Peptide immunogens from c-terminal end of alpha-synuclein protein and formulations thereof for treatment of synucleinopathies

Abstract

To provide alpha-synuclein peptide immunogen constructs, compositions containing the constructs, antibodies elicited by the constructs, and methods for making and using the constructs and compositions thereof.SOLUTION: An alpha-synuclein peptide immunogen construct is provided, which comprises: a B cell epitope containing about 10 to about 25 amino acid residues from the C-terminal fragment of alpha-synuclein corresponding to about amino acid G111 to around amino acid D135; a T helper epitope selected from the group of specific amino acid sequences; and an optional heterologous spacer selected from the group consisting of an amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys, wherein the B cell epitope is covalently linked to the T helper cell epitope directly or through the heterologous spacer.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application is a PCT international application claiming the benefit of U.S. Provisional Application No. 62 / 521,287, filed Jun. 16, 2017, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to a peptide immunogen construct based on the C-terminus of the alpha-synuclein (α-Syn) protein and its formulations for the treatment of synucleinopathies.

Background Art

[0003] The synuclein proteins (reviewed at the website: en.wikipedia.org / wiki / Synuclein) are a family of soluble proteins common to vertebrates that are expressed primarily in neural tissue and certain tumors. The synuclein family includes three known proteins: alpha-synuclein (reviewed at the website: en.wikipedia.org / wiki / Alpha-synuclein), beta-synuclein (reviewed at the website: en.wikipedia.org / wiki / Beta-synuclein), and gamma-synuclein. All synucleins share a highly conserved alpha-helical lipid-binding motif that is similar to the exchangeable apolipoprotein class A2 lipid-binding domain. Although some data suggest a role in the regulation of membrane stability and / or turnover, normal cellular function has not been determined for any of the synuclein proteins.

[0004] Full-length alpha-synuclein protein (α-Syn) is a 140-amino acid protein (accession number NP_000336) encoded by the SNCA gene. At least three α-Syn isoforms are generated by alternative splicing. The major form is the full-length protein. Other isoforms are α-Syn-126 lacking residues 41-54 due to the loss of exon 3, and α-Syn-112 lacking residues 103-130 due to the loss of exon 5.

[0005] The primary structure of α-Syn is usually divided into three different domains: (1) residues 1-60: a predominantly amphipathic N-terminal region containing four 11-residue repeats with a consensus sequence KTKEGV having a structural alpha-helix tendency similar to the apolipoprotein-binding domain; (2) residues 61-95: a central hydrophobic region containing the non-amyloid β component (NAC) region involved in protein aggregation; (3) residues 96-140: a highly acidic proline-rich region with no clear structural tendency. A 35-amino acid α-Syn fragment of the NAC region was found to be present together with Aβ in the amyloid-rich fraction. NAC was later shown to be a fragment of NACP, the precursor protein, a full-length human homolog of synuclein from Torpedo californica, now called human α-Syn.

[0006] The use of high-resolution ion mobility mass spectrometry (IMS-MS) in vitro of α-Syn purified by HPLC has shown that α-Syn is autoproteolytic (autoproteolytic) and generates various low-molecular-weight fragments during incubation. The full-length protein of 14.46 kDa generates a number of small fragments including a 12.16 kDa fragment (amino acids 14-133) and a 10.44 kDa fragment (amino acids 40-140) formed by C-terminal and N-terminal truncations, as well as a 7.27 kDa fragment (amino acids 72-140). The 7.27 kDa fragment, which contains most of the NAC region, has been shown to aggregate much faster than full-length α-Syn. These autoproteolytic products may play a role as intermediates or cofactors in the aggregation of α-Syn.

[0007] α-Syn is abundant in the human brain and accounts for 1% of all proteins in the cytoplasm of brain and glial cells. α-Syn is widely expressed in the neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus, and cerebellum. It is also highly expressed in hematopoietic cells including B cells, T cells, and NK cells, as well as monocytes and platelets. Small amounts of α-Syn are found in the heart, muscle, and other tissues. In the brain, α-Syn is mainly found at the tips of nerve cells (neurons) in a special structure called the presynaptic terminal. Within these structures, α-Syn interacts with phospholipids and proteins. The presynaptic terminal releases chemical messengers called neurotransmitters, such as dopamine, from compartments known as synaptic vesicles. The release of neurotransmitters relays signals between neurons and is important for normal brain function, including cognition.

[0008] α-Syn in solution is considered a natively unfolded protein in that it lacks a single stable 3D structure. α-Syn has been shown to interact prominently with tubulin, suggesting that α-Syn may have activity as a potential microtubule-associated protein like tau. α-Syn has classically been considered an unstructured soluble protein, but non-mutated α-Syn forms stable folded tetramers that resist aggregation. Nevertheless, α-Syn can aggregate to form insoluble fibrils in pathologies characterized by Lewy bodies. These disorders are known as synucleinopathies (reviewed at the website: en.wikipedia.org / wiki / Synucleinopathies).

[0009] Synucleinopathies are a diverse group of neurodegenerative disorders that share common pathological features, and neuropathological examination reveals characteristic lesions containing abnormal aggregates of insoluble α-Syn in selective vulnerable populations of neurons and glial cells. The most common synucleinopathies include Lewy body disorders (LBD) such as Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and dementia with Lewy bodies (DLB), as well as neurodegeneration in multiple system atrophy (MSA) or neuroferritinopathy type I (NBIA type I). Current treatment options for these diseases include symptomatic medications such as L-dopa, anticholinergic drugs, and inhibitors of monoamine oxidase. However, all current treatment opportunities only lead to symptomatic relief and do not induce a long-term disease-modifying effect in patients.

[0010] LBD is a progressive neurodegenerative disorder characterized by tremors, rigidity, bradykinesia, and loss of dopaminergic neurons in the brain. In the case of DLB and PDD, the symptoms also include cognitive impairment. Up to 2% of the population over 60 years old in Western European countries exhibit typical symptoms of PD / LBD. Genetic susceptibility and environmental factors seem to be involved in the onset of the disease. Patients suffering from this disease develop characteristic intracellular inclusions called Lewy bodies (LB) in the cerebral cortex and subcortical regions of the brain, especially in areas with high content of dopaminergic neurons or neuronal processes. In LBD, α-Syn accumulates in the LB throughout the affected brain regions. Furthermore, single point mutations, as well as duplications or expansions of the α-Syn gene, have been demonstrated to be associated with rare familial forms of Parkinson's syndrome.

[0011] Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder characterized by symptoms of L-dopa-resistant Parkinson's syndrome, cerebellar ataxia, and autonomic neuropathy. Patients suffering from multi-system nerve loss are affected in various brain regions including the striatum, substantia nigra, cerebellum, pons, as well as the inferior olivary nucleus, and spinal cord. MSA is characterized by α-Syn positive glial cytoplasmic inclusions (GCI) and rare neuronal inclusions throughout the central nervous system.

[0012] α-Syn pathology also exists in other rare disorders such as various axonal dystrophies, and α-Syn is the main structural component of Lewy body fibrils. Often, Lewy bodies contain tau protein, but α-Syn and tau constitute two characteristic subsets of filaments in the same inclusion. α-Syn pathology is found in both sporadic and familial cases of Alzheimer's disease.

[0013] The aggregation mechanism of α-Syn is unknown. Monomeric α-Syn can naturally unfold in solution, but can also bind to membranes in an α-helical form. Unfolded monomers first aggregate into small oligomeric species that can be stabilized by interactions such as β-sheets, and then into high-molecular-weight insoluble protofibrils. α-Syn exists as a mixture of conformational isomers rich in unstructured, alpha-helix, and beta-sheet in equilibrium. Mutations or buffer conditions known to improve aggregation strongly increase the population of beta conformers, thus suggesting that this is the conformation related to pathogenic aggregation. There is evidence of aggregates and, ultimately, beta-structured and structured intermediates that can be precursors of Lewy bodies.

[0014] (1) Phosphorylation by one or more kinases; (2) Truncation by proteases such as calpain; and (3) Nitration by nitric oxide (NO) or other reactive nitrogen species present during inflammation, some physiological factors may modify α-Syn and lead to the formation of aggregates. ER-Golgi transport, synaptic vesicles, mitochondria, lysosomes, and other proteolytic mechanisms are part of the proposed cellular targets related to α-Syn-mediated toxicity by such aggregation.

[0015] Strategies for treating synucleinopathies include compounds that inhibit the aggregation of α-Syn. Small-molecule cuminaldehyde has been shown to inhibit the fibrillation of α-Syn. In addition to small-molecule therapies, recent reports suggest that α-Syn aggregates may be targets for immunotherapy (review by Lee JS and Lee S-J, 2016). However, this report points out several potential problems or challenges associated with the development of α-Syn immunotherapy, including (1) potential interference with the normal physiological function of α-Syn; (2) the difficulty of delivering antibody drugs to the brain parenchyma; and (3) the effectiveness of immunotherapy.

[0016] To date, the need to develop site-specific peptide immunogens and their formulations for the cost-effective treatment of patients suffering from synucleinopathy has not yet been met.

[0017] References: 1. ”Alpha-synuclein,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org / w / index.php?title=Alpha-synuclein&oldid=781366541 (accessed on May 30, 2017). 2. ”Synucleinopathies,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org / w / index.php?title=Synucleinopathies&oldid=686287116 (accessed on May 30, 2017). 3. ”Beta-synuclein,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org / w / index.php?title=Beta-synuclein&oldid=763171134 (accessed on May 30, 2017). 4. ”Synucleinopathies,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org / w / index.php?title=Synucleinopathies&oldid=686287116 (accessed on May 30, 2017). 5. LEE, J.S., et al., “Mechanism of Anti-α-synuclein Immunotherapy”, J. / Mov Disord:, 9(1): 14-19(2016) 6. Traggiai, E., et al. “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus”, Nat Med:, 10(8):871 - 875(2004) 7. Wang, C., et al. “Versatile Structures of α - Synuclein”, Front Mol Neurosci. 9:48(2016)

Summary of the Invention

[0018] The present disclosure relates to a peptide immunogen construct of alpha - synuclein protein (α - Syn). The present disclosure also relates to a composition containing the peptide immunogen construct, methods of making and using the peptide immunogen construct, and antibodies produced by the peptide immunogen construct.

[0019] The disclosed peptide immunogen construct contains a B - cell epitope from α - Syn linked directly or, if necessary, via a heterologous spacer to a heterologous T - helper cell (Th) epitope. The B - cell epitope portion of the peptide immunogen construct contains from about 10 to about 25 amino acid residues from the C - terminal region of α - Syn corresponding to the sequence from around glycine (G111) at position 111 to around asparagine (D135) at position 135 of the full - length α - Syn (SEQ ID NO: 1). The heterologous Th epitope portion of the peptide immunogen construct is derived from an amino acid sequence derived from a pathogenic protein. The B - cell epitope portion and the Th epitope portion of the peptide immunogen construct act together when administered to a host to stimulate the generation of antibodies that specifically recognize and bind to the B - cell epitope portion of the α - Syn of the construct.

[0020] In some embodiments, the α-Syn peptide immunogen construct comprises: (a) a B cell epitope comprising about 10 to about 25 amino acid residues from the C-terminal fragment of α-Syn corresponding to the vicinity of amino acid G111 to the vicinity of amino acid D135 of SEQ ID NO: 1; (b) a T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 98; and (c) an optional heterologous spacer selected from the group consisting of an amino acid, Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), wherein the B cell epitope is covalently bound to the T helper epitope directly or via an optional heterologous spacer. In certain embodiments, the α-Syn peptide immunogen construct comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111 to 113, and 115 to 147.

[0021] The present disclosure also relates to a composition comprising a disclosed peptide immunogen construct, including a pharmaceutical composition. The disclosed pharmaceutical composition can induce an immune response and antibody production against the disclosed peptide immunogen construct in a host. The disclosed composition can include a mixture of one or more of the disclosed peptide immunogen constructs. In some embodiments, the composition comprises the disclosed peptide immunogen construct together with additional components including a carrier, an adjuvant, a buffer, and other suitable reagents. In certain embodiments, the composition comprises the disclosed peptide immunogen construct in the form of a stabilized immunostimulatory complex with a CpG oligomer optionally supplemented with an adjuvant.

[0022] In certain embodiments, the composition comprises an α-Syn peptide immunogen construct comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, 115-147. In certain embodiments, the composition is a pharmaceutical composition comprising an α-Syn peptide immunogen construct comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, 115-147, and a pharmaceutically acceptable carrier or adjuvant.

[0023] The present disclosure also relates to antibodies produced by a host immunized with the disclosed peptide immunogen constructs. The disclosed antibodies specifically recognize and bind to the B cell epitope portion of α-Syn of the peptide immunogen construct. The disclosed α-Syn antibodies unexpectedly have a high cross-reactivity to the β-sheet of α-Syn in monomeric, oligomeric, or fibrillar forms. Based on their unique features and properties, the disclosed antibodies can provide an immunotherapeutic approach for the targeting, identification, and treatment of synucleinopathies.

[0024] In certain embodiments, the antibody or an epitope-binding fragment thereof specifically binds to the B cell epitope of an α-Syn peptide immunogen construct selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, 115-147.

[0025] The present disclosure also relates to methods of making and using the disclosed peptide immunogen constructs, antibodies, and compositions. The disclosed methods provide for the low-cost manufacture and quality control of peptide immunogen constructs and compositions containing the constructs, which can be used in methods for preventing and treating synopathy.

[0026] The present disclosure also includes methods of treating and / or preventing synucleinopathy using the disclosed peptide immunogen constructs and / or antibodies against the peptide immunogen constructs. In some embodiments, the method of treating and / or preventing synucleinopathy includes administering to a host a composition comprising the disclosed peptide immunogen construct. In certain embodiments, the composition utilized in the method comprises the disclosed peptide immunogen construct in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, by electrostatic binding, and the complex is further supplemented, optionally, with an inorganic salt or an oil as an adjuvant for administration to a patient with synucleinopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen construct to a host at risk of synucleinopathy or having synucleinopathy.

[0027] In various embodiments, methods are described using α-Syn peptide immunogen constructs and / or antibodies elicited by the α-Syn peptide immunogen constructs. In certain embodiments, the methods are for producing antibodies, inhibiting α-Syn aggregation, reducing the amount of α-Syn aggregates, and identifying α-Syn aggregates of different sizes, as described. The various methods include administering to a host in need thereof a pharmacologically effective amount of the α-Syn peptide immunogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

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[0029] Detailed Description of the Invention The present disclosure relates to peptide immunogen constructs of alpha-synuclein protein (α-Syn). The present disclosure also relates to compositions comprising the peptide immunogen constructs, methods of making and using the peptide immunogen constructs, and antibodies produced by the peptide immunogen constructs.

[0030] The disclosed peptide immunogen constructs comprise B cell epitopes from α-Syn directly or, optionally via a heterologous spacer, linked to heterologous T helper cell (Th) epitopes. The B cell epitope portion of the peptide immunogen construct comprises from about 10 to about 25 amino acid residues from the C-terminus of α-Syn corresponding to the sequence from around glycine (G111) at position 111 to around asparagine (D135) at position 135 of the full-length α-Syn (SEQ ID NO: 1). The heterologous Th epitope portion of the peptide immunogen construct is derived from an amino acid sequence derived from a pathogenic protein. The B cell epitope portion and the Th epitope portion of the peptide immunogen construct act together when administered to a host to stimulate the production of antibodies that specifically recognize and bind to the B cell epitope portion of α-Syn of the construct.

[0031] The present disclosure also relates to compositions comprising the disclosed peptide immunogen constructs, including pharmaceutical compositions. The disclosed pharmaceutical compositions can induce an immune response and the production of antibodies against the disclosed peptide immunogen constructs in a host. The disclosed compositions can comprise a mixture of one or more of the disclosed peptide immunogen constructs. In some embodiments, the composition comprises the disclosed peptide immunogen constructs together with additional components including a carrier, an adjuvant, a buffer, and other suitable reagents. In certain embodiments, the composition comprises the disclosed peptide immunogen constructs in the form of a stabilized immunostimulatory complex with CpG oligomers optionally supplemented with an adjuvant.

[0032] The present disclosure also relates to antibodies produced by a host immunized with the disclosed peptide immunogen constructs. The disclosed antibodies specifically recognize and bind to the B cell epitope portion of α-Syn of the peptide immunogen construct. The disclosed α-Syn antibodies have unexpectedly high cross-reactivity against the β-sheet of α-Syn in the form of monomers, oligomers, or protofibrils. Based on their unique features and properties, the disclosed antibodies can provide an immunotherapeutic approach for the targeting, identification, and treatment of synucleinopathies.

[0033] The present disclosure also relates to methods of making and using the disclosed peptide immunogen constructs, antibodies, and compositions. The disclosed methods provide for low-cost manufacture and quality control of peptide immunogen constructs and compositions containing the constructs, which can be used in methods for preventing and treating synopathy.

[0034] The present disclosure also includes methods of treating and / or preventing synucleinopathy using the disclosed peptide immunogen constructs and / or antibodies against the peptide immunogen constructs. In some embodiments, the method of treating and / or preventing synucleinopathy includes administering to a host a composition comprising the disclosed peptide immunogen construct. In certain embodiments, the composition utilized in the method comprises the disclosed peptide immunogen construct in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, by electrostatic binding, and the complex is further supplemented with an inorganic salt or oil as an adjuvant for administration to a patient with synucleinopathy, if desired. The disclosed methods also include dosing schedules, dosage forms, and routes for administering the peptide immunogen construct to a host at risk of synucleinopathy or having synucleinopathy.

[0035] The headings of the sections used in this specification are for the purpose of organization only and are not to be construed as limiting the subject matter described. All references or portions of references cited in this application are hereby expressly incorporated by reference in their entirety for all purposes.

[0036] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, "including A or B" means including A, or B, or both A and B. Further, it is to be understood that all amino acid sizes, and all molecular weight or molecular mass values given for polypeptides are approximate and are provided for illustrative purposes. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, but the appropriate methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0037] α-Syn peptide immunogen construct The present disclosure provides a peptide immunogen construct comprising a B cell epitope from α-Syn covalently linked directly or, optionally via a heterologous spacer, to a heterologous T helper cell (Th) epitope.

[0038] As used herein, the phrase "α-Syn peptide immunogen construct" refers to a peptide comprising (a) a B cell epitope having from about 10 to about 25 amino acid residues from the C-terminus of α-Syn corresponding to the sequence from around glycine (G111) at position 111 to around asparagine (D135) at position 135 of full-length α-Syn (SEQ ID NO: 1), (b) a heterologous Th epitope, and (c) a heterologous spacer as required.

[0039] In certain embodiments, the peptide immunogen construct has the formula: (Th) m -(A) n -(C-terminal fragment of α-Syn)-X or (C-terminal fragment of α-Syn)-(A) n -(Th) m -X and can be represented by wherein Th is a heterologous T helper epitope, A is a heterologous spacer, (C-terminal fragment of α-Syn) is a B cell epitope having from about 10 to about 25 amino acid residues from the C-terminus of α-Syn, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4, n is from 0 to about 10.

[0040] The various components of the disclosed α-Syn peptide immunogen constructs are described below.

[0041] a. α-Syn and the C-terminal fragment of α-Syn As used herein, terms such as "α-Syn", "alpha-synuclein", and "α-synuclein" refer to (a) full-length α-Syn protein and / or (b) fragments thereof from any organism that expresses α-Syn. α-Syn is characterized by extreme conformational diversity, adapting to various conditions in membrane-bound, cytoplasmic, and amyloid-aggregated states and performing highly versatile functions. In some embodiments, the α-Syn protein is of human origin. In certain embodiments, the full-length human α-Syn protein (Accession No. NP_000336) (SEQ ID NO: 1) has 140 amino acids.

