The skilled person is well-aware of methods for providing binding molecule libraries and in particular antibody libraries. Alternatively, libraries may be obtained commercially before evaluation/screening.
The invention furthermore relates to an in vitro assay for screening for alpha-synuclein binding molecules for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, said assay comprising the steps of bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and selecting the alpha-synuclein binding molecule as being able to inhibit and/or delay seeded and/or spontaneous alpha-synuclein aggregation based on the signal of the detectable dye, in particular the fluorescent dye, determined in the absence and presence of the alpha-synuclein binding molecule. In an alternative or additional embodiment, the invention relates to an in vitro assay for evaluating an alpha-synuclein binding molecule for the capability of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation, said assay comprising the steps of: bringing an alpha-synuclein binding molecule in contact with alpha-synuclein aggregates (seeds); allowing the alpha-synuclein binding molecule to bind to alpha-synuclein aggregates, to form an immunological complex; adding alpha-synuclein monomeric protein and a detectable dye, in particular a fluorescent dye, to the immunological complex; and determining the time to reach half-maximum signal of the detectable dye, particularly the signal of fluorescent dye, wherein an increase in time to reach half-maximum signal of the detectable dye in the presence of binding molecule relative to the seeded aggregation in the absence of binding molecule indicates that the alpha-synuclein binding molecule is capable of inhibiting and/or delaying the seeded and/or spontaneous alpha-synuclein aggregation. In a particular embodiment, the fluorescent dye is thioflavin.
The invention also relates to kits for use in screening or evaluating alpha-synuclein binding molecules, in particular antibodies. Such kits may comprise all necessary components for performing the herein provided methods and/or assays, such as, for example, buffers, detectable dyes, laboratory equipment, reaction containers, instructions and the like.
The invention also relates to methods for the prevention, alleviation or treatment of diseases, disorders and/or abnormalities associated with alpha-synuclein, particularly with pathological alpha-synuclein and/or aggregated alpha-synuclein, comprising administering an effective amount of an alpha-synuclein binding molecule, in particular an antibody, of the invention to a subject in need thereof.
The invention will be further understood with reference to the following non-limiting examples:
Preparation of an Alpha-Synuclein Liposomal Vaccine Composition
The liposome-based antigenic constructs were prepared according to the protocols published in WO2012/055933. The liposomal vaccine with human full-length alpha-synuclein protein as antigen was used for antibody generation (Table 2, SEQ ID NO: 1) or liposomal vaccine with alpha-synuclein peptide as antigen was used for antibody generation.
Female C57BL/6JOIaHsd and BALB/cOIaHsd mice (Envigo, USA) were vaccinated at 10 weeks of age. C57BL/6JOIaHsd substrain is known to have a spontaneous deletion of the alpha-synuclein gene. Mice were vaccinated with vaccine containing human full-length alpha-synuclein protein or alpha-synuclein peptide presented on the surface of liposomes in the presence of synthetic monophosphoryl hexa-acyl Lipid A 3-deacyl (3D-(6-acyl) PHAD®) (Avanti Polar Lipids, USA) as adjuvant.
Mice were vaccinated by subcutaneous injection (s.c.) on days 0, 5, 8, 21, 35, 84, and in some cases on day 14, 28, 63, 73 and 398. Mice were bled and heparinized plasma prepared 7 days before immunization (pre-immune plasma) and on days 14, 28, 40, 84, 90 and in some cases on day 7, 21, 35, 37, 73, 77 and 308 after first immunization. Mice used for myeloma fusion were additionally vaccinated with three or four daily booster injections by intraperitoneal injection (i.p.) of liposomal vaccines without adjuvant. Very high antigen-specific IgG responses were obtained in all immunized mice.
Isolation of Clonal Mouse Hybridoma Cell Lines Producing Specific and High-Affinity Monoclonal Antibodies
Mice were euthanized and fusion with PAI myeloma cells was performed using splenocytes from immunized mice. For screening fusion products, cell culture supernatant was diluted 1:50 and analysed using Luminex bead-based multiplex assay (Luminex, The Netherlands). Luminex beads were conjugated to either full-length alpha-synuclein, alpha-synuclein peptide 1-60aa, alpha-synuclein peptide 1-95aa, alpha-synuclein peptide 61-140aa, or full-length beta-synuclein (irrelevant target), and with capturing IgGs with anti-mouse IgG-Fc antibodies specific for the IgG1, IgG2a, IgG2b, IgG2c, and IgG3 subclasses (Jackson Immunoresearch, USA). Luminex assay results binding to full-length alpha-synuclein identified 92 hits. In a second round of fusion of immunized mice splenocytes and PAI myeloma cells, 400 hits were identified by Luminex assay binding to full-length alpha-synuclein. Viable hybridomas were grown using serum-containing selection media, and the best hybridomas binding to full-length alpha-synuclein were then selected for subcloning. Following limiting dilution, the clonal hybridomas were grown in low immunoglobulin containing medium and stable colonies were selected for antibody screening and selection.
