Methods for preventing and treating synucleinopathies

Abstract

The present disclosure provides methods and compositions for preventing and treating synucleinopathy. In one aspect, the present invention provides a method of preventing, reducing, suppressing, or delaying the onset of one or more motor symptoms of synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of an immunotherapy that targets alpha-synuclein (alpha-syn). In some embodiments, the one or more motor symptoms of synucleinopathy are selected from the group consisting of muscle rigidity, bradykinesia, resting tremor, and postural instability. In another aspect, the present invention provides a method of treating, preventing, reducing, or suppressing one or more gastrointestinal symptoms of synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of an immunotherapy that targets alpha-syn.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. Said XML copy, created on August 31, 2022, is named 51615-002WO2_Sequence_Listing_8_31_22_ ​​and is 272,021 bytes in size.

[0002] The present disclosure relates to methods for preventing and treating synucleinopathies. [Background technology]

[0003] Synucleinopathies are chronic progressive neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain. Synucleinopathies include Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) (Outeiro et al., Mol. Neurodegener. 14(1):5, 2019). Although several treatment options are available for these diseases, they only provide symptomatic relief and do not directly target the underlying pathology. In the current aging population, PD and DLB cases are increasing, which highlights the urgent need to develop therapies that can prevent or slow the progression of neurodegeneration.

[0004] α-Syn is primarily an intracellular protein. However, many studies have shown that extracellular α-syn also plays a role in disease and is involved in the propagation of α-syn between neurons. α-Syn can be detected in the cerebrospinal fluid (CSF) and interstitial fluid (ISF) of the brain parenchyma (Emmanouilidou et al., PLoS One 6(7):e22225, 2011). α-Syn is primarily secreted from cells by exocytosis (Lee et al., J.Neurosci.25(25):6016-6024, 2005) or released directly into the extracellular space upon cell lysis and death. Internalization of proteins by neighboring cells leads to the formation of protein aggregates, which may result in the spread of disease to anatomically connected brain regions (Danzer et al., Mol. Neurodegener. 7:42, 2012; Lee et al., J. Biol. Chem. 285:9262-9272, 2010). This mechanism has been demonstrated in cell culture studies, where the addition of preformed fibrils of α-syn to primary neuronal cultures at concentrations equivalent to those found in CSF (0.1 ng / ml) induced the formation of LB-like inclusions by endogenous α-syn. This did not occur with monomeric α-syn, consistent with oligomeric and fibrillar α-syn species being the toxic species in PD (Volpicelli-Daley et al., Neuron 72:57-71, 2011).

[0005] The diagnosis of PD involves the observation of characteristic changes in motor function, including symptoms of bradykinesia, rigidity, and tremor. In PD, nonmotor or autonomic dysfunction typically precedes motor symptoms for up to 20 years (Yu et al., Scientific Reports 8(1):567, 2018; Postuma et al., Nat. Rev. Neurol. 12(11)622-634, 2016; Durcan et al., Eur. J. Neurol. 26(7):979-985, 2019). One of the most common nonmotor features of PD is gastrointestinal (GI) dysfunction (Fasano et al., The Lancet Neurology 14(6):625-639, 2015; Noyce et al., Annals of Neurology 72(6):893-901, 2012). Numerous independent population-based longitudinal studies of patients who develop PD disease have shown that over 50% of PD patients suffer from GI dysfunction (Mukhtar et al., BMJ Open 8(5):e019172, 2018). Additional features of PD that precede the onset of motor symptoms include, for example, hyposmia, REM sleep behavior disorder, excessive daytime sleepiness, depression, cognitive symptoms, and autonomic nervous system dysfunction (Crosiers et al., Front. Neurol. Doi.org / 10.3389 / fneur.2020634490, 2021), as well as anosmia, reduced color vision, impaired quantitative motor testing, and abnormal findings in substantia nigra neuroimaging. Approaches to treat the early premotor symptoms of synucleinopathies such as PD are needed, with the goal of reducing premotor symptoms and delaying, reducing, or preventing the onset of motor symptoms. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Outeiro et al.,Mol.Neurodegener.14(1):5,2019 [Non-Patent Document 2] Emmanouilidou et al.,PLoS One 6(7):e22225,2011 [Non-Patent Document 3] Lee et al.,J.Neurosci.25(25):6016-6024,2005 [Non-Patent Document 4] Danzer et al.,Mol.Neurodegener.7:42,2012 [Non-Patent Document 5] Lee et al.,J.Biol.Chem.285:9262-9272,2010 [Non-Patent Document 6] Volpicelli-Daley et al.,Neuron 72:57-71,2011 Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the invention provides a method for preventing, alleviating, inhibiting, or delaying the onset of one or more motor symptoms of a synucleinopathy in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunotherapy that targets alpha-synuclein (alpha-syn).

[0008] In some embodiments, the one or more motor symptoms of a synucleinopathy are selected from the group consisting of muscle rigidity, bradykinesia, resting tremor, and postural instability.

[0009] In another aspect, the present invention provides a method of treating, preventing, alleviating, or suppressing one or more gastrointestinal symptoms of a synucleinopathy in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunotherapy targeting alpha-syn.

[0010] In some embodiments, the one or more gastrointestinal symptoms are selected from the group consisting of sialorrhea, hypersalivation, dysphagia, nausea, vomiting, dyspepsia, constipation, abdominal pain, gastroparesis, and fecal incontinence.

[0011] In some embodiments, the gastrointestinal symptom occurs in the colon of the subject.

[0012] In another aspect, the present invention provides a method for reducing alpha-syn levels in the gastrointestinal tract (e.g., colon) in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunotherapy that targets alpha-syn.

[0013] In some embodiments, the subject does not have one or more motor symptoms of a synucleinopathy or exhibits only minimal motor symptoms of a synucleinopathy.

[0014] In some embodiments, the subject does not have one or more motor symptoms of a synucleinopathy selected from the group consisting of muscle rigidity, bradykinesia, resting tremor, and postural instability.

[0015] In some embodiments, the subject is suffering from an early, precursor stage of a synucleinopathy.

[0016] In another aspect, the invention provides a method for inducing an immune response to α-syn in a subject, inhibiting α-syn aggregation in a subject, or reducing the amount of α-syn aggregates in a subject, the method comprising administering to a subject an effective amount of an immunotherapy targeting α-syn, wherein the subject has an early precursor stage of a synucleinopathy.

[0017] In some embodiments, the synucleinopathy is selected from the group consisting of Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuroaxonal dystrophy, and pure autonomic failure (PAF).

[0018] In some embodiments, the immunotherapy comprises a peptide, a protein (e.g., an antibody), a fragment or fusion of a peptide or protein (e.g., an antibody), or a nucleic acid molecule (e.g., an mRNA or a nucleic acid in a vector) encoding one of these molecules.

[0019] In some embodiments, the immunotherapy comprises a peptide immunogen construct.

[0020] In some embodiments, a peptide immunogen construct comprises a B cell epitope, a heterologous T cell epitope, and an optional linker.

[0021] In some embodiments, the B cell epitope, for example, when present in a peptide immunogen construct, induces an immune response against α-syn.

[0022] In some embodiments, the B cell epitope comprises a peptide of the C-terminal region of an alpha-syn protein, which peptide is optionally about 10 to about 25 amino acids in length.

[0023] In some embodiments, the alpha-syn protein comprises the sequence of SEQ ID NO:1.

[0024] In some embodiments, the B cell epitope comprises a peptide selected from the sequences of Table 1 (e.g., any one of the peptides of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69).

[0025] In some embodiments, the heterologous T cell epitope is derived from a pathogenic protein.

[0026] In some embodiments, the heterologous T cell epitope comprises a sequence selected from a sequence in Table 2 (e.g., any one of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98).

[0027] In some embodiments, the peptide comprises a heterologous spacer or linker between the B cell epitope and the T cell epitope.

[0028] In some embodiments, the heterologous spacer or linker is selected from the group consisting of Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys.

[0029] In some embodiments, the B cell epitope is located N-terminal to the T cell epitope.

[0030] In some embodiments, the T cell epitope is located N-terminal to the B cell epitope.

[0031] In some embodiments, the peptide immunogen construct is selected from the sequences in Table 3 (e.g., any one of the constructs in SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147).

[0032] In some embodiments, the peptide immunogen construct comprises: (a) 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; (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 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 linked to the T helper epitope directly or via the optional heterologous spacer.

[0033] In some embodiments, the B cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17, and 49-63.

[0034] In some embodiments, the T helper epitope is selected from the group consisting of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98, e.g., selected from the group consisting of SEQ ID NOs: 81, 83, and 84.

[0035] In some embodiments, the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

[0036] In some embodiments, the T helper epitope is covalently linked to the amino terminus of the B cell epitope.

[0037] In some embodiments, the T helper epitope is covalently linked to the amino terminus of the B cell epitope via an optional heterologous spacer.

[0038] In some embodiments, the peptide immunogen construct has the following formula: m -(A) n-(α-Syn C-terminal fragment)-X or (α-Syn C-terminal fragment)-(A) n -(Th) m -X, where Th is a T helper epitope, A is a heterologous spacer, (α-Syn C-terminal fragment) is a B cell epitope, X is the amino acid α-COOH or α-CONH2, m is 1 to about 4, and n is 1 to about 10.

[0039] In some embodiments, the peptide immunogen construct comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

[0040] In some embodiments, the peptide immunogen construct is in a stabilized immune stimulatory complex with a CpG oligodeoxynucleotide (ODN).

[0041] In some embodiments, the immunotherapy is comprised in a composition that optionally comprises multiple immunotherapies, for example multiple peptide immunogen constructs.

[0042] In some embodiments, the composition comprises a peptide immunogen construct comprising the amino acid sequences of SEQ ID NOs:112 and 113.

[0043] In some embodiments, the composition is a pharmaceutical composition comprising the immunotherapy(s) and a pharma- ceutically acceptable delivery vehicle and / or adjuvant.

[0044] In some embodiments, the composition optionally comprises an adjuvant comprising an inorganic salt of aluminum selected from the group consisting of Al(OH) 3 and AlPO 4 .

[0045] In some embodiments, the (a) peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 1 SEQ ID NOs: 107, 108, 111-113, and 115-147; (b) the composition comprises an adjuvant which is an inorganic salt of aluminum selected from the group consisting of Al(OH)3 and AlPO4.

[0046] In some embodiments, the (a) peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 3 29, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147, for example, selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; (b) the peptide immunogen construct is in the form of a stabilized immune stimulatory complex with CpG ODN.

[0047] In some embodiments, the immunotherapy comprises an antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of a peptide immunogen construct described herein (e.g., SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 1 6, and 147), a B-cell epitope of SEQ ID NO: 1 (e.g., the C-terminal region of SEQ ID NO: 1), or a peptide of Table 1 (e.g., any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, and 69).

[0048] In some embodiments, the methods involve the use of two or more, three or more, four or more, or five or more immunotherapies.

[0049] In some embodiments, the subject is diagnosed with rapid eye movement (REM) sleep behavior disorder (RBD).

[0050] In some embodiments, the subject has one or more of hyposmia, REM sleep behavior disorder, excessive daytime sleepiness, depression, cognitive symptoms, autonomic nervous system dysfunction, loss of smell, reduced color vision, impaired quantitative motor testing, abnormal findings in substantia nigra neuroimaging, or other prodromal symptoms, e.g., as described herein.

[0051] In some embodiments, the subject does not have any or any significant bradykinesia, rigidity, and / or tremor, or other symptoms of a synucleinopathy that is not in the prodromal stage.

[0052] In some embodiments, the immunotherapy comprises or consists of a peptide immunogen construct comprising or consisting of SEQ ID NO:112.