[0042] As used herein, the phrase "C-terminal region" or "C-terminus" of α-Syn refers to any amino acid sequence from the carboxyl-terminal portion of α-Syn. In certain embodiments, the C-terminal region or C-terminus of α-Syn relates to the amino acid sequence between residues 96 - 140 of α-Syn, or fragments thereof. The C-terminal region of α-Syn is rich in proline and negatively charged residues, which are common characteristics found in native unfolded proteins that maintain solubility. The C-terminal region of α-Syn is generally present in a random coil structure due to its low hydrophobicity and large net negative charge. In vitro studies have revealed that α-Syn aggregation can be induced by a decrease in pH that neutralizes these negative charges.

[0043] As used herein, the phrase "C-terminal fragment of α-Syn" or "B cell epitope from the C-terminus of α-Syn" refers to a part of the full-length α-Syn sequence that contains approximately 10 to approximately 25 amino acid residues from the C-terminus of α-Syn corresponding to the sequence from around glycine (G111) at amino acid position 111 to around asparagine (D135) at amino acid position 135 of full-length α-Syn. The α-Syn C-terminal fragment is also referred to herein as the G111 - D135 peptide of α-Syn and fragments thereof. The various C-terminal fragments of α-Syn described herein are referred to by their amino acid positions relative to the full-length sequence of α-Syn represented by SEQ ID NO: 1.

[0044] The amino acid sequence of the C-terminal fragment of α-SynC used in the α-Syn peptide immunogen construct was selected based on a number of design principles. Some of these principles include the use of the following α-Syn peptide sequences: (i) Since β-Syn can bind to α-Syn and prevent its aggregation, it does not share significant sequence homology with beta-synuclein (β-Syn) to avoid the generation of antibodies that cross-react with β-Syn. (ii) It lacks self-T helper epitopes within α-Syn to prevent self-T cell activation that could lead to brain inflammation causing meningococcal encephalitis, as previously reported in clinical trials using the AN1792 vaccine targeting Aβ1-42 for the treatment of Alzheimer's disease. (iii) It is contained within regions of α-Syn that are susceptible to conformational changes from its native form. (iv) Since it is a self-molecule, it is non-immunogenic by itself. (v) It can be rendered immunogenic by a protein carrier or a strong T helper epitope(s). (vi) When rendered immunogenic and administered to a host: (a) It induces high-titer antibodies against the α-Syn peptide sequence (B cell epitope), rather than against the protein carrier or strong T helper epitope(s). (b) It induces high-titer antibodies that react with the misfolded β-sheets of α-Syn in its monomeric, oligomeric, or protofibrillar form, and such antibodies prevent the aggregation of α-Syn, deaggregate any α-Syn aggregates, and result in the removal of toxic α-Syn oligomers, aggregates, and / or protofibrils, thus reducing or preventing the burden of α-Syn aggregates in the brain. (c) It does not induce antibodies that react with native α-Syn, which is a major cellular protein with a wide tissue distribution, presenting high safety concerns.

[0045] Considering these design principles, the C-terminal region of α-Syn was selected as the target for peptide immunogen design. Furthermore, the C-terminal region of α-Syn was selected because, based on its structural properties, this region was considered to be the most susceptible to regulation by antibodies or other physical factors compared to other regions of α-Syn.

[0046] As further described in the examples, evaluation of a number of peptide sequences derived from α-Syn led to the identification and selection of multiple α-Syn peptides that satisfy the above design principles. Specifically, sequences that satisfy the design principles include peptides having from about 10 to about 25 amino acid residues from the C-terminal region of α-Syn corresponding to the sequence from around glycine (G111) at amino acid position 111 to around asparagine (D135) at amino acid position 135 of the full-length α-Syn.

[0047] In some embodiments, the C-terminal fragment of α-Syn is the 25-amino acid α-Syn G111-D135 peptide represented by SEQ ID NO: 12. In other embodiments, the C-terminal fragment of α-Syn comprises about 10 contiguous amino acids of the α-Syn G111-D135 peptide represented by SEQ ID NO: 12. In certain embodiments, the C-terminal fragment of α-Syn comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids of the α-Syn G111-D135 peptide represented by SEQ ID NO: 12. In specific embodiments, the C-terminal fragment of α-Syn has an amino acid sequence represented by SEQ ID NOs: 12-15, 17, or 49-64, as shown in Table 1.

[0048] The C-terminal fragment of α-Syn of the present disclosure also includes immunologically functional analogs or homologs of the α-SynG111-D135 peptide, and fragments thereof. Functional immunological analogs or homologs of the α-SynG111-D135 peptide and fragments thereof include variants that retain substantially the same immunogenicity as the original peptide. Immunologically functional analogs can have conservative substitutions at amino acid positions; changes in overall charge; covalent attachment to another moiety; or addition, insertion, or deletion of amino acids; and / or any combination thereof.

[0049] A conservative substitution is when one amino acid residue is replaced by another amino acid residue having similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0050] Functionally immunologically analogous bodies include amino acid sequences that contain conservative substitutions, additions, deletions, or insertions of 1 to about 4 amino acid residues that induce an immune response that cross-reacts with the α-SynG111-D135 peptide. Conservative substitutions, additions, and insertions can be achieved with natural or unnatural amino acids. Unnatural amino acids include, but are not limited to, ε-N lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitrotyrosine, pyroglutamic acid, and the like. Natural amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0051] In one embodiment, a functional immunological analog of a particular peptide contains the same amino acid sequence as the original peptide and further contains three lysine residues (Lys-Lys-Lys) added to the amino terminus of the B cell epitope peptide of the α-SynG111-D135 peptide and its fragments. In this embodiment, by including three lysine residues in the original peptide sequence, the overall charge of the original peptide changes, but the function of the original peptide does not change.

[0052] In certain embodiments, a functional analog of the C-terminal fragment of α-Syn has at least 50% identity with the original amino acid sequence. In other embodiments, the functional analog has at least 80% identity with the original amino acid sequence. In still other embodiments, the functional analog has at least 85% identity with the original amino acid sequence. In still other embodiments, the functional analog has at least 90% or at least 95% identity with the original amino acid sequence.

[0053] b. Heterologous T helper cell epitope (Th epitope) The present disclosure provides a peptide immunogen construct comprising a B cell epitope from α-Syn covalently linked directly or, optionally via a heterologous spacer, to a heterologous T helper cell (Th) epitope.

[0054] The heterologous Th epitope of the α-Syn peptide immunogen construct enhances the immunogenicity of the C-terminal fragment of α-Syn and promotes the production of specific high-titer antibodies against a rationally designed and optimized target B cell epitope (i.e., the C-terminal fragment of α-Syn).

[0055] As used herein, the term "heterologous" refers to an amino acid sequence that is not part of or homologous to the wild-type sequence of α-Syn. Thus, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that is not naturally found in α-Syn (i.e., the Th epitope is non-self with respect to α-Syn). Since the Th epitope is heterologous to α-Syn, when the heterologous Th epitope is covalently linked to the C-terminal fragment of α-Syn, the native amino acid sequence of α-Syn does not extend in either the N-terminal or C-terminal direction.

[0056] The heterologous Th epitopes of the present disclosure can be any Th epitope that does not have the amino acid sequence naturally found in α-Syn. The Th epitope can have an amino acid sequence derived from any species (e.g., human, pig, cow, dog, rat, mouse, guinea pig, etc.). The Th epitope can also have a promiscuous binding motif for MHC class II molecules of multiple species. In certain embodiments, the Th epitope comprises multiple promiscuous MHC class II binding motifs that allow for maximal activation of T helper cells that initiate and regulate an immune response. The Th epitope is preferably immunologically silent by itself, i.e., antibodies generated by the α-Syn peptide immunogen construct are mostly not against the Th epitope, if any, so that a highly focused immune response against the targeted B cell epitope of the C-terminal fragment of α-Syn is enabled.

[0057] The epitopes of the present disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens as exemplified in Table 2 (SEQ ID NOs: 70-98). Further, the Th epitopes include idealized artificial Th epitopes and combinations of idealized artificial Th epitopes (e.g., SEQ ID NOs: 71 and 78-84). Heterologous Th epitope peptides presented as combinatorial sequences (e.g., SEQ ID NOs: 79-82) contain a mixture of amino acid residues represented at specific positions within the peptide framework based on the variable residues of homologs of that particular peptide. A set of combinatorial peptides can be synthesized in one process by adding a mixture of designated protected amino acids at designated positions during the synthesis process instead of one specific amino acid. Such a set of combinatorial heterologous Th epitope peptides can allow for broad Th epitope coverage in animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 79-82 shown in Table 2. The epitope peptides of the present invention provide broad reactivity and immunogenicity to animals and patients in a genetically diverse population.

[0058] The α-Syn peptide immunogen construct containing the Th epitope is generated simultaneously by single solid-phase peptide synthesis in parallel with the C-terminal fragment of α-Syn. The Th epitope also includes immunological analogs of the Th epitope. Immunological Th analogs include immunopotentiating analogs, cross-reactive analogs, and any segment of these Th epitopes sufficient to enhance or stimulate an immune response against the C-terminal fragment of α-Syn.

[0059] Functional immunological analogs of the Th epitope peptide are also effective and are included as part of the present invention. Functional immunological Th analogs can include conservative substitutions, additions, deletions, and insertions of 1 to about 5 amino acid residues in the Th epitope that do not substantially modify the Th-stimulating function of the Th epitope. Conservative substitutions, additions, and insertions can be achieved with natural or non-natural amino acids, as described above for the C-terminal fragment of α-Syn. Table 2 identifies another variation of functional analogs of the Th epitope peptide. In particular, SEQ ID NOs: 71 and 78 of Th of MvF1 and MvF2 are functional analogs of SEQ ID NOs: 81 and 83 of MvF4 and MvF5, which differ in the amino acid frame by deletion (SEQ ID NOs: 71 and 78) or inclusion (SEQ ID NOs: 81 and 83) of two amino acids at the N-terminus and C-terminus, respectively. The differences between these two sets of similar sequences will not affect the function of the Th epitope contained within these sequences. Thus, functional immunological Th analogs include several versions of the Th epitope derived from measles virus fusion protein MvFl-4 Th (SEQ ID NOs: 71, 78, 79, 81, and 83), as well as hepatitis B surface protein HBsAg1-3 Th (SEQ ID NOs: 80, 82, and 84).

[0060] The Th epitope of the α-Syn peptide immunogen construct can be covalently linked to the N-terminus or C-terminus of the C-terminal peptide of α-Syn. In some embodiments, the Th epitope is covalently linked to the N-terminus of the C-terminal peptide of α-Syn. In other embodiments, the Th epitope is covalently linked to the C-terminus of the C-terminal peptide of α-Syn. In certain embodiments, more than one Th epitope is covalently linked to the C-terminal fragment of α-Syn. When more than one Th epitope is linked to the C-terminal fragment of α-Syn, each Th epitope can have the same amino acid sequence or a different amino acid sequence. Further, when more than one Th epitope is linked to the α-Syn C-terminal fragment, the Th epitopes can be arranged in any order. For example, the Th epitopes can bind consecutively to the N-terminus of the C-terminal fragment of α-Syn, or bind consecutively to the C-terminus of the C-terminal fragment of α-Syn, or a Th epitope can be covalently linked to the N-terminus of the C-terminal fragment of α-Syn while another Th epitope is covalently linked to the C-terminus of the C-terminal fragment of α-Syn. There is no limitation on the arrangement of the Th epitopes related to the C-terminal fragment of α-Syn.

[0061] In some embodiments, the Th epitope is directly covalently linked to the C-terminal fragment of α-Syn. In other embodiments, the Th epitope is covalently linked to the C-terminal fragment of α-Syn via a heterologous spacer as described in detail below.

[0062] c. Heterologous spacer The disclosed α-Syn peptide immunogen construct optionally includes a heterologous spacer that covalently links a B cell epitope from α-Syn to a heterologous T helper (Th) epitope.

[0063] As described above, the term "heterologous" refers to an amino acid sequence that is not part of or homologous to a wild-type sequence of α-Syn. Thus, when covalently attaching a heterologous spacer to a B cell epitope from α-Syn, since the spacer is heterologous to the α-Syn sequence, the native amino acid sequence of α-Syn does not extend in either the N-terminal or C-terminal direction.

[0064] A spacer is any molecule or chemical structure that can join two amino acids and / or peptides together. The spacer can vary in length and polarity depending on the application. The spacer linkage can be through an amide bond or a carboxyl bond, although other functional groups are possible as well. The spacer can include a compound, a naturally occurring amino acid, or a non-naturally occurring amino acid.

[0065] The spacer can provide structural features to the α-Syn peptide immunogen construct. Structurally, the spacer physically separates the Th epitope from the B cell epitope of the C-terminal fragment of α-Syn. The physical separation by the spacer may disrupt any artificial secondary structure created by binding the Th epitope to the B cell epitope. Furthermore, the physical separation of the epitopes by the spacer can eliminate interference between the Th cell response and / or the B cell response. Additionally, the spacer can be designed to create or modify the secondary structure of the peptide immunogen construct. For example, the spacer can be designed to function as a flexible hinge to enhance the separation of the Th epitope and the B cell epitope. The flexible hinge spacer enables more efficient interaction between the presented peptide immunogen and appropriate Th and B cells, and can enhance the immune response to the Th epitope and the B cell epitope. Examples of sequences encoding flexible hinges are found in the immunoglobulin heavy chain hinge region and are often proline-rich. One particularly useful flexible hinge that can be used as a spacer is provided by the Pro-Pro-Xaa-Pro-Xaa-Pro sequence (SEQ ID NO: 148), where Xaa is any amino acid, preferably aspartic acid.

[0066] The spacer can also provide functional features to the α-Syn peptide immunogen construct. For example, the spacer can be designed to alter the overall charge of the α-Syn peptide immunogen construct, which can affect the solubility of the peptide immunogen construct. Additionally, altering the overall charge of the α-Syn peptide immunogen construct can affect the ability of the peptide immunogen construct to bind to other compounds and reagents. As described in more detail below, the α-Syn peptide immunogen construct can form stable immunostimulatory complexes with highly charged oligonucleotides such as CpG oligomers via electrostatic binding. The overall charge of the α-Syn peptide immunogen construct is important for the formation of these stable immunostimulatory complexes.

[0067] Compounds that can be used as spacers include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (mini-PEG3), trioxatridecane-succinic acid (Ttds), 12-aminododecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

[0068] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0069] Non-natural amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitrotyrosine, pyroglutamic acid, and the like.

[0070] The spacer of the α-Syn peptide immunogen construct can be covalently bound to the C-terminus of the Th epitope and the N-terminus or C-terminus of the C-terminal peptide of α-Syn. In some embodiments, the spacer is covalently bound to the C-terminus of the Th epitope and the N-terminus of the C-terminal peptide of α-Syn. In other embodiments, the spacer is covalently bound to the C-terminus of the C-terminal peptide of α-Syn and the N-terminus of the Th epitope. In certain embodiments, for example, when there is more than one Th epitope in the peptide immunogen construct, more than one spacer can be used. When using more than one spacer, each spacer may be the same as or different from each other. Further, when there is more than one Th epitope in the peptide immunogen construct, the Th epitopes can be separated by spacers, and the Th epitopes may be the same as or different from the spacers used to separate the Th epitopes from the B cell epitopes. There are no restrictions on the arrangement of the spacers with respect to the Th epitope or the C-terminal fragment of α-Syn.

[0071] In certain embodiments, the heterologous spacer is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments, the spacer comprises more than one naturally occurring or non-naturally occurring amino acid. In specific embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

[0072] d. Specific embodiments of the α-Syn peptide immunogen construct The α-Syn peptide immunogen construct has the formula: (Th) m -(A) n -(C-terminal fragment of α-Syn)-X or (C-terminal fragment of α-Syn)-(A) n -(Th) m -X and can be represented by wherein Th is a heterologous T helper epitope, A is a heterologous spacer, (the C-terminal fragment of α-Syn) is a B cell epitope having from about 10 to about 25 amino acid residues from the C-terminus of α-Syn, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4, n is from 0 to about 10.

[0073] In certain embodiments, the heterologous Th epitope of the α-Syn peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 70-98 shown in Table 2, or a combination thereof. In certain embodiments, the Th epitope has an amino acid sequence selected from any of SEQ ID NOs: 78-84. In certain embodiments, the α-Syn peptide immunogen construct comprises more than 1 Th epitope.

[0074] In certain embodiments, the heterologous spacer as needed is selected from any of Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), and combinations thereof. In certain embodiments, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

[0075] In certain embodiments, the C-terminal fragment of α-Syn has from about 10 to about 25 amino acid residues from the C-terminal region of α-Syn corresponding to the sequence from around glycine (G111) at position 111 to around asparagine (D135) at position 135 of the full-length α-Syn. In certain embodiments, the C-terminal fragment of α-Syn has an amino acid sequence represented by SEQ ID NOs: 12-15, 17, or 49-64, as shown in Table 1.

[0076] In certain embodiments, the α-Syn peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 107-108, 111-113, and 115-147 shown in Table 3. In certain embodiments, the α-Syn peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 107-108 and 111-113.

[0077] Composition The present disclosure also provides a composition comprising the disclosed α-Syn peptide immunogen construct.

[0078] a. Peptide composition The composition comprising the disclosed α-Syn peptide immunogen construct can be in liquid or solid form. The liquid composition can include water, buffer, solvent, salts, and / or other acceptable reagents that do not change the structural or functional properties of the α-Syn peptide immunogen construct. The peptide composition can include one or more of the disclosed α-Syn peptide immunogen constructs.

[0079] b. Pharmaceutical composition The present disclosure also relates to a pharmaceutical composition comprising the disclosed α-Syn peptide immunogen construct.

[0080] The pharmaceutical composition can include a carrier and / or other additives in a pharmaceutically acceptable delivery system. Thus, the pharmaceutical composition can include a pharmaceutically effective amount of the α-Syn peptide immunogen construct together with other excipients such as pharmaceutically acceptable carriers, adjuvants, and / or diluents, additives, stabilizers, preservatives, solubilizers, buffers.

[0081] The pharmaceutical composition can include one or more adjuvants that do not have a specific antigenic effect per se but act to accelerate, prolong, or enhance the immune response to the α-Syn peptide immunogen construct. Adjuvants used in the pharmaceutical composition can include oils, aluminum salts, virosomes, aluminum phosphate (such as ADJU-PHOS®), aluminum hydroxide (such as ALHYDROGEL®), lipoxin, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA 35, ISA 206, ISA50V, ISA51, ISA 720, and other adjuvants and emulsifiers.

[0082] In some embodiments, the pharmaceutical composition includes Montanide™ ISA 51 (an oil adjuvant composition consisting of vegetable oil and mannide oleate for the production of water-in-oil emulsions), Tween® 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides, and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion containing Emulsigen or Emulsigen D as an adjuvant.

[0083] The pharmaceutical composition can be formulated as an immediate-release formulation or for sustained-release formulations. Further, the pharmaceutical composition can be formulated for the induction of systemic or local mucosal immunity by capture of the immunogen and co-administration with microparticles. Such delivery systems can be readily determined by those skilled in the art.

[0084] The pharmaceutical composition can be prepared as an injection, either as a liquid solution or a suspension. The liquid vehicle containing the α-Syn peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition can be administered by any suitable mode of application, such as i.d., i.v., i.p., i.m., intranasal, oral, subcutaneous, etc., and with any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration, including oral and intranasal applications, can also be prepared.

[0085] The pharmaceutical composition can be formulated as an immediate release formulation or for sustained release. Additionally, the pharmaceutical composition can be formulated for induction of systemic or local mucosal immunity by capture of the immunogen and co-administration with microparticles. Such delivery systems can be readily determined by those skilled in the art.

[0086] The pharmaceutical composition can also be formulated in suitable unit dosage forms. In some embodiments, the pharmaceutical composition contains from about 0.5 μg to about 1 mg of the α-Syn peptide immunogen construct per kg of body weight. The effective amount of the pharmaceutical composition will vary depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic animals, can also be treated. When delivered in multiple doses, the pharmaceutical composition can be conveniently divided into appropriate amounts per unit dosage form. The dosage administered will depend on the age, weight, and overall health of the subject, as is well known in the art of therapy.

