The present invention relates to α-synuclein antibodies and their use in the prevention or treatment of disease, in particular alpha-synucleinopathies, and more particularly Parkinson's disease (PD).
Alpha-synucleinopathies, also known as Lewy body diseases (LBDs), are a family of neurodegenerative diseases that all have at their core alpha-synuclein as the key pathological hallmark (Jellinger, Mov Disord (2003), 18 Suppl 6: S2-12, and Spillantini and Goedert, Ann N Y Acad Sci (2000), 920: 16-27; both of which are incorporated herein by reference). Alpha-synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA).
PD is a slowly progressive age-related movement disorder affecting greater than 1% of people over 65 years old. PD is the second most common neurodegenerative condition after Alzheimer's disease.
A defining hallmark pathology of alpha-synucleinopathies are Lewy bodies and Lewy neurites, which are insoluble inclusions of aggregated proteins found inside neurons of the brain revealed upon post-mortem histopathological examination.
The presence of Lewy pathology and neuronal loss in non-motor brain regions such as the basal forebrain, mesopontine system, amygdala, neocortex, dorsal motor nucleus of the vagus nerve, olfactory bulbs, locus coeruleus, and the brainstem, may cause cognitive deficits and dementia, hyposmia, sleep disturbances including rapid eye movement sleep behaviour disorder (RBD), mood disorders including depression and anxiety, autonomic dysfunction including cardiovascular and gastrointestinal problems such as constipation, and fatigue and somnolence. Some of these non-motor symptoms appear to characterise the premotor or prodromal phase of the disease (Kalia et al. Lancet (2015), 386(9996): 896-912; incorporated herein by reference).
The presence of Lewy pathology and neuronal loss in motor brain regions, including most notably the death of dopaminergic neurons in the substantia nigra, may cause resting tremor, rigidity, bradykinesia and postural instability (Spillantini and Goedert, Ann N Y Acad Sci (2000), 920: 16-27; incorporated herein by reference).
Alpha-synuclein (also called “α-synuclein” or “α-syn”) protein is the major structural component of Lewy bodies and Lewy neurites. Alpha-synuclein is a small acidic protein made up of 140 amino acids (14 kDa). Human natural wild-type alpha-synuclein has the amino acid sequence SEQ ID NO: 1 as described under UniProtKB accession number P37840. Unless otherwise apparent from the context, reference to alpha-synuclein or its fragments includes the natural human wild-type amino acid sequence indicated above, and human allelic variants thereof, in particular those associated with Lewy body disease (e.g., E46K, A30P, H50Q, G51D and A53T, where the first letter indicates the amino acid in SEQ ID NO: 1, the number is the codon position in SEQ ID NO: 1, and the second letter is the amino acid in the allelic variant). Such variants can optionally be present individually or in any combination. The induced mutations E83Q, A90V, A76T, which enhance alpha-synuclein aggregation, can also be present individually or in combination with each other and/or with human allelic variants E46K, A30P, H50Q, G51D and A53T. At the structural level, alpha-synuclein contains three distinct regions: an amphipathic N-terminal alpha-helix domain that has lipid and membrane binding properties (residues 1-60), a central hydrophobic amyloid-binding domain that encodes the non-amyloid-beta component (NAC) of plaques (residues 61-95), and an acidic proline-rich C-terminal tail (residues 96-140). Residues 71-82 of the NAC domain are believed to be key to the aggregation/fibrillation properties of alpha-synuclein by enabling the protein to switch from a random coil structure to a beta-sheet structure (Bisaglia et al. FASEB J (2009), 23(2): 329-40; incorporated herein by reference). Although the C-terminal domain is free of significant secondary structure it contains a key phosphorylation site at residue Ser129 and a number of tyrosine residues that are nitrated in cytosolic alpha-synuclein inclusions. N-terminal and C-terminal truncated forms of alpha-synuclein also exist. Post-translational modifications to the protein can affect alpha-synuclein aggregation and toxicity (Oueslati et al. Prog Brain Res (2010), 183: 115-45, incorporated herein by reference).
Alpha-synuclein is abundant in the central nervous system (CNS)/brain where it is found both intracellularly in neurons and glia and also extracellularly in cerebrospinal fluid (CSF) (Mollenhauer el al. J Neural Transm (2012), 119(7): 739-46; incorporated herein by reference) and the interstitial fluid (ISF) that bathes and surrounds the cells of the brain (Emmanouilidou et al. PLoS One (2011), 6(7): e22225; incorporated herein by reference). Alpha-synuclein is a synaptic protein predominantly expressed in neurons of the neocortex, hippocampus, substantia nigra, thalamus, and cerebellum (Iwai el al. Neuron (1995), 14: 467-475; incorporated herein by reference). Under physiological conditions, it is located in neuronal synaptic terminals and is specifically up-regulated at presynaptic terminals during acquisition-related synaptic rearrangement (Fortin et al. J Neurosci (2005), 25: 10913-10921; incorporated herein by reference).