In another round of fusion of immunized mice splenocytes or lymph nodes (popliteals, axial, brachials, and inguinals) and X63/AG.8653 myeloma cells, 279 hits were identified by ELISA assay binding to alpha-synuclein peptide 1-120aa. Viable hybridomas were grown using serum-containing selection media, and the best hybridomas binding to alpha-synuclein peptide were then selected for subcloning. Following limiting dilution, the clonal hybridomas were grown in low immunoglobulin containing medium and stable colonies were selected for antibody screening and selection.
Antibody Binding to Human Full-Length Alpha-Synuclein
Antibody binding to human full-length alpha-synuclein was determined using an indirect ELISA. Full-length alpha-synuclein was diluted in carbonate/bicarbonate buffer pH 9.6 (Sigma, C3041) to a final concentration of 2.5 μg/ml and coated onto ELISA plates overnight at 4° C. After washing with PBS/0.05% Polyethylene glycol sorbitan monolaurate (Tween® 20) and blocking for 1 hour at 37° C. (PBS/0.05% Tween® 20/1% BSA), plates were incubated for 2 hours at 37° C. with three-fold dilution series of alpha-synuclein antibodies from 1 μg/mL to 0.0005 μg/mL using PBS/0.05% Tween® 20/1% BSA as diluent. Dilution series (three-fold from 0.1 μg/mL to 0.0001 μg/mL) of Syn1 antibody (BD Biosciences, 610787; epitope 91-99aa) was used as positive control, where applicable. Next, plates were washed with PBS/0.05% Tween® 20 and incubated for 2 hours at 37° C. with the detection antibody, anti-mouse IgG conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories Inc., 115-055-164) at 1:1000 dilution. After final wash, plates were incubated 2 hours at 25° C. with 1 mg/mL of alkaline phosphatase substrate (p-nitrophenyl phosphate disodium hexahydrate; pNPP, S0942, Sigma) and read the absorbance optical density (O.D.) signal at 405 nm using an ELISA plate reader (Tecan, Switzerland). All generated antibodies show very good binding to human full-length alpha-synuclein (
Epitope Mapping on Alpha-Synuclein
Serum-free supernatants were harvested from stable hybridomas. The supernatants containing antibodies of interest were then screened by an indirect ELISA assay to determine epitopes. Epitopes were first determined using a library of 15-mer peptides covering the entire sequence of human alpha-synuclein protein, spanning amino acids (aa) 1-140 with 9aa offset and 6aa overlap. All peptides were synthesized biotinylated at N-terminus with aminohexanoic acid spacer except the N-terminal peptide 1-14aa (SEQ ID NO: 130) which was synthesized biotinylated at the C-terminus. Briefly, streptavidin-coated ELISA plates were blocked overnight at 4° C. (PBS/0.05% Tween® 20/1% BSA) and then incubated for 1 hour at 25° C. with 0.25 μM of biotinylated full-length alpha-synuclein protein or biotinylated 15-mer peptides. Peptide sequences are provided in Table 3. Plates were washed with PBS/0.05% Tween® 20 and then incubated with the hybridoma supernatants at 1/100 dilution for 1 hour at 25° C. Next, plates were washed with PBS/0.05% Tween® 20 and incubated for 1 hour at 25° C. with the detection antibody, anti-mouse IgG conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories Inc., 115-055-164) at 1:1000 dilution. After final wash, plates were incubated 2 hours at 25° C. with alkaline phosphatase substrate (p-nitrophenyl phosphate disodium hexahydrate; pNPP, S0942, Sigma) and read the absorbance optical density (O.D.) signal at 405 nm using an ELISA plate reader (Tecan, Switzerland). Tested antibodies were found to bind to one or more of the following peptides: 1-14aa, 1-15aa, 10-24aa, 28-42aa, 46-60aa, 64-78aa, 82-96aa, 91-105aa, 118-132aa, 127-140aa, or 81-120aa. For antibodies ACI-7079-2601B6-Ab1, ACI-7087-4125E6-Ab1, and ACI-7089-4415G5-Ab1 no linear epitope could be identified, no binding was observed to peptides of 15-mer length while antibodies bound to full-length alpha-synuclein. Results are shown in
Epitopes were further determined using a library of 8-mer peptides covering the alpha-synuclein sequences previously identified by indirect ELISA on a library of 15-mer peptides. The 8-mer peptides were designed with 1aa offset and 7aa overlap. Finally, for determining the critical residues for antibody binding an Alanine scanning library of peptides was utilized covering the alpha-synuclein sequences previously identified with the library of 15-mer peptides. The peptides of the Alanine scanning library were from 15 to 30 residues in length and synthesized with an alanine residue in each position substituting the natural residue in the sequence (except when the natural residue is alanine). All peptides were synthesized biotinylated at N-terminus with aminohexanoic acid spacer. For the indirect ELISA, streptavidin-coated ELISA plates were blocked overnight at 4° C. (PBS/0.05% Tween® 20/1% BSA) and then incubated for 1 hour at 25° C. with 0.25 μM of biotinylated peptides. Plates were washed with PBS/0.05% Tween® 20 and then incubated with the hybridoma supernatants at 1/100 dilution for 1 hour at 25° C. Next, plates were washed with PBS/0.05% Tween® 20 and incubated for 1 hour at 25° C. with the detection antibody, anti-mouse IgG conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories Inc., 115-055-164) at 1:1000 dilution. After final wash, plates were incubated 2 hours at 25° C. with alkaline phosphatase substrate (p-nitrophenyl phosphate disodium hexahydrate; pNPP, S0942, Sigma) and read the absorbance optical density (O.D.) signal at 405 nm using an ELISA plate reader (Tecan, Switzerland). The binding epitopes for the antibodies are shown in Table 4.