[0087] In some embodiments, the pharmaceutical composition comprises more than 1 α-Syn peptide immunogen construct. A pharmaceutical composition comprising a mixture of more than 1 α-Syn peptide immunogen construct can synergistically enhance the immunogenic effect of the construct. A pharmaceutical composition comprising more than 1 α-Syn peptide immunogen construct is more effective in a larger genetic population because of the broad range of MHC class II, and thus the immune response to the α-Syn peptide immunogen construct is improved.

[0088] In some embodiments, the pharmaceutical composition comprises an α-Syn peptide immunogen construct selected from SEQ ID NOs: 107-108, 111-113, 115-147, and homologs, analogs, and / or combinations thereof. In certain embodiments, the pharmaceutical composition comprises an α-Syn peptide immunogen construct selected from SEQ ID NOs: 107-108, 111-113, and any combination thereof.

[0089] A pharmaceutical composition comprising an α-Syn peptide immunogen construct can be used to induce an immune response and produce antibodies in a host upon administration.

[0090] c. Immunostimulatory complex The present disclosure also relates to a pharmaceutical composition comprising an α-Syn peptide immunogen construct in the form of an immunostimulatory complex with a CpG oligonucleotide. Such immunostimulatory complexes are particularly adapted to act as adjuvants and peptide immunogen stabilizers. The immunostimulatory complex is in the form of microparticles and can efficiently present the α-Syn peptide immunogen to cells of the immune system to generate an immune response. The immunostimulatory complex may be formulated as a suspension for parenteral administration. The immunostimulatory complex can also be formulated in the form of a w / o emulsion as a suspension in combination with an inorganic salt or an in situ gelling polymer for efficient delivery of the α-Syn peptide immunogen to cells of the host immune system after parenteral administration. The immunostimulatory complex can generate an immune response against the β-sheet of α-Syn that has a protective / therapeutic effect (e.g., FIGS. 8A, 8B, and 8C of Example 13).

[0091] The stabilized immunostimulatory complex can be formed by complexing an α-Syn peptide immunogen construct with an anionic molecule, oligonucleotide, polynucleotide, or a combination thereof via electrostatic binding. The stabilized immunostimulatory complex can be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0092] In certain embodiments, the α-Syn peptide immunogen construct is designed to include a positively charged cationic moiety at a pH in the range of 5.0 to 8.0. The net charge of the cationic moiety of the α-Syn peptide immunogen construct, or a mixture of constructs, is calculated by assigning a +1 charge to each lysine (K), arginine (R), or histidine (H), a -1 charge to each aspartic acid (D) or glutamic acid (E), and a 0 charge to the other amino acids within the sequence. The charges are summed within the cationic moiety of the α-Syn peptide immunogen construct and represented as the net average charge. Suitable peptide immunogens have a cationic moiety with a net average positive charge of +1. Preferably, the peptide immunogen is in the range where the net positive charge is greater than +2. In some embodiments, the cationic portion of the α-Syn peptide immunogen construct is a heterologous spacer. In certain embodiments, when the spacer sequence is (a,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), the cationic portion of the α-Syn peptide immunogen construct has a charge of +4.

[0093] As used herein, "anionic molecule" refers to any molecule that is negatively charged at a pH in the range of 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge of the oligomer or polymer is calculated by assigning a -1 charge to each phosphodiester or phosphorothioate group of the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules of 8 to 64 nucleotide bases, wherein the number of CpG motif repeats ranges from 1 to 10. Preferably, the CpG immunostimulatory single-stranded DNA molecule comprises 18 to 48 nucleotide bases, and the number of CpG motif repeats ranges from 3 to 8.

[0094] More preferably, the anionic oligonucleotide has the formula: 5’ X 1 CGX 2 3’, wherein C and G are not methylated; X 1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine); X 2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide has the formula: 5’ (X 3 )2CG(X 4 )23’, wherein C and G are not methylated; X 3 is selected from the group consisting of A, T, or G; X 4 is C or T.

[0095] The resulting immunostimulatory complex is typically in the form of particles in the size range of 1 to 50 microns and is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species. The micronized immunostimulatory complex has the advantage of providing adjuvantation and upregulation of specific immune responses in vivo. Additionally, the stabilized immunostimulatory complex is suitable for preparing pharmaceutical compositions by various processes including water-in-oil emulsions, inorganic salt suspensions, and polymer gels.

[0096] Antibody The present disclosure also provides antibodies induced by the α-Syn peptide immunogen construct.

[0097] An α-Syn C-terminal fragment having from about 10 to about 25 amino acid residues from the vicinity of glycine (G111) at amino acid position 111 to the vicinity of aspartic acid (D135) at amino acid position 135 of full-length α-Syn is non-immunogenic or weakly immunogenic by itself. However, the disclosed α-Syn peptide immunogen constructs, which include an α-Syn C-terminal fragment, a heterologous Th epitope, and, optionally, a heterologous spacer, can induce an immune response and antibody production when administered to a host. The design of the α-Syn peptide immunogen constructs can disrupt tolerance to self α-Syn and induce the production of site-specific antibodies that recognize conformational rather than linear epitopes.

[0098] Surprisingly, the antibodies produced by the α-Syn peptide immunogen constructs do not bind to the native alpha helix of native α-Syn monomers. Instead, the antibodies generated by the α-Syn peptide immunogen constructs recognize and bind to the denatured β-sheet of α-Syn in the form of monomers, oligomers, and protofibrils. Furthermore, the antibodies produced by the a-Syn peptide immunogen constructs do not bind to similar structures of other amyloidogenic proteins, namely, Aβ1-42 and Tau441. Thus, the specific design of the α-Syn peptide immunogen constructs (including an α-Syn C-terminal fragment, a heterologous Th epitope, and, optionally, a heterologous spacer) allows the conformational structure of the highly versatile α-Syn C-terminal fragment to be changed to enable a β-sheet-like conformational structure.

[0099] Extensive comparisons of antibodies derived from the immune sera of animals immunized with α-Syn peptide immunogen constructs were performed in a number of functional assays. These comparisons demonstrated the ability of the antibodies to bind to α-Syn in NGF-treated PC12 cells with high specificity only to β-sheet monomers and oligomers of α-Syn and not to amyloidogenic proteins of other species (see Example 9).

[0100] Antibodies induced by the α-Syn peptide immunogen construct can, surprisingly, prevent the aggregation of α-Syn (anti-aggregation activity) and dissociate pre-formed α-Syn aggregates (disaggregation activity). Furthermore, the antibodies can, surprisingly, reduce the production of TNF-α and IL6 induced by microglial cells, indicating that these antibodies can effectively reduce α-Syn aggregate- or fibril-mediated microglial activation. These antibodies have also been found to reduce neurodegeneration caused by both exogenous α-Syn aggregates and endogenous α-Syn aggregates in α-Syn overexpressing cells. Furthermore, such antibodies specifically recognize and bind to pathological α-Syn oligomeric aggregates or fibrils, but do not react with non-pathological α-Syn. Specifically, the antibodies react with Lewy bodies in brain sections taken from patients with Parkinson's disease of alpha-synucleinopathy, but do not react with normal human tissues.

[0101] Also, surprisingly, two Parkinson's mouse models (MPP+-induced mouse model and fibrillar α-Syn-inoculated mouse model) administered a composition containing the α-Syn peptide immunogen construct produced (a) antibodies highly cross-reactive to the β-sheet of α-Syn, (b) reduced α-Syn serum levels, (c) reduced oligomeric α-Syn levels in the brain, and (d) reduced neuropathology resulting in recovery of motor function.

[0102] The immune response obtained from animals immunized with the α-Syn peptide immunogen construct of the present invention demonstrated the ability of the construct to produce potent site-specific antibodies that react with the denatured β-sheets of α-Syn in the form of monomers, oligomers, and fibrils, rather than the random coil structure of native C-terminal α-Syn.

[0103] In vitro functional assays Antibodies produced by the α-Syn peptide immunogen construct can be used in in vitro functional assays. These functional assays include, but are not limited to: (a) In vitro inhibition of recombinant α-Syn aggregation; disaggregation of pre-formed recombinant α-Syn aggregates (see Example 8); (b) In vitro inhibition of intracellular α-Syn aggregation and dissociation of pre-formed α-Syn aggregates in cells (see Example 9); (c) Reduction of microglial TNF-α and IL6 secretion (see Example 10); (d) Reduction of neurodegeneration induced by exogenous α-Syn aggregates (see Example 11); (e) Reduction of neurodegeneration in α-Syn overexpressing cells (see Example 12); (f) In vivo demonstration of efficacy in a mouse model of fibrillar α-Syn inoculation and MPP+-induced Parkinson's disease showing reduction of serum α-Syn levels, reduction of brain oligomeric α-Syn levels, reduction of neuropathology, and recovery of motor activity (see Example 15).

[0104] Method The present disclosure also relates to methods of making and using α-Syn peptide immunogen constructs, compositions, and pharmaceutical compositions.

[0105] a. Method for manufacturing an α-Syn peptide immunogen construct The α-Syn peptide immunogen constructs of the present disclosure can be prepared by chemical synthesis methods well known to those skilled in the art (see, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W. H. Freeman & Co., New York, NY, 1992, p. 77). The α-Syn peptide immunogen constructs can be synthesized, for example, using the automated Merrifield technique of solid-phase synthesis with α-NH2 protected either by t-Boc or F-moc chemistry using side-chain protected amino acids of an Applied Biosystems peptide synthesizer model 430A or 431. Preparation of α-Syn peptide immunogen constructs containing combinatorial library peptides of Th epitopes can be achieved by providing a mixture of alternative amino acids for coupling at specific variable positions.

[0106] After the desired α-Syn peptide immunogen construct is fully assembled, the resin can be treated according to standard procedures to cleave the peptide from the resin and deblock the functional groups of the amino acid side chains. The free peptide can be purified by HPLC and biochemically characterized, for example, by amino acid analysis or sequencing. Methods for purification and characterization of peptides are well known to those skilled in the art.

[0107] The quality of the peptides produced by this chemical process can be controlled and defined, and as a result, the reproducibility, immunogenicity, and yield of the α-Syn peptide immunogen constructs can be ensured. A detailed description of the production of α-Syn peptide immunogen constructs by solid-phase peptide synthesis is shown in Example 1.

[0108] The range of structural variability that allows for the retention of the intended immunological activity is found to be far more adaptable than the range of structural variability that allows for the retention of specific drug activity by small molecule drugs, or the desirable activity and undesirable toxicity of large molecules co-produced with biologics. Thus, peptides analogs designed intentionally, or those inevitably produced by errors in the synthesis process as a mixture of deletion sequence by-products with chromatographic and immunological properties similar to the intended peptide, are often as effective as the purified preparation of the desired peptide. As long as distinguishable QC procedures are developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and effectiveness of the final product using these peptides, mixtures of designed and unintended analogs are effective.

[0109] The α-Syn peptide immunogen construct can also be made using recombinant DNA technology including nucleic acid molecules, vectors, and / or host cells. Thus, nucleic acid molecules encoding the α-Syn peptide immunogen construct and its immunologically functional analogs are also encompassed in the present disclosure as part of the invention. Similarly, vectors comprising expression vectors containing nucleic acid molecules, and host cells containing the vectors are also encompassed in the present disclosure as part of the invention.

[0110] Various exemplary embodiments also include methods for making immunologically functional analogs of α-Syn peptide immunogen constructs and α-SynG111-D135 fragment-derived peptide immunogen constructs. For example, the method can include incubating a host cell comprising an expression vector comprising a nucleic acid molecule encoding an α-Syn peptide immunogen construct and / or an immunologically functional analog thereof under conditions such that the peptide and / or analog is expressed. Longer synthetic peptide immunogens can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of the invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding an amino acid sequence having codons that are optimal for the organism in which the gene is to be expressed. Next, typically, a synthetic gene is created by synthesizing oligonucleotides encoding the peptide and optionally any regulatory elements. The synthetic gene is inserted into an appropriate cloning vector and transfected into a host cell. The peptide is then expressed under conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0111] b. Method for producing immunostimulatory complex Various exemplary embodiments also include a method of generating an immunostimulatory complex comprising an α-Syn peptide immunogen construct and a CpG oligodeoxynucleotide (ODN) molecule. The stabilized immunostimulatory complex (ISC) is derived from the cationic portion of the α-Syn peptide immunogen construct and the polyanionic CpG ODN molecule. The self-assembly system is driven by electrostatic neutralization of charges. The stoichiometry of the molar charge ratio of the cationic portion of the α-Syn peptide immunogen construct and the anionic oligomer determines the degree of association. The non-covalent electrostatic binding of the α-Syn peptide immunogen construct and CpG ODN is a completely reproducible process. Aggregates of the peptide / CpG ODN immunostimulatory complex facilitate presentation to “professional” antigen-presenting cells (APCs) of the immune system and thus further enhance the immunogenicity of the complex. These complexes are easily characterized for quality control during manufacture. Peptide / CpG ISC is well tolerated in vivo. This new particulate system comprising a CpG ODN and a peptide immunogen construct derived from the α-SynG111-D135 fragment is designed to promote a balanced Th-1 / Th-2 type response while taking advantage of the generalized B cell mitogenicity associated with the use of CpG ODN.

[0112] The CpG ODN in the disclosed pharmaceutical composition binds 100% to the immunogen in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particulates. The particulate morphology allows for a significant reduction in the dosage of CpG from the conventional use of CpG adjuvants, a reduction in the potential for harmful innate immune responses, and promotes alternative immunogen processing pathways including antigen-presenting cells (APCs). As a result, such formulations are conceptually novel and offer potential benefits by promoting the stimulation of immune responses by alternative mechanisms.

[0113] c. Method for manufacturing a pharmaceutical composition Various exemplary embodiments also include a pharmaceutical composition comprising an α-Syn peptide immunogen construct. In certain embodiments, the pharmaceutical composition uses an oil-in-water emulsion and a suspension comprising an inorganic salt.

[0114] When using pharmaceutical compositions in a large population and including the prevention of α-Syn aggregation as part of the administration goal, safety becomes another important factor to be considered. Despite many formulations in clinical trials using human oil-in-water emulsions, alum remains a major adjuvant for use in formulations due to its safety. Thus, alum or its inorganic salt aluminum phosphate (ADJUPHOS) is frequently used as an adjuvant in the preparation for clinical applications.

[0115] d. Method of using the pharmaceutical composition The present disclosure also includes a method of using a pharmaceutical composition comprising an α-Syn peptide immunogen construct.

[0116] In certain embodiments, a pharmaceutical composition comprising an α-Syn peptide immunogen construct can be used for the following: (a) Inhibiting α-Syn aggregation in a host; (b) Inducing the degradation of pre-formed α-Syn aggregates in a host; (c) Reducing the secretion of microglial TNF-α and IL6 in a host; (d) Reducing the neurodegeneration caused by exogenous α-Syn aggregates in a host; (e) Reducing the neurodegeneration of α-Syn overexpressing cells; (f) Reducing the serum α-Syn level in a host; (g) Reducing the oligomeric α-Syn level in the brain of a host; (h) Alleviating neuropathology and restoring the motor activity of a host; etc.

[0117] The methods described above include administering to a host in need thereof a pharmaceutical composition comprising a pharmacologically effective amount of an α-Syn peptide immunogen construct.

[0118] Specific embodiments Specific embodiments of the present invention include, but are not limited to, the following. (1) An α-synuclein (α-Syn) peptide immunogen construct, comprising: a B cell epitope comprising about 10 to about 25 amino acid residues from the C-terminal fragment of α-Syn corresponding to the vicinity of amino acid G111 to the vicinity of amino acid D135 of SEQ ID NO: 1; a T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 98; optionally, a heterologous spacer selected from the group consisting of amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), wherein the B cell epitope is covalently bound to the T helper cell epitope directly or via the optionally included heterologous spacer, the α-Syn peptide immunogen construct.

[0119] (2) The α-Syn peptide immunogen construct according to (1), wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 12 to 15, 17, and 49 to 63.

[0120] (3) The α-Syn peptide immunogen construct according to (1), wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 81, 83, and 84.

[0121] (4) The α-Syn peptide immunogen construct according to (1), wherein the optionally included heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

[0122] (5) The α-Syn peptide immunogen construct according to (1), wherein the T helper epitope is covalently bound to the amino terminus of the B cell epitope.

[0123] (6) The α-Syn peptide immunogen construct according to (1), wherein the T helper epitope is covalently bound to the amino terminus of the B cell epitope via the optionally included heterologous spacer.

[0124] (7) The following formula: (Th) m -(A) n -(C-terminal fragment of α-Syn)-X or (C-terminal fragment of α-Syn)-(A) n -(Th) m -X comprising, wherein, Th is the T helper epitope, A is the heterologous spacer, (C-terminal fragment of α-Syn) is the B cell epitope, X is α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4, n is from 1 to about 10, The α-Syn peptide immunogen construct according to (1).

[0125] (8) The α-Syn peptide immunogen construct according to (1), comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111 to 113, and 115 to 147.

[0126] (9) The α-Syn peptide immunogen construct according to (1), comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, and 111 to 113.

[0127] (10) A composition comprising the α-Syn peptide immunogen construct according to (1).

[0128] (11) A composition comprising more than 1 of the α-Syn peptide immunogen constructs according to (1).

[0129] (12) The composition according to (11), wherein the α-Syn peptide immunogen construct has the amino acid sequences of SEQ ID NOs: 112 and 113.

[0130] A pharmaceutical composition comprising the α-Syn peptide immunogen construct described in (13)(1) and a pharmaceutically acceptable delivery medium and / or adjuvant.

[0131] (14) a. The α-Syn peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. b. The adjuvant is an inorganic salt of aluminum selected from the group consisting of Al(OH)3 or AIPO4. The pharmaceutical composition according to (13).

[0132] (15) a. The α-Syn peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. b. The α-Syn peptide immunogen construct is mixed with CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex. The pharmaceutical composition according to (13).

[0133] (16) An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B cell epitope of the α-Syn peptide immunogen construct described in (1).

[0134] (17) The isolated antibody or an epitope-binding fragment thereof described in (16) that binds to the α-Syn peptide immunogen construct.

[0135] (18) An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B cell epitope of the α-Syn peptide immunogen construct described in (9).

[0136] (19) A composition comprising the isolated antibody or an epitope-binding fragment thereof described in (16).

[0137] (20) A composition comprising the isolated antibody or an epitope-binding fragment thereof described in (18).

[0138] (21) a. An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B cell epitope of SEQ ID NO: 112, and b. An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B cell epitope of SEQ ID NO: 113, and The composition according to (20), comprising a mixture of.

[0139] (22) A method for producing an antibody that recognizes α-Syn in a host, comprising administering to the host a composition comprising the α-Syn peptide immunogen according to (1) and a delivery medium and / or an adjuvant.

[0140] (23) A method for inhibiting α-Syn aggregation in an animal, comprising administering to the animal a pharmacologically effective amount of the α-Syn peptide immunogen of (1).

[0141] (24) A method for reducing the amount of α-Syn aggregates in an animal, comprising administering to the animal a pharmacologically effective amount of the α-Syn peptide immunogen of (1).

[0142] (25) A method for identifying α-Syn aggregates of different sizes in a biological sample, comprising a. exposing the biological sample to the antibody according to (16) or an epitope-binding fragment thereof under conditions that allow the antibody or the epitope-binding fragment thereof to bind to the α-Syn aggregates, and b. detecting the amount of the antibody or the epitope-binding fragment thereof bound to the α-Syn aggregates in the biological sample, and The method comprising.

[0143] A detailed description of the procedure used is provided in the following examples. Example 1 Synthesis of related peptides of alpha-synuclein and preparation of its formulation a. Synthesis of the C-terminal fragment of α-Syn Describe the method of synthesizing a designer α-Syn C-terminal fragment that was included in the development efforts of α-Syn peptide immunogen constructs. Peptides were synthesized in small amounts useful for serological assays, laboratory pilot and field studies, as well as in large scale (kilogram) amounts useful for industrial / commercial production of pharmaceutical compositions. A large repertoire of α-Syn related antigenic peptides having sequences of about 10 to 40 amino acids in length was designed for screening and selection of optimal peptide constructs for use in effective α-Syn peptide immunogen constructs.