[0053] In another aspect, the invention provides a composition or kit for use in practicing any one of the methods described herein.

[0054] In another aspect, the present invention provides a composition as described herein for use in treating, inhibiting, preventing, ameliorating, alleviating, suppressing, or delaying the onset of one or more diseases or conditions as described herein, or one or more symptoms thereof.

[0055] In another aspect, the present invention provides the use of one or more compositions described herein for the manufacture of a medicament for treating, inhibiting, reducing, preventing, ameliorating, alleviating, suppressing, or delaying the onset of one or more diseases or conditions described herein, or one or more symptoms thereof.

[0056] In other aspects, the invention provides a composition or kit as described herein for use in the preparation of a medicament for carrying out any of the methods described herein.

[0057] In another aspect, the invention provides methods of producing the compositions and kits described herein for use in treating, inhibiting, reducing, preventing, ameliorating, alleviating, suppressing, or delaying the onset of one or more diseases or conditions described herein, the methods comprising, for example, mixing the components thereof. [Brief description of the drawings]

[0058] [Figure 1]Schematic diagram of the immunization regime. Ten-week-old Thy1SNCA / 15 mice and wild-type controls were administered either UB312 or adjuvant by intramuscular injection at 3-week intervals. Blood samples were taken for antibody titer analysis before each injection and at weeks 10 and 15 (final time point). Behavioral testing was performed before the start of immunotherapy and at the end of the 15-week study period. [Diagram 2] Antibody titer analysis. Ten-week-old Thy1SNCA / 15 mice and wild-type littermates were administered three intramuscular injections of either UB312 or adjuvant at three-week intervals. Blood samples were collected before each injection, at weeks 9 and 15 (final time point), and antibody titers were measured for each of the collected sera. Data points represent the mean ± 95% CI. [Diagram 3] Behavioral analysis. 10-week-old Thy1SNCA / 15 mice and wild-type littermates were subjected to three different motor performance tests before immunization (pre-immunization) and 15 weeks after the first injection (post-immunization). These included the challenging beam crossing test, the pole test, and the wire hanging test. Bars represent the mean ± 95% CI. [Figure 4] Immunohistochemistry for α-syn. α-Syn immunoreactivity was quantified in the cortex, hippocampus, striatum, and substantia nigra of Thy1SNCA / 15 mice treated with UB312 (n=12) or adjuvant (n=11). Two-tailed T-tests showed no differences in the mean area percentage of α-syn immunoreactivity between treatment groups. DAPI was used to visualize cell nuclei. Scale bar: 50 μm. [Diagram 5] Western blot analysis of α-syn oligomers. α-Syn assemblies in brain homogenates from the cortex, striatum, and hippocampus of Thy1SNCA / 15 mice treated with UB312 (n=14) or adjuvant (n=16) were resolved by non-denaturing Western blot. Quantitation of oligomeric and monomeric immunoreactive bands showed a significant reduction in α-syn oligomers, but not monomers, upon UB312 treatment. Bars represent the mean ± 95% CI. [Figure 6]Immunohistochemistry of microglia. Iba1 immunoreactivity was quantified in the cortex, hippocampus, striatum, and substantia nigra of Thy1SNCA / 15 mice treated with UB312 (n=11) or adjuvant (n=9) or wild-type littermates treated with adjuvant (n=8). One-way ANOVA with Bonferroni correction showed a significant increase in the mean area percentage of Iba1 in the substantia nigra. Bars represent the mean ± 95% CI. [Figure 7] Immunohistochemistry for astrocytes. GFAP immunoreactivity was quantified in the cortex, hippocampus, striatum, and substantia nigra of Thy1SNCA / 15 mice treated with UB312 (n=11) or adjuvant (n=9) or wild-type littermates treated with adjuvant (n=8). One-way ANOVA showed no differences between treatment groups in each brain region. Bars represent the mean ± 95% CI. [Figure 8] Immunohistochemistry for endothelial activation and T cell infiltration. ICAM1 immunoreactivity was quantified in the cortex, hippocampus, striatum, and substantia nigra of Thy1SNCA / 15 mice treated with UB312 (n=11) or adjuvant (n=9) or wild-type littermates treated with adjuvant (n=8). One-way ANOVA showed no differences between treatment groups in each brain region. CD3+ T cells were counted in whole brain sections. Bars represent the mean ± 95% CI. [Figure 9]Immunohistochemistry of α-syn and enteric glial cell (EGC) activation in the gastrointestinal tract. Immunofluorescence shows α-syn immunoreactivity in the duodenum and colon muscle (arrows) with DAPI counterstain. Two-tailed t-tests showed a significant decrease in the mean area percentage of α-syn in the colonic muscle layer after UB312 immunotherapy compared to adjuvant in Thy1SNCA / 15 mice. EGC reactivity was measured by quantifying GFAP immunoreactivity in the myenteric ganglion. One-way ANOVA showed a significant decrease in the mean area percentage of GFAP in the colon of Thy1SNCA / 15 mice treated with UB312 when compared to adjuvant or wild-type littermates receiving adjuvant. There was no effect of UB312 immunotherapy in the duodenum on α-syn or GFAP expression levels. Bars represent the mean ± 95% CI. [Figure 10] Optimization of antibody concentrations for Western blot analysis. A shows the determination of the linear range of α-syn (MJFR1) and total protein (Revert, Licor). Brain homogenates were loaded at increasing concentrations from 1 to 50 μg protein in a 12% non-denaturing gel. Quantification of α-syn immunoreactive bands and total protein is shown on the right. B shows transmission electron micrographs (TEM) of autologous synthesized α-syn monomers (right) compared to after fibrillization (left). Monomeric α-syn was used as a molecular weight marker for Western blot analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The present disclosure is based, in part, on the discovery that immunotherapy directed against alpha-synuclein (alpha-syn) can be used to prevent or reduce the onset or early development of motor symptoms characteristic of synucleinopathies, as well as to reduce alpha-syn levels in the gastrointestinal tract.

[0060] Thus, the present disclosure relates to a method for preventing or reducing the onset of one or more motor symptoms of synucleinopathy in a subject in need thereof by using immunotherapy targeting alpha-syn. The present disclosure also relates to a method for treating, preventing, reducing, or suppressing one or more gastrointestinal symptoms of synucleinopathy or asymptomatic gastrointestinal disease using such an approach. Furthermore, the present disclosure relates to a method for reducing the level of alpha-syn in the gastrointestinal tract of a subject in need thereof using such an approach. As an advantage, in some embodiments, the method of the present disclosure can be used to treat subjects in the early stages of synucleinopathy development, which can provide a substantial benefit for preventing or delaying the onset of the disease, improving quality of life and extending the healthy period.

[0061] The methods of the disclosure, including the molecules and compositions used herein, are described in the exemplary methods below.

[0062] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references or portions of references cited in this application are expressly incorporated herein by reference in their entirety for all purposes.

[0063] Unless otherwise indicated, all 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 pertains. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, "comprising A or B" means including A or B, or A and B. Furthermore, it should 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, suitable methods and materials are described below.

[0064] 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. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0065] Immunotherapeutic agents and compositions The methods of the disclosure may include, for example, the use of one or more peptides, proteins (e.g., antibodies), fragments or fusions of peptides or proteins (e.g., antibodies), or nucleic acid molecules (e.g., mRNA or nucleic acid in a viral vector) encoding one of the above molecules, which molecules are directed against alpha-syn.

[0066] peptide In some embodiments, the methods of the disclosure use peptide immunogen constructs that include a-syn derived B cell epitopes linked directly or via any heterologous spacer to heterologous T helper cell (Th) epitopes. Constructs such as these are described, for example, in WO2018 / 232369, the contents of which are incorporated herein by reference.

[0067] The B cell epitope portion of the peptide immunogen construct may optionally comprise, for example, about 10 to about 25 amino acid residues from the C-terminus of α-syn, corresponding to the sequence from about glycine at amino acid position 111 (G111) to about asparagine at amino acid position 135 (D135) of full-length α-syn (SEQ ID NO: 1). The heterologous Th epitope portion of the peptide immunogen construct may optionally be derived from a pathogenic protein. The B cell epitope portion and the Th epitope portion of the peptide immunogen construct, when administered to a subject, act together to stimulate the production of antibodies that specifically recognize and bind to the α-syn B cell epitope portion of the construct.

[0068] Thus, as used herein, the phrase "α-syn peptide immunogen construct" refers to a peptide containing (a) a B cell epitope having about 10 to about 25 amino acid residues from the C-terminus of α-syn, corresponding to the sequence from about glycine at amino acid position 111 (G111) to about asparagine at amino acid position 135 (D135) of full-length α-syn (SEQ ID NO: 1), (b) a heterologous Th epitope, and (c) an optional heterologous spacer.

[0069] In certain embodiments, the peptide immunogenic construct has the formula: m -(A) n -(α-Syn C-terminal fragment)-X or (α-Syn C-terminal fragment)-(A) n -(Th) m-X, where Th is a heterologous T helper epitope, A is a heterologous spacer, (α-Syn C-terminal fragment) is a B cell epitope having about 10 to about 25 amino acid residues from the C-terminus of α-Syn, X is an amino acid α-COOH or α-CONH2, m is an integer from 1 to about 4, and n is an integer from 0 to about 10. In some embodiments, A is an amino acid and n indicates the number of amino acids, and each A can be identical to one another or one or more of the A can be different amino acids. The various components of the α-syn peptide immunogenic constructs of the present disclosure are described below.

[0070] α-Syn and α-Syn C-terminal fragments The terms "α-syn," "alpha-synuclein," "α-synuclein," and the like, as used herein, refer to (a) full-length α-syn protein and / or (b) fragments thereof from any organism that expresses α-Syn. In some embodiments, the α-syn protein is human. In certain embodiments, the full-length human α-syn protein has 140 amino acids (Accession No. NP_000336) (SEQ ID NO: 1).

[0071] The phrase "C-terminal region" or "C-terminus" of α-syn, as used herein, 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 refers to the amino acid sequence between residues 96-140 of α-syn, or a fragment thereof.

[0072] As used herein, the phrase "α-syn C-terminal fragment" or "B-cell epitope from the C-terminus of α-syn" refers to a portion of the full-length α-syn sequence comprising about 10 to about 25 amino acid residues from the C-terminus of α-syn, corresponding to the sequence from about glycine at amino acid position 111 (G111) to about asparagine at amino acid position 135 (D135) of full-length α-syn. The α-syn C-terminal fragment is also referred to herein as α-syn G111-D135 peptide and fragments thereof. The various α-syn C-terminal fragments 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.

[0073] In some embodiments, the α-syn C-terminal fragment is the 25 amino acid α-syn G111-D135 peptide represented by SEQ ID NO: 12. In other embodiments, the α-syn C-terminal fragment comprises about 10 contiguous amino acids of the α-syn G111-D135 peptide represented by SEQ ID NO: 12. In certain embodiments, the α-syn C-terminal fragment 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 some embodiments, the alpha-syn C-terminal fragment has a sequence selected from any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, and 69. In certain embodiments, the α-syn C-terminal fragment has an amino acid sequence represented by one of SEQ ID NOs: 12-15, 17, or 49-64, as shown in Table 1.

[0074] In some embodiments, the B cell epitope of the peptide immunogen construct comprises or consists of any one of the peptides of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69.

[0075] The alpha-syn C-terminal fragments of the present disclosure also include immunologically functional analogs or homologs of the alpha-syn G111-D135 peptide or fragments thereof. Functional immunological analogs or homologs of the alpha-syn G111-D135 peptide or fragments thereof include variants that retain substantially the same immunogenicity as the original peptide. Immunologically functional analogs may have one or more conservative substitutions at an amino acid position, an overall charge change, covalent linkage to another moiety, amino acid additions, insertions, or deletions, and / or any combination thereof.