Inhibition or Delay of Seeded Alpha-Synuclein Aggregation
Monoclonal anti-alpha-synuclein antibodies were evaluated for their ability to inhibit the aggregation of alpha-synuclein in vitro. The presence of alpha-synuclein pre-formed aggregates (seeds) increases the de novo aggregation propensity of monomeric a-synuclein. Alpha-synuclein antibodies were incubated with alpha-synuclein seeds prior to adding the monomeric alpha-synuclein for the aggregation assay. Kinetics of alpha-synuclein aggregation were monitored by thioflavin T (ThT) fluorescence. The ability of alpha-synuclein antibodies to inhibit the seeded aggregation was quantified by a percent change in the aggregation half-time (time to reach half-maximum ThT fluorescence signal).
Alpha-synuclein recombinant protein (rPeptide, S-1001-4) at concentration of 5 mg/mL was re-suspended and dialyzed against DPBS (Slide-A-Lyzer Mini Dialysis 10K MWCO, ThermoScientific, 88404) four times of 60 minutes each at 4° C. Higher molecular weight species were then removed by centrifugal filtration (Microcon DNA Fast Flow Centrifugal Filter Unit with Ultracel membrane, Sigma, MRCF0R100). Sonicated alpha-synuclein fibrils were diluted with PBS to a final concentration of 1.0 mg/mL. Aggregations were assembled in low-binding 96-well plates (ThermoScientific, 278752), in triplicate for each condition. Alpha-synuclein seeds were used at 1% the final concentration of monomeric alpha-synuclein (14 μM). Alpha-synuclein seeds (34.5 pmoles) were incubated with alpha-synuclein antibodies (787 pmoles, ˜22.8 equivalents) for 1 hour at 25° C. As a reference control, alpha-synuclein seeds were incubated without the addition of alpha-synuclein antibodies. The Syn303 antibody (BioLegend, 824301) was used as a reference standard (Tran et al., Cell Rep. 2014, 7(6):2054-65). To control for any non-alpha-synuclein specific effect from the antibodies, the mouse isotype control (IgG2a) was produced recombinantly or purchased (ThermoFisher, 02-6200) and was used as a negative control.
Monomeric aSyn and ThT (3 mM stock solution, Sigma, D8537) were added to reach a final concentration of 14 μM and 46 μM respectively. Each aggregation was then aliquoted into 3 separate wells (65 μL/well) of the 96-well plates. Kinetic measurements were performed using an M200 Infinite Pro Microplate Reader (Tecan, Switzerland).
ThT fluorescent measurements were obtained in triplicate for each aggregation condition (technical repeats) and run twice on independent days (for a total of N=6). A baseline correction was performed by subtraction of the initial ThT value (t=0) and data was then normalized as a percent maximum ThT signal (see Equation 1). Aggregation half-times (τ1/2) were calculated from non-linear regressions using either a sigmoidal dose-response (see Equation 2) or a one-phase association (see Equation 3) (GraphPad Prism 7) and represent the time taken to reach half the maximum ThT signal.
Where Bottom is a fit of the minimum ThT signal, Top is a fit of the maximum ThT signal, EC50 is the x value when the ThT signal is halfway between Bottom and Top, and the HillSlope is the steepness of the curve. Here, the aggregation half-time (τ1/2) is obtained directly from EC50.
% ThT(x)=ThT(x0)+((Plateau−ThT(x0))*(1−exp(−K*x)) Equation 3
Where ThT(x0) is the initial ThT signal, Plateau is the fit of the maximum ThT signal, and K is the rate constant. Here, the aggregation half-time (τ1/2) is calculated from In(2)/K
Where τno mab is the aggregation half-time in the absence of antibody (mAb) and τmab is the aggregation half-time in the presence of the indicated antibody.
Where % TmAb is the percent increase in τ1/2 from Equation 4, τno mab is the aggregation half-time in the absence of mAb, τmab is the aggregation half-time in the presence of the indicated mAb, and SEM is the standard error (calculations resulting from fitting of Equations 2 and 3).
Aggregation half-times (T1/2) were obtained using either a sigmoidal fit (Equation 2) or an exponential fit (Equation 3) dependent upon the kinetic profile and best fit. Varied time frames were used to obtain optimal fitting as ThT signals can decrease following completion of aggregation. Change in τ1/2 values, in the presence of the indicated antibodies, were normalized relative to the τ1/2 value in the absence of antibody.
The percent increase in τ1/2 values were calculated relative to the seeded aggregation in the absence of antibody (see Equation 4).