[0144] Representative full-length α-Syn (SEQ ID NO: 1) and β-Syn (SEQ ID NO: 2), α-Syn 111-132 α-Syn 126-135 such as α-Syn segments, 10mer peptides, etc. are shown in Table 1 (SEQ ID NOs: 1 and 3 - 69). The selected α-Syn fragments were made into α-Syn peptide immunogen constructs by synthetically conjugating them to carefully designed helper T cell (Th) epitopes derived from pathogen proteins including measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza, Clostridum tetani, and Epstein - Barr virus (EBV) identified in Table 2 (SEQ ID NOs: 70 - 98). The Th epitopes were used either as a single sequence (SEQ ID NOs: 70 - 78 and 83 - 98) or as a combinatorial library (SEQ ID NOs: 79 - 82), enhancing the immunogenicity of each α-Syn peptide immunogen construct.

[0145] Representative α-Syn peptide immunogen constructs selected from peptide constructs over 100 are identified in Table 3 (SEQ ID NOs: 99-147). All peptides used for immunogenic studies or related serological tests for the detection and / or measurement of anti-α-Syn antibodies were synthesized on a small scale using F-moc chemistry on Applied BioSystems Models 430A, 431, and / or 433 peptide synthesizers. Each peptide was generated by independent synthesis on a solid support using F-moc protection at the N-terminus and side chain protecting groups for trifunctional amino acids. The completed peptide was cleaved from the solid support and the side chain protecting groups were removed with 90% trifluoroacetic acid (TFA). Synthetic peptide preparations were evaluated by matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry to ensure correct amino acid content. Each synthetic peptide was also evaluated by reverse phase HPLC (RP-HPLC) to confirm the synthetic profile and the concentration of the preparation. Despite strict control of the synthetic process, including stepwise monitoring of coupling efficiency, peptide analogs were generated by unintended events during elongation cycles such as amino acid insertions, deletions, substitutions, and premature terminations. Thus, the synthetic preparations typically contained multiple peptide analogs along with the target peptide. Despite the inclusion of such unintended peptide analogs, the resulting synthetic peptide preparations were still suitable for use in immunological applications, including immunodiagnosis (as an antibody-capturing antigen) and pharmaceutical compositions (as a peptide immunogen). As long as distinguishable QC procedures have been developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and effectiveness of the final products using these peptides, such peptide analogs, whether intentionally designed or generated in the synthetic process as a mixture of by-products, are as often effective as purified preparations of the desired peptide. Large-scale peptide synthesis in amounts from several hundred grams to several kilograms was carried out on a scale of 15 mmole to 50 mmole using a customized automated peptide synthesizer such as UBI2003.For the active ingredient used in the final pharmaceutical composition of the clinical trial, the α-Syn peptide construct was purified by preparative RP-HPLC under a shallow elution gradient and characterized for purity and identity by MALDI-TOF mass spectrometry, amino acid analysis, and RP-HPLC.

[0146] b. Preparation of a composition containing an α-Syn peptide immunogen construct Formulations were prepared using oil-in-water emulsions and suspensions containing inorganic salts. Safety is another important factor to consider as the pharmaceutical composition is designed for use in a large population and prevention is also part of the administration goal. Despite many pharmaceutical compositions in clinical trials using human oil-in-water emulsions, alum remains a major adjuvant for use in pharmaceutical compositions due to its safety. Thus, alum or its inorganic salt ADJUPHOS (aluminum phosphate) is frequently used as an adjuvant in preparations for clinical applications.

[0147] Briefly stated, the formulations designated for each study group described below generally contained all types of designer α-Syn peptide immunogen constructs. Over 100 designer α-Syn peptide immunogen constructs were initially evaluated in guinea pigs for the evaluation of serological cross-reactivity between different homologous peptides by an ELISA assay using plates coated with the corresponding α-Syn peptides representing the B epitope peptides of the immunogen and different peptides having SEQ ID NOs: 1 to 153.

[0148] The α-Syn peptide immunogen constructs were prepared in a water-in-oil emulsion containing Seppic Montanide™ ISA 51 as an oil approved for use in humans (i), or (ii) mixed with the inorganic salt ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum) at various amounts of the designated peptide construct. The compositions were typically prepared by dissolving the α-Syn peptide immunogen construct in water at about 20 - 800 μg / mL and formulating into a water-in-oil emulsion (1:1 by volume) with Montanide™ ISA 51, or with an inorganic salt or ALHYDROGEL (alum) (1:1 by volume). The compositions were maintained at room temperature for about 30 minutes and mixed by vortexing for about 10 - 15 seconds prior to immunization. Some animals were immunized by the intramuscular route with two to three administrations of a specific composition, which were administered at 0 time (prime) and 3 weeks post-initial immunization (wpi) (booster), and at 5 or 6 wpi for a second boost if needed. These immunized animals were then tested with the selected B epitope peptide(s) to evaluate the immunogenicity of the various α-Syn peptide immunogen constructs present in the formulation and the cross-reactivity with the relevant target peptide or protein. These α-Syn peptide immunogen constructs that had strong immunogenicity in the initial screening of guinea pigs were further tested in primates with water-in-oil emulsion, inorganic salt, and alum-based formulations over the dosing schedule indicated in the immunization protocol for the specified period.

[0149] Only the most promising α-Syn peptide immunogen constructs were more extensively evaluated in GLP-guided preclinical studies for immunogenicity, duration, toxicity, and efficacy prior to incorporation into the final formulation for investigational new drug applications and submission of clinical trials in synucleinopathy patients.

[0150] Example 2 Preparation of Recombinant Alpha-Synuclein Protein Cloning of the α-Syn gene into pGEX-4T1 has been previously described in Neurotoxicology and teratology 2004, 26 (3):397-406. The target sequence (SEQ ID NO: 1) was inserted into the pGEX-4T1 vector between the BamHI and XhoI restriction sites. The fragment was generated by polymerase chain reaction (PCR) using KAPA HiFi DNA polymerase (Kapa Biosystems, Inc., Woburn, MA, USA). The primer sequences were as follows: forward primer 5’-cgggatccgatgtgtttatgaaaggtctgag-3’ (SEQ ID NO: 149); reverse primer 5’-ggaattccgatgtgtttatgaaaggtctgag-3’ (SEQ ID NO: 150). The PCR conditions were as follows: after denaturation at 94 °C for 1 minute, 30 cycles of denaturation at 94 °C for 15 seconds, annealing at 60 °C for 30 seconds, extension at 68 °C for 2 minutes, and a final extension at 68 °C for 5 minutes. Site-directed mutagenesis of A53T α-Syn was performed using the Q5 Site-Directed Mutagenesis Kit (New England BioLabs, Beverly, MA, USA). The primer sequences for the mutant α-Syn were as follows: forward primer 5’-tcatggtgtgaccaccgttgcag-3’ (SEQ ID NO: 151); reverse primer 5’-accacgccttctttggttttg-3’ (SEQ ID NO: 152).

[0151] α-Syn cloned into the pGEX-4T1 GST vector was transformed into E. coli BL21(DE3) for protein expression. E. coli was cultured in LB broth at 37°C, and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 4 mM when the OD600 reached 0.8. After 4 hours of incubation, cells were collected by centrifugation at 5,000×g for 20 minutes at 4°C. The collected cells were resuspended in PBS, sonicated on ice for disruption, and centrifuged at 5,000×g for 20 minutes. The supernatant fraction was loaded onto a glutathione sepharose-4B column (GE Healthcare) equilibrated with PBS. After washing three times with PBS, 1 mL of thrombin (20 U / mL in PBS) was added for overnight digestion at 4°C to release GST from the fusion protein. Subsequently, tag-free α-Syn was eluted, and then thrombin was removed using a HiTrap Benzamidine FF column (GE Healthcare). The dialyzed α-Syn was immediately frozen at -80°C. After separation by 10% SDS-PAGE, the purified α-Syn with a MW of 14 kDa was identified by Western blotting using an anti-α-Syn antibody (1:2000, Millipore, targeting α-Syn 111-131 ).

[0152] Example 3 Serological assays and reagents Serological assays and reagents for evaluating the functional immunogenicity of synthetic peptide constructs and their formulations are detailed below.

[0153] a. Peptide-based ELISA test for antibody specificity analysis An ELISA assay for evaluating the immune serum samples described in the following examples has been developed and is described below. The wells of a 96-well plate were individually coated with 100 μl of 2 μg / ml (unless otherwise specified) of the target peptides, α-Syn fragments A85-A140, A91-A140, A101-A140, A111-A140, D121-A140, E126-A140, K97-D135, G101-D135, G111-D135, D121-D135, E123-D135, E126-D135, G101-132, and G111-G132 peptides (SEQ ID NOs: 4-17) in 10 mM NaHCO3 buffer, pH 9.5 (unless otherwise specified) for 1 hour at 37°C.

[0154] b. Evaluation of antibody reactivity against Th peptides by Th peptide-based ELISA test Wells coated with the peptide (SEQ ID NOs: 70 to 98) were incubated with 250 μL of 3% gelatin in PBS at 37°C for 1 hour to block non-specific protein binding sites, and then washed three times with PBS containing 0.05% (v / v) TWEEN® 20 and dried. Serum to be analyzed was diluted 1:20 (unless otherwise specified) with PBS containing 20% (v / v) normal goat serum, 1% (w / v) gelatin, and 0.05% (v / v) TWEEN® 20. 100 microliters (100 μL) of the diluted specimen (e.g., serum, plasma) was added to each well and reacted at 37°C for 60 minutes. Then, the wells were washed six times with PBS containing 0.05% (v / v) TWEEN® 20 to remove unbound antibody. Goat anti-IgG specific for the species of horseradish peroxidase (HRP) conjugation (e.g., mouse, guinea pig, or human) was used as a labeled tracer that binds to the antibody / peptide antigen complex formed in the positive wells. 100 μL of peroxidase-labeled goat anti-IgG in 1% normal goat serum containing 0.05% (v / v) TWEEN® 20 in PBS was added to each well at a previously titrated optimal dilution and incubated at 37°C for an additional 30 minutes. The wells were washed six times with PBS containing 0.05% (v / v) TWEEN® 20 to remove unbound antibody and reacted with 100 μL of a substrate mixture containing 0.04% (w / v) 3’,3’,5’,5’-tetramethylbenzidine (TMB), and further reacted with 0.12% (v / v) hydrogen peroxide in sodium citrate buffer for 15 minutes. Using this substrate mixture, the peroxidase label was detected by forming a colored product. The reaction was stopped by adding 100 μL of 1.0 M H2SO4, and the absorbance (A 450 ) at 450 nm was determined. For determination of the antibody titers of immunized animals administered with various α-Syn-derived peptide immunogens, 10-fold serial dilutions of serum from 1:100 to 1:10,000 were tested, and the titer of the tested serum represented as Log 10 was calculated by linear regression analysis of A 450 with the cut-off A 450 set at 0.5.

[0155] c. Detailed specificity analysis and epitope mapping of α-Syn fragments by 10-mer peptide-based ELISA tests of B cell epitope clusters The detailed specificity analysis of anti-α-Syn antibodies in immunized hosts was determined by epitope mapping. Briefly, the wells of a 96-well plate were coated with individual 10-mer peptides of α-Syn (SEQ ID NOs: 18-69) at 0.5 μg per 0.1 mL per well, and then 100 μL of serum samples (1:100 dilution in PBS) were incubated in duplicate in the 10-mer plate wells according to the steps of the above antibody ELISA method. The B cell epitopes of the α-Syn peptide immunogen constructs and the relevant detailed specificity analysis of the anti-α-Syn antibodies in the immune sera of immunized hosts were also tested for the corresponding α-Syn peptides (SEQ ID NOs: 99, 102, 108, 110, 112, 113) or fragments thereof without spacer and Th sequences, or β-Syn (SEQ ID NO: 153) for confirmation of additional reactivity and specificity.

[0156] d. Immunogenicity evaluation Pre-immune and immune serum samples were collected from animals according to the experimental immunization protocol and heated at 56 °C for 30 minutes to inactivate serum complement factors. After administration of the pharmaceutical composition, blood samples were obtained according to the protocol and their immunogenicity against specific target site(s) was evaluated. Serial diluted sera were tested and positive titers were expressed as the Log of reciprocal dilution. 10 The immunogenicity of a particular pharmaceutical composition is evaluated by its ability to induce a high-titer B cell antibody response against the desired epitope specificity within the target antigen, while maintaining the antibody reactivity against the "helper T cell epitopes" utilized at low levels to negligible levels, resulting in an enhancement of the desired B cell response.

[0157] e. Immunoassay of α-Syn levels in mouse immune sera The serum α-Syn levels of mice administered with α-Syn-derived peptide immunogen were measured by sandwich ELISA (Cloud-clon, SEB222Mu) using an anti-α-Syn antibody as the capture antibody and a biotin-labeled anti-α-Syn antibody as the detection antibody. Briefly, the antibody was immobilized at 100 ng / well in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) on a 96-well plate and incubated overnight at 4°C. The coated wells were blocked with 200 μL / well of assay diluent (0.5% BSA, 0.05% TWEEN®-20, 0.02% ProClin 300 in PBS) for 1 hour at room temperature. The plate was washed three times with 200 μL / well of wash buffer (PBS containing 0.05% TWEEN®-20). A standard curve (range of 156 - 1250 ng / mL with 2-fold serial dilutions) was prepared with assay diluent containing 5% mouse serum using purified recombinant α-Syn. 50 μL of diluted serum (1:20) and standards were added to the coated wells. Incubation was carried out for 1 hour at room temperature. All wells were aspirated and washed six times with 200 μL / well of wash buffer. The captured human α-Syn was incubated with 100 μL of detection antibody solution (50 ng / ml biotin-labeled HP6029 in assay diluent) for 1 hour at room temperature. Then, streptavidin poly-HRP (1:10,000 dilution, Thermo Pierce) was used (100 μL / well) for 1 hour to detect the bound biotin-HP6029. All wells were aspirated and washed six times with 200 μL / well of wash buffer, and the reaction was stopped by the addition of 100 μL / well of 1 M H2SO4. The standard curve was generated using SoftMax Pro software (Molecular Devices), which produced a four-parameter logistic curve fit and was used to calculate the concentration of α-Syn in all tested samples. Data were compared using a Student's t-test by using Prism software.

[0158] f. Preparation of α-Syn Aggregates with Recombinant α-Syn To prepare aggregated α-Syn, purified wild-type or A53T mutant α-Syn [0.1 μg / μl in 100 μL of PBS / KCl aggregation buffer (1× PBS, pH 7.4, 2.5 mM MgCl2, 50 mM HEPES, and 150 mM KCl)] was incubated at 37 °C for 7 days in 1.5 mL Eppendorf tubes in a Thermomixer (Eppendorf) without shaking. The aggregated α-Syn was immediately frozen at -80 °C for later use.

[0159] g. Purification of anti-α-Syn antibody Anti-α-Syn antibody was purified from sera collected 3 - 15 weeks post-injection (WPI) of guinea pigs immunized with α-Syn peptide immunogen constructs containing peptides of different sequences (SEQ ID NOs: 99 - 121) by using an affinity column (Thermo Scientific, Rockford). Briefly, after equilibration with buffer (0.1 M phosphate and 0.15 M sodium chloride, pH 7.2), 400 μL of serum was added to a Nab Protein G spin column and subjected to 10 minutes of inversion mixing and centrifugation at 5,800 × g for 1 minute. The column was washed three times with binding buffer (400 μL). Subsequently, elution buffer (400 μL, 0.1 M glycine pH 2.0) was added to the spin column, and after centrifugation at 5,800 × g for 1 minute, the antibody was eluted. The eluted antibody was mixed with neutralization buffer (400 μL, 0.1 M Tris pH 8.0), and the concentrations of these purified antibodies were measured using a Nan-Drop at OD280 with BSA (bovine serum albumin) as a standard.

[0160] h. Specificity of anti-α-Syn antibodies purified from guinea pig antisera immunized with α-Syn peptide immunogen constructs of different sizes Using Western blot, anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs were screened for binding specificity to α-Syn molecular complexes of different sizes. 20 μM of α-Syn was separated by 12% Tris-glycine SDS-PAGE and transferred to a nitrocellulose (NC) membrane before photoinduced crosslinking (PICUP) treatment. The membrane was incubated with the anti-α-Syn antibody at 1 μg / mL purified from guinea pig antiserum, and then incubated with the donkey anti-guinea pig antibody conjugated with HRP (706-035-148, Jackson). The blot was visualized with the chemiluminescence reagent Western Lightning ECL Pro (PerkinElmer). As a result, the monomeric α-Syn (Mw 14,460 Da) was blotted near the size of 14 kDa, while the molecular weights of the dimer, trimer, or oligomer were several times larger than the size of the 14 kDa monomeric α-Syn. A commercially available antibody, Syn211 (Abeam), which can detect various oligomer species such as dimers, trimers, and larger oligomers, was used as a positive control.

[0161] i. Dot blot assay using different species of amyloidogenic proteins Aβ 1-42 , Tau, and the preparations of α-helix monomers, β-sheet monomers, β-sheet oligomers, and β-sheet protofibrils of α-Syn are as follows. 1. Aβ 1-42 α-helix monomer: 20 μg of AP 1-42 β-sheet monomer (50 μL) was added to 1× PBS containing 20% trifluoroacetic acid and 20% hexafluoroisopropanol (10 μL) and incubated at 4°C for 24 hours to form α-helix monomers. 2. Aβ 1-42 β-sheet monomer: 60 μg of Aβ in 120 μL of 1× PBS containing 5% TFA aggregated at 37°C for 24 hours 1-42 was transferred to a 10 kDa cut-off filter (Millipore) to recover the β-sheet monomer. 3. Aβ 1-42β-sheet oligomers: 60 μg of Aβ in 120 μL of 1×PBS aggregated at 37 °C for 3 days 1-42 was sonicated on ice and transferred to 10 and 30 kDa cut-off filters (Millipore) to recover β-sheet oligomer protofibrils with a molecular weight less than 35 kDa. 4. Aβ 1-42 β-sheet protofibrils: 60 μg of Aβ in 120 μL of 1×PBS aggregated at 37 °C for 3 days 1-42 was sonicated on ice and transferred to a 30 kDa cut-off filter (Millipore) to isolate β-sheet protofibrils. 5. α-Syn α-helix monomer: 40 μg of freshly prepared α-Syn was dissolved in 100 μL of cold 1×PBS at 4 °C and immediately transferred to a 10 kDa cut-off filter (Millipore) to recover α-helix monomers. 6. α-Syn β-sheet monomer: 40 μg of α-Syn incubated at 37 °C for 24 hours in 100 μL of PBS / KCl buffer was transferred to a 10 kDa cut-off filter (Millpore) to recover β-sheet monomers. 7. α-Syn β-sheet oligomers: 40 μg of α-Syn aggregated at 37 °C for 8 days in 100 μL of PBS / KCl buffer was sonicated on ice and then transferred to 30 and 100 kDa cut-off filters to recover β-sheet oligomers. 8. α-Syn β-sheet protofibrils: 40 μg of α-Syn aggregated at 37 °C for 8 days in 100 μL of PBS / KCl buffer was sonicated on ice and then transferred to 30 and 100 kDa cut-off filters to isolate β-sheet protofibrils. Tau441 α-helix monomer: 60 μg of Tau prepared in 100 μL of 1×PBS at 4 °C was transferred to a 100 kDa cut-off to recover α-helix monomers. 9. Tau441 β-sheet monomer: 60 μg of Tau aggregated at 25 °C for 48 hours in 100 μL of 1×PBS containing 10 units / mL heparin was transferred to a 100 kDa cut-off filter at 4 °C to recover β-sheet monomers. 10. Tau441 β-sheet oligomers: 60 μg of Tau aggregated with 100 μL of 1×PBS containing 10 units / mL heparin at 37°C for 48 hours was transferred to 100 and 300 kDa cut-off filters (Pall) at 4°C to recover the β-sheet oligomers. 11. Tau441 β-sheet protofibrils: 60 μg of Tau aggregated with 100 μL of 1×PBS containing 10 units / mL heparin at 37°C for 6 days was transferred to a 300 kDa cut-off filter (Pall) at 4°C to isolate the β-sheet protofibrils.