[0076] Conservative substitutions are those that replace one amino acid residue with another that has similar chemical properties, e.g., 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.

[0077] Immunologically functional analogs include amino acid sequences that contain conservative substitutions, additions, deletions, or insertions of one to about four amino acid residues that cross-react with the α-syn G111-D135 peptide and elicit an immune response. Conservative substitutions, additions, and insertions can be accomplished using natural or unnatural amino acids. Unnatural amino acids include 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-nitro-tyrosine, pyroglutamic acid, and the like. 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.

[0078] In one embodiment, an immunologically functional 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 α-syn G111-D135 peptide (or a fragment thereof). In this embodiment, the addition of the three lysine residues to the original peptide sequence changes the overall charge of the original peptide, but does not change the function of the original peptide.

[0079] Also included are functional analogs or homologs of other peptides described herein (see list above and Table 1; e.g., SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, and 69).

[0080] In certain embodiments, functional analogs of α-syn C-terminal fragments have at least 50% sequence identity to the original amino acid sequence. In other embodiments, functional analogs have at least 80% identity to the original amino acid sequence. In yet other embodiments, functional analogs have at least 85% identity to the original amino acid sequence. In yet other embodiments, functional analogs have at least 90% or at least 95% identity to the original amino acid sequence. The percentage of identity between two sequences can be determined manually by inspecting two optimally aligned sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters as known in the art.

[0081] Heterologous T helper cell epitopes (Th epitopes) The peptide immunogen constructs used in the disclosed methods comprise a-syn derived B cell epitopes covalently linked to heterologous T helper cell (Th) epitopes either directly or via an optional heterologous spacer. The heterologous Th epitopes in the a-syn peptide immunogen constructs enhance the immunogenicity of the a-syn C-terminal fragment, which promotes the production of specific, high titer antibodies against the targeted B cell epitopes (i.e., the a-syn C-terminal fragments) optimized through rational design.

[0082] As used herein, the term "heterologous" refers to an amino acid sequence that is not part of the wild-type sequence of α-syn or is derived from an amino acid sequence that is not homologous to the wild-type sequence. Thus, a heterologous Th epitope is a Th epitope that is derived from an amino acid sequence that is not naturally found in α-syn (i.e., the Th epitope is not autologous to α-syn). Because a Th epitope is heterologous to α-syn, when the heterologous Th epitope is covalently attached to the α-syn C-terminal fragment, the native amino acid sequence of α-syn is not extended in either the N-terminal or C-terminal direction.

[0083] A heterologous Th epitope of the present disclosure can be any Th epitope that does not have an amino acid sequence naturally found in α-syn. The Th epitope can have an amino acid sequence derived from any species (e.g., human, porcine, bovine, canine, rat, mouse, guinea pig, etc.) or pathogen (e.g., measles virus or hepatitis virus (e.g., hepatitis virus surface protein, see below). The Th epitope can also have promiscuous binding motifs for MHC class II molecules of multiple species. In certain embodiments, the Th epitope contains multiple promiscuous MHC class II binding motifs that allow for maximal activation of helper T cells that lead to the initiation and regulation of the immune response. The Th epitope is preferably immune silent by itself, i.e., few, if any, antibodies generated by the α-syn peptide immunogen construct are directed against the Th epitope, allowing for a highly focused immune response directed against the target B cell epitope of the α-syn C-terminal fragment.

[0084] Epitopes include, but are not limited to, amino acid sequences derived from foreign pathogens as exemplified in Table 2 (SEQ ID NOs: 70-98). Additionally, 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 shown at specific positions in a peptide framework based on the variable residues of homologs to that particular peptide. A collection of combinatorial peptides can be synthesized in one process by adding a mixture of designated protected amino acids in place of one specific amino acid to a designated position in the synthesis process. Such combinatorial heterologous Th epitope peptide collections can cover a wide range of Th epitopes for animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 79-82 and are shown in Table 2. The Th epitope peptides of the present invention provide broad reactivity and immunogenicity across animals and patients from genetically diverse populations.

[0085] Thus, the Th epitope of the peptide immunogen construct can be selected from any one of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98, and their immunologically functional analogs (see below).

[0086] α-Syn peptide immunogen constructs containing Th epitopes can be generated in tandem with the α-syn C-terminal fragments simultaneously in a single solid-phase peptide synthesis. Th epitopes also include immunological analogs of the Th epitopes. Immunological Th analogs include immune enhancing analogs, cross-reactive analogs, and fragments of any of these Th epitopes sufficient to enhance or stimulate an immune response to the α-syn C-terminal fragments.

[0087] Immunologically functional analogs of Th epitope peptides are also effective and can be used in the methods of the present disclosure. Immunologically functional 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 essentially alter the Th stimulatory function of the Th epitope. Conservative substitutions, additions, and insertions can be accomplished using natural or non-natural amino acids, as described above for α-syn C-terminal fragments. Table 2 identifies another variation of functional analogs to Th epitope peptides. In particular, MvF1 and MvF2 Th of SEQ ID NOs: 71 and 78 are functional analogs of MvF4 and MvF5 of SEQ ID NOs: 81 and 83, which differ in the amino acid frame by the 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 epitopes contained within these sequences. Thus, functional immunological Th analogs may include, for example, several versions of Th epitopes from measles virus fusion protein MvF1-4 Th (sequence numbers 71, 78, 79, 81, and 83), and from hepatitis surface protein HBsAg 1-3 Th (sequence numbers 80, 82, and 84).

[0088] The Th epitope in the α-syn peptide immunogen construct can be covalently attached to either the N-terminus or the C-terminus of the α-syn C-terminal peptide. In some embodiments, the Th epitope is covalently attached to the N-terminus of the α-syn C-terminal peptide. In other embodiments, the Th epitope is covalently attached to the C-terminus of the α-syn C-terminal peptide. In certain embodiments, multiple Th epitopes are covalently attached to the α-syn C-terminal fragment. When multiple Th epitopes are linked to the α-syn C-terminal fragment, each Th epitope can have the same or different amino acid sequences. Furthermore, when multiple Th epitopes are linked to the α-syn C-terminal fragment, the Th epitopes can be arranged in any order. For example, the Th epitopes can be linked contiguously to the N-terminus of the α-syn C-terminal fragment, or contiguously to the C-terminus of the α-syn C-terminal fragment, or one Th epitope can be covalently attached to the N-terminus of the α-syn C-terminal fragment while another Th epitope is covalently attached to the C-terminus of the α-syn C-terminal fragment. There are no limitations on the placement of the Th epitopes relative to the α-syn C-terminal fragment.

[0089] In some embodiments, the Th epitope is covalently linked directly to the α-syn C-terminal fragment, while in other embodiments, the Th epitope is covalently linked to the α-syn C-terminal fragment via a heterologous spacer, which is described in more detail below.

[0090] Heterogeneous Spacers The α-syn peptide immunogen constructs optionally contain a heterologous spacer that covalently links the α-syn-derived B cell epitope to a heterologous helper T cell (Th) epitope.

[0091] As stated above, the term "heterologous" refers to an amino acid sequence that is derived from an amino acid sequence that is not part of or homologous to the wild-type sequence of α-syn. Thus, the native amino acid sequence of α-syn is not extended in either the N- or C-terminal direction when a heterologous spacer is covalently linked to a B-cell epitope derived from α-syn, since the spacer is heterologous to α-syn.

[0092] A spacer is any molecule or chemical structure that can link two amino acids and / or peptides together. Spacers can vary in length or polarity depending on the application. The attachment of the spacer can be via an amide bond or a carboxyl-bond, although other functional groups are possible as well. Spacers can include chemical compounds, natural amino acids, or unnatural amino acids.

[0093] Spacers 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 α-syn C-terminal fragment. The physical separation by the spacer can disrupt any artificial secondary structure that arises from the binding of 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. Furthermore, the spacer can be designed to generate or modify the secondary structure of the peptide immunogen construct. For example, the spacer can be designed to act as a flexible hinge to facilitate the separation of the Th and B cell epitopes. The flexible hinge spacer can also allow for more efficient interaction between the presented peptide immunogen and the appropriate Th and B cells to enhance the immune response to the Th and B cell epitopes. An example of a flexible hinge sequence is found in the immunoglobulin heavy chain hinge region, which is often proline-rich.One particularly useful flexible hinge that can be used as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 149), where Xaa is any amino acid, for example, aspartic acid.

[0094] Spacers can also provide functional features to the α-syn peptide immunogen constructs. For example, spacers can be designed to change the overall charge of the α-syn peptide immunogen construct, which can affect the solubility of the peptide immunogen construct. In addition, changes in the overall charge of the α-syn peptide immunogen construct can affect the ability of the peptide immunogen construct to associate with other compounds and reagents. As discussed in more detail below, the α-syn peptide immunogen constructs can be formed into stable immunostimulatory complexes with highly charged oligonucleotides, such as CpG oligomers, via electrostatic binding. The overall charge of the α-syn peptide immunogen constructs is important for the formation of these stable immunostimulatory complexes.

[0095] 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-amino-dodecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

[0096] 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.

[0097] Unnatural amino acids include 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-nitro-tyrosine, pyroglutamic acid, and the like.

[0098] The spacer in the α-syn peptide immunogen construct may be covalently attached to either the N-terminus or C-terminus of the Th epitope and the α-syn C-terminal peptide. In some embodiments, the spacer is covalently attached to the C-terminus of the Th epitope and the N-terminus of the α-syn C-terminal peptide. In other embodiments, the spacer is covalently attached to the C-terminus of the α-syn C-terminal peptide and the N-terminus of the Th epitope. In certain embodiments, for example, when multiple Th epitopes are present in the peptide immunogen construct, multiple spacers can be used. When multiple spacers are used, each spacer can be the same as or different from each other. Furthermore, when multiple Th epitopes are present in the peptide immunogen construct, the Th epitopes can be separated by a spacer that can be the same as or different from the spacer used to separate the Th epitope from the B cell epitope. There are no limitations on the placement of the spacer relative to the Th epitope or the α-syn C-terminal fragment.

[0099] In certain embodiments, the heterologous spacer is a natural or unnatural amino acid. In other embodiments, the spacer contains multiple natural or unnatural amino acids (e.g., the spacer is a peptide). The spacer can include one or more Lys (e.g., 1, 2, 3, 4, 5, or 6) and / or one or more Gly (e.g., 1, 2, 3, 4, 5, or 6). 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).

[0100] Specific Embodiments of Alpha-Syn Peptide Immunogen Constructs The α-syn peptide immunogen construct can be represented by the following formula: (Th) m -(A) n -(α-syn C-terminal fragment)-X or (α-syn C-terminal fragment)-(A) n -(Th) m -X, where Th is a heterologous T helper epitope, A is a heterologous spacer, (α-Syn C-terminal fragment) is a B cell epitope having about 10 to about 25 amino acid residues from the C-terminus of α-Syn, X is an amino acid α-COOH or α-CONH2, m is an integer from 1 to about 4, and n is an integer from 0 to about 10. In some embodiments, A is an amino acid and n indicates the number of amino acids, and each A can be identical to one another, or one or more of the As can be different amino acids.

[0101] In certain embodiments, the heterologous Th epitope in the α-syn peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 70-98, or a combination thereof, as set forth in Table 2. 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 multiple Th epitopes.

[0102] In certain embodiments, the optional heterologous spacer is selected from 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).