Affinity Measurements on Alpha-Synuclein Monomers and Alpha-Synuclein Fibrils by SPR
Affinity measurements were performed on an surface plasmon resonance (SPR) instrument (Biacore T200, GE Healthcare Life Sciences) using CM5 Series S sensor chips (GE Healthcare, BR-1005-30). Flow channels (Fc) 1-4 were activated with a fresh solution of EDC/NHS (Amine Coupling Kit, 1:1 ratio of both reagents, GE Healthcare, BR-1006-33). The goat anti-mouse antibody (GE Healthcare, BR-1008-38) was captured at a concentration of 30 μg/mL diluted in 10 mM sodium acetate (pH 5.0). Following, all unreacted activated ester groups were capped with 1 M ethanolamine (GE Healthcare, BR-1006-33). Any non-covalently bound antibodies were removed by three successive regenerations of 10 mM Glycine pH 1.7 (GE Healthcare, 28-9950-84). Immobilization levels were evaluated following ethanolamine capping (Bound) and finally following regeneration (Final). Non-covalent immobilization of alpha-synuclein antibodies was performed using a target immobilization method of 2000 response units (RU). Antibodies were diluted in 10 mM sodium acetate pH 5.5 (GE Healthcare, BR-1003-52) to a final concentration of 5 μg/mL.
Binding affinity of alpha-synuclein antibodies to monomeric or fibrillar alpha-synuclein species was performed using a single-cycle kinetics method. The instrument was primed with 1×HBS-P+ buffer (10× stock from GE Healthcare, BR-1003-52 diluted in Milli-Q water). Injections of monomeric alpha-synuclein (aSyn) (Boston Biochem, SP-485), increasing in concentration from 0.62-50 nM prepared from serial 2-fold dilutions, were performed with contact times of 300 sec/injection at a flow rate of 30 μL/min. A dissociation phase of 900 sec followed the final 50 nM injection. Regeneration of the sensor to the goat anti-mouse antibody layer was achieved using 3 regenerations of 10 mM Glycine pH 1.7. Injections of alpha-synuclein fibrils of increasing concentration from 5.56-450 nM prepared from serial 2-fold dilutions, were performed with contact times of 300 sec/injection at a flow rate of 30 μL/min. A dissociation phase of 900 sec followed the final 450 nM injection. Regeneration of the sensor to the goat anti-mouse antibody layer was achieved using 3 regenerations of 10 mM Glycine pH 1.7. Results obtained from single-cycle kinetics were evaluated by Biacore T200 evaluation software with 1:1 binding homogenous Langmuir model (with a global Rmax) with Cycle 5 as a blank subtraction. The following kinetic parameters were obtained: on-rate (ka), off-rate (kd), affinity constant (KD, ratio of kd by ka), maximum response (Rmax), and goodness of fit (Chi2).
Non-covalent capture of the alpha-synuclein antibodies was performed in three separate runs. Capture levels ranged from 1800 to 2100 RU based on the target immobilization level of 2000 R U. Sensograms were obtained for responses to monomeric and fibrillar alpha-synuclein, representative examples for two antibodies are shown in
Target Engagement on Human Alpha-Synuclein Aggregates
Target engagement was evaluated in immunohistochemistry experiments on tissues from PD and Multiple System Atrophy (MSA) donor brains. Human brain tissues were obtained from the Netherlands Brain Bank. All tissues have been collected from donors for or from whom a written informed consent for a brain autopsy and the use of the material and clinical information for research purposes had been obtained by the Netherlands Brain Bank. Immunohistochemistry was performed on 10 μm thick frozen sections using fluorescent secondary antibody detection. An antibody recognizing alpha-synuclein phosphorylated at Ser129, [EP1536Y] (pSyn) (Abcam ab51253) was used as control for detecting pathological aggregated and phosphorylated alpha-synuclein. Antibodies ACI-7067-1101C8-Ab2, ACI-7067-1113D10-Ab1 and ACI-7067-1108B11-Ab2 bind to pathological alpha-synuclein aggregates in Lewy bodies and Lewy neurites in PD cases (
Antibody Variable Region Gene Sequencing
Clonal hybridoma cell lysates were used for variable region gene sequencing. Mouse hybridomas were harvested and lysed using a lysis buffer containing guanidinium salts that deactivates RNases. Genomic DNA was then eliminated by RNase-free DNase, and RNA was purified with a silica-based affinity column using multiple washes and eluted from the column using RNase-free water. Once the RNA was extracted, its purity and concentration was measured spectrophotometrically. The integrity of the RNA was assessed on a denaturing agarose gel and RNA was reverse transcribed into cDNA using reverse transcriptase (RT). Before adding the reaction mixture, the RNA was heated to 70° C. for 10 min in order to disrupt RNA secondary structures. The RT products were directly used for PCR amplification. For high-fidelity PCR amplification of the cDNA, each of the variable region primers corresponding to the different gene families encoding for antibodies were individually mixed with the constant primer, for variable heavy chain domain (VH) and variable light chain domain (VL) separately. In first intention, a degenerate primer pool was used (12 for VH and 12 for VL) and, depending on the results, a second pool was used to obtain PCR products. After the PCR reaction, the products were analyzed by gel electrophoresis on 2% agarose gels stained with ethidium bromide. The PCR products for VL and VH were individually purified on an agarose gel using tris-acetate-EDTA (TAE). The purified fragments excised from the gel were then sequenced using the dye-terminator sequencing method. The same primers as those used for PCR were used for the sequencing reaction. Sequencing was carried out in both directions to provide overlap at both ends. Sequencing data were analyzed on the Ig Blast/Kabat database. Nucleotide sequences for VH and VL are shown in Table 6. Protein sequences for VH and VL, and their complementarity-determining regions (CDRs) are shown in Table 7.