[0162] These monomers and oligomers were verified by thioflavin-T (ThT, Sigma) fluorescence or PAGE (polyacrylamide gel electrophoresis). The concentration of the amyloid-forming protein was measured by Nano-Drop using a commercially available amyloid-forming Aβ 1-42 stock as a standard. These monomers and oligomers were individually spotted onto PVDF membranes in amounts of 3 μg for Aβ, 4 μg for α-Syn, and 7 μg for Tau. After incubating the membrane with an anti-α-Syn antibody purified from guinea pig antiserum (1:1000 dilution) as the primary antibody, hybridization with an anti-guinea pig HRP-conjugated secondary antibody (1:5000; Vector Laboratories) was performed. The membrane was treated with Luminata Western HRP substrate (Bio-Rad, Hercules, CA, USA), and the signal was detected using a ChemiDoc-It 810 digital imaging system (UVP Inc., Upland, CA, USA). 1-42 i. Binding specificity to aggregated α-Syn in α-Syn overexpressing PC12 cells during nerve growth factor (NGF) treatment

[0163] Immunocytochemistry (ICC) was performed using anti-α-Syn antibodies purified from guinea pig antisera collected at 8 or 9 weeks post-immunization (WPI) in parental PC12 cells, mock control PC12 cells, and α-Syn overexpressing PC12 cells after NGF treatment to evaluate the binding affinity of the antibodies induced after immunization. Cell nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole). Photographs were taken with a fluorescence microscope, and the ratio of the number of positively stained cells to the total number of cells was scored and classified as -, +, ++, and +++ representing less than 1%, 1 - 15%, 16 - 50%, and more than 50%, respectively.

[0164] Example 4 Cells and Animals Used in Immunogenicity and Efficacy Studies a. α-Syn Overexpressing PC12 Cells: The pZD / XOL-L-α-Syn plasmid was constructed by inserting the cDNA sequence encoding full-length human wild-type α-Syn or A53T mutant α-Syn into the pZD / XOL-L vector with a CMV promoter. According to the manufacturer's procedure, the construct was transfected into PC12 cells using Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA, USA). A transfection mixture of 2.5 μL, 500 μL of Opti-MEM medium, 2.5 μL of PLUS reagent, and 8.75 μL of Lipofectamine LTX was mixed and incubated at room temperature for 25 minutes. After replacing the medium with 1.5 mL of RPMI1640 growth medium, 500 μL of the transfection mixture was added directly to each well and incubated at 37°C for 1 day. The transfection efficiency was confirmed by PCR and Western blotting.

[0165] b. Guinea Pig: The immunogenicity studies were performed in mature, naive, adult male and female Dunkin Hartley guinea pigs (300 - 350 g / BW). At least three guinea pigs were used per group in the experiments. The protocol involving Dunkin Hartley guinea pigs (8 - 12 weeks old; Covance Research Laboratories, Denver, PA, USA) was conducted at a contract animal facility and sponsored by UBI under an approved IACUC application.

[0166] c. Parkinson's mouse model inoculated with fibrillar α - Syn FVB female mice (body weight 25 - 30 g) were housed on a 12 - hour light: 12 - hour dark cycle, and animal care was in accordance with AAALAC - approved guidelines. Fibrillar α - Syn was prepared by incubating α - Syn peptide (5 mg / mL) in PBS / high KCl buffer containing 0.1% NaN3 at 37°C without shaking for 7 days. Fibrosis was monitored by measuring ThT fluorescence and confirmed when the signal increased more than three - fold over the original α - Syn monomer. Western blot was also used to verify the aggregation of α - Syn prior to inoculation into the unilateral substantia nigra (anterior - posterior; - 3.0 mm; medial - lateral: - 1.3 mm; dorsal - ventral: - 4.7 mm from bregma and dura) and the dorsal neostriatum (anterior - posterior; + 0.2 mm, medial - lateral; - 2 mm, dorsal - ventral: - 3.2 mm from bregma and dura) of isoflurane - anesthetized animals.

[0167] d. MPP+-induced Parkinson's mouse model Balb / c female mice (body weight 18 - 20 g) were housed on a 12 - hour light: 12 - hour dark cycle, and animal care was in accordance with AAALAC - approved guidelines. MPP+ iodide (Sigma, St. Luis, MO) was dissolved in saline, and a 10 - μl solution containing 18 μg of MPP+ iodide (0.8 mg / kg) was injected into the unilateral cerebral ventricle of anesthetized animals. The stereotaxic coordinates of the injection site were bregma - 1.0 mm, lateral 1.0 mm, depth 2.0 mm.

[0168] Example 5 Design principles, screening, identification, and optimization of multi-component pharmaceutical compositions incorporating alpha-synuclein peptide immunogens a. History of design Each α-Syn peptide immunogen construct or immunotherapy product requires a unique design focus and approach based on the specific disease mechanism and target protein(s) necessary for intervention. Targets for design can include cellular proteins involved in the disease pathway, or infectious agents where several proteins from the pathogen may be involved. The process from research to commercialization is very long and typically requires more than 10 years to achieve.

[0169] Once the target molecule is selected, an extensive serological validation process is required. Identification and distribution of B-cell and T-cell epitopes within the target molecule are important for the design of the molecular α-Syn peptide immunogen construct. When the target B-cell epitope is recognized, sequential pilot immunogenicity studies in small animals are conducted, and the functional properties of the antibodies induced by the designer peptide pharmaceutical composition are evaluated. Then, such serological applications are performed in animals of the target species for further validation of the immunogenicity and functional properties of the induced antibodies against the α-Syn peptide immunogen construct. All studies are conducted in multiple parallel groups, and sera are collected from the immunized hosts for evaluation. Initial immunogenicity studies in non-human primates for target species or human pharmaceutical compositions are also performed to further validate immunogenicity and design direction. Then, when preparing the target peptides with various mixtures and using them in combination to prepare each formulation design, subtle differences in the functional properties related to each interaction between the peptide constructs are evaluated. After additional evaluations, the final peptide construct, peptide composition, and its formulation are established along with the respective physical parameters of the formulation, leading to the final product development process.

[0170] b. Design and validation of α-Syn-derived peptide immunogen constructs for pharmaceutical compositions potentially treating synucleinopathy patients To generate the most potent peptide constructs for incorporation into pharmaceutical compositions, a large repertoire of promiscuous T-helper epitopes derived from various pathogens or artificial T-helper epitopes further engineered from the measles virus fusion (MVF) protein sequence or the hepatitis B surface antigen (HBsAg) protein were incorporated into immunogenicity studies in guinea pigs. 126-140 , α-Syn 121-140 , α-Syn 111-140 , α-Syn 101-140 , α-Syn 91-140 , α-Syn 85-140 , α-Syn 121-135 , α-Syn 111-135 , α-Syn 101-135 , α-Syn 97-135 , α-Syn 123-135 , α-Syn 126-135 , α-Syn 111-132 , and α-Syn 101-132 Representative studies of derived peptide constructs are shown in Table 3 (SEQ ID NOs: 99-121), in which α-Syn peptides were linked to individual promiscuous T helper epitopes via εK and / or KKK as spacer(s).

[0171] i) Selection of the C-terminal portion of α-Syn as a target for peptide immunogen design. α-Syn is a natively unfolded protein. It consists of 140 amino acids and is divided into three regions. The N-terminal region (residues 1 - 60) can form an amphipathic helix, a typical conformation for membrane recognition and binding. The central region, including residues 61 - 95, is well-known as the non-amyloid-β component (NAC), first identified in AD senile plaques. This region has a high tendency to form β-rich conformations and to aggregate. Different types of post-translational modifications within this region show clearly different effects on the regulation of α-Syn aggregation. The C-terminal region of residues 96 - 140 is rich in proline and negatively charged residues, which is a common characteristic found in natively unfolded proteins that maintain solubility. This C-terminal domain generally exists in a random coil structure due to its low hydrophobicity and large net negative charge. In vitro studies have revealed that α-Syn aggregation can be induced by a decrease in pH that neutralizes these negative charges. α-Syn is characterized by an extreme conformational diversity that adapts to various conditions in the states of membrane binding, cytoplasm, and amyloid aggregation, performing highly versatile functions. The random coil and natively unfolded region at the C-terminus, which is important for the protein to maintain solubility, was selected as a target for peptide immunogen design because this region was considered to be the most susceptible to regulation by antibodies or other physical factors compared to the N-terminal amphipathic helix and the central β-rich conformational region.

[0172] ii) Identification of self-Th epitopes for exclusion in α-Syn B epitope design. Preliminary immunogenicity analysis showed that deletion of peptide sequences from the N-terminus of the α-Syn sequence made α-Syn 126-140 (SEQ ID NO: 9), α-Syn 121-140 (SEQ ID NO: 8), α-Syn 111-140 (SEQ ID NO: 7) peptides completely non-immunogenic, but some moderate immunogenicity was observed for α-Syn 101-140 (SEQ ID NO: 6), α-Syn 91-140 (SEQ ID NO: 5), and α-Syn 85-140(SEQ ID NO: 4) Peptide (Table 4) was observed, indicating the presence of a potential self-Th-like structure within the C-terminal sequence, and the presence of a helper T cell epitope(s) structure was confirmed at the C-terminus of α-Syn. Inclusion of such sequences in the B epitope(s) design may cause brain inflammation upon booster immunization due to activation of self T cells, similar to the previous Alzheimer's disease vaccine AN1792. Therefore, this discovery necessitates the design of α-Syn peptide immunogen constructs with B cell epitope(s) starting from amino acid residue G111 to avoid the potential inclusion of self T cell epitope(s) in the B epitope design.

[0173] iii) Ranking of heterologous T helper epitopes and their inclusion in the design of α-Syn peptide immunogen constructs to restore and enhance the immunogenicity of the selected α-Syn B epitope peptides. Table 2 lists a total of 29 heterologous Th epitopes (SEQ ID NOs: 70 - 98) tested within the group for various relative potencies obtained from mice, rats, guinea pigs, baboons, macaques, etc. to enhance B cell epitope immunogenicity. As shown in Table 5, the UBITh1 (SEQ ID NO: 83) and UBITh2 (SEQ ID NO: 84) T cell epitopes derived from the MvF protein can enhance the non-immunogenic α-Syn 101-140 (SEQ ID NO: 6) peptide to strong and moderate immunogenicity, respectively. Extensive testing of multiple α-Syn-derived peptide immunogen constructs was performed, enabling the ranking of the relative immunogenicity among these immunogen constructs. UBITh3 (SEQ ID NO: 81) also shows similar immunopotentiating activity when tested by ELISA on plates coated with the long α-Syn peptide A91 - A140 (SEQ ID NO: 5) and when covalently bound to various C-terminal α-Syn peptides (SEQ ID NOs: 4 - 9) via a spacer, as shown in Table 6.

[0174] iv) Evaluation of the immunogenicity of C-terminal α-Syn peptide immunogen constructs with respect to antibody reactivity against the corresponding α-Syn and β-Syn. The synuclein family includes three known proteins, α-Syn, β-Syn, and gamma-synuclein. All synucleins share a highly conserved alpha-helical lipid-binding motif that is similar to the exchangeable apolipoprotein class A2 lipid-binding domain. β-Syn is highly homologous to α-Syn. β-Syn has been suggested to be an inhibitor of α-Syn aggregation that occurs in neurodegenerative diseases such as Parkinson's disease. Therefore, β-Syn may protect the central nervous system from the neurotoxic effects of α-Syn. Therefore, it is preferred to have an α-Syn peptide immunogen construct to induce antibodies that preferentially react with α-synuclein rather than the corresponding aggregation-protective β-Syn. When testing six peptide immunogen constructs with a C-terminus ending in A140, all antibodies derived from the immune sera of these constructs showed significant cross-reactivity with β-Syn of the corresponding size, as shown in Table 6. A close examination of the sequence homology between α-Syn and β-Syn (SEQ ID NOs: 1 and 2) showed that the sequence corresponding to the five C-terminal amino acids YEPEA is identical between the two proteins. Therefore, it is desirable to design B epitope(s) excluding the sequence containing these five amino acids of YEPEA. Therefore, the findings from the immunogenicity studies shown in Table 6 result in the deletion of YEPEA (Y136 - A140) in B epitope(s) design. When incorporating a spacer sequence and, for example, the artificial T helper peptide UBITh1 (SEQ ID NO: 83) into the α-Syn peptide immunogen construct, the design using the B cell epitope sequence excluding the YEPEA tail shown in the α-Syn peptide immunogens of Table 7 (SEQ ID NOs: 107 - 114) all showed high immunogenicity when evaluated with the long α-Syn peptide K97 - A140 (SEQ ID NO: 110). None of the immune sera reacted with β-Syn. Therefore, from the data obtained in Tables 6 and 7, the B epitope design of the peptide immunogen construct is limited to α-Syn from G111 to D135 and its fragments.

[0175] v) Antibodies induced by the αSyn peptide immunogen construct reacted only with beta sheet monomers, oligomers, or protofibrils, not with alpha helix monomers. Although appropriate principles were used in the design of the α-Syn peptide immunogen, it was surprisingly found that antibodies generated from the designed α-Syn peptide immunogen construct had a B epitope with a sequence starting at G111 and ending at D135 or a fragment thereof, and the induced antibodies specifically reacted with beta sheet α-Syn monomers, oligomers, and protofibrils, but not with beta sheet Aβ 1-42 or Tau1-441. Thus, as typically shown by the α-Syn peptide immunogen constructs (SEQ ID NOs: 112 and 113) in Figure 8, candidate ideal α-Syn peptide immunogen constructs are provided.

[0176] vi) Expansion of MHC coverage by using non-specific T helper epitopes different from the α-Syn-derived peptide immunogen construct. When designing pharmaceutical compositions for treating patients with diverse genetic backgrounds, it is important to design them to cover the largest population with diverse genetic backgrounds. Thus, the synergistic immunogenic effects of α-Syn-derived peptide immunogen constructs on such combinations were investigated. Mixtures of peptide constructs containing helper T epitopes were designed for such exploration because non-specific T helper epitopes derived from MVF or HBsAg are one of the most potent in providing such enhancement of immunogenicity. A mixture of two peptide immunogen constructs with the same B epitope was found to induce a significant immune response when compared to the immune responses induced by each individual peptide construct.

[0177] Example 6 Focused antibody response induced by the α-Syn peptide immunogen construct only against the targeted B cell epitope All carrier proteins (e.g., keyhole limpet hemocyanin (KLH), or other carrier proteins such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins) used to enhance the immune response to a targeted B cell epitope peptide by chemical conjugation of such B cell epitope peptides to their respective carrier proteins induce, in the immunized host, antibodies to more than 90% of the enhancing carrier protein and less than 10% of the antibodies to the targeted B cell epitope. Therefore, it is interesting to evaluate the specificity of the α-Syn peptide immunogen constructs of the present invention. For immunogenicity evaluation, a series of eight α-Syn peptide immunogen constructs (SEQ ID NOs: 107 to 114) having B cell epitopes of various lengths conjugated to the heterologous T cell epitope UBITh1 (SEQ ID NO: 83) via a spacer sequence were prepared. UBITh1 (a T helper peptide used for B epitope immune enhancement) was coated on plates, and guinea pig immune sera were used to test for cross-reactivity with the UBITh1 peptide used for immune enhancement. As shown in Tables 6 and 7, in contrast to the high immunogenicity of these constructs to the corresponding targeted B epitopes, as shown by the high-titer antibodies generated against the B epitope(s), many, but not all, of the immune sera were found to be non-reactive to the UBITh1 peptide, as shown in Table 8.

[0178] In summary, a simple immunogen design incorporating a targeted B cell epitope conjugated to a carefully selected T helper epitope enables the generation of a focused and non-defective immune response targeting only the α-Syn B cell epitope. In the design of pharmaceutical compositions, the more specific the immune response, the higher the safety profile provided by the composition. Therefore, the α-Syn peptide immunogen constructs of the present invention are very specific but very powerful for their target.

[0179] Example 7 Epitope mapping for detailed specificity analysis of immune sera (9 WPI) against various alpha-synuclein peptide immunogen constructs In a detailed epitope mapping study (Table 9) to determine the antibody binding site(s) to specific residues within the C-terminal region of α-Syn, 52 overlapping 10-mers (SEQ ID NOs: 18 - 69) encompassing the α-Syn amino acid sequence of (K80 - A140) were synthesized. Two longer peptides of (97 - 135, SEQ ID NO: 10) and (111 - 132, SEQ ID NO: 17) were used as positive controls. These 10-mer peptides and the two longer peptides were individually coated onto the wells of a 96-well microtiter plate as solid-phase immunosorbents. Pooled guinea pig antisera were diluted 1:100 in sample dilution buffer and added to the plate wells coated with 2.0 μg / mL of the 10-mer peptides, and then incubated at 37 °C for 1 hour. After washing the plate wells with wash buffer, horseradish peroxidase-conjugated protein A / G was added and incubated for 30 minutes. After washing again with PBS, substrate was added to the wells to measure the absorbance at 450 nm with an ELISA plate reader, and the samples were analyzed in duplicate. The binding of the antiserum to the corresponding long α-Syn peptide of the B epitope immunogen construct represents the maximum binding.

[0180] As shown in Table 9, the pooled 9wpi guinea pig immune sera obtained from six α-Syn peptide immunogen constructs [(K97-D135, SEQ ID NO: 110), (G111-D135, SEQ ID NO: 108), (G111-G132, SEQ ID NO: 113), (E126-D135, SEQ ID NO: 112), (G101-A140, SEQ ID NO: 104), and (E126-A140, SEQ ID NO: 99)] were selected for detailed epitope mapping. These six B-epitope fragments of various lengths completely encompass the sequence of 97-140 in the C-terminal region of α-synuclein. The results of ELISA showed that all six immune sera strongly reacted with the representative long peptide of α-Syn (97-135, SEQ ID NO: 10). As a result of the detailed epitope mapping study of the 10mer, an immunogenic epitope encompassing the region around AA114 to 125 (peptides 114-123, 115-124, 116-125 of SEQ ID NO: 52, 53, 54) and a highly immunogenic region at the C-terminus represented by peptide 131-140 (SEQ ID NO: 69) were revealed. Interestingly, many immune sera derived from the C-terminal α-Syn peptide immunogen construct induced antibodies that recognize conformational epitopes rather than linear epitopes, except for the epitope located at the C-terminus of α-Syn with the sequence of EGYQDYEPEA (SEQ ID NO: 69) and responsible for cross-reactivity with the β-Syn protein.

[0181] The results of this epitope mapping were somewhat unexpected, but they correlated well with the discovery that these antibodies were derived from α-Syn peptide immunogen constructs represented by α-Syn111-132 (SEQ ID NO: 113) and α-Syn126-135 (SEQ ID NO: 112) from the C-terminal random coil region of α-Syn that bind to a heterologous Th epitope structure that resembles the denatured β-sheet of α-Syn and results in a conformation that is non-cross-reactive with the α-helix of native α-Syn.

[0182] Example 8 Antibodies Induced by α-Syn Peptide Immunogen Constructs and Their Formulations: Anti-Aggregation and Disaggregation Effects on Recombinant Alpha-Synuclein Protein Using anti-α-Syn antibodies purified from guinea pig antisera against recombinant α-Syn, the effects of α-Syn peptide immunogen constructs in in vitro anti-aggregation assays and disaggregation assays were evaluated.