[0103] In certain embodiments, the α-syn C-terminal fragment has about 10 to about 25 amino acid residues from the C-terminus of α-syn, corresponding to the sequence from about glycine at amino acid position 111 (G111) to about asparagine at amino acid position 135 (D135) of full-length α-syn. In some embodiments, the alpha-syn C-terminal fragment has the amino acid sequence of any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, and 69. In certain embodiments, the α-syn C-terminal fragment has an amino acid sequence represented by SEQ ID NO: 12-15, 17, or 49-64, as shown in Table 1.

[0104] In certain embodiments, the α-syn peptide immunogenic construct has an amino acid sequence in Table 3, e.g., a 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 immunogenic construct has an amino acid sequence selected from any of SEQ ID NOs: 107-108 and 111-113.

[0105] In some embodiments, an alpha-syn peptide immunogen construct may comprise or consist of any one of SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147, as well as homologs, analogs, fragments, and / or combinations thereof.

[0106] composition The peptide immunogen constructs can be included in compositions, such as pharmaceutical compositions, that can induce an immune response and antibody production against the peptide immunogen constructs in a subject (e.g., a human patient). The disclosed compositions can include one peptide immunogen construct or a mixture of multiple peptide immunogen constructs. Furthermore, in some embodiments, the compositions include the peptide immunogen construct(s) together with one or more additional components, such as carriers, adjuvants, buffers, and other suitable reagents. In some embodiments, the compositions include the peptide immunogen constructs in the form of stabilized immune stimulatory complexes with CpG oligomers, optionally with the addition of adjuvants.

[0107] Compositions containing the disclosed alpha-syn peptide immunogenic constructs may be in liquid or solid form. Liquid compositions may contain water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structural or functional properties of the alpha-syn peptide immunogenic constructs. Peptide compositions may contain one or more of the disclosed alpha-syn peptide immunogenic constructs.

[0108] Pharmaceutical Compositions The methods of the disclosure can utilize pharmaceutical compositions containing the disclosed alpha-syn peptide immunogenic construct(s).

[0109] A pharmaceutical composition may contain a carrier and / or other additives in a pharma- ceutically acceptable delivery system. Thus, a pharmaceutical composition may contain a pharma- ceutically effective amount of an α-syn peptide immunogen construct together with a pharma- ceutically acceptable carrier, adjuvant, and / or other excipients, such as diluents, additives, stabilizers, preservatives, solubilizers, buffers, and the like.

[0110] The pharmaceutical composition may contain one or more adjuvants that act to accelerate, prolong, or enhance the immune response to the α-syn peptide immunogen construct without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition may include oils, aluminum salts, virosomes, aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), liposin, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA35, ISA206, ISA50V, ISA51, ISA720, and other adjuvants and emulsifiers.

[0111] In some embodiments, the pharmaceutical composition contains Montanide™ ISA51 (an oil adjuvant composition consisting of vegetable oil and mannide oleate to produce a water-in-oil emulsion), 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 that includes Emulsigen or Emulsigen D as an adjuvant.

[0112] The pharmaceutical compositions can be formulated for immediate or sustained release. Additionally, the pharmaceutical compositions can be formulated to induce systemic or local mucosal immunity through entrapment of immunogens and co-administration with microparticles. Such delivery systems are readily determined by those skilled in the art.

[0113] The pharmaceutical composition can be prepared as an injection, either as a liquid solution or suspension. A 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 application mode, such as intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intranasal, oral, etc., and by using any suitable formulation or delivery device.

[0114] The pharmaceutical composition may also be formulated in a suitable dosage unit. In some embodiments, the pharmaceutical composition contains about 0.5 μg to about 1 mg of α-syn peptide immunogenic construct per kg body weight. In some embodiments, the pharmaceutical composition contains 10-1000 μg, e.g., 20-500 μg, 50-400 μg, or 100-300 μg of an immunotherapy (e.g., peptide immunogenic construct) described herein. The effective dose of the pharmaceutical composition will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other agents administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. When delivered in multiple doses, the pharmaceutical composition may be conveniently divided into appropriate amounts per dosage unit, as determined to be appropriate by one of skill in the art. As is well known in the therapeutic arts, the dosage will depend on the age, weight, and overall health of the subject.

[0115] In some embodiments, the pharmaceutical composition contains multiple α-syn peptide immunogenic constructs and / or antibodies. The pharmaceutical composition may contain a mixture of multiple α-syn peptide immunogenic constructs (and / or antibodies) to synergistically enhance the immunological efficacy of the constructs. Pharmaceutical compositions containing multiple α-syn peptide immunogenic constructs may be more effective in a larger genetic population due to broader MHC class II coverage, thus improving the immune response to the α-syn peptide immunogenic constructs.

[0116] In some embodiments, the pharmaceutical composition contains an alpha-syn peptide immunogenic construct selected from SEQ ID NOs: 107, 108, 111-113, and 115-147, and homologs, analogs, fragments, and / or combinations thereof. In certain embodiments, the pharmaceutical composition contains an alpha-syn peptide immunogenic construct selected from SEQ ID NOs: 107, 108, 111-113, and any combination thereof.

[0117] In some embodiments, an alpha-syn peptide immunogen construct may comprise or consist of any one of SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147, as well as homologs, analogs, fragments, and / or combinations thereof.

[0118] A pharmaceutical composition containing an α-syn peptide immunogenic construct can be used to elicit an immune response and generate antibodies in a subject upon administration. In some embodiments, the pharmaceutical composition described herein is administered to a subject one, two, three, four, five, or more times, as determined by one of skill in the art. The composition can be administered, for example, at an initial dose, followed by one or more (e.g., two, three, four, five, or more) booster doses. In some embodiments, an initial dose is administered at week 1, followed by a dose at week 5, and an additional dose at week 13. In some embodiments, the amount of each dose is the same (e.g., 100 μg or 300 μg, see also above). In some embodiments, the amount of each dose can vary as may be determined by one of skill in the art. For example, an initial dose of 40 μg can be administered, followed by doses of 100 μg, 300 μg, or 1000 μg in subsequent administrations (e.g., at weeks 5 and 13).

[0119] immune stimulating complex The method of the present disclosure can also utilize pharmaceutical compositions containing α-syn peptide immunogen constructs in the form of immune stimulating complexes with CpG oligonucleotides. Such immune stimulating complexes are specifically adapted to act as adjuvants and peptide immunogen stabilizers. The immune stimulating complexes are in particulate form, which can efficiently present the α-syn peptide immunogen to cells of the immune system to generate an immune response. The immune stimulating complexes may be formulated as suspensions for parenteral administration. The immune stimulating complexes can also be formulated in the form of water-in-oil emulsions, as suspensions combined with inorganic salts or in-situ gelling polymers, to efficiently deliver the α-syn peptide immunogen to cells of the immune system of a subject after parenteral administration.

[0120] The stabilized immune stimulating complex may be formed by complexing the α-syn peptide immunogen construct with an anionic molecule, an oligonucleotide, a polynucleotide, or a combination thereof via electrostatic binding. The stabilized immune stimulating complex may be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0121] In certain embodiments, the α-syn peptide immunogen construct is designed to contain a cationic moiety that is positively charged at a pH ranging from 5.0 to 8.0. The net charge of the cationic moiety of the α-syn peptide immunogen construct or mixture of constructs is calculated by assigning a charge of +1 to each lysine (K), arginine (R), or histidine (H) in the sequence, a charge of -1 to each aspartic acid (D) or glutamic acid (E), and a charge of 0 to other amino acids. The charges are summed within the cationic moiety of the α-syn peptide immunogen construct and expressed as a net average charge. A suitable peptide immunogen has a cationic moiety with a net average positive charge of +1. In some embodiments, the peptide immunogen has a net positive charge in the range of greater than +2. In some embodiments, the cationic moiety of the α-syn peptide immunogen construct is a heterologous spacer. In a specific embodiment, the cationic moiety of the α-syn peptide immunogen construct has a charge of +4 when the spacer sequence is (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

[0122] As used herein, an "anionic molecule" refers to any molecule that is negatively charged at a pH ranging from 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules having 8 to 64 nucleotide bases, and the number of repeats of the CpG motif ranges from 1 to 10. In some embodiments, the CpG immunostimulatory single-stranded DNA molecule contains 18 to 48 nucleotide bases, and the number of repeats of the CpG motif ranges from 3 to 8.

[0123] In some embodiments, the anionic oligonucleotide has the formula: 5′ X 1 CGX 2 3', where C and G are unmethylated, and X 1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine); 2 is C (cytosine) or T (thymine). In other embodiments, the anionic oligonucleotide has the formula: 5' (X 3 )2CG(X 4 ) 23', in which C and G are not methylated, and X 3 is selected from the group consisting of A, T, or G; 4 is C or T.

[0124] The resulting immunostimulating complex is usually in the form of particles with sizes ranging from 1 to 50 microns, which is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species. Micronized immunostimulating complexes have the advantage of providing in vivo adjuvantization and upregulation of specific immune responses. Furthermore, the stabilized immunostimulating complexes are suitable for preparing pharmaceutical compositions by a variety of processes including water-in-oil emulsions, inorganic salt suspensions, and polymeric gels.

[0125] The α-syn peptide immunogen constructs used in the methods of the present disclosure can be made using chemical synthesis methods well known in the art (see, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77). For example, the α-syn peptide immunogen constructs can be synthesized using automated Merrifield techniques of solid phase synthesis, for example, using α-NH2 protected by either t-Boc or F-moc chemistry with side-chain protected amino acids on an Applied Biosystems Peptide Synthesizer Model 430A or 431. Preparation of α-syn peptide immunogen constructs, including combinatorial library peptides against Th epitopes, can be accomplished by providing a mixture of alternative amino acids for coupling at a given variable position. 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 on the amino acid side chains. The free peptide can be purified by HPLC and characterized biochemically, for example, by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to those of skill in the art.

[0126] The quality of the peptides produced by this chemical process can be controlled and defined, thereby ensuring the reproducibility, immunogenicity, and yield of the α-syn peptide immunogen constructs. A detailed description of the production of α-syn peptide immunogen constructs by solid phase peptide synthesis is provided in Example 1 of WO2018 / 232369.

[0127] The range of structural variability that allows for the retention of the intended immunological activity has been found to be much more flexible than that that allows for the retention of specific drug activity by small molecule drugs, or the desired activity and undesired toxicity observed in macromolecular drugs co-produced with biologically derived drugs. Thus, intentionally designed peptide analogs, or those inevitably generated by errors in the synthesis process as a mixture of deleted sequence by-products with similar chromatographic and immunological properties as the intended peptide, are often as effective as purified preparations of the desired peptide. Mixtures of designed and unintended analogs are effective if discriminatory QC procedures are developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and efficacy of the final product using these peptides.

[0128] The alpha-syn peptide immunogenic constructs can also be produced using recombinant DNA technology, including the use of nucleic acid molecules, vectors, and / or host cells. Thus, nucleic acid molecules encoding the alpha-syn peptide immunogenic constructs and immunologically functional analogs thereof are also included in this disclosure as part of the invention. Similarly, vectors, such as expression vectors, that contain the nucleic acid molecules, and host cells harboring the vectors are also included in this disclosure as part of the invention.