Efficacy of Alpha-Synuclein Antibodies in an In Vivo Mouse Model of Parkinson's Disease
Animal studies were performed in accordance with all local Animal Care guidelines. Male, hemizygous transgenic-M83 mice were inoculated with human alpha-synuclein pre-formed fibrils (hPFFs) or phosphate buffered saline (PBS) as negative control via stereotactic injection into the anterior olfactory nucleus as described in Luk et al., 2012. Vehicle control (formulation buffer comprising of: 25 mM histidine, 150 mM NaCl, 0.02% poloxamer 188, pH 5.5) or antibodies (ACI-7067-1101C8-Ab2, ACI-7067-1108B11-Ab2, or ACI-7067-1113D10-Ab1) against alpha-synuclein were injected intraperitoneally (i.p.) at 30 mg/kg/week, for 17 weeks starting at the week of surgery (Week 0) to Week 16 following stereotaxic surgery. The health status of mice was monitored daily and body weight was recorded on a weekly basis. No adverse effects were observed post-dosing; animals showed no distress or discomfort and had normal activity level. ACI-7067-1101C8-Ab2 and ACI-7067-1108B11-Ab2 demonstrated a significant reduction in the rate of body weight loss as compared to the vehicle treated control group injected with human pre-formed fibrils, while for ACI-7067-1113D10-Ab1 a trend of reduction in the rate of body weight was observed (
After 17 weeks post inoculation, mice were sacrificed by perfusion with 20 mL of phosphate buffered saline, followed by transcardiac infusion of 20 mL of 10% neutral-buffered formalin. Brains were immersion-fixed in 10% neutral-buffered formalin for 72 hours. Fixed brains for paraffin embedding were dehydrated through graded ethanol and xylene, and then infiltrated with paraffin wax. Processed brains were oriented and embedded in paraffin blocks followed by sectioning at 5 microns. For quantification of pathological alpha-synuclein, slides initially underwent a two-step epitope retrieval and were treated with mild PK digestion prior to staining with an antibody directed against phosphorylated alpha-synuclein [EP1536Y]. Neuronal density measurements were performed by staining for NeuN, a neuronal specific protein, by IHC with the antibody clone A60 (Millipore). Data for all IHC measurements were acquired by an Axio Scan.Z1 digital whole slide scanner (Carl Zeiss). Regions of interest, brain areas interconnected with the injection site, were manually delimited and quantification of IHC staining, percent area stained, was performed on each of the slides using an automated software algorithm. The IHC analysis and quantification was performed in a blinded manner with respect to the treatment groups. Disease spreading and propagation of pathology in the M83 mouse model was monitored by an increase in pathological phosphorylated alpha-synuclein IHC staining (normalized by neuronal density) and a decrease in NeuN IHC staining for the human pre-formed fibril injected groups. ACI-7067-1101C8-Ab2 and ACI-7067-1108B11-Ab2 significantly delayed the aggregation and seeding of pathological alpha-synuclein indicated by the significantly reduced levels of alpha-synuclein pathology in the piriform cortex and brainstem contralateral to the injection site (
Inhibiting a-Syn Propagation in Cells
Monoclonal anti-alpha-synuclein antibodies were evaluated for their ability to inhibit the uptake and seeding of alpha-synuclein aggregation in an in vitro cellular model that is susceptible to alpha-synuclein seeding and in mouse primary cortical neurons. The addition of alpha-synuclein seeds to the cellular model or primary neurons initiates the de novo aggregation of monomeric a-synuclein. The formation of de novo a-syn aggregates or de novo pathological alpha-synuclein (phosphorylated alpha-synuclein) was assessed in the presence or absence of alpha-synuclein antibodies relative to an isotype control antibody. The ability of alpha-synuclein antibodies to inhibit uptake or seeded aggregation was quantified as a percent change in the number of alpha-synuclein aggregates observed.
