Peptide immunogens from the C-terminus of alpha-synuclein protein and formulations thereof for the treatment of synucleinopathies

Abstract

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

Description

[Technical Field]

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

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

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

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

[0005] The primary structure of α-Syn is generally divided into three distinct domains: (1) residues 1–60: an amphipathic N-terminal region dominated by four 11-residue repeats containing the consensus sequence KTKEGV, which has a structural alpha-helical propensity similar to apolipoprotein-binding domains; (2) residues 61–95: a central hydrophobic region containing the non-amyloid-β component (NAC) region involved in protein aggregation; and (3) residues 96–140: a highly acidic proline-rich region with no clear structural propensity. A 35-amino acid α-Syn fragment of the NAC region was discovered to coexist with Aβ in amyloid-enriched fractions. NAC was later shown to be a fragment of its precursor protein, NACP, now called human α-Syn, which was later determined to be the full-length human homolog of synuclein from the spotted ray (Torpedo californica).

[0006] In vitro high-resolution ion mobility mass spectrometry (IMS-MS) of HPLC-purified α-Syn demonstrated that α-Syn is autoproteolytic (self-proteolytic) and generates various low molecular weight fragments upon incubation. The 14.46 kDa full-length protein was found to generate numerous smaller fragments, including a 12.16 kDa fragment (amino acids 14–133) and a 10.44 kDa fragment (amino acids 40–140) formed by C- and N-terminal truncation, as well as a 7.27 kDa fragment (amino acids 72–140). The 7.27 kDa fragment, which contains most of the NAC region, was shown to aggregate significantly faster than full-length α-Syn. These autoproteolytic products may serve as intermediates or cofactors in α-Syn aggregation.

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

[0008] In solution, α-Syn is considered an intrinsically disordered protein, lacking a single, stable 3D structure. α-Syn interacts significantly with tubulin, suggesting that α-Syn may have potential microtubule-associated protein activity, similar to tau. While α-Syn has classically been considered an unstructured, soluble protein, unmutated α-Syn forms stably folded tetramers that resist aggregation. Nevertheless, α-Syn can aggregate to form insoluble fibrils in pathologies characterized by Lewy bodies. These disorders are known as synucleinopathies (reviewed at en.wikipedia.org / wiki / Synucleinopathies).

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

[0010] LBD is a progressive neurodegenerative disorder characterized by tremor, rigidity, bradykinesia, and loss of dopaminergic neurons in the brain. In the cases of DLB ​​and PDD, symptoms also include cognitive impairment. Up to 2% of the population over 60 years of age in Western countries exhibit typical signs of PD / LBD. Genetic susceptibility and environmental factors appear to be involved in the development of the disease. Patients suffering from this disease develop characteristic intracellular inclusions called Lewy bodies (LBs) in the cortical and subcortical regions of the brain, particularly in areas with a high content of dopaminergic neurons or neuronal processes. In LBD, α-Syn accumulates in LBs throughout the affected brain regions. Furthermore, single point mutations, duplications, or amplifications of the α-Syn gene have been demonstrated to be associated with rare familial forms of parkinsonism.

[0011] Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder characterized by symptoms of L-dopa-resistant parkinsonism, cerebellar ataxia, and dysautonomia. Patients suffer from multisystem neuronal loss in various brain regions, including the striatum, substantia nigra, cerebellum, pons, and inferior olivary nucleus, and spinal cord. MSA is characterized by α-Syn-positive glial cytoplasm (GCIs) and rare neuronal inclusions throughout the central nervous system.

[0012] Other rare disorders, such as various neuroaxonal dystrophies, also contain α-Syn pathology, and α-Syn is the major structural component of Lewy body fibrils. While Lewy bodies often contain tau protein, α-Syn and tau constitute two distinct subsets of filaments within the same inclusion. α-Syn pathology is also found in both sporadic and familial cases of Alzheimer's disease.

[0013] The aggregation mechanism of α-Syn is unclear. Monomeric α-Syn spontaneously unfolds in solution but can also bind to membranes in an α-helical conformation. The unfolded monomer first aggregates into small oligomeric species that can be stabilized by β-sheet-like interactions, and then into high-molecular-weight insoluble fibrils. α-Syn exists as an equilibrium mixture of unstructured, alpha-helical, and beta-sheet-rich conformers. Mutations or buffer conditions known to improve aggregation strongly increase the population of the beta conformer, thus suggesting that this may be the conformation associated with pathogenic aggregation. Evidence exists for beta-rich, structured intermediates that may be precursors to aggregation and ultimately to Lewy bodies.

[0014] Several physiological factors can modify α-Syn, leading to the formation of aggregates, including (1) phosphorylation by one or more kinases; (2) truncation by proteases such as calpain; and (3) nitration by nitric oxide (NO) or other reactive nitrogen species present during inflammation. ER-Golgi transport, synaptic vesicles, mitochondria, lysosomes, and other proteolytic machinery are some of the proposed cellular targets associated with α-Syn-mediated toxicity due to such aggregation.

[0015] One strategy for treating synucleinopathies is to use compounds that inhibit α-Syn aggregation. The small molecule cuminaldehyde has been shown to inhibit α-Syn fibrillation. In addition to small molecule therapy, recent reports suggest that α-Syn aggregates may be a potential target for immunotherapy (reviewed in Lee JS and Lee SJ, 2016). However, this report points out several potential issues or challenges associated with the development of α-Syn immunotherapy, including (1) potential interference with the normal physiological function of α-Syn; (2) difficulties in delivering antibody drugs to the brain parenchyma; and (3) efficacy of immunotherapy.

[0016] To date, there is an unmet need to develop site-specific peptide immunogens and formulations thereof for the cost-effective treatment of patients suffering from synucleinopathies.

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

[0018] The present disclosure relates to peptide immunogenic constructs of alpha-synuclein protein (α-Syn). The disclosure also relates to compositions comprising the peptide immunogenic constructs, methods of making and using the peptide immunogenic constructs, and antibodies produced by the peptide immunogenic constructs.

[0019] The disclosed peptide immunogen constructs comprise a B cell epitope from α-Syn linked to a heterologous T helper cell (Th) epitope, either directly or, optionally, via a heterologous spacer. The B cell epitope portion of the peptide immunogen construct comprises approximately 10 to approximately 25 amino acid residues from the C-terminal region of α-Syn, corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately 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 is derived from an amino acid sequence derived from a pathogenic protein. When administered to a host, the B cell epitope portion and the Th epitope portion of the peptide immunogen construct act together to stimulate the production of antibodies that specifically recognize and bind to the α-Syn B cell epitope portion of the construct.

[0020] In some embodiments, the α-Syn peptide immunogen construct comprises: (a) a B cell epitope comprising about 10 to about 25 amino acid residues from 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: 70-98; 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), wherein the B cell epitope is covalently linked to the T helper epitope directly or via an optional heterologous spacer. In certain embodiments, the alpha-Syn peptide immunogenic construct comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

[0021] The present disclosure also relates to compositions comprising the disclosed peptide immunogenic constructs, including pharmaceutical compositions. The disclosed pharmaceutical compositions can elicit an immune response and antibody production in a host against the disclosed peptide immunogenic constructs. The disclosed compositions can include a mixture of one or more of the disclosed peptide immunogenic constructs. In some embodiments, the compositions include the disclosed peptide immunogenic constructs along with additional components, including carriers, adjuvants, buffers, and other suitable reagents. In certain embodiments, the compositions include the disclosed peptide immunogenic constructs in the form of stabilized immunostimulatory complexes with CpG oligomers, optionally supplemented with adjuvants.

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

[0023] The present disclosure also relates to antibodies produced by hosts immunized with the disclosed peptide immunogenic constructs. The disclosed antibodies specifically recognize and bind to the B-cell epitope portion of α-Syn in the peptide immunogenic constructs. The disclosed α-Syn antibodies have unexpectedly high cross-reactivity with the β-sheets of α-Syn in the form of monomers, oligomers, or fibrils. Based on their unique characteristics and properties, the disclosed antibodies can provide an immunotherapeutic approach to targeting, identifying, and treating synucleinopathies.

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

[0025] The present disclosure also relates to methods of making and using the disclosed peptide immunogenic constructs, antibodies, and compositions. The disclosed methods provide low-cost manufacturing and quality control of peptide immunogenic constructs and compositions comprising the constructs, which can be used in methods of preventing and treating synopathies.

[0026] The present disclosure also includes methods for treating and / or preventing synucleinopathy using the disclosed peptide immunogenic constructs and / or antibodies against the peptide immunogenic constructs. In some embodiments, the methods for treating and / or preventing synucleinopathy include administering to a host a composition comprising the disclosed peptide immunogenic constructs. In certain embodiments, the composition utilized in the methods comprises the disclosed peptide immunogenic constructs in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, via electrostatic binding, optionally further supplemented with mineral salts or oils as adjuvants for administration to patients with synucleinopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogenic constructs to a host at risk for or with synucleinopathy.

[0027] In various embodiments, methods are described that use α-Syn peptide immunogen constructs and / or antibodies elicited by the α-Syn peptide immunogen constructs. In certain embodiments, methods are described for producing antibodies, inhibiting α-Syn aggregation, reducing the amount of α-Syn aggregates, and identifying α-Syn aggregates of different sizes. Various methods include administering a pharmacologically effective amount of an α-Syn peptide immunogen to a host in need thereof. [Brief explanation of the drawings]