[0129] Various exemplary embodiments also encompass methods of producing immunologically functional analogs of the α-syn peptide immunogen constructs and α-syn G111-D135 fragment derived peptide immunogen constructs. For example, the methods may include incubating a host cell containing an expression vector containing a nucleic acid molecule encoding the α-syn peptide immunogen construct and / or its immunologically functional analog under conditions such that the peptide and / or analog is expressed. Longer synthetic peptide immunogens may 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 the peptide of the invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with optimal codons for the organism in which the gene is to be expressed. The synthetic gene is then typically made by synthesizing oligonucleotides encoding the peptide and any regulatory elements as required. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0130] Methods for producing immune stimulating complexes As mentioned above, the method of the present disclosure can further use immune stimulating complexes comprising α-syn peptide immunogen constructs and CpG oligodeoxynucleotide (ODN) molecules. Stabilized immune stimulating complexes (ISCs) are derived from the cationic portion of the α-syn peptide immunogen construct and polyanionic CpG ODN molecules. The self-assembly system is driven by electrostatic neutralization of charges. The extent of association is determined by the stoichiometry of the molar charge ratio of the cationic portion of the α-syn peptide immunogen construct to the anionic oligomer. The non-covalent electrostatic binding of the α-syn peptide immunogen construct to the CpG ODN is a completely reproducible process. The peptide / CpG ODN immune stimulating complex aggregates promote presentation to "professional" antigen presenting cells (APCs) of the immune system, thus further enhancing the immunogenicity of the complex. These complexes are easily characterized for quality control during manufacturing. The peptide / CpG ISCs are well tolerated in vivo. This particle system, containing CpG ODN and an α-syn G111-D135 fragment-derived peptide immunogen construct, is designed to take advantage of the generalized B cell mitogenicity associated with the use of CpG ODN, while also promoting a balanced Th-1 / Th-2 type response.

[0131] The CpG ODN in the disclosed pharmaceutical composition is 100% bound to the immunogen in a process mediated by electrostatic neutralization of the opposite charge, resulting in the formation of micron-sized particles. This particulate form allows the dose of CpG to be significantly reduced compared to the conventional use of CpG adjuvants, reducing the possibility of adverse innate immune responses and promoting alternative immunogen processing pathways involving antigen-presenting cells (APCs). Thus, such formulations are conceptually novel and offer potential advantages by promoting stimulation of immune responses by alternative mechanisms.

[0132] antibody The methods of the present disclosure can utilize antibodies that specifically recognize and bind to α-syn, e.g., the C-terminal peptide of α-syn, e.g., the B-cell epitope portion of the peptide immunogen construct described herein (see also WO2018 / 232369). Antibodies for use in therapy can be generated using standard methods in the art and include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and trispecific antibodies), and antibody fragments (so long as the desired antigen-binding activity and specificity are maintained). Antibody fragments include, for example, Fv, single chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody), and (Fab')2 (including chemically linked F(ab')2). Antibodies also include, for example, chimeric antibodies, humanized antibodies, and antibodies of various species, such as mouse, human, and cynomolgus monkey. In addition, antibody variants with sequences derived from other organisms are also included. Antibody fragments also include single chain scFvs in any orientation, tandem di-scFvs, diabodies, tandem tri-sdcFvs, minibodies, etc. Antibody fragments further include nanobodies (sdAbs, i.e., antibodies with no light chains and a single monomer domain, such as a pair of variable domains of a heavy chain). Antibody fragments may in some embodiments be referred to as being species specific (e.g., human scFv or mouse scFv). This indicates the sequence of at least a portion of the non-CDR regions, not the source of the construct.

[0133] Treatment method The present disclosure provides methods for treating, delaying, alleviating, and / or preventing synucleinopathy using the disclosed immunotherapies (e.g., peptide immunogen constructs and / or antibodies against peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing the disclosed peptide immunogen constructs and / or antibodies. In certain embodiments, the compositions utilized in the methods contain the disclosed peptide immunogen constructs in the form of stable immune stimulatory complexes with negatively charged oligonucleotides, such as CpG oligomers, via electrostatic association, the complexes being optionally further supplemented with mineral salts or oils as adjuvants for administration to a subject suffering from a synucleinopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen constructs to a subject at risk for or suffering from a synucleinopathy.

[0134] Subjects that can be treated according to the methods of the present disclosure include patients, such as human patients suffering from or at risk of developing a synucleinopathy, such as Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuroaxonal dystrophies, pure autonomic failure (PAF), and the like.

[0135] In some embodiments, the subject treated according to the method of the present disclosure is in an early stage of the development of synucleinopathy. For example, the subject may be in what is known in the art as a "prodromal" stage, where early signs and symptoms of the disease may appear, but major symptoms of the disease (e.g., motor symptoms) are not yet present. Identification of subjects in the prodromal stage usually involves considering a combination of clinical, humoral, histological, genetic, and imaging features or markers. For example, imaging by positron emission tomography (PET), single photon emission computed tomography (SPECT), or magnetic resonance imaging (MRI) can be used. In some embodiments, PET or SPECT is used to detect dopamine transporter (DAT) (DAT-PET or DAT-SPECT). An additional approach that can be used is the detection of pathological α-syn in cerebrospinal fluid or tissue biopsy by protein misfolding circulating amplification (PMCA). In other examples, skin tests can be used to detect, for example, phosphorylated α-syn (e.g., Syn-One Test™) or sebum lipids (Sinclair et al., Nature 12:1592, 2021). In addition to these tests, subjects can be identified by detecting prodromal symptoms of synucleinopathy, including, for example, REM sleep behavior disorder, hyposmia, constipation, mood disorders, excessive daytime somnolence, global agnosia, micrographia, restless legs syndrome, orthostatic hypotension, sexual dysfunction, dysuria, vocal and facial akinesia, or a combination thereof. Furthermore, family history can be a useful consideration. In addition, subthreshold parkinsonism scores can be measured, such as UPDRS scores for prodromal PD (Goetz et al., Mov. Disord. 27:1239-1242, 2012). Markers such as α-syn, neurofilament light chain (NfL), and plasma urate levels can also be assessed. Genetic markers (e.g., mutations in the LRRK2, GBA, SNCA, and / or VPS35 genes) can further be used. Subjects at an early (e.g., precursor) stage of a synucleinopathy can be treated by the methods of the present disclosure.In some embodiments, the subject is diagnosed with REM sleep behavior disorder, but does not have any or any significant motor symptoms (e.g., bradykinesia, rigidity, and / or tremor).In some embodiments, the subject has one or more of hyposmia, REM sleep behavior disorder, excessive daytime sleepiness, depression, cognitive symptoms, autonomic nervous system dysfunction, anosmia, reduced color vision, reduced quantitative motor test, and abnormal findings in substantia nigra neuroimaging.In some embodiments, the subject does not have any or any significant motor symptoms (e.g., bradykinesia, rigidity, and / or tremor).

[0136] The present disclosure also includes methods of using pharmaceutical compositions containing α-syn peptide immunogenic constructs. In certain embodiments, pharmaceutical compositions containing α-syn peptide immunogenic constructs can be used to (a) inhibit α-syn aggregation in a subject, (b) induce disaggregation of preformed α-syn aggregates in a subject, (c) reduce microglial TNF-α and IL6 secretion in a subject, (d) reduce neurodegeneration induced by exogenous α-syn aggregates in a subject, (e) reduce neurodegeneration in α-syn overexpressing cells, (f) reduce serum α-syn levels in a subject, (g) reduce oligomeric α-syn levels in the brain of a subject, (h) reduce neuropathology and restore motor activity in a subject, etc., where the subject is in an early precursor stage of a synucleinopathy.

[0137] The above methods include administering to a subject in need thereof a pharmaceutical composition comprising a pharmacologically effective amount of an immunotherapy (e.g., one or more peptide immunogen constructs and / or antibodies (e.g., as described herein)) targeting α-syn. The amounts and regimens used in the methods can be consistent with the information provided above in the section on compositions or can be determined as appropriate by one of skill in the art.

[0138] The present invention also provides compositions and kits as described herein for use in preventing, ameliorating, inhibiting, delaying, or treating any of the diseases or conditions described herein.

[0139] The following examples illustrate certain features and aspects of the present disclosure and should not be construed as limiting the scope of the disclosure in any way. EXAMPLES

[0140] Alpha-synuclein (α-syn) has a key role in the pathogenesis of Parkinson's disease (PD), dementia with Lewy bodies (LBD), and multiple system atrophy (MSA). Immunotherapies aimed at neutralizing toxic α-syn species are being investigated in the clinic as potential disease-modifying therapies for PD and other synucleinopathies. In this study, the effect of active immunization against α-syn with the UB312 vaccine was investigated in the Thy1SNCA / 15 mouse model of PD. Young transgenic and wild-type mice were subjected to an immunization regimen for 6 weeks and then observed for an additional 9 weeks. Behavioral assessments were performed before immunization and 15 weeks after the first dose.

[0141] UB312 immunization prevented the development of motor deficits in the wire test and challenged beam test, which was associated with reduced levels of α-syn oligomers in the cerebral cortex, hippocampus, and striatum of Thy1SNCA / 15 mice.UB312 immunotherapy caused a significant reduction in α-syn burden in the colon, accompanied by reduced reactivity of enteric glial cells in the colonic ganglion.

[0142] Our results show that immunization with UB312 prevents functional deficits and both central and peripheral pathology in Thy1SNCA / 15 mice.

[0143] Materials and Methods animal Thy1SNCA / 15 mice (stock number 017682) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA) and re-derived at the University of Southampton to establish and maintain a colony. Thy1SNCA / 15 mice overexpress one to two copies of the gene encoding human wild-type α-syn driven by the mouse thymocyte antigen 1 (Thy1) promoter (Choi et al., Nat. Commun. 11(1):1386, 2020). Thy1SNCA / 15 mice show widespread α-syn expression, primarily at synapses, up to 10 months of age, with no reported LB-like aggregates or phosphorylated α-syn (Rabl et al., BMC Neurosci. 18:22, 2017; Choi et al., Nat. Commun. 11(1):1386, 2020). Non-transgenic (C57BL / 6J background) littermate mice were used as controls. No behavioral studies have been performed to date in Thy1SNCA / 15 mice.

[0144] All mice were housed in groups of 5–10 and maintained under a standard 12-h light / dark cycle with standard RM1 chow (SDS, UK) and water ad libitum. All procedures were performed in accordance with the animal care guidelines stipulated by the United Kingdom Animals (Scientific Procedures) Act 1986, Home Office license.

[0145] Vaccination of mice with UB312 and antibody titers The immunization regimen is summarized in Figure 1. Ten-week-old Thy1SNCA / 15 mice received three intramuscular injections (at 3-week intervals) of either UB312 (40 μg per injection, n=29) or adjuvants (Adju-Phos® and CpG1) (n=27). Ten-week-old non-transgenic C57BL / 6J mice were also subjected to equivalent immunizations with adjuvants (n=22). Serum was collected for antibody titer analysis before each injection, as well as at 10 and 15 weeks after the first injection. Antibody titers were measured using an anti-α-syn enzyme immunoassay (EIA) kit (United Biomedical, Inc.) using a synthetic target peptide immunoadsorbent against the region K97-D135 of α-syn. UB312: UBITh1-εK-KKK-α-synuclein126-135 (SEQ ID NO: 112; UBITh1-εk-kkk-EMPSEEGYQD).

[0146] Fifteen weeks after the first injection, mice were terminally anesthetized with pentobarbitone (200 mg / kg) and perfused for immunohistochemistry (Tg-UB-312, n = 12; Tg-Adj, n = 11; WT-Adj, n = 9) or biochemical analysis (Tg-UB-312, n = 17; Tg-Adj, n = 16; WT-Adj, n = 13). For immunohistochemistry analysis, mice were perfused intracardially with PBS (0.01 M) followed by 4% paraformaldehyde (PFA) (0.01 M in PBS, pH 7.4). Brains and intestines (duodenum and proximal colon) were excised and immersed in 4% PFA for an additional 4 h, then transferred to 30% sucrose for cryoprotection. For Western blot analysis, mice were perfused with ice-cold PBS (0.01 M) and the cortex, hippocampus, and striatum were immediately dissected onto ice-cold PBS and snap frozen on dry ice for further processing.