[0183] a. Inhibition of α-Syn aggregation For potential anti-aggregation ability, an initial screening assay of different anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogen constructs was performed by quantifying the level of change in α-Syn aggregation by thioflavin T measurement as described in Example 3. Recombinant α-Syn prepared at 100 μM in PBS was incubated for an additional 6 days at a concentration of 5 μM in a 384-well plate with 40 μL of PBS / KCl buffer (1× PBS, 2.5 mM MgCl2, 50 mM HEPES, and 150 mM KCl in pH 7.4) to induce aggregation. Different concentrations (0.05, 0.5, or 5 μg / mL) of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs collected at different time points were added to the incubation mixture, and the respective effects of inhibiting α-Syn aggregation were evaluated. By the end of the incubation, the aggregation level was determined using the ThT assay. The measured values obtained from each test run were standardized with the aggregation level of the media control set as 100% and the measured values obtained in the absence of α-Syn set as 0%.

[0184] As summarized in Table 10, the three anti-α-Syn antibodies induced by α-Syn 111-132 , α-Syn 121-135 , or α-Syn 126-135 revealed a more potent, concentration-dependent inhibition against α-Syn aggregation. Among all the anti-α-Syn antibodies assayed, α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), α-Syn 123-135 (SEQ ID NO: 111), or α-Syn 126-135Four selected antibodies (collected at 9 WPI) induced by [SEQ ID NO: 112] showed an inhibitory effect on α-Syn aggregation of approximately 40% compared to the aggregation level of 100% media control (Figure 1).

[0185] b. Dissociation of pre-formed α-Syn aggregates From the above studies, it was found that anti-α-Syn antibodies purified from guinea pig antisera immunized with specific α-Syn peptide immunogen constructs were effective in inhibiting α-Syn aggregation. To further evaluate whether the antibodies induced by the α-Syn peptide immunogen construct are effective in dissociating pre-formed α-Syn aggregates, an in vitro disaggregation assay was performed using anti-α-Syn antibodies purified from guinea pig antisera.

[0186] α-Syn was aggregated at a concentration of 5 μM in 200 μL of PBS / KCl buffer for 3 days. After centrifugation (13,000×g, 4°C, 30 minutes), the α-Syn aggregates were recovered and confirmed by ThT assay. The pre-formed α-Syn aggregates were then incubated for 3 days in 100 μL of PBS / KCl buffer with or without anti-α-Syn antibodies (5 μg / mL) purified from guinea pig antisera. After incubation, the aggregates were collected after centrifugation at 13,000×g for 30 minutes at 4°C and quantified by ThT assay as described in Example 3. The residual α-Syn aggregates after spontaneous dissociation in the media control were normalized to 100%.

[0187] α-Syn 111-132 (SEQ ID NO: 113) or α-Syn 126-135 (SEQ ID NO: 112), two selected anti-α-Syn antibodies induced thereby, and α-Syn 111-132 (SEQ ID NO: 113) induced and α-Syn 126-135 (SEQ ID NO: 112) induced anti-α-Syn antibody combinations were tested in this in vitro disaggregation assay. As a result, α-Syn 126-135 (SEQ ID NO: 112) and α-Syn 111-132The anti-α-Syn antibody induced by [[SEQ ID NO: 113]] demonstrated an approximately 50% dissociation effect on pre-formed α-Syn aggregates compared to the vehicle control as 100%, while other anti-α-Syn antibodies and antibodies purified from pre-immunized animals were unable to show an equivalent effect (Figure 2).

[0188] Example 9 Antibodies induced by α-Syn peptide immunogen constructs and their formulations: Anti-aggregation and disaggregation effects on the kinetics of α-Syn aggregation in α-Syn overexpressing cells α-Syn aggregation is known to accelerate during neuronal differentiation. To evaluate the effect of α-Syn peptide immunogen constructs on either inhibiting α-Syn aggregation or dissociating pre-formed α-Syn aggregates under cell-based conditions, anti-α-Syn antibodies generated from guinea pig antisera immunized with different α-Syn peptide immunogen constructs were evaluated in an anti-aggregation assay and a disaggregation assay based on NGF-treated, neuronal differentiated α-Syn overexpressing PC12 cells.

[0189] a. Inhibition of α-Syn aggregation α-Syn overexpressing PC12 cells were seeded in poly-D-lysine pre-coated 96-well plates and then treated with nerve growth factor (NGF) (100 ng / mL) for 4 days together with anti-α-Syn antibodies (0 or 0.5 μg / mL) purified from guinea pigs immunized with different α-Syn peptide immunogen constructs to verify the anti-aggregation activity.

[0190] The treated cells were lysed, 20 μg of cell lysates were separated by SDS-PAGE and then detected with an α-Syn antibody (BD). The amount of α-Syn signal detected in the high molecular weight region was quantified and normalized to the vehicle control group as 100%. As also shown in Figure 3, α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), α-Syn 123-135 (SEQ ID NO: 111), or α-Syn 126-135For all four selected anti-α-Syn antibodies induced by [[SEQ ID NO: 112]], an inhibitory effect of 80 - 90% on the amount of aggregated α-Syn was observed compared to the amount of aggregated α-Syn in the vehicle control.

[0191] b. Dissociation of pre-formed α-Syn aggregates To verify the disaggregation activity of pre-formed α-Syn aggregates, α-Syn overexpressing PC12 cells were neurodifferentiated and treated with NGF (100 ng / mL) for 3 days to initiate aggregation of α-Syn, and further treated with anti-α-Syn antibodies (0 or 0.5 μg / mL) purified from guinea pigs immunized with different α-Syn peptide immunogen constructs for an additional 4 days.

[0192] The treated cells were lysed, 20 μg of cell lysates were separated by SDS-PAGE, and then detected with an α-Syn antibody (BD). The amount of α-Syn signal detected in the high molecular weight region was quantified and normalized to the vehicle control group as 100%. As also shown in Figure 3, anti-α-Syn antibodies induced by α-Syn 111-135 (SEQ ID NO: 107), α-Syn 123-135 (SEQ ID NO: 111), or α-Syn 126-135 (SEQ ID NO: 112) peptide immunogen constructs showed a 50 - 60% decrease in the amount of aggregated α-Syn, while anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 113) showed a decrease of more than 90% in the amount of aggregated α-Syn.

[0193] Example 10 Antibodies induced by α-Syn peptide immunogen constructs and their formulations: Effects on the reduction of microglial TNF-α and IL-6 secretion Neuronal damage in the substantia nigra is thought to release aggregated α-Syn into the substantia nigra, activate microglia through the production of pro-inflammatory mediators, thereby leading to persistent and progressive substantia nigra neurodegeneration in PD. To evaluate the effect of reducing microglial activation by anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogen constructs, the amounts of pro-inflammatory mediators, TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6), released by microglia when treated with α-Syn aggregates in the presence or absence of different anti-α-Syn antibodies were measured.

[0194] Mouse BV2 cells or human SVG p12 cells were seeded at 5,000 cells / well in RPMI1640 medium supplemented with 1% FBS. The cells were treated with 1 μM α-Syn and incubated for 24 hours at 37 °C in a 5% CO2, humidified atmosphere. Then, the medium was collected, centrifuged, and the supernatant was separated. The concentrations of IL-6 secreted by BV2 cells and TNF-α secreted by SVG p12 cells in the supernatant were analyzed in triplicate using a mouse IL-6 or human TNF-α mouse ELISA kit (Thermofisher), respectively. The signals were normalized to the media control as 100%.

[0195] Data showed that the anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 113) and α-Syn 123-135 (SEQ ID NO: 111) decreased TNF-α release via α-Syn aggregates by SVG p12 cells by 30 - 50%, while the anti-α-Syn antibody induced by α-Syn 123-135 (SEQ ID NO: 111) decreased IL-6 release by SVG p12 cells by approximately 30% (Figure 4). The results indicated that the anti-α-Syn antibody induced by α-Syn 123-135 (SEQ ID NO: 111) was more potent than the other anti-α-Syn antibodies tested in alleviating α-Syn aggregate-mediated microglial activation.

[0196] Example 11 Antibodies Induced by α-Syn Peptide Immunogen Constructs and Their Formulations: Effects on the Reduction of Neurodegeneration Induced by Exogenous Alpha-Synuclein To evaluate the neuroprotective effects of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs, an in vitro neurodegeneration model with exogenous pre-formed α-Syn aggregates in NGF-treated neuronally differentiated PC12 cells was employed.

[0197] PC12 cells were treated with NGF (100 ng / mL) for 6 days to induce neuronal differentiation. The morphology of the neuronally differentiated cells was confirmed and analyzed using an InCell high-content image analysis system (GE Healthcare). The neurotrophic effect of NGF was reflected in neurite outgrowth, and the number of neuronally differentiated cells was quantified. The level of neurite outgrowth and the number of neuronally differentiated cells were shown as percentages (mean ± SEM) after normalization. The neurite lengths of PC12 cells with and without NGF treatment were set at 100% and 0%, respectively. The number of neuronally differentiated PC12 cells after 6 days of NGF treatment was normalized to 100%.

[0198] Neurodegeneration was observed by adding exogenous pre-formed α-Syn aggregates to neuronally differentiated PC12 cells. In the presence of pre-formed α-Syn aggregates, neurite length was shortened and the number of neuronally differentiated PC12 cells decreased. This α-Syn aggregate-driven neurodegeneration was proportional to the amount of exogenous α-Syn aggregates added and could be blocked by curcumin, which is widely known for its neuroprotective effect against the neurotoxicity of α-Syn aggregates, in a concentration-dependent manner. A commercially available anti-α-Syn antibody (BD bioscience) attenuated α-Syn aggregate-driven neurodegeneration, but an antibody purified from naive guinea pigs did not. This model was employed as a screening platform to identify which anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs have a neuroprotective effect of restoring neurite growth and neuronal survival in a concentration-dependent manner (Tables 11 and 12).

[0199] As a result, anti-α-Syn antibodies purified from guinea pig antisera immunized with more than half of the different α-Syn peptide immunogen constructs restored neurite outgrowth in a concentration-dependent manner (Table 11), and anti-α-Syn antibodies purified from guinea pig antisera immunized with almost all of the different α-Syn peptide immunogen constructs protected neuronal differentiated PC12 cells from neuronal cell death induced by α-Syn aggregates (Table 12). Combining the two different parameters, it was found that nearly one-third of the anti-α-Syn antibodies assayed were effective in both neurite length and cell survival against the neurotoxicity of α-Syn aggregates. α-Syn 111-132 (SEQ ID NO: 113), α-Syn 126-135 (SEQ ID NO: 112), and the anti-neurodegenerative effects of pre-immune antibodies from naive guinea pigs were observed, and neurite length and cell number with the fluorescent live cell labeling dye calcein AM (Life Technologies) were quantified. In neurite-rich neuronal differentiated PC12 cells, anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 113) (Figure 5B) and α-Syn 126-135 (SEQ ID NO: 112) (Figure 5C) showed a protective effect against the shortening of neurite outgrowth mediated by α-Syn aggregates, but this was not the case with pre-immune antibodies purified from naive guinea pigs (Figure 5A).

[0200] Example 12 Antibodies Induced by α-Syn Peptide Immunogen Constructs and Their Formulations: Effects on the Reduction of Neurodegeneration in α-Syn Overexpressing Cells To evaluate the neuroprotective effects of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs, in vitro neurodegeneration models with wild-type α-Syn overexpressing PC12 cells and A53T mutant α-Syn overexpressing PC12 cells were employed.

[0201] After incubation with NGF, mock control cells (transfected with plasmid vector) developed long neurite outgrowth and increased cell number similar to parental wild-type PC12 cells, while wild-type α-Syn overexpressing PC12 cells and A53T mutant α-Syn overexpressing PC12 cells did not exhibit equivalent neurite outgrowth or increased cell number, and a neurodegenerative effect with aggregated α-Syn upon NGF treatment was confirmed. In the characterization of overexpressed α-Syn in wild-type α-Syn overexpressing PC12 cells upon NGF treatment, Western blot and ThT assay were performed using cell lysates of wild-type α-Syn overexpressing PC12 cells after NGF treatment. The results of Western blot showed overexpression of α-Syn in cell lysates of PC12 cells overexpressing wild-type α-Syn upon NGF treatment, and the results of ThT assay showed that α-Syn in cell lysates of PC12 cells overexpressing wild-type α-Syn upon NGF treatment was in β-sheet structure (i.e., an increase in ThT fluorescence signal). Compared with the results of Western blot and ThT assay of wild-type α-Syn overexpressing PC12 cells without NGF treatment, it was suggested that a structural transition of overexpressed α-Syn from α-helix to β-sheet, which may lead to a subsequent neurodegenerative effect of β-sheet oligomeric α-Syn, occurred during NGF-induced neuronal differentiation. Furthermore, compared with wild-type α-Syn overexpressing PC12 cells, overexpressed A53T mutant α-Syn brought about a strong neurodegenerative effect reflected in both shortening of neurite length and decrease in cell number upon NGF treatment, indicating that A53T mutant α-Syn caused a stronger neurodegenerative effect than wild-type α-Syn in α-Syn overexpressing PC12 cells.

[0202] α-Syn 101-132 (SEQ ID NO: 114), α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), α-Syn 123-135 (SEQ ID NO: 111), or α-Syn 126-135 (SEQ ID NO: 112)-induced anti-α-Syn antibody, and α-Syn 111-132 (SEQ ID NO: 113) and α-Syn126-135 (SEQ ID NO: 112)-induced anti-α-Syn antibody combinations were assayed by an in vitro neurodegeneration model using wild-type α-Syn overexpressing PC12 cells to evaluate the individual protective effects against neurodegeneration. Wild-type α-Syn overexpressing PC12 cells were treated with NGF for 3 days to initiate neuronal differentiation and then further incubated for 3 days with both anti-α-Syn antibody (final concentration 5 μg / mL) and NGF. Microscopic observation of the cells until the end of the incubation period revealed that co-incubation with the selected anti-α-Syn antibodies restored neurite length and increased cell number compared to the vehicle control. Quantification of neurite length and cell number was performed using the readings of parental PC12 cells treated with NGF for 6 days normalized to 100%. As a result, when compared to the vehicle control, α-Syn 101-132 (SEQ ID NO: 114), α-Syn 111-132 (SEQ ID NO: 113), or α-Syn 123-135 (SEQ ID NO: 111)-induced anti-α-Syn antibodies, and combinations of anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 113) and α-Syn 126-135 (SEQ ID NO: 112) showed a significantly higher cell number, while α-Syn 101-132 (SEQ ID NO: 114), α-Syn 111-132 (SEQ ID NO: 113), α-Syn 123-135 (SEQ ID NO: 111), or α-Syn 126-135 (SEQ ID NO: 112)-induced anti-α-Syn antibodies, and combinations of anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 115) and α-Syn 126-135 (SEQ ID NO: 114) showed a significantly longer neurite length (FIGS. 6A and 6B).

[0203] Example 13 Antibodies Induced by α-Syn Peptide Immunogen Constructs and Their Formulations: Specificity for Beta-Sheet Oligomers and Fibrillar Alpha-Synuclein Protein To better characterize the specificity of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs, a series of in vitro assays were performed with α-Syn molecular complexes of different sizes, different amyloid-forming proteins including α-Syn, Aβ, and tau proteins, and α-Syn aggregated in α-Syn overexpressing PC12 cells upon NGF treatment. a. Specificity for large α-Syn molecular complexes Western blots of α-Syn molecular complexes of different sizes were performed using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs as primary antibodies. The results showed that all anti-α-Syn antibodies strongly reacted with α-Syn molecular complexes of larger sizes, including dimers, trimers, tetramers, and oligomers, in addition to the smaller-sized monomeric α-Syn. When compared with the commercially available anti-α-Syn antibody, Syn211 (Abcam), the anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), α-Syn 123-135 (SEQ ID NO: 111), and α-Syn 126-135 (SEQ ID NO: 112) showed a higher ratio of the signal of α-Syn molecular complexes of larger sizes (including dimers, trimers, tetramers, and oligomers) to the signal of the smaller-sized monomeric α-Syn (Figures 7A and 7B), suggesting that the anti-α-Syn antibodies have specificity for larger α-Syn molecular complexes.

[0204] b. Specificity for α-Syn among different amyloid-forming proteins Different amyloidogenic proteins prepared as described in Example 3 (i.e., α-Syn, Aβ 1-42Dot blot assays with different species of α-Syn (i.e., α-helix monomer, β-sheet monomer, β-sheet oligomer, and β-sheet protofibril), including Tau441, were performed using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs as primary antibodies. As a result, 126-135 (SEQ ID NO: 112) and α-Syn 111-132 (SEQ ID NO: 113)-induced anti-α-Syn antibodies were shown to specifically react with all β-sheet forms of α-Syn (monomer, oligomer, protofibril species), but not with the α-helix monomer (FIGS. 8A, 8B, and 8C). Furthermore, α-Syn 126-135 (SEQ ID NO: 112) and α-Syn 111-132 (SEQ ID NO: 113)-induced anti-α-Syn antibodies reacted more strongly with the β-sheet protofibrils of α-Syn and the β-sheet oligomers of α-Syn than with the β-sheet monomers of α-Syn. In contrast, α-Syn 126-135 (SEQ ID NO: 112) and α-Syn 111-132 (SEQ ID NO: 113)-induced anti-α-Syn antibodies showed no detectable reactivity against different species of β-Syn or amyloid-forming protein Aβ 1-42 and Tau441 (i.e., α-helix monomer, β-sheet monomer, β-sheet oligomer, and β-sheet protofibril) (FIGS. 8A, 8B, and 8C). This finding suggested that the anti-α-Syn antibodies induced by α-Syn 126-135 (SEQ ID NO: 112) and α-Syn 111-132 (SEQ ID NO: 113) have specificity for α-Syn in the β-sheet monomer, β-sheet oligomer, and β-sheet fibrillar forms.

[0205] Binding specificity to aggregated α-Syn in α-Syn overexpressing PC12 cells during NGF treatment Immunocytochemistry (ICC) using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs was performed on parental PC12 cells, mock control PC12 cells, wild-type α-Syn overexpressing PC12 cells, and A53T mutant α-Syn overexpressing PC12 cells, and as described in Example 3, the binding affinity of the antibodies for α-Syn aggregated upon NGF treatment was evaluated. As shown in Figure 9, α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), or α-Syn 126-135 (SEQ ID NO: 112)-induced anti-α-Syn antibodies demonstrated stronger reactivity in wild-type α-Syn overexpressing PC12 cells and A53T mutant α-Syn overexpressing PC12 cells than in parental PC12 cells or mock control PC12 cells upon NGF treatment. Since overexpressed α-Syn aggregation was induced by NGF treatment, this finding suggested that the anti-α-Syn antibodies induced by α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), or α-Syn 126-135 (SEQ ID NO: 112) had specificity for aggregated α-Syn in wild-type α-Syn overexpressing PC12 cells and A53T mutant α-Syn overexpressing PC12 cells upon NGF treatment.

[0206] Example 14 Immunohistochemical staining of human brains from Parkinson's disease for the evaluation of the tissue specificity of α-Syn peptide immunogen constructs and their formulations Before immunization, α-Syn 126-135 (SEQ ID NO: 112) or α-Syn 111-132Immunohistopathological studies using the anti-α-Syn antibody induced by [Accession No. 113] and a 1:1 combination of both anti-α-Syn antibodies were performed on normal human tissues to monitor specificity and autoreactivity of unwanted antibodies. Human tissue panels (Pantomics) were deparaffinized in xylene, rehydrated in ethanol, then treated with a 0.25% trypsin solution containing 0.5% CaCl2 in PBS for 30 minutes, incubated with 1% hydrogen peroxide in methanol to block endogenous peroxidase activity, and then incubated with 10% Block Ace (Sigma) in PBS, α-Syn 126-135 ([Accession No. 112]) or α-Syn 111-132 Anti-α-Syn antibodies from guinea pigs immunized with [Accession No. 113], and a 1:1 combination (1:300 dilution) of both antibodies were applied. Sections were developed with 3-3’ diaminobenzidine (DAB) and counterstained with hematoxylin before examination under a microscope. In contrast to the positive reaction of a commercially available anti-α-Syn antibody (BD, 610708), the anti-α-Syn antibodies purified from guinea pigs immunized with α-Syn 126-135 ([Accession No. 112]) or α-Syn 111-132 ([Accession No. 113]), and a 1:1 combination of both antibodies showed negative reactivity against normal human tissues, which was consistent with the pattern of pre-immune antibodies of naive guinea pigs (Figure 10A).