[0028] [Figure 1]This graph shows the level of α-Syn aggregation after 6 days in vitro in the presence of antibodies against the C-terminus of α-Syn (Samples 1-4) or in the presence of a vehicle control (Sample 5). Specifically, α-Syn aggregation was induced by α-Syn111-132 (Sample 1); α-Syn121-135 (Sample 2); α-Syn123-135 (Sample 3); and α-Syn126-135 (Sample 4) in the presence of an anti-α-Syn antibody or a vehicle control (Sample 5). The level of α-Syn aggregation was measured by thioflavin-T (ThT) staining of the aggregates. Samples 1-4 were normalized to the vehicle control in Sample 5. Error bars represent the SEM (standard error of the mean) of each duplicated study. [Figure 2] This graph shows the dissociation level of preformed in vitro α-Syn aggregates after incubating the aggregates for 3 days in the presence of antibodies against the C-terminus of α-Syn (Samples 1-3) or a preimmune serum control (Sample 4). Specifically, preformed α-Syn aggregates were incubated with anti-α-Syn antibodies elicited by α-Syn111-132 (Sample 1); α-Syn126-135 (Sample 2), a combination of antibodies elicited by α-Syn111-132 and α-Syn126-135 (Sample 3), or a preimmune serum control (Sample 4). The level of α-Syn aggregation was measured by thioflavin-T (ThT) staining of the aggregates. Samples 1-3 were normalized to the preimmune serum control (Sample 4). Error bars represent the standard error of the mean (SEM) of each duplicated study. [Figure 3]This graph shows the levels of α-Syn aggregation and α-Syn disaggregation in α-Syn-overexpressing PC12 cells incubated with nerve growth factor (NGF) in the presence of antibodies against the C-terminus of α-Syn (Samples 1-4) or a vehicle control (Sample 5). Specifically, PC12 cells were incubated with anti-α-Syn antibodies induced by α-Syn111-132 (Sample 1); α-Syn121-135 (Sample 2); α-Syn123-135 (Sample 3); or α-Syn126-135 (Sample 4) or a vehicle control (Sample 5). Samples 1-4 were normalized to the vehicle control in Sample 5. Error bars represent the standard error of each triplicate study. [Figure 4] This graph shows the levels of α-Syn aggregate-mediated TNF-α and IL-6 release from cells incubated in the presence of antibodies against the C-terminus of α-Syn (Samples 1-4) or a vehicle control (Sample 5). Specifically, microglial cells were incubated with anti-α-Syn antibodies induced by α-Syn111-132 (Sample 1); α-Syn121-135 (Sample 2); α-Syn123-135 (Sample 3); or α-Syn126-135 (Sample 4) or a vehicle control (Sample 5). Samples 1-4 were normalized to the vehicle control in Sample 5. Error bars represent the standard error of each triplicate study. [Figure 5]Figures 5A-5C are graphs showing the effect of anti-α-Syn antibodies in an in vitro neurodegeneration model with exogenous preformed α-Syn aggregates in NGF-induced neuronally differentiated PC12 cells. Figure 5A evaluates neurite length in PC12 cells treated with NGF alone (dark solid line), NGF with exogenous preformed α-Syn aggregates (dotted line), NGF with preimmune serum (light solid line), and NGF with exogenous preformed α-Syn aggregates and preimmune serum (dashed line). Figure 5B assesses neurite length in PC12 cells treated with NGF with vehicle (dark solid line); NGF with exogenous preformed α-Syn aggregates (dotted line); NGF with anti-α-Syn antibody induced by α-Syn111-132 (SEQ ID NO: 113) (light solid line); and NGF with exogenous preformed α-Syn aggregates and anti-α-Syn antibody induced by α-Syn111-132 (SEQ ID NO: 113) (dashed line). [Figure 6] Figure 5C assesses neurite length in PC12 cells treated with NGF alone with vehicle (dark solid line); NGF with exogenous preformed α-Syn aggregates (dotted line); NGF with anti-α-Syn antibody induced by α-Syn126-135 (SEQ ID NO: 112) (light solid line); and NGF with exogenous preformed α-Syn aggregates and anti-α-Syn antibody induced by α-Syn126-135 (SEQ ID NO: 112) (dashed line). [Figure 7]Figures 6A and 6B are graphs showing the effects of anti-α-Syn antibodies on cell number and neurite length in an in vitro neurodegeneration model using NGF-induced neuronally differentiated wild-type α-Syn-overexpressing PC12 cells. Cells were treated with a vehicle control (Sample 1); anti-α-Syn antibodies induced by α-Syn101-132 (Sample 2), α-Syn111-132 (Sample 3), α-Syn121-135 (Sample 4), α-Syn123-135 (Sample 5), or α-Syn126-135 (Sample 6); a combination of anti-α-Syn antibodies induced by α-Syn111-132 and α-Syn126-135 (Sample 7); or a pre-immune serum control (Sample 8). Figure 6A evaluates the protective effect of each sample on the recovery of PC12 cell number. Figure 6B evaluates the neurite length of cells treated with each sample. Samples 1–8 were normalized to NGF-induced neuronally differentiated wild-type PC12 cells. A t-test was used to test for significance (a p-value of less than 0.05 was defined as statistically significant and is indicated by an asterisk (*)). [Figure 8] Figures 7A and 7B show the ability of anti-α-Syn antibodies to recognize and bind α-Syn aggregates of different sizes by Western blot analysis. Figure 7A shows a Western blot image comparing the commercial anti-α-Syn antibody Syn211 (lane 1); preimmune serum control (lane 2); anti-α-Syn antibodies elicited by Syn111-132 (lane 3); anti-α-Syn antibodies elicited by Syn111-135 (lane 4); anti-α-Syn antibodies elicited by Syn121-135 (lane 5); anti-α-Syn antibodies elicited by Syn123-135 (lane 6); and anti-α-Syn antibodies elicited by α-Syn126-135 (lane 7). [Figure 9] Figure 7B is a bar graph showing the relative ability of each antibody to bind to α-Syn molecular complexes of various sizes (including monomers, dimers, trimers, tetramers, and oligomers). The chemiluminescent signals of the Western blot bands shown in Figure 7A were quantified and reported in the bar graph in Figure 7B. [Figure 10]Figures 8A-8C are dot blot images showing that antibodies against the C-terminus of α-Syn recognize and bind only different species of α-Syn (i.e., α-helical monomers, β-sheet monomers, β-sheet oligomers, and β-sheet fibrils) but do not recognize or bind to the same species of other amyloidogenic proteins (i.e., Aβ1-42 and Tau441). Figure 8A is a control sample showing that antibodies purified from preimmune serum of guinea pigs showed undetectable levels of all protein species assayed. Figure 8B evaluates the ability of anti-α-Syn antibodies elicited by α-Syn111-132 (SEQ ID NO: 113) to recognize and bind to different species of α-Syn, Aβ1-42, and Tau441 proteins. FIG. 8C assesses the ability of anti-α-Syn antibodies elicited by α-Syn126-135 (SEQ ID NO: 112) to recognize and bind to different species of α-Syn, Aβ1-42, and Tau441 proteins. [Figure 11] Figure 9 shows the relative binding affinities of antibodies against the C-terminus of α-Syn to intracellular α-Syn in various PC12 cell lines, as measured by positive signals in immunocytochemistry (ICC) studies. Specifically, the relative binding affinities of anti-α-Syn antibodies elicited by α-Syn111-132, α-Syn121-135, and α-Syn126-135, or preimmune serum control samples, were evaluated in parental, mock-control, wild-type, and A53T mutant α-Syn-overexpressing PC12 cells upon NGF treatment. [Figure 12] Figures 10A-10C show that antibodies against the C-terminus of α-Syn bind only to α-Syn in PD brain sections, but not in healthy brain sections. Figure 10A shows that α-Syn antibodies and pre-immune antibodies elicited by α-Syn peptide immunogen constructs showed no detectable immunoreactivity in a panel of normal human tissues, including brain sections. [Figure 13] FIG. 10B shows antibody immunoreactivity to α-Syn aggregates in PD thalamic sections, as indicated by arrowheads. [Figure 14]Figure 10C is a table reporting the immunoreactivity of antibodies against the C-terminus of α-Syn and pre-immune serum controls to α-Syn aggregates in sections from PD and healthy brains, as determined by counting positive staining under a microscope. [Figure 15] Figures 11A and 11B are graphs showing anti-α-Syn IgG levels in the serum of PD mouse models after three immunizations with peptide immunogens containing adjuvant alone (open circles) or α-Syn111-132 (open squares); α-Syn126-135 (filled circles); or a combination of α-Syn111-132 and α-Syn126-135 (filled squares). Figure 11A shows IgG levels in an MPP+-induced mouse model. Figure 11B shows IgG levels in a mouse model inoculated with fibrillar α-Syn. [Figure 16] Figures 12A and 12B are graphs showing α-Syn levels in the peripheral circulation of PD mouse models after three immunizations with peptide immunogens containing adjuvant alone (open circles) or α-Syn111-132 (open squares); α-Syn126-135 (filled circles); or a combination of α-Syn111-132 and α-Syn126-135 (filled squares). Figure 12A shows α-Syn levels in an MPP+-induced mouse model. Figure 12B shows α-Syn levels in a mouse model inoculated with fibrillar α-Syn. [Figure 17] Figures 13A and 13B show the levels of oligomeric α-Syn in brain samples from untreated healthy mouse models (lane 1) or PD mouse models (lanes 2-3) immunized three times with adjuvant alone (lane 2) or a peptide immunogen containing α-Syn (lane 3). Untreated Balb / c mice represent the healthy mouse model, while MPP+-induced mice represent the PD mouse model. Figure 13A is a Western blot showing the levels of oligomeric α-Syn in the samples and the levels of GAPDH as a protein loading control. Figure 13B is a graph comparing the relative oligomeric α-Syn levels shown in the Western blot in Figure 13A after normalizing the protein levels to GAPDH levels and further normalizing the ratio of untreated healthy mouse model lysates to a level of 1.00 for comparison. [Figure 18] Figures 14A-14G show the levels of oligomeric α-Syn and tyrosine hydroxylase in brain samples from untreated healthy (lane 1) or PD (lanes 2-4) mice immunized three times with adjuvant alone (lane 2) or peptide immunogens containing α-Syn111-132 (lane 3) or α-Syn126-135 (lane 4). Untreated FVB mice represent the healthy mouse model, while fibrillar α-Syn-inoculated mice represent the PD mouse model. Figure 14A is a Western blot showing the levels of oligomeric α-Syn and tyrosine hydroxylase in lysates from ipsilateral substantia nigra, as well as the level of GAPDH as a protein loading control. Figure 14B is a graph comparing the relative oligomeric α-Syn levels shown in the Western blot in Figure 14A after normalizing protein levels to GAPDH levels. FIG. 14C is a graph comparing the relative tyrosine hydroxylase protein levels shown in the Western blot of FIG. 14A after normalizing the protein levels to GAPDH levels. [Figure 19] Figure 14D is a Western blot showing the levels of oligomeric α-Syn and GAPDH as a protein loading control in lysates from the ipsilateral striatum, and Figure 14E is a graph comparing the relative oligomeric α-Syn levels shown in the Western blots in Figure 14C after normalizing protein levels to GAPDH levels. [Figure 20] Figure 14F is a Western blot showing the levels of oligomeric α-Syn and GAPDH as a protein loading control in lysates from contralateral striatum, and Figure 14G is a graph comparing the relative oligomeric α-Syn levels shown in the Western blots in Figure 14E after normalizing protein levels to GAPDH levels. [Figure 21]Figures 15A-15C are