[0147] Behavioral testing Before immunization and 15 weeks after the first immunization dose, mice were subjected to three different behavioral tests, each performed on a separate day, including the habituation period, so that the behavioral tests did not overlap on any day. The order of the tests and habituation periods was kept the same before and after treatment (Tg-UB-312, n=29; Tg-Adj, n=27; WT-Adj, n=22). Evaluators were blinded to the treatment status of the animals.

[0148] Challenging Beam Crossing Test Mice were trained to cross a 1 m long beam consisting of four equal sections (3.5, 2.5, 1.5, 0.5 cm wide) that narrowed towards the ends. Mice were placed at the wide end of the beam and encouraged to cross the beam to a clean cage on the other side. Mice were given 5 trials per day for 3 days, followed by test days. On test days, a 1 cm 2 A wire mesh of 100 mm was placed on the beam and the mouse was allowed to freely traverse the beam in five trials. Video recordings were analyzed for each trial and the number of errors was recorded. An error was considered if the mouse moved forward and one of its paws slid halfway down the wire mesh. The average number of errors over the five trials was calculated.

[0149] Pole Exam The pole test consisted of a vertical pole (1.5 cm diameter and 55 cm height) fixed in a clean cage. Mice were placed with their heads facing upwards on the side of the pole and the time it took them to reorient themselves 180° downwards and descend the pole was recorded. Each mouse was allowed 3 days of habituation, up to 5 trials per session, followed by a test day.

[0150] Wire hanging test Before and after immunotherapy, mice were given only one trial in the wire test. They were placed upside down on a 6 mm thick wire loop (20 cm diameter) that could rotate freely on a pivot. Inappropriate behaviors such as balancing on the top of the wire or intentionally jumping off the wire were prevented and the trial was either discarded or repeated. The total time to fall off the wire was recorded with a cutoff of 5 min.

[0151] immunohistochemistry Sagittal sections 20 μm thick from the brain (1800 μm from the midline) or intestine were cut using a Leica Cryostat. Immunofluorescence was used to detect α-Syn. Briefly, tissue sections were rehydrated in 0.01M PBS (Sigma, 1002795531) and blocked in 15% normal goat serum (Fisher Scientific, 1002817944) for 1 hour. Sections were incubated overnight at 4°C in anti-α-syn antibody MJFR1 (1:2000, Abcam, ab138501) in 0.01M PBS, 0.1% Triton®X [1001466726, ThermoFisher]. Sections were then incubated at room temperature in Alexa-Fluor555-conjugated goat anti-rabbit secondary antibody (Molecular Probes life technologies). Sections were counterstained with DAPI and mounted in Mowiol and Citifluor (ThermoFisher).

[0152] To analyze the inflammatory state in the brain and gut, markers of astrocytes (GFAP, 1:400, Dako), microglia (Iba1, 1:400, Wako, 019-19741), T cells (CD3(KT3), 1:200, BioRad, MCA500G), and endothelial activation (ICAM1, 1:200, Bioledgend, 116101) were selected. Endogenous peroxidase activity was quenched with 3% H2O2 (H1009-500ml, Sigma Aldrich) for 10 min. For Iba1 staining, heat-induced antigen retrieval was performed by heating the tissue in citrate buffer (15 mM Tris sodium citrate [101578237, Sigma Aldrich], 0.1% tween, pH 6 [P1379, Sigma Aldrich]) using a Panasonic 800W microwave at medium heat for 25 minutes. Nonspecific binding sites were blocked with 15% normal goat serum (Fisher Scientific) for 1 hour. The tissue was then incubated with primary antibody in 0.01M PBS, 0.1% tritonX overnight at 4°C. The tissue was then incubated in biotinylated secondary antibody for 1 hour at room temperature. The tissue was incubated in avidin biotin complex (ABC) for 1 hour at room temperature (PK-6100 Vectastain ABC kit). Chromogen development was performed with Nickel DAB. Before mounting in Distyrene Plasticizer Xylene (DPX, 12658646 Fisher Scientific), tissues were dehydrated in 50%, 70%, 95%, and 100% IMS for 2 min each, counterstained with eosin, and incubated in xylene for 5 min.

[0153] Western blot Tissue samples were homogenized on ice using a Kontes pellet pestle homogenizer in 10% W / V radioimmunoprecipitation analysis (RIPA) buffer (ThermoFisher, 89901) using HALT protease and phosphatase inhibitor cocktail (ThermoScientific, 78442). Homogenates were centrifuged at 14000 rpm at 4°C in an Eppendorf 5417R benchtop centrifuge. The pellet was discarded and the supernatant was retained for analysis. The protein concentration of each supernatant was determined using the Pierce Bovine Serum Albumin (BSA) Assay Kit (ThermoFisher, 23227) according to the manufacturer's instructions.

[0154] A Mini-PROTEIN Tetra vertical electrophoresis cell (BioRad; 1568004) was used to separate proteins from brain homogenates. 1 mm thick polyacrylamide gels for either denaturing or non-denaturing conditions were prepared.

[0155] For non-denaturing polyacrylamide gel electrophoresis (PAGE), brain homogenates were diluted in 4x Laemmli sample buffer (BioRad, 1620112) and 20 μg of protein was loaded on a 10% or 12% non-denaturing gel. Highly purified monomeric α-syn (Figure 10A) was run in parallel with the brain homogenates as a molecular weight marker. Protein concentrations for loading were determined from the linear range of the antibodies used (Figure 10A). Electrophoresis was performed in Laemmli buffer (192 mM glycine [Sigma Aldrich, G8898], 25 mM Tris base [ThermoFisher, 10103203]) at 100-150 V for 2 h. Semi-dry transfer was performed using the Trans Blot turbo system (BioRad, 1704150) and the Mini Transfer Kit (BioRad, 1704270). Proteins were transferred to 0.2 μm nitrocellulose membranes at 2.5 V, 2 A, and 15 min. Membranes were blocked with 3% bovine serum albumin (BSA) (Sigma Aldrich, 102052095) for 1 h at room temperature. After washing the membranes 3×5 min in Tris-buffered saline (TBS) (0.25 M Tris base, 1.5 M NaCl, pH 7.2), 0.1% Tween® 20 (Sigma, P1379), they were incubated in MJFR1 (1:5000; ab138501, Abcam) overnight at 4° C. To normalize protein loading, Revert 700 total protein stain (LiCor, 926-11015) was applied followed by blocking in BSA.

[0156] Image Analysis and Statistics Immunoblots were imaged on a LiCor Odyssey Fc scanner and analyzed using Image Studio Lite V5.2. Immunoreactive α-syn bands were normalized to GAPDH for SDS-PAGE and to Revert for non-denaturing PAGE. Immunostained tissue sections processed for fluorescence microscopy were visualized and images were captured at 20x using an SP8 confocal laser scanning microscope (Milton Keys, UK). DAB immunostained tissue sections were scanned for analysis at 20x using an Olympus VS110 high-throughput virtual microscope system. From the scanned images, images (each 0.16 mm ) were extracted using Olympus VS software. 2 ) were captured. For each marker tested, the area percentage of immunoreactivity across two serial sections per animal was calculated using FIJI software. Mean area percentages were calculated for each brain region and statistical analysis was performed using GraphPad Prism software. Two-way analysis of variance (ANOVA) was used for behavioral analysis with Bonferroni correction for post hoc multiple comparisons. For analysis of α-syn immunoreactivity in Western blots and immunohistochemistry, T-tests were performed unless otherwise stated. For analysis of inflammatory markers, one-way analysis of variance was used. Post hoc analysis was performed with Bonferroni correction for multiple comparisons analysis when applicable. Differences were considered significant when p<0.05. Number (n) refers to the number of mice used for each experiment.

[0157] result Antibody titers All transgenic mice produced high levels of anti-α-syn97-135 antibody titers after the first injection. Antibody titer levels increased rapidly over the first 6 weeks, peaked between weeks 6 and 10, and remained stable for the remainder of the 15-week study period (Figure 2). Unexpectedly, some adjuvanted wild-type and transgenic mice also produced background antibody titers, although these were 2-3 orders of magnitude lower than those induced by UB312.

[0158] UB312 immunization improves exercise performance The effect of UB312 immunotherapy on functional outcomes in Thy1SNCA / 15 mice was examined using three behavioral tests designed to assess motor function. These included the hanging wire test to measure grip strength, the challenging beam test for sensorimotor ability, and the pole test for locomotor control (Fleming et al., J.Neurosci.24:9434-9440,2004). As shown in Figure 3, at 10 weeks of age before the start of immunotherapy, Thy1SNCA / 15 mice showed no difference in motor performance compared to wild-type mice in any test. The motor performance of Thy1SNCA / 15 mice deteriorated with age in the beam and wire tests (at 26 weeks of age), but this deterioration was prevented by 15 weeks of UB312 immunotherapy.

[0159] In the challenging beam crossing test, a two-way analysis of variance was used to evaluate the correlation between age (F) and number of paw errors. (1,83) = 22.46, p < 0.0001) and treatment (F (2,83) = 5.72, p = 0.0047). Post hoc analysis of multiple comparisons showed that control adjuvanted Thy1SNCA / 15 mice made significantly more errors per trial at 6 months of age compared to 10-week-old mice (P < 0.0001) and compared to wild-type mice at 6 months of age (Wt-Adj: 3.1, Tg-Adj: 5.1; p < 0.0001). The number of errors per trial was not significantly different between wild-type and UB312-treated Thy1SNCA / 15 mice (p = 0.38).

[0160] In the hanging wire test, a significant effect of treatment on the age-related decline observed in adjuvanted Thy1SNCA / 15 mice was observed (F (2,80)= 4.03, P = 0.022). Post-hoc analysis showed a trend towards a decrease in the latency to fall in the control group of adjuvant-treated Thy1SNCA / 15 mice compared to wild-type mice (Wt-Adj: 3.85, Tg-Adj: 2.98; p = 0.102), which was significantly lower than in UB312-treated Thy1SNCA / 15 mice (Tg-Adj: 2.98, Tg-UB312: 4.2; p = 0.0095). At the end of the treatment period, there was no significant difference between wild-type and UB312-treated Thy1SNCA / 15 mice (Wt-Adj: 3.85, Tg-UB312: 4.20; p > 0.99).

[0161] Regarding the pole test, the time it took for mice to turn and descend the pole was similar in Thy1SNCA / 15 and wild-type mice, indicating a relationship between age and motor ability (F (1,64) = 0.156, p = 0.6941) or treatment (F (2,64) = 2.688, p = 0.076).

[0162] UB312 immunization reduces α-syn oligomers in the brain At the completion of the 15-week treatment period, 6-month-old mice were anesthetized and tissues were harvested to evaluate the effect of UB312 immunotherapy on α-syn-mediated pathology. α-syn pathology was analyzed by immunohistochemistry and western blot using the MJFR1 anti-α-syn antibody, which is specific for human α-syn overexpressed by Thy1SNCA / 15 mice. As expected, wild-type mice did not show immunoreactivity for human α-syn and were not included in the quantitative analysis. Immunohistochemical staining of brain sections for α-syn in Thy1SNCA / 15 mice showed widespread granular or punctate patterns in the gray matter, consistent with synaptic locations. α-syn inclusions such as Lewy bodies were not detectable in the brains of 6-month-old Thy1SNCA / 15 mice. Quantitative analysis of the percentage of area covered by α-syn immunoreactivity in each region of interest (cortex, striatum, hippocampus, substantia nigra, and cerebellum; Figure 4) showed no differences between UB312 and adjuvant-treated mice. Similarly, total levels of α-syn detected by Western blot analysis (Figure 5) showed no differences between UB312 and adjuvant-treated mice. To examine whether UB312 specifically reduces higher molecular weight α-syn oligomers, native non-denaturing Western blots were performed. Highly purified monomeric α-syn was used as a molecular weight marker, corresponding to the lowest band in the gel. The results, shown in Figure 5, show that UB312 significantly reduced α-syn oligomers, but not monomers, in the hippocampus of Thy1SNCA / 15 mice by 27.8% (p=0.049), in the striatum by 27.9% (p=0.045), and in the cortex by 49.8% (p=0.035) compared to control Thy1SNCA / 15 mice receiving adjuvant.