[0207] Before immunization, another immunohistopathological study using the anti-α-Syn antibody induced by α-Syn 126-135 ([Accession No. 112]) or α-Syn 111-132 ([Accession No. 113]), and a 1:1 combination of both anti-α-Syn antibodies was performed to test reactivity with the brains of human Parkinson's disease. Tissue sections (BioChain) of three regions (i.e., cerebellum, corpus callosum, and thalamus) were assayed. As a result, α-Syn 126-135 ([Accession No. 112]) or α-Syn 111-132The anti-α-Syn antibody induced by [SEQ ID NO: 113], and the 1:1 combination of both anti-α-Syn antibodies showed positive reactivity against PD brain sections in all three regions (indicated by arrows) compared to the negative reactivity of healthy brain sections (Figure 10B and Figure 10C). Quantification of the reactivity against α-Syn aggregates in PD brain sections was performed by counting positive staining under microscopic observation. The results show that α-Syn 126-135 (SEQ ID NO: 112) or α-Syn 111-132 The anti-α-Syn antibody induced by [SEQ ID NO: 113], and the 1:1 combination of both anti-α-Syn antibodies showed strong positivity in PD brain sections compared to healthy human brain sections. Among the three different anti-α-Syn antibodies assayed, the antibody induced by α-Syn 111-132 (SEQ ID NO: 113) had the strongest immunoreactivity against α-Syn aggregates in PD brain sections.

[0208] Example 15 Proof of the efficacy of α-Syn peptide immunogen constructs and their formulations in animal models a. Immunization and collection of blood / brain tissues The Parkinson's disease (PD) mouse model was established as described in Example 4. Two weeks after MPP + injection, or seven weeks after fibrillar α-Syn inoculation, the mice were randomly divided into three groups, in addition to the adjuvant group (immunized with the adjuvant and solvent used in the preparation of the composition (ISA 51 VG, CpG3, 0.2% TWEEN®-80)), including UBITh1-conjugated α-Syn 111-132 (SEQ ID NO: 113) peptide, UBITh1-conjugated α-Syn 126-135 (SEQ ID NO: 112) peptide, and the combination of both peptides. Intramuscular (IM) immunization was administered three times at a dose of 40 μg at three-week intervals. The schedule of administration and blood collection was carried out according to Table 13.

[0209] At each time point, 200 μL of blood was collected by facial vein puncture. The blood dripping from the punctured submandibular vein was collected into a microtube and centrifuged at 300 rpm for 10 minutes to prepare serum. After sacrificing the animals, brain tissue samples were collected for Western blotting.

[0210] b.α-Syn 111-132 (SEQ ID NO: 113) or / and α-Syn 126-135 Immune response in PD model mice administered with a composition containing a (SEQ ID NO: 112) peptide immunogen construct Pooled serum samples from each treatment group were diluted with 1% BSA (in PBST), and then applied to ELISA plates coated with 200 μL of full-length α-Syn peptide (Cloud-clone) in 0.1 M sodium bicarbonate (α-Syn concentration 4.4 μg / μl, pH 9.6). Incubated at room temperature for 2 hours, washed 3 times with PBST, then 100 μl of HRP-conjugated anti-mouse IgG antibody diluted 1:3000 with 1% BSA was added and reacted at room temperature for 2 hours. Then, the plates were washed 3 times with PBST, incubated with 100 μl of 3,3,5,5-tetramethylbenzidine (TMB) in the dark for 10 minutes. Next, 100 μL of 2 M H2SO4 was added, incubated for 15 - 30 minutes, and then the optical density (OD) value at 450 nm was measured with SpectraMax i3x multimode detection (Molecular Devices).

[0211] Formulated α-Syn 111-132 (SEQ ID NO: 113), formulated α-Syn 126-135 (SEQ ID NO: 112), or two PD mouse models immunized with a combination of both peptide immunogen constructs, respectively, received MPP + In the induction model (Figure 11A) or the fibrillar α-Syn inoculation model (Figure 11B), after the second immunization, there were anti-α-Syn antibody optical density (OD) values exceeding 3.0 and the increase was maintained until the end of the 15-week and 19-week studies after the initial immunization, while the animals administered with adjuvant did not induce a measurable anti-α-Syn immune response.

[0212] In the fibrillar α-Syn inoculation model, α-Syn 111-132 construct induced a stronger immune response than the α-Syn 126-135 construct (Figure 11B), but it should be noted that no difference in immunogenicity was observed in the MPP + induced model (Figure 11A).

[0213] c. Decrease in serum α-Syn levels The α-Syn levels in serum pooled from animals in each group were assayed using an ELISA kit (SEB222Mu, USCN) that can detect both the α-helix and β-sheet forms of α-Syn, as described in Example 3.

[0214] The α-Syn quantitative ELISA was used to test whether the anti-α-Syn antibody response in the immunized groups was associated with a decrease in the amount of peripheral α-Syn compared to untreated animals. In both the MPP + induced model (Figure 12A) and the fibrillar α-Syn inoculation model (Figure 12B), immunization with α-Syn 126-135 (SEQ ID NO: 112), α-Syn 111-132 (SEQ ID NO: 113), or a combination of these constructs was shown to result in a decrease in the optical density (OD) value of α-Syn levels compared to adjuvant-administered animals. The results suggested that when an anti-α-Syn antibody response occurred upon immunization with the α-Syn peptide immunogen construct, the amount of α-Syn in the peripheral circulation decreased accordingly.

[0215] d. Decrease in oligomeric α-Syn levels in the brain After sacrificing the animals, brain tissue samples were collected for Western blotting. In the MPP +In the induced mice, the brains were removed and homogenized, while in the mice inoculated with fibrillar α-Syn, the striatum and substantia nigra regions were first separated and then homogenized. Brain tissue lysates were prepared by adding lysis buffer (Amresco) and 1× protease inhibitor (Roche) to the homogenate. The lysates were then separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred to a polyvinylidene fluoride (PVDF) membrane, and incubated overnight with 5% milk in PBS. To detect the abundance of dopaminergic neurons, the membrane was incubated with an anti-tyrosine hydroxylase antibody (1:1000 dilution, Abcam), followed by hybridization with a goat anti-rabbit IgG (H+L) HRP-conjugated secondary antibody (1:5000 dilution, Jackson Immunoresearch). For visualization, Luminata Western HRP Substrates were used, and the resulting signals were captured with a ChemiDoc-It 810 digital imaging system. Quantification of the oligomeric α-Syn level was performed by normalizing to the GAPDH level, and the ratio of non-lesioned lysates was further normalized to 100% for comparison.

[0216] MPP + In the induced model, α-Syn 111-132 A decrease in the oligomeric α-Syn fraction was shown in animals immunized with the peptide immunogen construct (Figure 13A). Similarly, in mice inoculated with fibrillar α-Syn, Western blots using lysates from the substantia nigra and striatum ipsilateral to the fibrillar α-Syn inoculation (Figures 14A and 14D) and from the striatum contralateral to the fibrillar α-Syn inoculation (Figure 14F) showed an increase in the oligomeric α-Syn level up to 2- to 3-fold that seen in adjuvant control mice, which was reduced after treatment with the α-Syn 111-132 (SEQ ID NO: 113) and α-Syn 126-135 (SEQ ID NO: 112) constructs, as shown by the quantification of the Western blotting results in Figures 13B, 14B, 14C, 14D, and 14G.

[0217] e. Decrease in neuropathology In the case of mice inoculated with fibrillar α-Syn, the substantia nigra region was first isolated and then homogenized. Tissue lysates were prepared by adding lysis buffer (Amresco) and 1× protease inhibitor (Roche) to the homogenate. The lysates were then separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred to a polyvinylidene fluoride (PVDF) membrane, and incubated overnight with 5% milk in PBS. To detect the abundance of dopaminergic neurons, the membrane was incubated with an anti-tyrosine hydroxylase antibody (1:1000 dilution, Abcam), followed by hybridization with a goat anti-rabbit IgG (H+L) HRP-conjugated secondary antibody (1:5000 dilution, Jackson Immunoresearch). For visualization, Luminata Western HRP Substrates were used, and the resulting signals were captured with a ChemiDoc-It 810 digital imaging system. The expression level of α-Syn was normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase), which was used as a protein loading control.

[0218] As a result, α-Syn 111-132 Immunization with the construct demonstrated that the amount of tyrosine hydroxylase was restored to a level equivalent to that of non-lesioned normal animals (Figures 14C-14D), suggesting a neuroprotective effect of the α-Syn peptide immunogen construct against the neurotoxicity associated with aggregated α-Syn inoculated into the mice.

[0219] f. Recovery of motor activity CatWalk (trademark) XT (Noldus Information Technology, Wageningen, Netherlands) is a video-based analysis system that dynamically and objectively measures various aspects of footfalls based on the position, pressure, and surface area of each footfall. All mice were trained to cross the runway in a consistent manner at least three times a day for at least one day prior to the experiment. A successful run was defined as the animal running through the runway without interruption or hesitation, and mice that failed the training were excluded from the study.

[0220] The average of five crossings per mouse was analyzed. Since the fibrillar α-Syn inoculation was performed on the right brain, the left hind limb stance time was considered as a reference parameter only for the running time.

[0221] In the fibrillar α-Syn inoculation model, α-Syn 126-135 (SEQ ID NO: 112) or α-Syn 111-132 (SEQ ID NO: 113) after treatment with the composition containing, a significant difference was observed in the measurement of the left hind limb stance time (Figure 15A). On the other hand, in both the fibrillar α-Syn inoculation model and the MPP+-induced model, α-Syn 111-132 (SEQ ID NO: 113) after treatment with the composition containing, a significant difference was observed in the measured running time (Figures 15B and 15C). The results suggested a relationship between treatment with the formulated α-Syn 126-135 (SEQ ID NO: 112) or the formulated α-Syn 111-132 (SEQ ID NO: 113) α-Syn peptide immunogen constructs and the improvement of motor function in two PD mouse models.

[0222] Example 16 Reactivity of antibodies generated by α-Syn peptide immunogen constructs having different α-Syn strains found in neurodegenerative diseases

[0223] α-Syn drives Parkinson's disease and other synucleinopathies. Each of these diseases is caused by one or more distinct types of aggregates because the α-Syn protein can form clearly distinguishable types of aggregates that differ in size and structure and have different effects on cells. α-Syn aggregates of different shapes cause different patterns of damage in the brain and can even cause distinct brain diseases. This study was designed to evaluate how antibodies generated by α-Syn peptide immunogen constructs interact with the various α-Syn strains found in neurodegenerative diseases.

[0224] Dr. Ronald Melki was a co-investigator on this study. In the lab, the following different types of α-Syn aggregates were generated: (a) protofibrils - long, twisted, zipper-like chains of α-Syn protein; (b) ribbons - wider, flatter structures; and (c) α-Syn oligomers (O550), dopamine-stabilized (ODA), and glutaraldehyde-stabilized (OGA) oligomers.

[0225] Antibodies generated in guinea pigs by the various α-Syn peptide immunogen constructs disclosed herein were tested for their relative affinities. Representative samples from PD-021514 (α-Syn 85-140 , wpi 08), PD-021522 (α-Syn 85-140 , wpi 13), PD-100806 (α-Syn 126-135 , wpi 09), PRX002, and the commercially available monoclonal antibody Syn1 (clone 42) were tested in individual α-Syn assemblies containing protofibrils, ribbons, protofibril 65, protofibril 91, protofibril 110, fibrillar assembly pathway α-Syn oligomers (O550), dopamine-stabilized (ODA), and glutaraldehyde-stabilized (OGA) oligomers, along with a control monomer using a filter trap assay.

[0226] Methods and Materials a. Assembly of α-Syn into Protofibrils and Ribbons For protofibril formation, soluble WTα-Syn was incubated at 37°C with continuous shaking in a buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl) in an Eppendorf Thermomixer set at 600 r.p.m. Using a magnetic stir bar (6×3 mm), the assembly was continuously monitored by a Cary Eclipse spectrofluorometer (Varian Inc., Palo Alto, CA, USA) with an excitation wavelength set at 440 nm and emission wavelengths set at 440 nm and 480 nm, and an average time of 1 second, in the presence of thioflavin T (15 μM) in a 1×1 cm cuvette under stirring (100 r.p.m.). For ribbon formation, WTα-Syn was dialyzed against 1000-fold buffer B (5 mM Tris-HCl pH 7.5) at 4°C for 16 h and then incubated at 37°C with continuous shaking in an Eppendorf Thermomixer set at 600 r.p.m. The assembly was monitored by measuring the scattered light at 440 nm. Alternatively, the amount of protein remaining in the supernatant after sedimentation at 35,000×g was determined by measuring the absorbance at 280 nm with a Hewlett Packard 8453 diode array spectrophotometer. The nature of the oligomeric species was evaluated using a Jeol1400 (Jeol Ltd.) TEM with adsorption of the sample onto a carbon-coated 200 mesh grid and negative staining with 1% uranyl acetate. Images were recorded with a Gatan Orius CCD camera (Gatan). The binding ability of α-Syn assemblies to Congo Red was evaluated as follows: α-Syn protofibrils and ribbons were incubated with 100 μM Congo Red (Sigma-Aldrich, St Louis, MO, USA) in 20 mM Tris buffer (pH 7.5) for 1 h. The polymers were then precipitated at 20°C and 25,000 g for 30 min in a TL100 tabletop Beckman ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA). The pellet was washed 4 times with an equal volume of water. After resuspension of the pellet, aliquots were placed on a coverslip and imaged immediately or dried.The samples were observed under bright field and cross-polarized light using a polarized light microscope equipped with a cross-polarizer, a Leica (MZ12.5) microscope (Leica Microsystems, Ltd., Heerbrugg, Switzerland).

[0227] b. Determination of α-Syn fibril and ribbon concentration The length heterogeneity of α-Syn fibrils and ribbons was reduced by sonication on ice for 20 minutes in a 2 ml Eppendorf tube of VialTweeter equipped with an ultrasonic processor UIS250v (250 W, 2.4 kHz, Hielscher Ultrasonic, Teltow, Germany) set to 75% amplitude and a 0.5 second pulse. The sedimentation rates of α-Syn fibrils and ribbons were measured. The sedimentation boundary was analyzed with Sedfit software using the least-squares boundary modeling 1s-g*(s) optimal for a heterogeneous mixture of large particles. This gave a particle distribution in the sedimentation coefficient range of 50 - 150 S for α-Syn ribbons and 100 - 1,000 S for α-Syn fibrils. For α-Syn ribbons and fibrils, the species with sedimentation coefficients of approximately 90 S and 375 S were centered respectively. In the case of α-Syn ribbons, it corresponded to particles with a molecular weight of approximately 11,500 kDa, composed of, for example, approximately 800 α-Syn molecules (12,000 kDa / 14.5 kDa). In the case of α-Syn fibrils, it corresponded to particles with a molecular weight of approximately 102,000 kDa, composed of, for example, approximately 7,000 α-Syn molecules (102,000 kDa / 14.5 kDa). Therefore, assuming that 100% of the protein is found in the pellet fraction by centrifugation of the sample and that 100% of α-Syn assembles into ribbons or fibrils in the steady state, at a usage concentration of 20 μM, the total concentration of α-Syn ribbon and fibril particles is approximately 20 μM / approximately 800 = approximately 0.02 μM and 20 μM / approximately 7,000 = approximately 0.003 μM for α-Syn ribbons and fibrils respectively.

[0228] c. Evaluation of the affinity of end bodies for various α-Syn fibrils and ribbons The affinity of the antibodies generated by the α-Syn peptide immunogen constructs disclosed herein was evaluated for distinct α-Syn assemblies using a filter trap assay with the antibody used as reference. α-Syn assemblies (protofibrils, ribbons, protofibril 65, protofibril 91, protofibril 110, protofibril assembly pathway α-Syn oligomers (O550), dopamine-stabilized (ODA) and glutaraldehyde-stabilized (OGA) oligomers) are described in Bousset L. et al., 2013 Nat Commun 4:2575; Makky A. et al., 2016 Sci Rep 6:37970; and Pieri L. et al, 2016 Sci. Rep 6:24526. Control monomeric α-Syn was also used.

[0229] Using a slot blot filtration apparatus, increasing amounts of fibrillar, oligomeric, or monomeric α-Syn in the range of 20 pg to 200 ng were spotted onto nitrocellulose filters. The filters were then blocked with skim milk and incubated with PRX002 or Syn1 antibody, or the test GP antibody of the present disclosure, at the designated dilutions. After thorough washing, the primary antibody binding profile was detected using secondary anti-human or anti-guinea pig IgG-HRP. A control with secondary antibody only was also tested. Super Signal ECL (Pierce #34096) was used for the blot, and then the blot was imaged with a BioRad imager (Chemidoc MP imaging system / BioRad imagelab software). The exposure time and dynamic range are shown in FIGS. 16A - 16H. In this series of measurements, human brain homogenates from DLB cases were spotted onto the membrane.

[0230] d. Results Guinea pig (GP) antibodies PD-021514 (α-Syn 85-140 , wpi 08), PD-021522 (α-Syn 85-140 , wpi 13), PD-100806 (α-Syn 126-135, the affinities of wpi 09), PRX002, and the commercially available antibody Syn1 (clone 42) were compared for different α-Syn assemblies using a filter trap assay. The α-Syn assemblies used included protofibrils, ribbons, protofibril 65, protofibril 91, protofibril 110, oligomers on the protofibril assembly pathway α-Syn (O550), dopamine stabilization (ODA), and glutaraldehyde stabilization (OGA) oligomers, along with control monomeric α-Syn.

[0231] Figures 16A - 16H show that, when compared to monomeric α-Syn, the reference antibody PRX002 recognizes fibrillar α-Syn with slightly better affinity, while both recognize the α-Syn of the present disclosure 126-135 PD-100806 and PD-021514 for the peptide construct have much higher affinity for fibrillar α-Syn compared to monomeric α-Syn, indicating that both have preferential binding to fibrillar α-Syn. The affinity of PRX002 for oligomeric and fibrillar α-Syn was found to be similar. The Syn1 monoclonal antibody bound to fibrillar α-Syn as well as oligomeric and monomeric α-Syn, but had no highly differentiated selectivity.

[0232] Example 17 Immunohistochemical study of antibodies derived from an α-Syn peptide immunogen construct on brain sections of patients with Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) Representative α-Syn of the present invention 126-135Antibodies obtained from immunizing guinea pigs with peptide immunogen constructs were used in immunochemical studies and characterized for their ability to bind to α-Syn present in brain sections of patients with α-synucleinopathy. This study was conducted in collaboration with Professor Roxana Carare. The ability of antibodies to bind to α-Syn present in brain sections obtained from patients with PD, LBD, and MSA was evaluated. Healthy tissue was included in the study as a negative control. NCL-L-ASYN, a commercially available monoclonal antibody used for postmortem diagnosis of α-synucleinopathy, was included as a positive control. This investigation was on α-Syn on tissue sections of brains of human patients with PD, LBD, and MSA 126-135 Provides evidence of positive immunoreactivity of antibodies against peptide immunogen constructs. Binding was particularly seen in the brains of synucleinopathy patients but not in non-patient brains, and the test antibody had more prominent binding than the commercially available diagnostic antibody.