graphs evaluating the motor function of mice measured with CatWalk™ XT in healthy mice (lanes 1-2) treated with saline (lane 1) or adjuvant alone (lane 2), or in PD mice (lanes 3-5) immunized with either adjuvant alone (lane 3) or peptide immunogens containing α-Syn126-135 (lane 4) or α-Syn111-132 (lane 5). A t-test was used to test significance (a p-value of less than 0.05 was defined as statistically significant and is indicated by an asterisk). Figure 15A evaluates the left hindlimb rearing time (seconds) of treated mice, with untreated FVB mice representing the healthy mouse model and fibrotic α-Syn-inoculated mice representing the PD mouse model. Figure 15B evaluates the running time (seconds) of treated mice, with untreated FVB mice representing the healthy mouse model and fibrotic α-Syn-inoculated mice representing the PD mouse model. FIG. 15C evaluates the running time (seconds) of treated mice, with untreated Balb / c mice representing a healthy mouse model and MPP+ induced mice representing a PD mouse model. [Figure 22] Figure 16A shows that PD-021514 (α-Syn 85-140, wpi 08) recognizes α-Syn strain fibrils with the highest affinity. Good binding to strain ribbons and fibrils-91 is observed. Poor binding to oligomers and fibrils-65. Poor binding to α-Syn monomers and fibrils lacking the C-terminal 30 amino acid residues (Fib-110). [Figure 23] Figure 16B shows that PD-021522 (α-Syn 85-140, wpi 13) binds to all strains / oligomers but not to monomers, and no concentration-dependent increase in signal is observed. The antibody binds to a fibril lacking the C-terminal 30 amino acid residues (Fib-110). Therefore, the epitope is not within this region. [Figure 24]Figure 16C shows that PD-100806 (α-Syn 126-135, wpi 09) binds to all strains and exhibits the highest affinity for ribbons. The antibody binds to native oligomeric α-Syn with low efficiency. Little binding is observed to glutaraldehyde- and dopamine-crosslinked oligomers and monomeric α-Syn. The antibody does not bind to fibrils (Fib-110) lacking the C-terminal 30 amino acid residues, presumably directed against the α-Syn 30 C-terminal amino acid residues. [Figure 25] Figure 16D shows that the commercially available antibody Syn1 (clone 42, BD bioscience) binds to all α-Syn strains and oligomers except glutaraldehyde crosslinks. The antibody also binds to monomeric asyn. Its epitope has been described to span residues 91 to 96 / 99. Consistently, the antibody binds to fibrils (Fib-110) lacking the C-terminal 30 amino acid residues. [Figure 26] Figure 16E shows that PRX002 recognizes fibrillar α-Syn with slightly better affinity than monomeric α-Syn. [Figure 27] FIG. 16F shows a background control of antibodies produced in guinea pigs. [Figure 28] FIG. 16G shows the background control for antibody Syn1. [Figure 29] FIG. 16H shows a background control of PRX002. [Figure 30]Figures 17A-17D show IHC analysis of the specificity of UNS antibodies against α-Syn in the basal ganglia of patients with dementia with Lewy bodies (DLB). The mean percentage area of ​​α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 in the putamen (Figure 17A), internal capsule (Figure 17B), and insular cortex (Figure 17C). Representative microscopic images of immunostaining of the putamen with each antibody are shown in Figure 17D. The UNS antibody detected a higher percentage area of ​​α-Syn aggregates in the putamen (F(3,7) = 1.550, p = 0.284 by ANOVA), internal capsule (F(3,7) = 1.356, p = 0.332 by ANOVA), and insular cortex (F(3,8) = 2.050, p = 0.195 by ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001. Data are shown as mean + SD (error bars). [Figure 31] Figures 18A-18D show IHC analysis of the specificity of UNS antibodies for α-Syn in the basal ganglia of Parkinson's disease (PD) patients. The mean percentage area of ​​α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 in the putamen (Figure 18A), internal capsule (Figure 18B), and insular cortex (Figure 18C) of three PD cases. A representative microscopic image of immunostaining is shown in Figure 18D for the putamen. The UNS antibody detected a higher percentage area of ​​α-Syn aggregates in the putamen (F(3,18) = 4.152, p = 0.047 by ANOVA), internal capsule (F(3,8) = 1.995, p = 0.1934 by ANOVA), and insular cortex (F(3,8) = 0.4044, p = 0.754 by ANOVA). PD100806 detected a significantly higher percentage area of ​​α-Syn compared with NCL-L-ASYN (p = 0.023 for PD100806 vs. NCL-L-ASYN; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA followed by Dunnett's test was performed. Data are shown as mean + SD (error bars). [Figure 32]Figures 19A-19C show IHC analysis of the specificity of the UNS antibody for α-Syn in the basal ganglia of patients with multiple system atrophy (MSA). The mean percentage area of ​​α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 in the putamen (Figure 19A) and internal capsule (Figure 19B) of three MSA cases. Because no pathology was detected in the insular cortex of MSA patients, it was not quantified. The UNS antibody detected a higher percentage area of ​​α-Syn aggregates in the putamen (F(3,8) = 1.56, p = 0.273 by ANOVA) and internal capsule (F(3,8) = 1.126, p = 0.395 by ANOVA). Representative microscopic images of immunostaining are shown for the putamen in Figure 19C, with each antibody indicated in C. P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are presented as mean + SD (error bars). [Figure 33] Figures 20A-20E show IHC analyses of the specificity of UNS antibodies for α-Syn in the midbrain of patients with different synucleinopathies. The mean percentage area of ​​α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 in the substantia nigra of patients with PD (Figure 20A), DLB (Figure 20B), and MSA (Figure 20C). The percentage area stained by each antibody was compared to the diagnostic antibody, NCL-L-ASYN. The UNS antibody detected a higher percentage area of ​​α-Syn aggregates in the substantia nigra of patients with MSA (F(3,8) = 0.830, p = 0.51 by ANOVA), DLB (F(3,7) = 2.493, p = 0.144 by ANOVA), and PD (F(3,7) = 0.189, p = 0.900 by ANOVA). [Figure 34] Representative microscopic images of immunostaining with each antibody are shown in Figure 20D (MSA) and Figure 20E (DLB). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean + SD (error bars). [Figure 35]Figures 21A-21F show IHC analyses of the specificity of UNS antibodies against α-Syn in the white and gray matter of the temporal cortex of patients with different synucleinopathies. The mean percentage area of ​​α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 of cortical gray matter and subcortical white matter in patients with PD (Figures 21A and 21D), DLB (Figures 21B and 21E), and MSA (Figures 21C and 21F). The percentage area stained by each antibody was compared to the diagnostic antibody, NCL-L-ASYN. *P<0.05; **P<0.01; ***P<0.001. One-way ANOVA followed by Dunnett's test was performed. Data are shown as mean + SD (error bars). [Figure 36] Figures 22A-C show IHC analyses of the specificity of the UNS antibody for α-Syn in the cerebellum of patients with different synucleinopathies. The mean percentage area of ​​α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 of cerebellar white matter from patients with PD (Figure 22A), DLB (Figure 22B), and MSA (Figure 22C). The UNS antibody detected a higher percentage area of ​​α-Syn aggregates in MSA (F(3,8) = 0.929, p = 0.469 by ANOVA), DLB (F(3,6) = 1.426, p = 0.325 by ANOVA), and PD (F(3,6) = 2.509, p = 0.157 by ANOVA). The percentage area stained by each antibody was compared with the diagnostic antibody NCL-L-ASYN. P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean + SD (error bars). [Figure 37] Figures 23A and 23B show representative images of immunostaining of the substantia nigra (Figure 23A) and putamen (Figure 23B) from the brain of an unaffected control patient with each antibody. Neither UNS antibody detected any α-Syn pathology comparable to the NCL-L-ASYN diagnostic antibody. [Figure 38]Figures 24A-24D show IHC analyses of the specificity of the UNS antibody for LBs in the insular cortex of the basal ganglia of patients with DLB or PD. The mean percentage area of ​​immunopositive LBs detected with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 in the insular cortex of patients with PD (Figure 24A) and DLB (Figure 24B). The percentage area of ​​LBs is expressed as a percentage of total α-Syn detected by each antibody. The UNS antibody detected a lower percentage of LBs (or a higher percentage of LNs) in the insular cortex of patients with DLB (F(3,7) = 0.836, p = 0.516 by ANOVA) and PD (F(3,4) = 0.913, p = 0.510 by ANOVA). The percentage area stained by each antibody was compared to the diagnostic antibody, NCL-L-ASYN. [Figure 39] Representative microscopic images of immunostaining with each antibody are shown in Figure 24C (PD) and Figure 24D (DLB). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean + SD (error bars). [Figure 40] Figures 25A-25D show IHC analyses of the specificity of the UNS antibody for LBs in the gray matter of the temporal cortex of patients with DLB or PD. The mean percentage area of ​​immunopositive LBs detected with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was determined for a total area of ​​7.5 mm2 of gray matter in patients with PD (Figure 25A) and DLB (Figure 25B). The percentage area of ​​LBs is expressed as a percentage of total α-synuclein detected by each antibody. The UNS antibody detected a lower percentage of LBs (or a higher percentage of LNs) in the gray matter of patients with PD (F(2,3) = 1.983, p = 0.282 by ANOVA) and DLB (F(3,7) = 1.906, p = 0.217 by ANOVA). The percentage area stained by each antibody was compared to the diagnostic antibody, NCL-L-ASYN. [Figure 41]Representative microscopic images of immunostaining with each antibody are shown in Figure 25C (PD) and Figure 25D (DLB). P<0.05 (*); P<0.01 (**); P<0.001 (***). Data are shown as mean + SD (error bars). [Figure 42] Figures 26A and 26B show representative images of immunostaining with UNS antibody and NCL-L-ASYN in the substantia nigra of the midbrain of a patient with DLB (Figure 26A) and PD (Figure 26B). Higher detection of LN by UNS antibody is observed compared to NCL-L-ASYN. [Figure 43] Figures 27A-C show cell-specific aggregation of α-Syn. Maximum projection overlay confocal images of α-Syn aggregates from the basal ganglia and midbrain of human cases with PD (Figure 27A), DLB (Figure 27B), and MSA (Figure 27C). α-Syn (PD062205, red) aggregates within neurons (HuD, green) in PD and DLB cases, but not in MSA. α-Syn (PD062205) and HuD are labeled in the grayscale drawings submitted with the application; color copies are available upon request. Scale bar: 10 μM. [Figure 44] Figures 28A-C show cell-specific aggregation of α-Syn. Maximum projection overlay confocal images of α-Syn aggregates from human cases of PD (Figure 28A), DLB (Figure 28B), and MSA (Figure 28C). α-Syn (PD062205, red) aggregates are located within oligodendrocytes (Olig2, green) in the case of MSA, but not in the case of PD or DLB. α-Syn (PD062205) and Olig2 are labeled in the grayscale drawings submitted with the application; color copies are available upon request. Scale bar: 10 μM.