[0163] UB312 does not induce widespread glial cell responses Immunohistochemistry was performed on adjacent tissue sections for the glial cell markers microglia (Iba1) and astrocytes (GFAP). Figure 6 shows representative images of Iba1, and Figure 7 shows GFAP immunostaining in each brain region (cortex, hippocampus, striatum, and substantia nigra). One-way ANOVA of Iba1 and GFAP immunoreactivity showed no differences between groups, with the exception of the SN (F (2,25) = 4.989) showed that Iba1 immunostaining was significantly increased in UB312-treated Thy1SNCA / 15 mice compared to control adjuvant-treated Thy1SNCA / 15 mice or wild-type mice (Wt-Adj: 0.29%, Tg-UB312: 0.65%; p = 0.015). Iba1 and GFAP immunoreactivity was comparable in adjuvant-treated Thy1SNCA / 15 and wild-type mice across all brain regions.

[0164] UB312 does not induce T cell infiltration The effect of UB312 treatment on T cell infiltration was examined by counting the number of parenchymal CD3 positive T cells across three consecutive brain sections of 20 μm thickness. The majority of brain sections were negative for CD3 T cells, and T cell numbers were not increased in UB312-treated Thy1SNCA / 15 mice (Figure 8). To assess the activation status of the endothelium, ICAM1 immunoreactivity on brain endothelial cells was quantified. ICAM1 is expressed on endothelial cells and is upregulated during inflammation, promoting T cell extravasation. The results are shown in Figure 8 and show no difference in ICAM1 immunoreactivity between UB312-treated and adjuvant-treated Thy1SNCA / 15 or wild-type mice.

[0165] UB312 reduces α-syn and enteric glial cell activation in the colon Gastrointestinal (GI) dysfunction is a common precursor feature of PD, and LBs have been identified in colonic biopsies from PD patients. Thy1SNCA / 15 mice show accumulation of α-syn in nerve fibers and synapses in the muscularis of the intestinal wall at 10 weeks of age (Figure 9). Two-tailed t-tests of α-syn immunoreactivity rates in the intestinal wall showed a significant reduction in Thy1SNCA / 15 mice treated with UB312 when compared to adjuvant controls in the colon (Tg-Adj: 2.65%, Tg-UB312: 0.98%; p=0.0093), but not in the duodenum (Tg-Adj: 1.12%, Tg-UB312: 1.18%; p=0.91).

[0166] A marker of ganglionic enteric glial cell activation (GFAP) was used to examine the pattern of glial cell reactivity within the gut. Figure 9 shows representative images of GFAP immunostaining and subsequent quantification of GFAP immunoreactivity within the myenteric ganglion. One-way ANOVA revealed a significant treatment effect on GFAP expression (F (2,20) =7.007; p=0.0049). UB312 immunotherapy in Thy1SNCA / 15 mice significantly reduced GFAP expression levels in colonic myenteric ganglia when compared to adjuvant-treated Thy1SNCA / 15 mice (Tg-Adj: 16.86%, Tg-UB312: 8.47%; P=0.014). There was no difference in GFAP immunoreactivity in control wild-type and Thy1SNCA / 15 adjuvant-treated mice (Wt-Adj: 16.73, Tg-Adj: 16.86; p>0.99), whereas UB312-treated Thy1SNCA / 15 mice showed a significant reduction in GFAP when compared to wild-type mice (Wt-Adj: 16.73%, Tg-Adj: 8.47%; p>0.012). There was no difference in GFAP expression between treatment groups in the duodenum. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 3-3]

[0167] Other embodiments Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as described in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

[0168] Some embodiments are within the scope of the following numbered paragraphs.

[0169] 1. A method for preventing, reducing, suppressing or delaying the onset of one or more motor symptoms of a synucleinopathy in a subject in need thereof, said method comprising administering to said subject an effective amount of an immunotherapy that targets alpha-synuclein (alpha-syn).

[0170] 2. The method of claim 1, wherein the one or more motor symptoms of the synucleinopathy are selected from the group consisting of muscle rigidity, bradykinesia, resting tremor, and postural instability.

[0171] 3. A method for treating, preventing, reducing, or suppressing one or more gastrointestinal symptoms of a synucleinopathy in a subject in need thereof, said method comprising administering to said subject an effective amount of an immunotherapy targeting alpha-syn.

[0172] 4. The method of claim 3, wherein the one or more gastrointestinal symptoms are selected from the group consisting of sialorrhea, hypersalivation, dysphagia, nausea, vomiting, dyspepsia, constipation, abdominal pain, gastroparesis, and fecal incontinence.

[0173] 5. The method of paragraph 3 or paragraph 4, wherein the gastrointestinal condition occurs in the colon of the subject.

[0174] 6. A method for reducing alpha-syn levels in the gastrointestinal tract (e.g., colon) in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunotherapy that targets alpha-syn.

[0175] 7. The method of any one of clauses 1 to 6, wherein the subject does not have one or more motor symptoms of a synucleinopathy or exhibits only minimal motor symptoms of a synucleinopathy.

[0176] 8. The method of claim 7, wherein the subject does not have one or more motor symptoms of a synucleinopathy selected from the group consisting of muscle rigidity, bradykinesia, resting tremor, and postural instability.

[0177] 9. The method according to any one of claims 1 to 8, wherein the subject has an early precursor stage of synucleinopathy.

[0178] 10. A method for inducing an immune response against α-syn in a subject, inhibiting α-syn aggregation in a subject, or reducing the amount of α-syn aggregates in a subject, the method comprising administering to the subject an effective amount of an immunotherapy that targets α-syn, wherein the subject has an early precursor stage of a synucleinopathy.

[0179] 11. The method according to any one of claims 1 to 10, wherein the synucleinopathy is selected from the group consisting of Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuroaxonal dystrophy, and pure autonomic failure (PAF).

[0180] 12. The method of any one of claims 1 to 11, wherein the immunotherapy comprises a peptide, a protein (e.g., an antibody), a fragment or fusion of a peptide or protein (e.g., an antibody), or a nucleic acid molecule (e.g., an mRNA or nucleic acid in a vector) encoding one of the above molecules.

[0181] 13. The method of any one of claims 1 to 12, wherein the immunotherapy comprises a peptide immunogen construct.

[0182] 14. The method of claim 13, wherein the peptide immunogen construct comprises a B cell epitope, a heterologous T cell epitope, and an optional linker.

[0183] 15. The method of claim 14, wherein the B cell epitope induces an immune response against α-syn.

[0184] 16. The method of claim 15, wherein the B cell epitope comprises a peptide of the C-terminal region of alpha-syn protein, the peptide being optionally about 10 to about 25 amino acids in length.

[0185] 17. The method of claim 16, wherein the alpha-syn protein comprises the sequence of SEQ ID NO:1.

[0186] 18. The method of any one of clauses 14-17, wherein the B cell epitope comprises a peptide selected from a sequence in Table 1 (e.g., any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, and 69).

[0187] 19. The method of any one of clauses 14 to 18, wherein the heterologous T cell epitope is derived from a pathogenic protein.

[0188] 20. The method of any one of clauses 14 to 19, wherein the heterologous T cell epitope comprises a sequence selected from the sequences in Table 2.

[0189] 21. The method of any one of clauses 14 to 20, wherein the peptide comprises a heterologous spacer or linker between the B cell epitope and the T cell epitope.

[0190] 22. The method of claim 21, wherein the heterologous spacer or linker is selected from the group consisting of Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys, or combinations thereof.

[0191] 23. The method according to any one of items 14 to 22, wherein the B cell epitope is located N-terminally to the T cell epitope.

[0192] 24. The method according to any one of items 14 to 22, wherein the T cell epitope is located N-terminally to the B cell epitope.

[0193] 25. The method of any one of clauses 13 to 24, wherein the peptide immunogenic construct is selected from the sequences in Table 3.

[0194] 26. The peptide immunogen construct comprises: (a) a B cell epitope comprising about 10 to about 25 amino acid residues derived from the C-terminal fragment of α-Syn, corresponding to about amino acid G111 to about 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 the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), or a combination thereof; Including, 26. The method of any one of clauses 13 to 25, wherein said B cell epitope is covalently linked to said T helper epitope directly or via said optional heterologous spacer.

[0195] 27. The method according to claim 26, wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 12 to 15, 17, and 49 to 63.

[0196] 28. The method of claim 26 or 27, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98, for example, selected from any one of SEQ ID NOs: 81, 83, and 84.

[0197] 29. The method of any one of clauses 26 to 28, wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

[0198] 30. The method of any one of clauses 26 to 29, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope.

[0199] 31. The method of any one of clauses 26 to 30, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope via the optional heterologous spacer.

[0200] 32. The peptide immunogen construct has the following formula: (Th) m -(A) n -(α-Syn C-terminal fragment)-X or (α-Syn C-terminal fragment)-(A) n -(Th) m -X wherein Th is the T helper epitope; A is the heterologous spacer; (α-Syn C-terminal fragment) is the B cell epitope, X is an amino acid α-COOH or α-CONH2; m is from 1 to about 4; 32. The method of any one of items 26 to 31, wherein n is from 1 to about 10.

[0201] 33. The method of any one of items 26 to 32, wherein the 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.

[0202] 34. The method of any one of clauses 13 to 33, wherein the peptide immunogen construct is in a stabilized immune stimulatory complex with a CpG oligodeoxynucleotide (ODN).

[0203] 35. The method of any one of clauses 1 to 34, wherein the immunotherapy is comprised in a composition that optionally comprises multiple immunotherapies, e.g., multiple peptide immunogen constructs.

[0204] 36. The method of claim 35, wherein the composition comprises a peptide immunogen construct comprising the amino acid sequences of SEQ ID NOs: 112 and 113.

[0205] 37. The method of paragraph 35 or paragraph 36, wherein the composition is a pharmaceutical composition comprising the immunotherapy(s) and a pharma- ceutically acceptable delivery vehicle and / or adjuvant.

[0206] 38. The method of claim 37, wherein the composition optionally comprises an adjuvant comprising an inorganic salt of aluminum selected from the group consisting of Al(OH)3 and AlPO4.

[0207] 39. (a) the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147, for example, from the group consisting of SEQ ID NOs: 107, 108, 111 to 113, and 115 to 147; (b) the composition comprises an adjuvant which is an inorganic salt of aluminum selected from the group consisting of Al(OH)3 and AlPO4; 39. The method according to paragraph 37 or 38.

[0208] 40. (a) the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147, for example, from the group consisting of SEQ ID NOs: 107, 108, 111 to 113, and 115 to 147; (b) the peptide immunogen construct is in the form of a stabilized immune stimulatory complex with CpG ODN; 40. The method according to any one of items 37 to 39.

[0209] 41. The immunotherapy comprises administering to a patient a peptide immunogen construct according to any one of claims 13 to 40, a B cell epitope of SEQ ID NO: 1 (e.g., the C-terminal region of SEQ ID NO: 1), or a peptide of Table 1 (e.g., SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 13. The method of any one of claims 1 to 12, comprising an antibody or epitope-binding fragment thereof that specifically binds to any one of the following:

[0210] 42. The method of any one of clauses 1-41, comprising the use of two or more, three or more, four or more, or five or more immunotherapies.