[0233] Methods and Materials a. Description of Reagents Used and Their Suppliers Representative α-Syn 126-135 Antibodies obtained from immunizing guinea pigs with peptide immunogen constructs were used at a 1:100 dilution. PD062220-09-1-2-Syn; PD062205-09-1-2-Syn; PD100806-09-1-2-Syn were provided by United NeuroScience (UNS), NCL-L-ASYN (mouse monoclonal antibody used at a 1:100 dilution) was provided by Leica Biosystems, HuD (E-I) (mouse monoclonal antibody at a 1:100 dilution) was provided by Santa Cruz Biotechnology, Olig2 (rabbit antibody at a 1:100 dilution) was provided by Millipore, Alexa Flour 594 (goat anti-guinea pig at a 1:200 dilution) and Alexa Flour 488 (goat anti-mouse at a 1:200 dilution) as well as Alexa Flour 488 (goat-rabbit at a 1:200 dilution) were provided by Molecular Probes life technologies.

[0234] b. Human brain tissue In this study, μm-thick sections were obtained from the UCL Brain Bank. All samples were collected and prepared according to procedures approved by the National Research Ethics Service.

[0235] Tissues were obtained from subjects with primary α-synucleinopathies, including multiple system atrophy (MSA; n = 3), dementia with Lewy bodies (DLB; n = 3), and Parkinson's disease (PD; n = 3) (Table 15). Subjects were postmortem diagnosed according to published criteria**.

[0236] c. Immunohistochemistry of human subjects with synucleinopathy To quantitatively compare the specificity of three antibodies produced by United Neuroscience (UNS) against α-synuclein aggregates, immunohistochemistry (IHC) was performed on human subjects with three different synucleinopathies (MSA, DLB, and PD). The specificity of the UNS antibodies (PD062220, PD062205, and PD100806) against α-synuclein aggregates was compared with that of a commercially available diagnostic antibody (NCL-L-ASYN). Antibody specificity was analyzed in the following four brain regions for each patient's subject and disease type. (1) Putamen, internal capsule, and insular cortex; (2) midbrain: substantia nigra; (3) temporal cortex: cortical gray matter; (4) cerebellum: subcortical white matter; cerebellar white matter.

[0237] These brain regions are known to be affected by α-Syn aggregation to varying degrees at different stages of disease progression for each disease type. Generally, the basal ganglia and midbrain are affected in the early stages of DLB, PD, and MSA, and also have the highest total burden of aggregates. The temporal cortex and cerebellum are affected in the later stages of the disease, and there are few cerebellar aggregates in PD and DLB. To confirm the absence of non-specific binding of the secondary antibody, negative controls (without using the primary antibody) were run together with each IHC protocol. Paraffin-embedded slides were deparaffinized in an oven at 60 °C for 15 - 20 minutes and then immersed in xylene I and II for 5 minutes each. The tissues were rehydrated with 4 dilutions of 100% - 50% IMS for 5 minutes each. The tissues were washed 3 times with 1×PBS for 5 minutes each and then incubated with 100% formic acid for 3 minutes for antigen retrieval. Before quenching the endogenous peroxidase activity with 3% H2O2 for 10 minutes, the tissues were thoroughly washed with 1×PBS. After cooling the tissues and washing them 3 more times (5 minutes each) with 1×PBS, the non-specific binding sites were blocked with 15% normal goat serum. The tissues were incubated with the primary antibody (1:100 in 0.1% TBS / t) at 4 °C overnight. The tissues were washed 3×5 minutes with 1×PBS and incubated in the biotinylated secondary antibody for 1 hour (RT). The ABC solution was prepared 30 minutes before its application. After washing the tissues 3×5 minutes with 1×PBS, they were incubated with ABC at room temperature for 1 hour. The VIP peroxidase substrate was prepared using the ImmPACTVIP peroxidase kit as detailed in the manufacturer's instructions. The VIP peroxidase substrate was added for 7 minutes at RT and washed with dH2O. Before mounting with DPX, the tissues were dehydrated with IMS 50%, 70%, 95%, 100%, 100% and xylene I and II for 2 minutes each. In double immunofluorescence staining, the tissues were not quenched with 3% H2O2 before applying the primary antibody.After applying the initial primary antibody and equivalent secondary antibody, the tissue was blocked with 15% normal goat serum for 30 minutes and incubated with the second primary antibody and secondary antibody as described above. After the final application of the fluorescently labeled secondary antibody, the tissue was incubated with 1% Sudan Black for 5 minutes to quench autofluorescence, washed with 0.1% TBS / T, and immediately mounted in mowiol cituflour. Fluorescently stained tissues were stored at 4°C until imaged.

[0238] d. Image analysis and statistics Slides were scanned using an Olympus VS110 high-throughput Virtual Microscopy System or an Olympus dot Slide Virtual Microscopy System for analysis at 20x magnification. Thirty images (each 500 μm 2 ) were captured from the scanned images using Olympus VS software from equivalent areas of each region of each subject (see Figures 17A–17D, 18A–18D, 19A–19C, 20A–20E, 21A–21F, 22A–22C, 24A–24D, and 25A–25D). This enabled analysis of a total area of 7.5 mm 2 of each brain region. ImageJ version Fiji windows-64 software was used for quantitative analysis of α-Syn immunoreactivity in each image.

[0239] For analysis of the total amount of α-Syn detected by each antibody, immunoreactivity was reported as a percentage of the total area of the image. The threshold applied to the selection of α-Syn positive immunoreactivity was adjusted for each brain region analyzed to account for differences in background staining that could affect the results. The mean percentage area encompassed by α-Syn positive aggregates was calculated for each antibody and brain region analyzed.

[0240] For the analysis of the relative specificity of each antibody against LB or LN, Fiji software was used to quantify the immunoreactivity of LB based on the parameters of size and roundness to distinguish it from LN (see Figures 24A - 24D, Figures 25A - 25D, and Figures 26A - 26B). To avoid false positives, brain regions with different morphologies of LB and LN were selected for this analysis, including the cortico - subcortical gray matter of the insular cortex and the temporal cortex in the basal ganglia. LB immunoreactivity was expressed as a percentage of the total α - Syn immunoreactivity.

[0241] Statistical analysis was performed using GraphPad Prism v7.01 software and reported as mean ± SD (unless otherwise specified). Results were analyzed by one - way analysis of variance (ANOVA), followed by post - hoc analysis with Dunnett's correction when appropriate. Differences were considered significant when p < 0.05 (*). Numbers (n) refer to the number of subjects used in each experiment.

[0242] Qualitative analysis of the location of α - Syn within neurons or glia was achieved by double immunofluorescence staining as described above. Slides were observed with a Leica SP8 laser scanning confocal microscope. Maximum projection overlay images were continuously acquired with a 40x objective lens. These images consisted of a series of z - stack images that were stacked together with both color channels overlaid to show the relative positions.

[0243] e.α - Syn 126-135 The antibody detected different patterns of α - Syn aggregates compared to NCL - L - ASYN The cell type and intracellular localization of α-Syn aggregates vary among different synucleinopathies. MSA is characterized by glial cytoplasmic inclusions (GCIs), while in DLB and PD, α-Syn aggregation occurs within neuronal cell bodies (LBs) and axonal processes (LNs). Analysis of the percentage of stained area enabled quantification of the total α-Syn aggregates detected by each antibody. However, this did not account for differences in the type or intracellular location of the detected aggregates. The distinct patterns of α-Syn aggregates within cell bodies and neurites in the cases of PD and DLB allowed quantification of the relative sensitivity of the UNS antibodies of the present disclosure to these different types of α-Syn aggregates.

[0244] To examine this, for each antibody in the cases of DLB and PD, the proportion of aggregates detected within cell bodies was estimated. Using FIJI software, aggregates within cell bodies were selected based on their size and circularity. The mean percentage area of cell body aggregates was then calculated as the proportion of the total α-Syn detected, and the results are shown in FIGS. 24A-24D and 25A-25D. The difference in the percentage of area of total α-Syn and cell body α-Syn was due to axonal aggregates (LNs) of α-Syn based on qualitative analysis of the tissue. A decrease in the proportion of cell body α-Syn detection results in an increase in LN detection. This analysis was performed in the gray and white matter of the temporal and insular cortices because these regions showed both pathologies such as LBs and LNs. LNs were very sparse and spread unevenly throughout the putamen and globus pallidus, so these regions of the basal ganglia were not selected in this analysis. Similar correlations were observed in the substantia nigra of the midbrain (FIGS. 26A and 26B), and the UNS antibodies of the present disclosure detected higher levels of LNs compared to NCL-L-ASYN in DLB and PD. However, due to the complex morphology of LNs and LBs, they could not be reliably distinguished and quantified in the same way.

[0245] The results in Figures 24A - 24D show that among the total α - Syn detected by each antibody, the proportion of aggregates detected in the cell bodies decreased with the UNS antibody compared to NCL - L - ASYN. This means that the ratio of the inclusion bodies in the cell bodies to LN decreased, and a higher proportion of LN was detected by the UNS antibody. Among the UNS antibodies, PD062205 was consistent between DLB and PD in the detection of a high proportion of LN in the insular cortex (Figures 17A - 17D and 18A - 18D). In contrast, all α - Syn 126-135 antibodies detected a higher proportion of cell body aggregates compared to NCL - L - ASYN in the temporal cortex gray - white matter of DLB and PD cases (Figures 25A - 25B).

[0246] f. Aggregation of α - Syn is cell - type specific α - Syn - containing aggregates are characteristic pathogenic features of synucleinopathies including MSA, DLB, and PD. α - Syn aggregation is the main causative protein of synucleinopathies, but the pattern of aggregation and the cell types prone to aggregation formation differ between specific disease subtypes. The clinical features of MSA, DLB, and PD explain the accumulation of α - Syn in cell bodies and in the superficial neurites of neurons in both DLB and PD, but in MSA, it is mainly found in glial cells and oligodendrocytes.

[0247] To establish the selectivity of α - Syn 126-135 antibodies for cell - specific α - Syn aggregates, double - immunofluorescence was performed using PD062205 and a marker for either neurons (HuD) or oligodendrocytes (Olig2).

[0248] The results in Figures 27A - 27C show that α - Syn detected by PD062205 co - localizes in the neuronal cell bodies of the basal ganglia and midbrain (high - pathology regions) of PD and DLB, but not in MSA. Using the oligodendrocyte marker (Olig2), Figures 28A - 28C show that α - Syn aggregates in glial cells in MSA, but not in PD or DL. These results show that α - Syn 126-135The antibodies are consistent with the clinical features of these synucleinopathies and show that the specificity of these antibodies for the pathological aggregates of α-Syn is confirmed.

[0249] Results a. Quantitative analysis of antibodies derived from immunization of guinea pigs with representative α-Syn 126-135 peptide immunogen constructs for immunotherapy To investigate the use of novel anti-α-Syn antibodies for immunotherapy, quantitative analysis of the relative specificity of each antibody against α-Syn was performed by immunohistochemistry (IHC) in human cases of three synucleinopathies (MSA, DLB, and PD).

[0250] b. Representative α-Syn 126-135 Antibodies derived from immunization of guinea pigs with peptide immunogen constructs are more sensitive than commercially available diagnostic antibodies in binding to α-Syn aggregates Disclosed α-Syn 126-135 To examine the relative antigenicity of the disclosed α-Syn antibodies, the α-Syn load detected with each antibody was compared with a commercially available diagnostic antibody for synucleinopathies (NCL-L-ASYN). By first examining the overall pattern of the results shown in FIGS. 17A - D to FIGS. 22A - 22C, a significant increase in the mean percentage area of α-Syn detected by the α-Syn 126-135 antibodies compared to NCL-L-ASYN can be found. This trend is consistent across each brain region and disease type, indicating that the disclosed α-Syn 126-135 antibodies are more sensitive or selective in binding to aggregated α-Syn than NCL-L-ASYN. In this study, the sample size was relatively small (n = 3), but a clear trend is still visible in the data. The specificity of the disclosed α-Syn 126-135 antibodies against α-Syn was confirmed in the same brain regions of brains from non-affected control patients. These results shown in FIGS. 23A - 23B indicate that there is no immunopositive staining with each antibody, including NCL-L-ASYN. These data show that the disclosed α-Syn 126-135The antibody has been shown to be specific for the pathological form of α-Syn.

[0251] c.α-Syn 126-135 Higher levels of α-Syn detected using the antibody indicate improved sensitivity and specificity when compared to commercially available antibodies. α-Syn of the present disclosure 126-135 The antibody detects more α-Syn compared to NCL-L-ASYN, indicating that the disclosed antibody is more preferable for use in immunotherapy to promote clearance of these α-Syn aggregates.

[0252] The first step in selecting an appropriate antibody for use as an immunotherapy reagent is to establish the selectivity of the antibody for the target antigen (α-Syn) in human brain tissue with primary α-Syn lesions. Different synucleinopathies differ in the mechanism of α-Syn aggregation and neuroanatomical pattern, as well as the vulnerability of specific cell types to aggregation.

[0253] To investigate the use of reagents as immunotherapy for common synucleinopathies, the selectivity of α-Syn antibodies against α-Syn in different synucleinopathies with different neuropathologies 126-135 It is important to evaluate. Clinically confirmed cases of PD, DLB, and MSA were selected for this purpose. PD and DLB are the second most common forms of dementia, mainly caused by the accumulation of α-Syn within neurons (LB and LN). In contrast to PD, amyloid beta and tau pathologies are known to contribute to neurodegeneration in DLB2. Different patterns of α-Syn aggregation are seen in MSA, and aggregates are mainly formed within glial cells rather than neurons (Figures 27A - 27C and Figures 28A - 28B). Furthermore, the progression of α-Syn pathology differs between disease types, and the midbrain and basal ganglia are common regions of initial pathology. By examining the antigenicity of each antibody in brain regions affected at different stages of the disease, insights can be gained into which antibody is more effective for the treatment of the early stages of the disease.

[0254] d. α-Syn 126-135 Antibodies (PD062220, PD062205, and PD100806) can specifically bind to the pathological aggregates of α-Syn in human brain tissues of PD, DLB, and MSA (from Figure 17A - D to Figure 22A - 22C), and do not detect any synuclein pathology in healthy controls (Figure 23A - 23B). Disclosed α-Syn 126-135 Detection of α-Syn by antibodies was achieved with the same cell type specificity as described in clinical neuropathology (Figure 27A - 27B and Figure 28A - 28B). Importantly, the disclosed α-Syn 126-135 antibodies did not show equivalent antigenicity to all forms of human α-Syn.

[0255] The specificity of PD062205 and PD100806 was further verified by the ability of each antibody to detect a greater proportion of LN than NCL-L-ASYN in the basal ganglia (Figure 24A - 24D). This was also visually observed in the midbrain (Figure 26A - 26B). Overall, due to the high percentage area of α-Syn detected by PD062205 and PD100806, these results indicate that the additional α-Syn 126-135 detected by the disclosed α-Syn antibodies may be partially attributed to the increased specificity of these antibodies for LN. Since LN is the major form of α-Syn aggregation in the basal ganglia at the early stage of the disease, these results are beneficial for immunotherapy. Other reagents for treating synucleinopathies in preclinical development do not provide IHC detection of LN. Therefore, the disclosed peptide immunogen constructs and α-Syn 126-135 antibodies generated from the peptide immunogen constructs have unique properties and characteristics compared to other commercial products.

[0256] In this study, by measuring the average amount of α-Syn aggregates in diseased brain regions, the α-Syn 126-135The sensitivity of the antibody was analyzed using IHC. This study, which quantified the mean percentage area of α-Syn in brain samples, demonstrated that the 126-135 disclosed α-Syn

[0257] antibody is highly sensitive for α-Syn detection at the early stages of disease progression in MSA, DLB, and PD, compared to commercially available antibodies. The higher sensitivity found in this study is due to the higher specificity of the disclosed antibody for LN compared to the diagnostic antibody NCL-L-ASYN. These results suggest that the disclosed α-Syn 126-135 antibody is likely to be the most effective candidate for investigating antibody-assisted clearance of α-Syn aggregates in synucleinopathies.

[0258]

Table 1

[0259]

Table 2

[0260]

Table 3

[0261]

Table 4

[0262]

Table 5

[0263]

Table 6

[0264]

Table 7

[0265]

Table 8

[0266]

Table 9

[0267]

Table 10

[0268]

Table 11

[0269]

Table 12

[0270]

Table 13

[0271]

Table 14

[0272]

Table 15

[0273]

Table 16

[0274]

Table 17

[0275]

Table 18

Claims

1. 1. An alpha-synuclein (α-Syn) peptide immunogen construct comprising: a B-cell epitope comprising about 10 to about 25 amino acid residues from a C-terminal fragment of α-Syn corresponding to about amino acid G111 to about amino acid D135 of SEQ ID NO:1; A T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-98; and an optional heterologous spacer selected from the group consisting of the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148); The α-Syn peptide immunogen construct, wherein the B cell epitope is covalently linked to the T helper cell epitope directly or via the optional heterologous spacer.

2. The α-Syn peptide immunogenic construct of claim 1, wherein the B-cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17, and 49-63.

3. The α-Syn peptide immunogenic construct of claim 1, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 81, 83, and 84.

4. 2. The α-Syn peptide immunogen construct of claim 1, wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

5. The α-Syn peptide immunogenic construct of claim 1, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope.

6. The α-Syn peptide immunogenic construct of claim 1, wherein said T helper epitope is covalently linked to the amino terminus of said B cell epitope via said optional heterologous spacer.

7. The formula: (Th) ｍ - (A) ｎ -(C-terminal fragment of α-Syn)-X or (C-terminal fragment of α-Syn)-(A) ｎ - (Th) ｍ -X Including, During the ceremony, Th is the T helper epitope, A is the heterologous spacer; (C-terminal fragment of α-Syn) is the B-cell epitope, X is the α-COOH or α-CONH of an amino acid ２ and m is from 1 to about 4; n is 1 to about 10; The α-Syn peptide immunogenic construct of claim 1.

8. 2. The α-Syn peptide immunogenic construct of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

9. 2. The α-Syn peptide immunogenic construct of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, and 111-113.

10. A composition comprising the α-Syn peptide immunogenic construct of claim 1.

11. A composition comprising more than one α-Syn peptide immunogenic construct of claim 1.

12. The composition of claim 11, wherein the α-Syn peptide immunogenic construct has the amino acid sequence of SEQ ID NOs: 112 and 113.

13. A pharmaceutical composition comprising the α-Syn peptide immunogenic construct of claim 1 and a pharma- ceutically acceptable delivery vehicle and / or adjuvant.

14. a. the α-Syn peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; b. The adjuvant is Al(OH) ３ Or AlPO ４ is an inorganic salt of aluminum selected from the group consisting of The pharmaceutical composition of claim 13.

15. a. the α-Syn peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; b. The α-Syn peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immune stimulatory complex; The pharmaceutical composition of claim 13.

16. 2. An isolated antibody or epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogenic construct of claim 1.

17. 17. The isolated antibody or epitope-binding fragment thereof of claim 16 bound to the α-Syn peptide immunogenic construct.

18. 10. An isolated antibody or epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogenic construct of claim 9.

19. A composition comprising the isolated antibody or epitope-binding fragment thereof described in claim 16.

20. 20. A composition comprising the isolated antibody or epitope-binding fragment thereof of claim 18.

21. a. an isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of SEQ ID NO: 112; and b. an isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of SEQ ID NO: 113; and 21. The composition of claim 20 comprising a mixture of:

22. A method for producing antibodies that recognize α-Syn in a host, the method comprising administering to the host a composition comprising the α-Syn peptide immunogen of claim 1 and a delivery vehicle and / or adjuvant.

23. 13. A method for inhibiting α-Syn aggregation in an animal, comprising administering to said animal a pharmacologically effective amount of the α-Syn peptide immunogen of claim 1.

24. 13. A method for reducing the amount of α-Syn aggregates in an animal, comprising administering to said animal a pharmacologically effective amount of the α-Syn peptide immunogen of claim 1.

25. 1. A method for identifying α-Syn aggregates of different sizes in a biological sample, comprising: a. exposing a biological sample to an antibody or epitope-binding fragment thereof according to claim 16 under conditions that allow the antibody or epitope-binding fragment thereof to bind to the α-Syn aggregates; b. detecting the amount of said antibody or epitope-binding fragment thereof bound to said α-Syn aggregates in said biological sample; The method comprising:

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