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

[0030] The disclosed peptide immunogen constructs comprise a B cell epitope from α-Syn linked to a heterologous T helper cell (Th) epitope, either directly or, optionally, via a heterologous spacer. The B cell epitope portion of the peptide immunogen construct comprises approximately 10 to approximately 25 amino acid residues from the C-terminus of α-Syn, corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately 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 is derived from an amino acid sequence derived from a pathogenic protein. When administered to a host, the B cell epitope portion and the Th epitope portion of the peptide immunogen construct act together to stimulate the production of antibodies that specifically recognize and bind to the α-Syn B cell epitope portion of the construct.

[0031] The present disclosure also relates to compositions comprising the disclosed peptide immunogenic constructs, including pharmaceutical compositions. The disclosed pharmaceutical compositions can elicit an immune response and antibody production in a host against the disclosed peptide immunogenic constructs. The disclosed compositions can include a mixture of one or more of the disclosed peptide immunogenic constructs. In some embodiments, the compositions include the disclosed peptide immunogenic constructs along with additional components, including carriers, adjuvants, buffers, and other suitable reagents. In certain embodiments, the compositions include the disclosed peptide immunogenic constructs in the form of stabilized immunostimulatory complexes with CpG oligomers, optionally supplemented with adjuvants.

[0032] The present disclosure also relates to antibodies produced by hosts immunized with the disclosed peptide immunogenic constructs. The disclosed antibodies specifically recognize and bind to the B-cell epitope portion of α-Syn in the peptide immunogenic constructs. The disclosed α-Syn antibodies have unexpectedly high cross-reactivity with the β-sheets of α-Syn in the form of monomers, oligomers, or fibrils. Based on their unique characteristics and properties, the disclosed antibodies can provide an immunotherapeutic approach to targeting, identifying, and treating synucleinopathies.

[0033] The present disclosure also relates to methods of making and using the disclosed peptide immunogenic constructs, antibodies, and compositions. The disclosed methods provide low-cost manufacturing and quality control of peptide immunogenic constructs and compositions comprising the constructs, which can be used in methods of preventing and treating synopathies.

[0034] The present disclosure also includes methods for treating and / or preventing synucleinopathy using the disclosed peptide immunogenic constructs and / or antibodies against the peptide immunogenic constructs. In some embodiments, the methods for treating and / or preventing synucleinopathy include administering to a host a composition comprising the disclosed peptide immunogenic constructs. In certain embodiments, the composition utilized in the methods comprises the disclosed peptide immunogenic constructs in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, via electrostatic binding, optionally further supplemented with mineral salts or oils as adjuvants for administration to patients with synucleinopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogenic constructs to a host at risk for or with synucleinopathy.

[0035] 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.

[0036] Unless otherwise explained, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates 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. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

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

[0038] As used herein, the phrase "α-Syn peptide immunogen construct" refers to a peptide comprising (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.

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

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

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

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

[0043] As used herein, the phrase "C-terminal fragment of α-Syn" or "B-cell epitope from the C-terminus of α-Syn" refers to a 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 the 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 α-Syn sequence represented by SEQ ID NO:1.

[0044] The amino acid sequences of the terminal fragments of α-SynC used in the α-Syn peptide immunogen constructs were selected based on a number of design principles. Some of these principles include the use of the following α-Syn peptide sequences: (i) It does not share significant sequence homology with beta-synuclein (β-Syn) to avoid the generation of antibodies that cross-react with β-Syn, since β-Syn can bind to α-Syn and prevent its aggregation. (ii) lacking self T-helper epitopes within α-Syn to prevent autologous T-cell activation that could lead to brain inflammation causing meningococcal encephalitis, as previously reported in clinical trials using the AN1792 vaccine targeting Aβ1-42 for the treatment of Alzheimer's disease; (iii) falls within a region of α-Syn that is susceptible to conformational changes from its native form. (iv) Being a self molecule, it is non-immunogenic in itself. (v) Immunogenicity can be conferred by a protein carrier or strong T-helper epitope(s). (vi) When rendered immunogenic and administered to a host: (a) Elicit high titer antibodies against the α-Syn peptide sequence (B cell epitope) but not against the protein carrier or strong T helper epitope(s). (b) Eliciting high-titer antibodies that react with denatured β-sheets of α-Syn in the form of monomers, oligomers, or fibrils, such that such antibodies prevent α-Syn aggregation and disaggregate any α-Syn aggregates, resulting in the removal of toxic α-Syn oligomers, aggregates, and / or fibrils, thus reducing or preventing α-Syn aggregate burden in the brain. (c) It does not induce antibodies reactive with native α-Syn, which poses a high safety concern because native α-Syn is a major cellular protein with wide tissue distribution.

[0045] Taking these design principles into consideration, the C-terminal region of α-Syn was selected as the target for peptide immunogen design. Furthermore, this region was chosen because, based on its structural characteristics, this region was thought to be the most susceptible to modulation by antibodies or other physical factors compared with other regions of α-Syn.

[0046] As further described in the Examples, evaluation of numerous peptide sequences derived from α-Syn led to the identification and selection of several α-Syn peptides that satisfy the above-described design principles. Specifically, sequences that satisfy the design principles include peptides having about 10 to about 25 amino acid residues from the C-terminal region 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.

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

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

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

[0050] Immunologically functional analogs include amino acid sequences containing conservative substitutions, additions, deletions, or insertions of one to about four amino acid residues that elicit an immune response that cross-reacts with the α-SynG111-D135 peptide. Conservative substitutions, additions, and insertions can be achieved with natural or unnatural amino acids. Unnatural amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitrotyrosine, and pyroglutamic acid. 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.

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

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

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

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

[0055] As used herein, the term "heterologous" refers to an amino acid sequence derived from an amino acid sequence that is not part of or homologous to the wild-type sequence of α-Syn. Thus, a heterologous Th epitope is a Th epitope derived from an amino acid sequence 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 C-terminal fragment of α-Syn, the native amino acid sequence of α-Syn is not extended in either the N- or C-terminal direction.

[0056] The 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, pig, cow, dog, rat, mouse, guinea pig, etc.). 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, allowing for maximal activation of T helper cells, which lead to the initiation and regulation of immune responses. The Th epitope is preferably immunosilent by itself, i.e., most, if any, antibodies generated by the α-Syn peptide immunogen construct are not directed against the Th epitope, thereby allowing for a highly focused immune response to the targeted B cell epitope of the C-terminal fragment of α-Syn.

[0057] Epitopes of the present disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens, as exemplified in Table 2 (SEQ ID NOS: 70-98). 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 mixtures of amino acid residues represented at specific positions within the peptide framework based on the variable residues of homologs of that particular peptide. A collection of combinatorial peptides can be synthesized in a single process by adding a mixture of designated protected amino acids at designated positions during the synthesis process, instead of one specific amino acid. Such a collection of combinatorial heterologous Th epitope peptides can enable broad Th epitope coverage in animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOS: 79-82, as shown in Table 2. The epitopic peptides of the present invention provide broad reactivity and immunogenicity across a genetically diverse population of animals and patients.

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

[0059] Functional immunological analogs of Th epitope peptides are also useful and are included as part of the present invention. Functional immunological Th analogs can include conservative substitutions, additions, deletions, and insertions of one to about five 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 achieved with natural or unnatural amino acids, as described above for the C-terminal fragment of α-Syn. Table 2 identifies additional variations of functional analogs of Th epitope peptides. In particular, SEQ ID NOS: 71 and 78 of Th epitopes MvF1 and MvF2 are functional analogs of SEQ ID NOS: 81 and 83 of MvF4 and MvF5, which differ in 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- and C-termini, respectively. 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 include several versions of Th epitopes derived from the measles virus fusion protein MvFl-4 Th (SEQ ID NOs: 71, 78, 79, 81, and 83) and the hepatitis surface protein HBsAg1-3 Th (SEQ ID NOs: 80, 82, and 84).

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

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

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

[0063] As noted above, the term "heterologous" refers to an amino acid sequence derived from an amino acid sequence that is not part of or homologous to the wild-type sequence of α-Syn. Thus, when a heterologous spacer is covalently attached to a B-cell epitope from α-Syn, the native amino acid sequence of α-Syn is not extended at either the N- or C-terminus because the spacer is heterologous to the α-Syn sequence.

[0064] A spacer is any molecule or chemical structure that can link two amino acids and / or peptides together. Spacers can vary in length and polarity depending on the application. Spacer bonds can be via amide or carboxyl bonds, although other functional groups are possible as well. Spacers can include chemical compounds, naturally occurring amino acids, or non-naturally occurring amino acids.

[0065] Spacers can provide structural features to α-Syn peptide immunogen constructs. Structurally, spacers physically separate the Th epitope from the B cell epitope of the C-terminal fragment of α-Syn. This physical separation can disrupt any artificial secondary structure created by linking the Th epitope to the B cell epitope. Furthermore, physical separation of epitopes by a spacer can eliminate interference between Th and / or B cell responses. Furthermore, spacers can be designed to create or modify secondary structure in peptide immunogen constructs. For example, to enhance the separation of Th and B cell epitopes, spacers can be designed to function as flexible hinges. Flexible hinge spacers allow more efficient interaction between the presented peptide immunogen and appropriate Th and B cells, enhancing immune responses against the Th and B cell epitopes. Examples of sequences encoding flexible hinges are found in the hinge region of immunoglobulin heavy chains, which are 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: 148), where Xaa is any amino acid, preferably aspartic acid.

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

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

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

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

[0070] The spacer in the α-syn peptide immunogen construct can be covalently attached to the N- or C-terminus of the Th epitope and the C-terminal peptide of α-syn. In some embodiments, the spacer is covalently attached to the C-terminus of the Th epitope and the N-terminus of the C-terminal peptide of α-syn. In other embodiments, the spacer is covalently attached to the C-terminus of the C-terminal peptide of α-syn and the N-terminus of the Th epitope. In certain embodiments, for example, when more than one Th epitope is present in the peptide immunogen construct, more than one spacer can be used. When more than one spacer is used, each spacer can be the same or different from the others. Furthermore, when more than one Th epitope is present in the peptide immunogen construct, the Th epitopes can be separated by a spacer, which can be the same or different from the spacer used to separate the Th epitope from the B cell epitope. There are no restrictions on the placement of the spacer relative to the Th epitope or the C-terminal fragment of α-syn.

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

[0072] d. Specific Embodiments of Alpha-Syn Peptide Immunogen Constructs The α-Syn peptide immunogen construct has the formula: (Th) m -(A) n -(C-terminal fragment of α-Syn)-X or (C-terminal fragment of α-Syn)-(A) n -(Th) m -X It can be expressed as During the ceremony, Th is a heterologous T helper epitope, A is a heterologous spacer, (C-terminal fragment of α-Syn) is a B cell epitope having about 10 to about 25 amino acid residues from the C-terminus of α-Syn, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4; n is 0 to about 10.

[0073] In certain embodiments, the heterologous Th epitope of the α-syn peptide immunogenic construct has an amino acid sequence selected from any one 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 one of SEQ ID NOs: 78-84. In certain embodiments, the α-syn peptide immunogenic construct comprises more than one Th epitope.

[0074] 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 any combination thereof. In certain embodiments, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

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

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

[0077] composition The present disclosure also provides compositions comprising the disclosed α-Syn peptide immunogenic constructs.

[0078] a. Peptide composition Compositions containing the disclosed α-Syn peptide immunogenic constructs can be in liquid or solid form. Liquid compositions can contain water, buffers, solvents, salts, and / or other acceptable reagents that do not alter the structural or functional properties of the α-Syn peptide immunogenic constructs. Peptide compositions can include one or more of the disclosed α-Syn peptide immunogenic constructs.

[0079] b. Pharmaceutical Compositions The present disclosure also relates to pharmaceutical compositions comprising the disclosed α-Syn peptide immunogenic constructs.