[0211] 43. The method of any one of claims 1 to 42, wherein the subject is diagnosed with rapid eye movement (REM) sleep behavior disorder (RBD).

[0212] 44. The method of any one of clauses 1 to 43, wherein the subject has one or more of the following prodromal symptoms: hyposmia, REM sleep behavior disorder, excessive daytime sleepiness, depression, cognitive symptoms, autonomic nervous system dysfunction, anosmia, reduced color vision, impaired quantitative motor testing, abnormal findings in substantia nigra neuroimaging, or other prodromal symptoms, e.g., as described herein.

[0213] 45. The method of any one of clauses 1 to 44, wherein the subject does not have any or any significant bradykinesia, rigidity, and / or tremor, or other symptoms of a synucleinopathy that is not in the prodromal stage.

[0214] 46. ​​The method of any one of clauses 1 to 45, wherein the immunotherapy comprises or consists of a peptide immunogen construct comprising or consisting of SEQ ID NO:112.

[0215] 47. A composition or kit for use in carrying out any one of the methods according to any one of claims 1 to 46.

[0216] 48. Use of a peptide immunogen construct or composition described herein in the preparation of a medicament for treating, preventing, suppressing, reducing or delaying the onset of one or more motor symptoms of a synucleinopathy in a subject in need thereof.

[0217] Other embodiments are within the scope of the claims.

Claims

1. A composition for treating, preventing, alleviating, or suppressing one or more gastrointestinal symptoms of a synucleinopathy in a subject or for reducing the level of alpha-synuclein in the gastrointestinal tract of a subject having a synucleinopathy, the composition comprising a peptide immunogen construct, wherein the subject is a mammal, and the peptide immunogen construct is: (a) a B-cell epitope comprising 10 to 25 amino acid residues comprising SEQ ID NO: 15, or an immunologically functional analog thereof; (b) a T helper cell epitope selected from SEQ ID NOs: 81, 83, and 84; and (c) a heterologous spacer selected from Lys-Lys-Lys, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys, or a combination thereof; Including, The composition, wherein said B cell epitope is covalently linked to said T helper cell epitope via said heterologous spacer.

2. The composition described in claim 1, wherein the B cell epitope is that of sequence number 15.

3. The composition described in claim 2, wherein the T helper cell epitope is of sequence number 83.

4. The peptide immunogen construct of claim 3, wherein the peptide immunogen construct has the following formula: (Th)-(A)-(B cell epitope)-X or (B cell epitope)-(A)-(Th)-X wherein Th is the T helper cell epitope; A is the heterologous spacer; The composition of claim 3, wherein X is α-COOH or α-CONH 2 of an amino acid.

5. The composition described in claim 3, wherein the peptide immunogen construct comprises or consists of SEQ ID NO:

112.

6. A composition described in any one of claims 1 to 5, wherein the peptide immunogen construct is in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition comprises an inorganic salt of aluminum.

7. The composition of claim 6, wherein the inorganic salt of aluminum is selected from aluminum hydroxide and aluminum phosphate.

8. A composition described in any one of claims 1 to 5, wherein the subject has a precursory stage of synucleinopathy.

9. The composition described in claim 8, wherein the subject does not have motor symptoms of synucleinopathy selected from muscle rigidity, bradykinesia, resting tremor, and postural instability.

10. A composition described in any one of claims 1 to 5, wherein the synucleinopathy is selected from Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuroaxonal dystrophy, and pure autonomic failure (PAF).

11. The composition described in claim 10, wherein the synucleinopathy is Parkinson's disease (PD).

12. A composition described in any one of claims 1 to 5, wherein the composition is for treating, preventing, alleviating, or suppressing one or more gastrointestinal symptoms of a synucleinopathy in a subject, and the one or more gastrointestinal symptoms are selected from sialorrhea, hypersalivation, dysphagia, nausea, vomiting, dyspepsia, constipation, abdominal pain, gastroparesis, and fecal incontinence.

13. A composition described in any one of claims 1 to 5, wherein the composition is for treating, preventing, alleviating, or suppressing one or more gastrointestinal symptoms of a synucleinopathy in a subject, and the gastrointestinal symptoms occur in the colon of the subject.

14. A composition described in any one of claims 1 to 5, wherein the composition is for reducing alpha-syn levels in the gastrointestinal tract of a subject with a synucleinopathy, and the gastrointestinal tract is the colon.

15. A composition described in any one of claims 1 to 5, wherein the composition is for administration at a dose of 50 to 400 μg of the peptide immunogen construct.

16. The composition described in claim 15 for intramuscular administration.

17. A composition described in any one of claims 1 to 5, wherein the subject is a human.

18. The composition of claim 1, wherein the composition is for treating, preventing, alleviating, or suppressing one or more gastrointestinal symptoms of a synucleinopathy in a subject, wherein the one or more gastrointestinal symptoms occur in the colon of the subject, the synucleinopathy is Parkinson's disease (PD), the subject is human, α-synuclein is detectable in cerebrospinal fluid or tissue biopsy of the subject, the peptide immunogen construct is of SEQ ID NO: 112, the composition is for administration at a dose of 50 to 400 μg of the peptide immunogen construct, the peptide immunogen construct is in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition is for intramuscular administration.

19. The composition of claim 1, wherein the composition is for reducing alpha-synuclein levels in the gastrointestinal tract of a subject having a synucleinopathy, the gastrointestinal tract being the colon, the synucleinopathy being Parkinson's disease (PD), the subject being human, alpha-synuclein being detectable in cerebrospinal fluid or a tissue biopsy of the subject, the peptide immunogen construct being of SEQ ID NO: 112, the composition being for administration at a dose of 50 to 400 μg of the peptide immunogen construct, the peptide immunogen construct being in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition being for intramuscular administration.

20. A composition for preventing, delaying the onset of, or delaying the onset of one or more motor symptoms of a synucleinopathy in a subject, the subject being a mammal with a precursor stage of a synucleinopathy, the composition comprising a peptide immunogen construct comprising: (a) a B-cell epitope comprising about 10 to about 25 amino acid residues comprising SEQ ID NO: 15, or an immunologically functional analog thereof; (b) a T helper cell epitope selected from SEQ ID NOs: 81, 83, and 84; and (c) a heterologous spacer selected from Lys-Lys-Lys, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys, or a combination thereof; Including, The composition, wherein said B cell epitope is covalently linked to said T helper cell epitope via said heterologous spacer.

21. The composition described in claim 20, wherein the B cell epitope is that of sequence number 15.

22. The composition described in claim 21, wherein the T helper epitope is of sequence number 83.

23. The peptide immunogen construct of claim 23, wherein the peptide immunogen construct has the following formula: (Th)-(A)-(B cell epitope)-X or (B cell epitope)-(A)-(Th)-X wherein Th is the T helper cell epitope; A is the heterologous spacer; 23. The composition of claim 22, wherein X is α-COOH or α-CONH 2 of an amino acid.

24. The composition of claim 22, wherein the peptide immunogen construct comprises or consists of sequence number 112.

25. A composition described in any one of claims 20 to 24, wherein the peptide immunogen construct is in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition comprises an inorganic salt of aluminum.

26. The composition of claim 25, wherein the inorganic salt of aluminum is selected from aluminum hydroxide and aluminum phosphate.

27. ​​A composition described in any one of claims 20 to 24, wherein one or more motor symptoms of the synucleinopathy are selected from muscle rigidity, bradykinesia, resting tremor, and postural instability.

28. The composition described in any one of claims 20 to 24, wherein the synucleinopathy is selected from Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuroaxonal dystrophy, and pure autonomic failure (PAF).

29. The composition described in claim 28, wherein the synucleinopathy is Parkinson's disease (PD).

30. A composition described in any one of claims 20 to 24, wherein the composition is for administration at a dose of 50 to 400 μg of the peptide immunogen construct.

31. The composition described in claim 30 for intramuscular administration.

32. A composition described in any one of claims 20 to 24, wherein the subject is a human.

33. The composition of any one of claims 20 to 24, wherein the synucleinopathy is Parkinson's disease (PD), the subject is human, α-synuclein is detectable in cerebrospinal fluid or tissue biopsy of the subject, one or more motor symptoms of the synucleinopathy are selected from muscle rigidity, bradykinesia, resting tremor, and postural instability, the peptide immunogen construct is of SEQ ID NO: 112, the composition is for administration at a dose of 50 to 400 μg of the peptide immunogen construct, the peptide immunogen construct is in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition is for intramuscular administration.

34. A composition for treating a precursor synucleinopathy in a subject, the composition comprising a peptide immunogenic construct, wherein the subject is a mammal, and the peptide immunogenic construct comprises: (a) a B-cell epitope comprising about 10 to about 25 amino acid residues comprising SEQ ID NO: 15, or an immunologically functional analog thereof; (b) a T helper cell epitope selected from SEQ ID NOs: 81, 83, and 84; and (c) a heterologous spacer selected from Lys-Lys-Lys, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys, or a combination thereof; Including, The composition, wherein said B cell epitope is covalently linked to said T helper cell epitope via said heterologous spacer.

35. The composition described in claim 34, wherein the B cell epitope is that of sequence number 15.

36. The composition described in claim 35, wherein the T helper cell epitope is of sequence number 83.

37. The peptide immunogen construct of claim 37, wherein the peptide immunogen construct has the following formula: (Th)-(A)-(B cell epitope)-X or (B cell epitope)-(A)-(Th)-X wherein Th is the T helper cell epitope; A is the heterologous spacer; 37. The composition of claim 36, wherein X is α-COOH or α-CONH 2 of an amino acid.

38. The composition of claim 36, wherein the peptide immunogen construct comprises or consists of sequence number 112.

39. A composition described in any one of claims 34 to 38, wherein the peptide immunogen construct is in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition comprises an inorganic salt of aluminum.

40. The composition of claim 39, wherein the inorganic salt of aluminum is selected from aluminum hydroxide and aluminum phosphate.

41. A composition described in any one of claims 34 to 38, wherein the subject does not have motor symptoms of the synucleinopathy or exhibits only minimal motor symptoms of the synucleinopathy.

42. The composition described in claim 41, wherein the subject does not have muscle rigidity, bradykinesia, resting tremor, or postural instability.

43. The composition described in any one of claims 34 to 38, wherein the subject has one or more symptoms of the prodromal stage of synucleinopathy selected from hyposmia, REM sleep behavior disorder (RBD), excessive daytime sleepiness, depression, cognitive symptoms, autonomic nervous system dysfunction, loss of smell, reduced color vision, impaired quantitative motor testing, and abnormal findings in substantia nigra neuroimaging.

44. The composition described in claim 43, wherein the subject has been diagnosed with rapid eye movement (REM) sleep behavior disorder (RBD).

45. The composition described in any one of claims 34 to 38, wherein the synucleinopathy is selected from Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), neuroaxonal dystrophy, and pure autonomic failure (PAF).

46. The composition described in claim 45, wherein the synucleinopathy is Parkinson's disease (PD).

47. A composition described in any one of claims 34 to 38, wherein the composition is for administration at a dose of 50 to 400 μg of the peptide immunogen construct.

48. The composition described in claim 47 for intramuscular administration.

49. A composition described in any one of claims 34 to 38, wherein the subject is a human.

50. The composition of claim 34, wherein the synucleinopathy is Parkinson's disease (PD), the subject is human, α-synuclein is detectable in cerebrospinal fluid or tissue biopsy of the subject, the peptide immunogen construct is of SEQ ID NO: 112, the composition is for administration at a dose of 50 to 400 μg of the peptide immunogen construct, the peptide immunogen construct is in a stabilized immunostimulatory complex with a CpG oligodeoxynucleotide (ODN), and the composition is for intramuscular administration.

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