For the in vitro cellular model, alpha-synuclein antibodies (ACI-7067-1101C8-Ab2, ACI-7067-1108B11-Ab2, or ACI-7067-1113D10-Ab1) or an isotype control antibody were incubated with 0.4 μL/well Ab-DeliverIN™ Transfection Reagent (OZ Biosciences, A121000) for 30 min at room temperature in low-binding 96-well plates (Eppendorf Microplate 96/V-PP, Sigma, EP951040227). Antibodies/Ab-DeliverIN were then added to the cells, plated at a density of 8,000 cells/well 24 hours prior to treatment, and placed back in the incubator (at 37° C. with 5% CO2) for 5 hours. Alpha-synuclein seeds (0.05 μg/well) were diluted in a reduced-serum medium (Opti-MEM™, Life Technologies, 31985070) and incubated with 0.2 μL/well Lipofectamine™ 2000 Transfection Reagent (Life Technologies, 11668019) for 30 min at 25° C. in a low-binding 96-well plate. Alpha-synuclein seeds/lipofectamine were then added to cells. As a reference control, cells were also transduced with lipofectamine without alpha-synuclein seeds. Cells were placed back in the incubator (at 37° C. with 5% CO2). Cells were then supplemented at 24 hours post-transduction with 100 μL of DMEM/glutamax (Gibco, 31966-021), supplemented with 5% Fetal Bovine Serum (qualified and heat inactivated; Gibco, 10500-064) and 1% Penicillin-Streptomycin (10,000 U/mL; Gibco, 15140-122). At 96 hours, post initial transduction, cells were fixed with an equal volume of cold 2% Triton X-100, 8% PFA in PBS, and Hoechst 33342 (1:10,000). Media was removed and washed three times with PBS, fixed cells were left in PBS, kept protected from light, and high-content imaging analysis was performed to detect and quantify the formation of de novo alpha-synuclein aggregates. Use of an intrinsically fluorescent reporter protein allowed for the detection of de novo alpha-synuclein aggregates. The percent aggregates formed were then calculated relative to conditions in the absence of antibodies. IC50 values were obtained from fitting using Equation 6 (Graph Pad Prism 7).
ACI-7067-1101C8-Ab2, ACI-7067-1108B11-Ab2, and ACI-7067-1113D10-Ab1 reduced the seeding capacity of alpha-synuclein aggregates in a dose-dependent fashion (
For the mouse primary cortical neurons, cells were cultured in 384-well plates. At 6 days in vitro (DIV), alpha-synuclein antibodies (ACI-7067-1101C8-Ab2, ACI-7067-1108B11-Ab2, or ACI-7067-1113D10-Ab1) or an isotype control antibody were added to cells plated at a density of 40,000 cells/well and incubated for 30 min. Alpha-synuclein seeds (8 μg) were then added to the cells. At 13 DIV (7 days after alpha-synuclein seed addition) the cells were fixated with PFA and stained with an antibody directed against phosphorylated alpha-synuclein (EP1536Y) and Hoechst stain. High-content image analysis was performed to detect and quantify the formation of de novo alpha-synuclein aggregates/cell. The percent aggregates formed were then calculated relative to conditions in the absence of antibodies. Data was combined from three independent experiments and IC50 values were obtained from fitting using Equation 7 (GraphPad Prism 7).
ACI-7067-1101C8-Ab2, ACI-7067-1108B11-Ab2, and ACI-7067-1113D10-Ab1 reduced the uptake and seeding capacity of alpha-synuclein aggregates in a dose-dependent fashion (
Humanization of Anti-Human a Synuclein Mouse Monoclonal Antibody
Design of the Humanized Variable Regions
Homology matching was used to choose human acceptor frameworks on which to graft ACI-7067-1101C8-Ab2 CDRs. Databases of human and mouse germline variable genes such as the IMGT database (Ehren mann, F et al, (2010) Nucl. Acids Res., 38 (S1):D301-D307) or IgBlast (Ye J. et al, (2013), Nucleic Acids Res. 2013 July; 41 (Web Server issue): W34-W40) or the VBASE2 (Retter I et al, (2005) Nucleic Acids Res. 33, Database issue D671-D674) may be used to identify the closest human variable domain subfamilies to the murine heavy and light chain V regions (SEQ ID NO: 10 and SEQ ID NO: 14, respectively). Selection of heavy and light chain variable sequences (VH and VL) within these subfamilies to be used as acceptor may be based upon sequence homology and/or a match of canonical structure of the CDR1 and CDR2 loop regions to help preserve the correct conformation of the six CDRs after grafting.
For example, use of the IMGT database indicates the best sequence homology between ACI-7067-1101C8-Ab2 heavy chain variable domain framework and the members of the human heavy chain variable domain subfamily 3. Highest homologies and identities of both CDRs and framework sequences were observed for germline sequences: IGHV3-73*01, IGHV3-73*02, IGHV3-72*01, IGHV3-49*01, all of which had sequence identity above 75% for the whole sequence up to CDR3. IGHV3-73*01 and IGHV3-73*02 showed 79% sequence identity while IGHV3-72*01 and IGHV3-49′01 showed a sequence identity of 76% and 75% respectively. IGHV3-73*01 was selected as the VH framework due to its high sequence homology and known stability.
Using the same approach, ACI-7067-1101C8-Ab2 light chain variable domain sequence showed the best sequence homology to the members of the human light chain variable domain kappa subfamily 2. Highest homologies and identities of both CDRs and framework sequences were observed for germline sequences: IGKV2-30*02, IGKV2-30*01, IGKV2D-29*02, IGKV2-24*01 all of which had sequence identity above 79% for the whole sequence up to CDR3. IGKV2-30*02 showed the highest sequence identity with 81%, while IGKV2-30*01, IGKV2D-29*02 had sequence identity of 80%. IGKV2-30*02 was selected as the VL framework due to its high sequence homology.