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

[0081] Pharmaceutical compositions can include one or more adjuvants that act to accelerate, prolong, or enhance the immune response to the α-Syn peptide immunogen construct without having a specific antigenic effect themselves. Adjuvants used in pharmaceutical compositions can 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, ISA 35, ISA 206, ISA50V, ISA51, ISA 720, and other adjuvants and emulsifiers.

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

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

[0084] Pharmaceutical compositions can be prepared as injections, either as liquid solutions or suspensions. Liquid vehicles containing the α-Syn peptide immunogen constructs can also be prepared prior to injection. Pharmaceutical compositions can be administered by any suitable mode of administration, such as id, iv, ip, im, intranasal, oral, or subcutaneous, and with any suitable delivery device. In certain embodiments, pharmaceutical compositions are formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration, including oral and intranasal administration, can also be prepared.

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

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

[0087] In some embodiments, pharmaceutical compositions contain more than one α-Syn peptide immunogen construct. Pharmaceutical compositions containing a mixture of more than one α-Syn peptide immunogen construct can synergistically enhance the immunological effects of the constructs. Pharmaceutical compositions containing more than one α-Syn peptide immunogen construct are more effective in larger genetic populations due to broader MHC class II coverage, thereby improving the immune response to the α-Syn peptide immunogen constructs.

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

[0089] Pharmaceutical compositions comprising α-Syn peptide immunogenic constructs can be used to elicit an immune response and produce antibodies in a host upon administration.

[0090] c. Immune stimulating complex The present disclosure also relates to pharmaceutical compositions comprising α-syn peptide immunogen constructs in the form of immune stimulating complexes with CpG oligonucleotides. Such immune stimulating complexes are particularly adapted to act as adjuvants and peptide immunogen stabilizers. The immune stimulating complexes are in the form of microparticles and can efficiently present the α-syn peptide immunogen to cells of the immune system to generate an immune response. The immune stimulating complexes may be formulated as suspensions for parenteral administration. The immune stimulating complexes can also be formulated as w / o emulsions, as suspensions combined with inorganic salts or in situ gelling polymers, for efficient delivery of the α-syn peptide immunogen to host immune system cells after parenteral administration. The immune stimulating complexes can generate a protective / therapeutic immune response against the β-sheet of α-syn (e.g., Figures 8A, 8B, and 8C in Example 13).

[0091] Stabilized immune stimulating complexes can be formed by complexing α-Syn peptide immunogen constructs with anionic molecules, oligonucleotides, polynucleotides, or combinations thereof via electrostatic binding. The stabilized immune stimulating complexes can be incorporated into pharmaceutical compositions as immunogen delivery systems.

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

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

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

[0095] The resulting immunostimulating complexes are typically in the form of particles ranging in size from 1 to 50 microns, 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 adjuvant and upregulation of specific immune responses in vivo. Furthermore, stabilized immunostimulating complexes are suitable for preparing pharmaceutical compositions by a variety of processes, including water-in-oil emulsions, inorganic salt suspensions, and polymer gels.

[0096] antibody The present disclosure also provides antibodies elicited by the α-Syn peptide immunogen constructs.

[0097] C-terminal fragments of α-Syn, comprising approximately 10 to approximately 25 amino acid residues from the C-terminal region of α-Syn, corresponding to the sequence from approximately glycine at amino acid position 111 (G111) to approximately asparagine at amino acid position 135 (D135) of full-length α-Syn, are non- or weakly immunogenic by themselves. However, the disclosed α-Syn peptide immunogen constructs, comprising a C-terminal fragment of α-Syn, a heterologous Th epitope, and an optional heterologous spacer, can elicit an immune response and antibody production when administered to a host. The design of the α-Syn peptide immunogen constructs can break tolerance to self-α-Syn and elicit the production of site-specific antibodies that recognize conformational, rather than linear, epitopes.

[0098] Surprisingly, antibodies generated by α-Syn peptide immunogen constructs do not bind to the native alpha helices of native α-Syn monomers. Instead, they recognize and bind to the denatured β-sheets of α-Syn in monomeric, oligomeric, and fibril forms. Furthermore, antibodies generated by α-Syn peptide immunogen constructs do not bind to similar structures in other amyloidogenic proteins (i.e., Aβ1-42 and Tau441). Therefore, the specific design of α-Syn peptide immunogen constructs (containing a C-terminal fragment of α-Syn, a heterologous Th epitope, and an optional heterologous spacer) allows for the conformational alteration of the versatile α-Syn C-terminal fragment to enable a β-sheet-like conformation.

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

[0100] Antibodies elicited by α-Syn peptide immunogen constructs surprisingly prevent α-Syn aggregation (anti-aggregation activity) and dissociate preformed α-Syn aggregates (disaggregation activity). Furthermore, the antibodies surprisingly reduce microglial cell-induced TNF-α and IL-6 production, indicating that these antibodies can effectively reduce α-Syn aggregate- or fibril-mediated microglial activation. These antibodies were also found to reduce neurodegeneration caused by both exogenous α-Syn aggregates and endogenous α-Syn aggregates in α-Syn-overexpressing cells. Furthermore, such antibodies specifically recognize and bind to pathological α-Syn oligomeric aggregates or fibrils, but do not react with non-pathological α-Syn. Specifically, the antibodies react with Lewy bodies in brain sections taken from patients with alpha-synucleinopathy and Parkinson's disease, but not with normal human tissue.

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

[0102] The immune responses obtained from animals immunized with the α-Syn peptide immunogen constructs of the present invention demonstrated the ability of the constructs to generate potent, site-specific antibodies that react with the denatured β-sheets of α-Syn in the monomeric, oligomeric, and fibril forms, but not with the random coil structure of the native C-terminal α-Syn.

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

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

[0105] a. Method for producing α-Syn peptide immunogen constructs The α-syn peptide immunogen constructs of the present disclosure can be produced by chemical synthesis methods familiar to those skilled in the art (see, e.g., Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W.H. Freeman & Co., New York, NY, 1992, p. 77). The α-syn peptide immunogen constructs can be synthesized, for example, using automated Merrifield techniques of solid-phase synthesis with α-NH2 protected with either t-Boc or F-moc chemistry using 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 of Th epitopes, can be achieved by providing a mixture of alternative amino acids for coupling at specific variable positions.

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

[0107] This chemical process allows for the control and definition of the quality of the peptides produced, thereby ensuring the reproducibility, immunogenicity, and yield of 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.

[0108] The range of structural variability that allows for the retention of intended immunological activity has been found to be much more flexible than the range of structural variability that allows for the retention of specific drug activity by small molecule drugs, or the range of structural variability that allows for the retention of desired activity and undesired toxicity by large molecules co-produced with biologically derived drugs. Thus, intentionally designed peptide analogs, or those inevitably produced by errors in the synthesis process as mixtures of deletion sequence by-products with chromatographic and immunological properties similar to the intended peptide, are often just as effective as purified preparations of the desired peptide. Mixtures of designed and unintended analogs can be effective as long as distinct QC procedures are developed to monitor both the manufacturing and product evaluation processes to ensure the reproducibility and efficacy of final products using these peptides.

[0109] Alpha-Syn peptide immunogenic constructs can also be produced using recombinant DNA technology, including nucleic acid molecules, vectors, and / or host cells. Accordingly, nucleic acid molecules encoding the alpha-Syn peptide immunogenic constructs and immunologically functional analogs thereof are also encompassed by this disclosure as part of the invention. Similarly, vectors, including expression vectors, containing nucleic acid molecules, and host cells containing vectors are also encompassed by this disclosure as part of the invention.

[0110] Various exemplary embodiments also encompass methods for producing immunologically functional analogs of α-Syn peptide immunogen constructs and α-Syn G111-D135 fragment-derived peptide immunogen constructs. For example, the methods can include incubating host cells containing expression vectors containing nucleic acid molecules encoding the α-Syn peptide immunogen constructs and / or immunologically functional analogs thereof under conditions such that the peptides and / or analogs are expressed. Longer synthetic peptide immunogens can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of the invention, the amino acid sequence is reverse-translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with optimal codons for the organism in which the gene will be expressed. A synthetic gene is then typically created by synthesizing oligonucleotides encoding the peptide and any regulatory elements, as needed. The synthetic gene is inserted into an appropriate cloning vector and transfected into a host cell. The peptide is then expressed under conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0111] b. Methods of producing immunostimulating complexes Various exemplary embodiments also encompass methods for producing immune stimulating complexes (ISCs) containing 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 stoichiometry of the molar charge ratio between the cationic portion of the α-Syn peptide immunogen construct and the anionic oligomer determines the degree of association. The non-covalent electrostatic binding of the α-Syn peptide immunogen construct to CpG ODN is a fully reproducible process. The aggregation of peptide / CpG ODN immune stimulating complexes facilitates presentation to "professional" antigen-presenting cells (APCs) of the immune system, thus further enhancing the immunogenicity of the complexes. These complexes are easily characterized for quality control during manufacturing. Peptide / CpG ISCs are well tolerated in vivo. This novel particulate system, comprising CpG ODN and an α-SynG111-D135 fragment-derived peptide immunogen construct, was designed to promote a balanced Th-1 / Th-2 type response while taking advantage of the generalized B cell mitogenicity associated with the use of CpG ODN.

[0112] The CpG ODN in the disclosed pharmaceutical compositions binds 100% to immunogens in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particles. The particulate form allows for a significant reduction in the CpG dosage from traditional use of CpG adjuvants, reducing the potential for adverse innate immune responses and promoting alternative immunogen processing pathways involving antigen-presenting cells (APCs). As a result, such formulations are conceptually novel and offer potential advantages by promoting stimulation of immune responses through alternative mechanisms.

[0113] c. Method of manufacturing pharmaceutical compositions Various exemplary embodiments also encompass pharmaceutical compositions comprising the α-Syn peptide immunogenic constructs. In certain embodiments, the pharmaceutical composition employs a water-in-oil emulsion and a suspension comprising an inorganic salt.

[0114] Safety is another important factor to consider when using pharmaceutical compositions in large populations and preventing α-Syn aggregation is part of the administration goal. Despite the use of human water-in-oil emulsions in many formulations in clinical trials, alum remains the primary adjuvant for use in formulations due to its safety. Therefore, alum or its inorganic salt, aluminum phosphate (ADJUPHOS), is frequently used as an adjuvant in preparations for clinical applications.

[0115] d. Methods of Using the Pharmaceutical Composition The present disclosure also includes methods of using pharmaceutical compositions comprising the α-Syn peptide immunogenic constructs.

[0116] In certain embodiments, pharmaceutical compositions comprising α-Syn peptide immunogenic constructs can be used to: (a) inhibiting α-Syn aggregation in the host; (b) induce the degradation of preformed α-Syn aggregates in the host; (c) reducing host microglial TNF-α and IL6 secretion; (d) reducing neurodegeneration caused by exogenous α-Syn aggregates in the host; (e) reducing neurodegeneration in α-Syn-overexpressing cells; (f) reducing host serum α-Syn levels; (g) reducing oligomeric α-Syn levels in the host brain; (h) Alleviating neuropathology and restoring host motor activity; etc.