In-vitro studies have shown that alpha-synuclein monomers may form the starting point for the aggregation process. The monomer can aggregate into a variety of small oligomeric species that are then stabilised by beta-sheet interactions, going on to form protofibrils which can polymerise into insoluble fibrillary structures reminiscent of those identified in Lewy bodies (Cremades et al. Cell (2012), 149(5): 1048-59; incorporated herein by reference).
Under pathological conditions, aberrant alpha-synuclein aggregation may be key to the pathological changes seen in alpha-synucleinopathies (Lashuel et al. Nature (2002), 418: 291, and Tsigelny et al. FEBS Journal (2007), 274: 1862-1877; both of which are incorporated herein by reference). In vitro and in vivo studies have shown that the neurotoxic effects of alpha-synuclein appear to be elicited by small soluble oligomeric conformers or protofibrils (Winner et al. Proc Natl Acad Sci USA (2011), 108(10): 4194-9; and Danzer el al. J Neurosci (2007), 27(34): 9220-32; both of which are incorporated herein by reference). While fibrillar aggregates of alpha-synuclein are characteristic of PD, oligomeric forms of alpha-synuclein are the toxic species (Danzer et al. J Neurosci (2007), 27(34): 9220-32; Lashuel et al. Nature (2002), 418; 291, and Winner et al. Proc Natl Acad Sci USA (2011), 108; 4194-4199; each of which are incorporated herein by reference).
Alpha-synuclein oligomers can be released to the extracellular environment and taken up by neighboring cells in a “propagation” mechanism (Angot and Brundin, Parkinsonism Relat Disord (2009), 15 Suppl 3: S143-147; Desplats el al. Proc Natl Acad Sci USA (2009), 106: 13010-13015; and Lee el al. J Biol Chem (2010), 285: 9262-9272; each of which are incorporated herein by reference). Aggregates of alpha-synuclein can propagate misfolding through a prion-like spreading mechanism (Lee et al. Nat Rev Neurol (2010), 6: 702-706; Luk et al. J Exp Med (2012), 209(5): 975-86; and Luk et al. Science (2012), 338(6109): 949-53; each of which are incorporated herein by reference). Alpha-synuclein can therefore induce neurodegeneration by either oligomer toxicity or propagation and prion-like spreading.
It is now well established and accepted that cells including neurons can secrete various forms of alpha-synuclein (monomers, oligomers, aggregates) under normal conditions and also under conditions of cellular stress, the secretion of monomeric and aggregated forms of alpha-synuclein is elevated under conditions of cellular stress, and through this release of alpha-synuclein into the extracellular milieu, pathological transmissible forms of alpha-synuclein may be propagated between neurons (Recasens and Dehay, Front Neuroanat (2014), 8: 159; incorporated herein by reference).
The effects of alpha-synuclein in PD may extend beyond the immediate damage to vulnerable neuronal cells. Like most neurodegenerative diseases there is also a pro-inflammatory cellular response observed (Lee et al. J Biol Chem (2010), 285: 9262-9272; incorporated herein by reference). Circulating alpha-synuclein and/or activated astrocytes can activate microglia, leading to increased generation of reactive oxygen species, nitric oxide and cytokine production, and further exacerbating neurodegeneration (Lee et al. J Biol Chem (2010), 285: 9262-9272; incorporated herein by reference).
A variety of different experimental models have demonstrated cell-to-cell transmission of alpha-synuclein in cultured cells, or in vivo spreading and propagation of alpha-synuclein pathologies. Lewy body pathology has been observed within embryonic mesencephalic neuronal grafts more than 10 years after the grafts were therapeutically transplanted into the striatum of PD patients. Specifically, grafted neurons contained a number of Lewy body-like inclusions that stained positively for alpha-synuclein, indicating that host-to-graft transmission of alpha-synuclein pathology had occurred (Li et al. Nat Med (2008), 14(5): 501-3; and Kordower et al. Nat Med (2008), 14(5): 504-6, both of which are incorporated herein by reference).