Potential deamidation and isomerization sites were identified within ACI-7067-1101C8-Ab2 CDR sequences at positions N53 and D61 in the variable heavy chain and position N28 in the variable light chain (according to Kabat numbering system). By 3D homology modelling, these PIM sites were confirmed to be solvent accessible. Point mutations N54A and D61A were introduced in the VH region whereas G29A was introduce in the VL region to remove the deamidation site in CDR L1. When combined all mutations retained the binding of ACI-7067-1101C8-Ab2 to its target; this set of mutations was included in the first humanized variant of ACI-7067-1101C8-Aba2.
As starting point for the humanization process, murine CDRs were grafted on human acceptor frameworks for both VH and VL regions. The resulting human-mouse hybrid heavy chain variable sequence had human IGHV3-73*01 framework regions, ACI-7067-1101C8-Ab2 mouse CDRs, and the best matching JH segment identified from the IMGT searches mentioned above. Similarly, the human-mouse hybrid light chain variable domain had human IGKV2-30*02 framework regions, ACI-7067-1101C8-Ab2 mouse CDRs, and the best matching JK segment.
To accommodate CDRs on to the human acceptor framework key positions were modified by substituting human residues to mouse residues. This process is called back-mutation and is the most unpredictable procedure in the humanization of monoclonal antibodies. It requires the identification and selection of critical framework residues from the mouse antibody that need to be retained in order to preserve affinity while at the same time minimizing potential immunogenicity in the humanized antibody.
To identify residues that may impact most greatly the CDR conformation and/or VH/VL orientation, a 3D model for the human-mouse hybrid VH-VL pair was generated by homology modelling using the Abodybuilder server (Dunbar, J. et al (2016). Nucleic Acids Res. 44. W474-W478) and PBD: 1 NBV as a template for the framework structure and VH/VL orientation. Model analysis allowed the selection of a subset of positions based on their putative influence on CDR loop conformation and/or heavy chain-light chain variable domain packing. This subset of positions consisted of positions 28, 49, 78, 93 and 100b in the variable heavy chain and positions 27B and 36 in the variable light chain.
From this first design, new sets of variable heavy and light chains were generated by introducing backmutation from human to mouse residues at the positions described above. Table 8 and 11 show the combination of backmutations in each different variable domain according to Kabat numbering system.
Production of Humanized Antibody Variants
DNA coding sequence for both heavy and light variable domains were synthesized and cloned using standard molecular biology techniques into plasmid allowing the expression in mammalian cells. Heavy chain variable domains were fused to the human Immunoglobulin IgG1 constant domain and light chain variable domains were cloned into plasmid containing the constant Kappa light chain domain. The chimeric antibody and the humanized variants were transiently expressed in Expi293F cells by cotransfecting heavy and light chain plasmid using the ExpiFectamine™ 293 transfection kit (ThermoFischer scientific, A14524). Post transfection, cells were maintained at 37° C. under 150 rpm agitation and 8% 002 level. Six days after transfection, supernatants were harvested and purified onto Protein A column pre-packed with 1 mL MabSelect Sure resin (GE Healthcare Life Sciences, 17543803). The column was equilibrated with 0.1 M Iris, pH7.0 before being loaded with the cell culture fluid. Following loading, the column was washed with 0.1 M Tris, pH7.0 followed by elution using 0.1 M citrate, pH3.5. The elution was then neutralized by adding 0.1 M Iris, pH9.0. The samples were then dialyzed in PBS buffer.
Characterization of ACI-7067-1101C8-Ab2 Humanized Variants by Surface Plasmon Resonance (SPR)
All variants were screened by SPA for binding against both a-synuclein aggregates and monomers. Single concentration measurements were performed on an SPR instrument (Biacore 8K, GE Healthcare Life Sciences) using CM5 Series S sensor chips (GE Healthcare Life Sciences, 29-1496-03). Flow channels (Fc) 1-8 were activated with a fresh solution of EDC/NHS (Amine Coupling Kit, 1:1 ratio of both reagents, GE Healthcare Life Sciences, BR100050). 30 μg/mL of an F(ab)′2 Goat anti-human IgG Fc (Jackson ImmunoResearch Europe Ltd, 109-006-098) diluted in 10 mM NaAc (pH 4.5) was then injected to channel 1-8 for 420 s at a flow rate of 10 μL/min. The chip was deactivated by 1 M ethanolamine-HCl (GE Healthcare Life Sciences, BR100050) at a flow rate of 10 μL/min for 420 s.