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

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

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

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

[0121] (4) The α-Syn peptide immunogen construct described in (1), wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (sequence number 148).

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

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

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

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

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

[0127] (10) A composition comprising the α-Syn peptide immunogenic construct described in (1).

[0128] (11) A composition comprising more than one α-Syn peptide immunogenic construct according to (1).

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

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

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

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

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

[0134] (17) The isolated antibody or epitope-binding fragment thereof according to (16), bound to the α-Syn peptide immunogenic construct.

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

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

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

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

[0139] (22) A method for producing antibodies that recognize α-Syn in a host, comprising administering to the host a composition comprising the α-Syn peptide immunogen described in (1) and a delivery vehicle and / or adjuvant.

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

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

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

[0143] A detailed description of the procedures used is provided in the following examples. Example 1 Synthesis of alpha-synuclein-related peptides and preparation of their formulations a. Synthesis of the C-terminal fragment of α-Syn We describe the synthesis of designer α-Syn C-terminal fragments that were included in the development effort for α-Syn peptide immunogen constructs. Peptides were synthesized in small quantities useful for serological assays, laboratory pilot and field studies, as well as in large-scale (kilogram) quantities useful for industrial / commercial production of pharmaceutical compositions. A large repertoire of α-Syn-related antigenic peptides with sequences ranging in length from approximately 10 to 40 amino acids was designed to screen and select optimal peptide constructs for use in effective α-Syn peptide immunogen constructs.

[0144] Representative full-length α-Syn (SEQ ID NO: 1) and β-Syn (SEQ ID NO: 2) used for epitope mapping in various serological assays. 111-132 , α-Syn 126-135 α-Syn segments, such as 10-mer peptides, are shown in Table 1 (SEQ ID NOS: 1 and 3-69). Selected α-Syn fragments were engineered into α-Syn peptide immunogen constructs by synthetically linking them to carefully designed helper T cell (Th) epitopes derived from pathogen proteins, including measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza, Clostridium tetani, and Epstein-Barr virus (EBV), as identified in Table 2 (SEQ ID NOS: 70-98). The Th epitopes were used either individually (SEQ ID NOS: 70-78 and 83-98) or in combinatorial libraries (SEQ ID NOS: 79-82) to enhance the immunogenicity of each α-Syn peptide immunogen construct.

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

[0146] b. Preparation of Compositions Comprising α-Syn Peptide Immunogen Constructs Formulations using water-in-oil emulsions and suspensions containing inorganic salts have been prepared. Because pharmaceutical compositions are designed for use in large populations and prevention is part of the administration goal, safety is another important factor to consider. Despite the use of human water-in-oil emulsions in many pharmaceutical compositions in clinical trials, alum remains the primary adjuvant for use in pharmaceutical compositions due to its safety. Therefore, alum or its inorganic salt, ADJUPHOS (aluminum phosphate), is frequently used as an adjuvant in formulations for clinical applications.

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

[0148] The α-Syn peptide immunogen constructs were prepared in various amounts of the designated peptide construct (i) in a water-in-oil emulsion containing Seppic Montanide™ ISA 51 as an oil approved for human use, or (ii) mixed with the inorganic salts ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum). Compositions were typically prepared by dissolving the α-Syn peptide immunogen construct in water at approximately 20-800 μg / mL and formulating it into a water-in-oil emulsion (1:1 by volume) with Montanide™ ISA 51 or with inorganic salts or ALHYDROGEL (alum) (1:1 by volume). The compositions were kept at room temperature for approximately 30 minutes and vortex-mixed for approximately 10-15 seconds prior to immunization. Some animals were immunized intramuscularly with two to three doses of a particular composition, administered at time 0 (prime) and 3 weeks post-immunization (wpi) (booster), with a second booster at 5 or 6 wpi, if needed. These immunized animals were then tested with selected B epitope peptide(s) to assess the immunogenicity of the various α-Syn peptide immunogen constructs present in the formulations and their cross-reactivity with relevant target peptides or proteins. Those α-Syn peptide immunogen constructs with strong immunogenicity in the initial guinea pig screening were further tested in primates in water-in-oil emulsion, inorganic salt, and alum-based formulations over the specified time periods and dosing regimens, as directed in the immunization protocol.

[0149] Only the most promising α-Syn peptide immunogen constructs were further extensively evaluated before being incorporated into the final formulation for immunogenicity, duration, toxicity, and efficacy studies in GLP-guided preclinical studies in preparation for submission of an Investigational New Drug application and clinical trials in patients with synucleinopathy.

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

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

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

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

[0154] b. Evaluation of antibody reactivity to Th peptides by Th peptide-based ELISA test The wells coated with peptides (SEQ ID NOS: 70-98) were incubated with 250 μL of 3% (wt.) gelatin in PBS for 1 hour at 37°C to block nonspecific protein binding sites, then washed three times with PBS containing 0.05% (v / v) TWEEN® 20 and dried. The serum to be analyzed was diluted 1:20 with PBS containing 20% ​​(v / v) normal goat serum, 1% (wt.) gelatin, and 0.05% (v / v) TWEEN® 20 (unless otherwise specified). One hundred microliters (100 μL) of the diluted sample (e.g., serum, plasma) was added to each well and incubated for 60 minutes at 37°C. The wells were then washed six times with PBS containing 0.05% (v / v) TWEEN® 20 to remove unbound antibody. Horseradish peroxidase (HRP)-conjugated goat anti-IgG specific for a species (e.g., mouse, guinea pig, or human) was used as a labeled tracer to bind to the antibody / peptide antigen complexes formed in positive wells. One hundred microliters of peroxidase-labeled goat anti-IgG in 1% (v / v) normal goat serum containing 0.05% (v / v) TWEEN® 20 in PBS at a pre-titered optimal dilution was added to each well and incubated for an additional 30 minutes at 37°C. The wells were washed six times with 0.05% (v / v) TWEEN® 20 in PBS to remove unbound antibody, and then reacted with 100 μL of a substrate mixture containing 0.04% (w / w) 3',3',5',5'-tetramethylbenzidine (TMB) and 0.12% (v / v) hydrogen peroxide in sodium citrate buffer for an additional 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. The reaction was stopped by adding 100 μL of 1.0 M H2SO4 and the absorbance at 450 nm (A 450 For the determination of antibody titers in immunized animals receiving various α-Syn-derived peptide immunogens, 10-fold serial dilutions of sera from 1:100 to 1:10,000 were tested, and Log 10 The titer of the tested serum, expressed as a cut-off A 450 A set to 0.5 450 was calculated by linear regression analysis.

[0155] Detailed specificity analysis and epitope mapping of cB cell epitope clusters against α-Syn fragments by 10-mer peptide-based ELISA assays Detailed specificity analysis of anti-α-Syn antibodies in immunized hosts was determined by epitope mapping. Briefly, wells of a 96-well plate were coated with individual α-Syn 10-mer peptides (SEQ ID NOS: 18–69) at 0.5 μg per 0.1 mL per well. Then, 100 μL of serum samples (1:100 dilution in PBS) were incubated in duplicate in the 10-mer plate wells, following the steps of the antibody ELISA method described above. Detailed specificity analysis of B cell epitopes of the α-Syn peptide immunogen constructs and associated anti-α-Syn antibodies in immune sera from immunized hosts was also performed using the corresponding α-Syn peptides (SEQ ID NOS: 99, 102, 108, 110, 112, and 113) or their fragments without the spacer and Th sequence, or β-Syn (SEQ ID NOS: 153) for additional reactivity and specificity confirmation.

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

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

[0158] f. Preparation of α-Syn aggregates using recombinant α-Syn To prepare aggregated α-Syn, purified wild-type or A53T mutant α-Syn (0.1 μg / μl in 100 μL of PBS / KCl aggregation buffer (2.5 mM MgCl, 50 mM HEPES, and 150 mM KCl in 1x PBS, pH 7.4)) was incubated in a 1.5 mL Eppendorf tube at 37°C for 7 days in a thermomixer (Eppendorf) without shaking. Aggregated α-Syn was immediately frozen at -80°C for later use.

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

[0160] h. Specificity of anti-α-Syn antibodies purified from antisera of guinea pigs immunized with α-Syn peptide immunogen constructs of different sizes. Anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs were screened for binding specificity to α-Syn molecular complexes of different sizes using Western blot analysis. 20 μM α-Syn was separated by 12% Tris-glycine SDS-PAGE and transferred to a nitrocellulose (NC) membrane prior to photoinduced cross-linking (PICUP) treatment. The membrane was incubated with 1 μg / mL anti-α-Syn antibody purified from the guinea pig antisera, followed by incubation with donkey anti-guinea pig antibody conjugated with HRP (706-035-148, Jackson). Blots were visualized with the chemiluminescent reagent Western Lightning ECL Pro (PerkinElmer). As a result, monomeric α-Syn (MW 14,460 Da) blotted at a size close to 14 kDa, whereas the molecular weights of dimers, trimers, and oligomers were several times larger than the 14 kDa size of monomeric α-Syn. Syn211 (Abeam), a commercially available antibody that can detect various oligomeric species, including dimers, trimers, and larger oligomers, was used as a positive control.

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

[0162] These monomers and oligomers were verified by thioflavin-T (ThT, Sigma) fluorescence or PAGE (polyacrylamide gel electrophoresis). The concentration of amyloidogenic proteins was measured using commercially available amyloidogenic Aβ 1-42 The stock was used as a standard for the Nano-Drop assay. These monomers and oligomers were analyzed using the Aβ 1-42 Three μg of α-Syn, 4 μg of α-Syn, and 7 μg of tau were individually spotted onto PVDF membranes. The membranes were incubated with anti-α-Syn antibody purified from guinea pig antiserum (1:1000 dilution) as the primary antibody, followed by hybridization with anti-guinea pig HRP-conjugated secondary antibody (1:5000; Vector Laboratories). The membranes were treated with Luminata Western HRP substrate (Bio-Rad, Hercules, CA, USA), and the signal was detected using a ChemiDoc-It 810 digital imaging system (UVP Inc., Upland, CA, USA).

[0163] i. Binding specificity to aggregated α-Syn in α-Syn-overexpressing PC12 cells upon nerve growth factor (NGF) treatment Immunocytochemistry (ICC) was performed on NGF-treated parental PC12, mock-control PC12, and α-Syn-overexpressing PC12 cells using anti-α-Syn antibodies purified from guinea pig antisera collected 8 or 9 weeks post-immunization to assess the binding affinity of the antibody elicited after immunization. Cell nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Images were taken under a fluorescence microscope and scored based on the ratio of positively stained cells to the total number of cells, with a grade of -, +, ++, and +++ representing <1%, 1-15%, 16-50%, and >50%.