Further, preformed recombinant alpha-synuclein fibrils and alpha-synuclein oligomers can be internalised by cultured cells and neurons, and the direct transfer of alpha-synuclein from donor to recipient cells with the formation of alpha-synuclein inclusions similar to Lewy pathology has been demonstrated (Danzer et al. J Neurosci (2007), 27(34): 9220-32; Volpicelli-Daley et al. Neuron (2011), 72(1): 57-71; and Luk et al. Proc Natl Acad Sci USA (2009), 106(47): 20051-6; each of which are incorporated herein by reference). Injection of preformed synthetic alpha-synuclein fibrils or Lewy body-like alpha-synuclein containing material extracted from the brains of aged alpha-synuclein transgenic mice into the brains of asymptomatic recipient mice promotes the formation of Lewy body-like pathology in host neurons of the recipient animals along with neurodegeneration and neurological deficits (Luk el al. J Exp Med (2012), 209(5); 975-86; and Luk et al. Science (2012), 338(6109): 949-53; both of which are incorporated herein by reference). Alpha-synuclein containing Lewy body extracts isolated from PD brains inoculated into the substantia nigra or striatum of macaque monkeys and mice is rapidly taken up by host cells (within 24 hours) followed by a slower loss of striatal dopaminergic terminals, with cell loss evident after more than a year (Recasens et al. Ann Neurol (2014), 75(3): 351-62; incorporated herein by reference). Similarly, inoculation of mice with brain homogenates derived from patients with the synucleinopathies DLB or MSA triggers alpha-synuclein Lewy-like pathology in the host mice (Watts et al. Proc Natl Acad Sci USA (2013), 110(48): 19555-60; and Masuda-Suzukake el al. Brain (2013), 136(Pt 4): 1128-38; both of which are incorporated herein by reference). Finally, transfer and transmission of both monomeric and oligomeric alpha-synuclein from the olfactory bulb to interconnected brain structures has been demonstrated in mice (Rey et al. Acta Neuropathol (2013), 126(4): 555-73; incorporated herein by reference).
Passive immunotherapy approaches with antibodies targeting alpha-synuclein have been tested in numerous preclinical alpha-synucleinopathy mouse models (Lawand et al. Expert Opin Ther Targets (2015): 1-10; incorporated herein by reference). Specifically, a study using a monoclonal antibody directed against alpha-synuclein (9E4) has shown in vivo clearance of alpha-synuclein aggregates and pathology, behavioural motor improvements, and neuroprotective effects (WO 2014/058924; which is incorporated herein by reference).
Further studies using passive immunisation of alpha-synuclein transgenic mice developed as experimental models of PD/DLB, with the 9E4 monoclonal antibody have shown the antibody to clear alpha-synuclein pathology, decrease synaptic and axonal deficits, abrogate loss of striatal tyrosine hydroxylase fibres, and significantly reduce memory deficits and motor function impairments (Games et al. J Neurosci (2014), 34(28): 9441-54; Bae et al. J Neurosci (2012), 32(39): 13454-69; and Masliah et al. PLoS One (2011), 6(4): e19338; each of which are incorporated herein by reference). Further it has been demonstrated that passive administration of anti-alpha-synuclein monoclonal antibodies in wild-type mice that were injected intrastriatally with synthetic alpha-synuclein preformed fibrils (pffs) led to robust reduction in Lewy pathology, prevention of dopamine neuron loss in the substantia nigra, and a significant improvement in motor impairments that are manifest in the mouse model after pffs treatment (Tran et al. Cell Rep (2014), 7(6): 2054-65; incorporated herein by reference).
Additionally, one of the major challenges associated with treating disorders of the CNS with large molecule therapeutics, such as antibodies, is getting these drugs into the affected tissue. The passage of large molecules into the brain and spinal cord is largely restricted by the blood-brain barrier (BBB). The BBB protects and regulates the homeostasis of the brain and prevents the free passage of molecules into most parts of the brain, thereby limiting the treatment of many brain diseases. Transport of essential molecules such as nutrients, growth factors, and hormones is achieved via a series of specific transporters and receptors that regulate passage across the brain endothelial cells. The delivery of biologics and other drugs to the brain therefore represents a significant challenge. Additionally, transport mechanisms appear to exist that rapidly remove antibodies from the brain, presumably to prevent inflammatory responses due to engagement of Fc with effector ligands that promote a pro-inflammatory response.
Over the last decade, reports of antibody transport across the BBB have emerged where binding to the extracellular domain of the transporter molecules facilitates transcytosis of the receptor antibody complex across the endothelial cell layer.
The BBB is mainly comprised of brain capillary endothelial cells, which have specialized characteristics, such as tight junctions, to limit transport of molecules into the brain (Reese et al. 1967, J. Cell Biol. 34: 207-217; Brightman et al. 1969, J. Cell Biol. 40: 648-677; Rubin et al. 1999, Ann. Rev. Neurosci. 22: 11-28), although other cell types, such as pericytes, astrocytes, and neuronal cells, also play an important role in the function of the BBB. Typically, less than 0.1% of a peripherally dosed antibody reaches the brain (Boado et al. 2010, Mol. Pharm. 7: 237-244, Pepinsky et al. 2011, Nat. Neurosci. 8: 745-751). The BBB functions as a physical, metabolic and immunological barrier (Gaillard et al. 2003, Microvasc. Res. 65: 24-31).
Antibody transport across the BBB can be enhanced by triggering receptor mediated transcytosis on brain endothelial cells. Through this process, engagement of antigens on the luminal side of the endothelial cell can induce the internalization and shuttling of the antibody across the cell, and then its subsequent release into the tissue.
Current drug therapies for PD are mainly focused on treating the motor-related symptoms of the disease. There are currently no marketed or available therapies that can treat or prevent alpha-synucleinopathy.
Accordingly, there is a need in the art for a therapy for treating alpha-synucleinopathies, particularly in humans.