A human IgG1 isotype control diluted in running buffer 1×HBS-EP+ (GE Healthcare Life Sciences, BR100669) was captured onto Fc1 via anti-human Fc IgG at a flow rate of 10 μL/min. ACI-7067-1101C8-Ab2 humanized variants diluted in running buffer 1×HBS-EP+ were captured onto Fc2 via anti-human Fc IgG at a flow rate of 10 μL/min. 100 nM of analyte a-syn monomers (Boston Biochem, SP-485) and running buffer were injected orderly to Fc1-Fc2 at a flow rate of 30 μL/min for an association phase of 400 s, followed by 600 of dissociation. 450 nM of analyte a-syn aggregates and running buffer were injected orderly to Fc1-Fc2 at a flow rate of 30 μL/min for an association phase of 400 s, followed by 3600 s of dissociation. 10 mM glycine pH 1.5 as regeneration buffer was injected following every dissociation phase.
The data for reference channel Fc1 and buffer channel were subtracted to generate the sensorgrams. The experimental data was fitted by 1:1 binding model or heterogeneous ligand model. The relative koff of the chimeric antibodies was determined for each humanized variant Results are shown in Table 14.
Ten variants were selected for full kinetics measurement by SPR based on their affinity to alpha-synuclein, expression level and/or sequence identity to the human acceptor framework.
Affinity measurements were performed on an surface plasmon resonance (SPR) instrument (Biacore 8K, GE Healthcare Life Sciences) using CM5 Series S sensor chips (GE Healthcare, BR-1005-30). Flow channels (Fc) 1-8 were activated with a fresh solution of EDC/NHS (Amine Coupling Kit, 1:1 ratio of both reagents, GE Healthcare Life Sciences, BR-1006-33). The anti-human antibody (GE Healthcare Life Sciences, BR-1008-39) was captured at a concentration of 30 μg/mL diluted in 10 mM sodium acetate (pH 5.0). Following, all unreacted activated ester groups were capped with 1 M ethanolamine (GE Healthcare Life Sciences, BR-1006-33). Any non-covalently bound antibodies were removed by three successive regenerations of 10 mM Glycine pH 1.7 (GE Healthcare Life Sciences, 28-9950-84). Immobilization levels were evaluated following ethanolamine capping (Bound) and finally following regeneration (Final). Non-covalent immobilization of alpha-synuclein antibodies was performed using a target immobilization method of 200 response units (RU). Antibodies were diluted in 10 mM sodium acetate pH 5.5 (GE Healthcare, BR-1003-52) to a final concentration of 2 μg/mL. Binding affinity of alpha-synuclein antibodies to monomeric or fibrillar alpha-synuclein species was performed using a single-cycle kinetics method. The instrument was primed with 1×HBS−P+ buffer (10× stock from GE Healthcare, BR-1003-52 diluted in Milli-Q water). Injections of monomeric alpha-synuclein (aSyn) (Boston Biochem, SP-485), increasing in concentration from 0.62-50 nM prepared from serial 2-fold dilutions, were performed with contact times of 300 sec/injection at a flow rate of 30 μL/min. A dissociation phase of 900 sec followed the final 50 nM injection. Regeneration of the sensor to the goat anti-human antibody layer was achieved using 3 regenerations of 10 mM Glycine pH 1.7. Injections of alpha-synuclein fibrils of increasing in concentration from 5.56-450 nM prepared from serial 2-fold dilutions, were performed with contact times of 300 sec/injection at a flow rate of 30 μL/min. A dissociation phase of 900 sec followed the final 450 nM injection. Regeneration of the sensor to the goat anti-human antibody layer was achieved using 3 regenerations of 10 mM Glycine pH 1.7. Results obtained from single-cycle kinetics were evaluated by Biacore K8 evaluation software with 1:1 binding homogenous Langmuir model (with a global Rmax) with Cycle 5 as a blank subtraction. The following kinetic parameters were obtained: on-rate (ka), off-rate (kd), affinity constant (KD, ratio of kd by ka), maximum response (Rmax), and goodness of fit (Chi2).
Kinetic constants were determined from 1:1 homogenous binding models for binding versus the aggregated form, while a steady state fit was used to determine KD values versus the monomeric form of alpha-synuclein. Kinetic constants are shown in Table 15.
Overall all humanized variants retained affinity to alpha-synuclein with binding preference to fibrillar alpha-synuclein. hACI-7067-1101C8-Ab2_H5L1, hACI-7067-1101C8-Ab2_H8L1, hACI-7067-1101C8-Ab2_H9L1, hACI-7067-1101C8-Ab2_H9L2 and hACI-7067-1101C8-Ab2_H10L1 demonstrated an improved affinity against the aggregated form of alpha synuclein compared to the chimeric antibody cACI-7067-1101C8-Ab2.
Inhibition or Delay of Seeded Alpha-Synuclein Aggregation of ACI-7067-1101C8-Ab2 Humanized Variants
Antibodies were tested for their ability to inhibit or delay alpha synuclein seeded aggregation in the seeded alpha-synuclein aggregation assay previously described. Antibodies were compared to the chimeric antibodies to identify the best performing humanized variants.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes in connection with the invention.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19170207.5 | Apr 2019 | EP | regional |
| 19207105.8 | Nov 2019 | EP | regional |
| 20165055.3 | Mar 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/060898 | 4/17/2020 | WO |