[0164] Example 4 Cells and animals used in immunogenicity and efficacy studies a. α-Syn overexpressing PC12 cells: The pZD / XOL-L-α-Syn plasmid was constructed by inserting a cDNA sequence encoding full-length human wild-type α-Syn or the A53T mutant α-Syn into the pZD / XOL-L vector, which contains a CMV promoter. The constructs were transfected into PC12 cells using Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. 2.5 μL of the transfection mixture, 500 μL of Opti-MEM medium, 2.5 μL of PLUS Reagent, and 8.75 μL of Lipofectamine LTX were mixed and incubated at room temperature for 25 minutes. After replacing the medium with 1.5 mL of RPMI 1640 growth medium, 500 μL of the transfection mixture was added directly to each well and incubated at 37°C for 1 day. Transfection efficiency was confirmed by PCR and Western blotting.

[0165] b. Guinea pig: Immunogenicity studies were conducted in adult, naive, male and female Duncan-Hartley guinea pigs (300–350 g / BW). Experiments included at least three guinea pigs per group. Protocols involving Duncan-Hartley guinea pigs (8–12 weeks old; Covance Research Laboratories, Denver, PA, USA) were performed in a contract animal facility under an approved IACUC application, sponsored by UBI.

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

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

[0168] Example 5 Design principles, screening, identification, and optimization of multicomponent pharmaceutical compositions incorporating alpha-synuclein peptide immunogenic constructs a. Design history Each α-Syn peptide immunogen construct or immunotherapy product requires a unique design focus and approach based on the specific disease mechanism and target protein(s) required for intervention. Targets of interest may include cellular proteins involved in disease pathways or infectious agents that may involve several proteins from the pathogen. The process from research to commercialization is very long, typically requiring more than 10 years to achieve.

[0169] Once a target molecule is selected, an extensive serological validation process is required. Identification and distribution of B- and T-cell epitopes within the target molecule are critical for the design of molecular α-Syn peptide immunogen constructs. Once the target B-cell epitope is identified, serial pilot immunogenicity studies in small animals are conducted to evaluate the functional properties of antibodies elicited by the designer peptide pharmaceutical composition. These serological applications are then conducted in animals of the target species to further validate the immunogenicity and functional properties of the α-Syn peptide immunogen constructs. All studies are conducted in multiple parallel groups, with sera collected from immunized hosts for evaluation. Initial immunogenicity studies in the target species, or in the case of human pharmaceutical compositions, in non-human primates, are also conducted to further validate immunogenicity and design direction. The target peptides are then prepared in various mixtures and used in combination to prepare each formulation, evaluating subtle differences in functional properties associated with the respective interactions between the peptide constructs. After further evaluation, the final peptide construct, peptide composition, and its formulation, along with the respective physical parameters of the formulation, are established, leading to the final product development process.

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

[0171] i) Selection of the C-terminal portion of α-Syn as a target for peptide immunogen design. α-Syn is an intrinsically disordered protein. It consists of 140 amino acids and is divided into three regions. The N-terminal region (residues 1–60) can form an amphipathic helix, a typical conformation for membrane recognition and binding. The central region, including residues 61–95, is well known as the non-amyloid-β component (NAC), which was first identified in AD senile plaques. This region has a high tendency to form a β-rich conformation and is prone to aggregation. Different types of post-translational modifications within this region have distinct effects on regulating α-Syn aggregation. The C-terminal region, from residues 96–140, is rich in proline and negatively charged residues, a common characteristic of intrinsically disordered proteins that maintain solubility. This C-terminal domain generally exists in a random coil conformation due to its low hydrophobicity and large net negative charge. In vitro studies have revealed that α-Syn aggregation can be induced by decreasing the pH, which neutralizes these negative charges. α-Syn is characterized by extreme conformational diversity, allowing it to adapt to various conditions and perform versatile functions in membrane-bound, cytosolic, and amyloid-aggregated states. The C-terminal random coil and intrinsically disordered region, which is important for maintaining protein solubility, was selected as a target for peptide immunogen design because this region was thought to be most susceptible to modulation by antibodies or other physical factors compared with the N-terminal amphipathic helix and central β-rich conformational region.

[0172] ii) Identification of self-Th epitopes for exclusion in α-Syn B epitope design. Preliminary immunogenicity analysis demonstrated that deletion of a peptide sequence from the N-terminus of the α-Syn sequence significantly inhibited the immunogenicity of α-Syn. 126-140 (SEQ ID NO: 9), α-Syn 121-140 (SEQ ID NO: 8), α-Syn 111-140 (SEQ ID NO: 7) peptide is completely non-immunogenic, but some moderate immunogenicity is observed. 101-140 (SEQ ID NO: 6), α-Syn 91-140 (SEQ ID NO: 5), and α-Syn 85-140This finding, observed with the (SEQ ID NO: 4) peptide (Table 4), indicates the presence of a potential self-Th-like structure within the C-terminal sequence and confirms the presence of helper T cell epitope(s) at the C-terminus of α-Syn. Inclusion of such a sequence in B epitope(s) design may lead to booster brain inflammation through activation of autologous T cells, as in the prior Alzheimer's disease vaccine AN1792. Therefore, this finding necessitates the design of α-Syn peptide immunogen constructs with B cell epitope(s) beginning at amino acid residue G111 to avoid the potential inclusion of self T cell epitope(s) in the B epitope design.

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

[0174] iv) Evaluation of the immunogenicity of C-terminal α-Syn peptide immunogen constructs with respect to antibody reactivity with the corresponding α-Syn and β-Syn. The synuclein family includes three known proteins: α-synuclein, β-synuclein, and gamma-synuclein. All synucleins share a highly conserved alpha-helical lipid-binding motif that is similar to the exchangeable apolipoprotein class A2 lipid-binding domain. β-synuclein is highly homologous to α-synuclein. β-synuclein has been suggested to be an inhibitor of α-synuclein aggregation, which occurs in neurodegenerative diseases such as Parkinson's disease. β-synuclein may therefore protect the central nervous system from the neurotoxic effects of α-synuclein. Therefore, it is preferable to have an α-synuclein peptide immunogen construct to elicit antibodies that preferentially react with α-synuclein rather than the corresponding aggregation-protecting β-synuclein. When six peptide immunogen constructs ending in C-terminus A140 were tested, all antibodies derived from sera immune to these constructs showed significant cross-reactivity with β-synuclein of the corresponding size, as shown in Table 6. A closer examination of the sequence homology between α-Syn and β-Syn (SEQ ID NOS: 1 and 2) showed that the sequences corresponding to the C-terminal five amino acids of YEPEA are identical between the two proteins. Therefore, it is desirable to design B epitopes that exclude these five amino acids of YEPEA. Thus, findings from the immunogenicity studies shown in Table 6 led to the deletion of YEPEA (Y136-A140) in the B epitope(s) design. Upon incorporation of a spacer sequence and, for example, the artificial T helper peptide UBITh1 (SEQ ID NOS: 83) into the α-Syn peptide immunogen construct, designs using the B cell epitope sequence excluding the YEPEA tail shown in Table 7 (SEQ ID NOS: 107-114) were all highly immunogenic when evaluated with the long α-Syn peptide K97-A140 (SEQ ID NOS: 110). None of the immune sera reacted with β-Syn. Therefore, from the data obtained from Tables 6 and 7, the B epitope design of peptide immunogen constructs is limited to α-SynG111 to D135 and fragments thereof.

[0175] v) Antibodies elicited by αSyn peptide immunogen constructs reacted only with beta-sheet monomers, oligomers, or fibrils, but not with α-helical monomers. Using sound principles for the design of α-Syn peptide immunogens, it was surprisingly found that antibodies generated from the designed α-Syn peptide immunogen constructs possess a B epitope with the sequence starting at G111 and ending at D135, or a fragment thereof, and the elicited antibodies react specifically with β-sheet α-Syn monomers, oligomers, and fibrils, but not with β-sheet Aβ. 1-42 Since it does not react with Tau1-441, it provides an ideal candidate for an α-Syn peptide immunogen construct, as representatively shown by the α-Syn peptide immunogen construct in Figure 8 (SEQ ID NOs: 112 and 113).

[0176] vi) Expanded MHC coverage by using α-Syn derived peptide immunogen constructs and different promiscuous T helper epitopes. When designing pharmaceutical compositions for treating patients with diverse genetic backgrounds, it is important to maximize the coverage of the genetically diverse population. Therefore, the synergistic immunogenic effects of α-Syn-derived peptide immunogen constructs in such combinations were investigated. Because promiscuous T-helper epitopes derived from MVF or HBsAg are among the most potent for providing such enhanced immunogenicity, combinations of peptide constructs containing T-helper epitopes were designed for such exploration. A mixture of two peptide immunogen constructs bearing the same B epitope was found to elicit a significant immune response compared to the immune responses elicited by each individual peptide construct.

[0177] Example 6 α-Syn peptide immunogen constructs elicit focused antibody responses solely against targeted B cell epitopes All carrier proteins used to enhance immune responses to targeted B cell epitope peptides by chemical conjugation of such B cell epitope peptides to the respective carrier proteins (e.g., keyhole limpet hemocyanin (KLH) or other carrier proteins such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins) elicit more than 90% of antibodies directed against the enhanced carrier protein and less than 10% of antibodies directed against the targeted B cell epitope in immunized hosts. Therefore, it is of interest to evaluate the specificity of the α-Syn peptide immunogen constructs of the present invention. For immunogenicity evaluation, a series of eight α-Syn peptide immunogen constructs (SEQ ID NOS: 107 to 114) were prepared, each carrying a B cell epitope of varying length linked via a spacer sequence to the heterologous T cell epitope UBITh1 (SEQ ID NOS: 83). UBITh1 (a T-helper peptide used for B epitope boosting) was coated onto plates, and guinea pig immune sera were used to test for cross-reactivity with the UBITh1 peptides used for boosting. In contrast to the high immunogenicity of these constructs against the corresponding targeted B epitopes, as shown by the high titers of antibodies generated against the B epitopes as shown in Tables 6 and 7, many, if not all, of the immune sera were found to be unreactive against the UBITh1 peptides, as shown in Table 8.

[0178] In summary, simple immunogen design incorporating a target B cell epitope linked to a carefully selected T helper epitope allows for the generation of a focused, clean immune response that targets only the α-Syn B cell epitope. In designing pharmaceutical compositions, the more specific the immune response, the higher the safety profile provided by the composition. Thus, the α-Syn peptide immunogen constructs of the present invention are highly specific for their target yet highly potent.

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

[0180] As shown in Table 9, pooled 9wpi guinea pig immune sera obtained from six α-Syn peptide immunogen constructs [(K97-D135, SEQ ID NO: 110), (G111-D135, SEQ ID NO: 108), (G111-G132, SEQ ID NO: 113), (E126-D135, SEQ ID NO: 112), (G101-A140, SEQ ID NO: 104), and (E126-A140, SEQ ID NO: 99)] were selected for detailed epitope mapping. These six B epitope fragments of various lengths completely encompass the 97-140 sequence of the C-terminal region of α-synuclein. ELISA results showed that all six immune sera reacted strongly with a representative long α-Syn peptide (97-135, SEQ ID NO: 10). Detailed epitope mapping studies of 10-mer peptides revealed immunogenic epitopes encompassing the region around AA114 to 125 (peptides 114-123, 115-124, and 116-125 of SEQ ID NOs: 52, 53, and 54), as well as a highly immunogenic region at the C-terminus represented by peptide 131-140 (SEQ ID NO: 69). Interestingly, most immune sera derived from the C-terminal α-Syn peptide immunogen constructs elicited antibodies that recognized conformational, but not linear, epitopes, with the exception of an epitope located at the C-terminus of α-Syn with the sequence EGYQDYEPEA (SEQ ID NO: 69) responsible for cross-reactivity with the β-Syn protein.

[0181] Although this epitope mapping result was largely unexpected, it correlated well with the finding that these antibodies were derived from α-Syn peptide immunogen constructs represented by α-Syn111-132 (SEQ ID NO: 113) and α-Syn126-135 (SEQ ID NO: 112) from the C-terminal random coil region of α-Syn bound to heterologous Th epitope structures that resemble the denatured β-sheet of α-Syn and result in a conformation that is non-cross-reactive with the α-helix of native α-Syn.

[0182] Example 8 Antibodies elicited by alpha-Syn peptide immunogen constructs and their formulations: Anti-aggregation and disaggregation effects on recombinant alpha-synuclein protein Anti-α-Syn antibodies purified from guinea pig antisera raised against recombinant α-Syn were used to evaluate the efficacy of α-Syn peptide immunogen constructs in in vitro antiaggregation and disaggregation assays.

[0183] a. Inhibition of α-Syn aggregation Initial screening assays for potential anti-α-syn antibodies purified from guinea pigs immunized with different α-syn peptide immunogen constructs were performed by quantifying the level of α-syn aggregation changes by thiofabin T measurement, as described in Example 3. Recombinant α-syn prepared at 100 μM in PBS was incubated at a concentration of 5 μM in 384-well plates with 40 μL of PBS / KCl buffer (2.5 mM MgCl, 50 mM HEPES, and 150 mM KCl in 1× PBS, pH 7.4) for an additional 6 days to induce aggregation. Different concentrations (0.05, 0.5, or 5 μg / mL) of anti-α-syn antibodies purified from guinea pig antisera immunized with different α-syn peptide immunogen constructs collected at different time points were added to the incubation mixture to evaluate their respective effects on inhibiting α-syn aggregation. By the end of the incubation, aggregation levels were determined using the ThT assay, and measurements from each run were normalized to the aggregation level of the vehicle control, which was set at 100%, and measurements obtained in the absence of α-Syn, which was set at 0%.

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

[0185] b. Dissociation of preformed α-Syn aggregates The above studies demonstrated that anti-α-Syn antibodies purified from guinea pig antisera immunized with specific α-Syn peptide immunogen constructs were effective in inhibiting α-Syn aggregation. To further evaluate whether antibodies elicited by α-Syn peptide immunogen constructs were effective in dissociating preformed α-Syn aggregates, we performed an in vitro disaggregation assay using anti-α-Syn antibodies purified from guinea pig antisera.

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

[0187] α-Syn 111-132 (SEQ ID NO: 113) or α-Syn 126-135 Two selected anti-α-Syn antibodies elicited by (SEQ ID NO: 112), as well as α-Syn 111-132 (SEQ ID NO: 113) Induction and α-Syn 126-135 (SEQ ID NO: 112) and combinations of elicited anti-α-Syn antibodies were tested in this in vitro disaggregation assay. 126-135 (SEQ ID NO: 112) and α-Syn 111-132The anti-α-Syn antibody elicited by (SEQ ID NO: 113) demonstrated a dissociation effect on preformed α-Syn aggregates of approximately 50% compared to the vehicle control as 100%, whereas other anti-α-Syn antibodies and antibodies purified from pre-immunized animals failed to show comparable effects (Figure 2).

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

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

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

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

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

[0193] Example 10 Antibodies elicited by α-Syn peptide immunogen constructs and their formulations: Effects on reducing microglial TNF-α and IL-6 secretion Neuronal injury in the substantia nigra is thought to release aggregated α-Syn into the substantia nigra, activating microglia through the production of proinflammatory mediators, thereby leading to the persistent and progressive neurodegeneration of the substantia nigra in PD. To evaluate the effectiveness of anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogen constructs in reducing microglial activation, we measured the levels of proinflammatory mediators, TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6), released by microglia upon treatment with α-Syn aggregates in the presence or absence of different anti-α-Syn antibodies.

[0194] Mouse BV2 cells or human SVG p12 cells were seeded at 5,000 cells / well in RPMI 1640 medium supplemented with 1% FBS. Cells were treated with 1 μM α-Syn and incubated at 37°C, 5% CO2, and a humidified atmosphere for 24 hours. The medium was then collected, centrifuged, and the supernatant separated. The concentrations of IL-6 secreted by BV2 cells and TNF-α secreted by SVG p12 cells in the supernatant were analyzed in triplicate using mouse IL-6 or human TNF-α mouse ELISA kits (Thermofisher), respectively. Signals were normalized to the vehicle control as 100%.

[0195] Data are from α-Syn 111-132 (SEQ ID NO: 113) and α-Syn 123-135 Anti-α-Syn antibodies elicited by (SEQ ID NO: 111) reduced α-Syn aggregate-mediated TNF-α release by SVG p12 cells by 30-50%; whereas α-Syn 123-135 Anti-α-Syn antibodies elicited by (SEQ ID NO: 111) reduced IL-6 release by SVGp12 cells by approximately 30% (Figure 4). 123-135 Anti-α-Syn antibodies elicited by (SEQ ID NO: 111) were shown to be more potent than other anti-α-Syn antibodies tested in attenuating α-Syn aggregate-mediated microglial activation.

[0196] Example 11 Antibodies elicited by alpha-Syn peptide immunogen constructs and their formulations: Impact on reducing neurodegeneration induced by exogenous alpha-synuclein To evaluate the neuroprotective effects of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs, an in vitro neurodegeneration model with exogenous preformed α-Syn aggregates in NGF-treated neuronally differentiated PC12 cells was employed.

[0197] PC12 cells were treated with NGF (100 ng / mL) for 6 days to induce neuronal differentiation. The morphology of neuronally differentiated cells was confirmed and analyzed using an InCell High-Content Image Analysis System (GE Healthcare). The neurotrophic effect of NGF was reflected in neurite outgrowth, and the number of neuronally differentiated cells was quantified. The level of neurite outgrowth and the number of neuronally differentiated cells were expressed as normalized percentages (mean ± SEM). The neurite length of PC12 cells with and without NGF treatment was set to 100% and 0%, respectively. The number of neuronally differentiated PC12 cells with 6 days of NGF treatment was normalized to 100%.

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

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

[0200] Example 12 Antibodies elicited by α-Syn peptide immunogen constructs and their formulations: Effect on reducing neurodegeneration in α-Syn overexpressing cells To evaluate the neuroprotective effects of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs, an in vitro neurodegeneration model using wild-type and A53T mutant α-Syn-overexpressing PC12 cells was employed.

[0201] After incubation with NGF, mock control cells (transfected with the plasmid vector) developed long neurite outgrowths and increased in cell number similar to parental wild-type PC12 cells, whereas wild-type α-Syn-overexpressing PC12 cells and A53T mutant α-Syn-overexpressing PC12 cells did not exhibit comparable neurite outgrowths or increased cell number, confirming the neurodegenerative effects associated with aggregated α-Syn upon NGF treatment. To characterize overexpressed α-Syn in wild-type α-Syn-overexpressing PC12 cells upon NGF treatment, Western blot and ThT assays were performed using cell lysates from wild-type α-Syn-overexpressing PC12 cells after NGF treatment. Western blotting results showed that α-Syn was overexpressed in the cell lysates of wild-type α-Syn-overexpressing PC12 cells upon NGF treatment, and ThT assay results showed that α-Syn in the cell lysates of wild-type α-Syn-overexpressing PC12 cells upon NGF treatment was in a β-sheet structure (i.e., an increase in ThT fluorescent signal). Compared with the results of Western blotting and ThT assays of wild-type α-Syn-overexpressing PC12 cells without NGF treatment, these results suggest that a structural transition from α-helical to β-sheet structure of overexpressed α-Syn occurs during NGF-induced neuronal differentiation, which may subsequently lead to the neurodegenerative effects of β-sheet oligomeric α-Syn. Furthermore, compared with wild-type α-Syn-overexpressing PC12 cells, overexpression of A53T mutant α-Syn resulted in stronger neurodegenerative effects, reflected by both shortened neurite length and decreased cell number upon NGF treatment, indicating that A53T mutant α-Syn caused stronger neurodegenerative effects than wild-type α-Syn in α-Syn-overexpressing PC12 cells.

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

[0203] Example 13 Antibodies elicited by alpha-Syn peptide immunogen constructs and their formulation: Specificity for beta-sheet oligomers and fibrillar alpha-synuclein protein To better characterize the specificity of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs, a series of in vitro assays were performed with α-Syn molecular complexes of different sizes, different amyloidogenic proteins including α-Syn, Aβ, and tau proteins, and α-Syn aggregated in α-Syn-overexpressing PC12 cells upon NGF treatment. a. Specificity for large α-Syn molecular complexes Western blots of α-Syn molecular complexes of different sizes were performed using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs as primary antibodies. The results showed that all anti-α-Syn antibodies reacted strongly with larger α-Syn molecular complexes, including dimers, trimers, tetramers, and oligomers, in addition to smaller monomeric α-Syn. Compared with a commercially available anti-α-Syn antibody, Syn211 (Abcam), α-Syn 111-132 (SEQ ID NO: 113), α-Syn 121-135 (SEQ ID NO: 107), α-Syn 123-135 (SEQ ID NO: 111), and α-Syn 126-135 The anti-α-Syn antibody elicited by (SEQ ID NO: 112) showed a high ratio of signals from larger α-Syn molecular complexes (including dimers, trimers, tetramers, and oligomers) to signals from smaller monomeric α-Syn (Figures 7A and 7B), suggesting that the anti-α-Syn antibody has specificity for larger α-Syn molecular complexes.

[0204] b. Specificity for α-Syn among different amyloidogenic proteins Different amyloidogenic proteins (i.e., α-Syn, Aβ) were prepared as described in Example 3. 1-42Dot blot assays with different species of α-Syn (i.e., α-helical monomers, β-sheet monomers, β-sheet oligomers, and β-sheet fibrils) were performed using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen constructs as primary antibodies. 126-135 (SEQ ID NO: 112) and α-Syn 111-132 Anti-α-Syn antibodies elicited by (SEQ ID NO: 113) were shown to specifically react with all β-sheet forms of α-Syn (monomer, oligomer, and fibrillar species), but not with α-helical monomers (Figures 8A, 8B, and 8C). 126-135 (SEQ ID NO: 112) and α-Syn 111-132 The anti-α-Syn antibodies elicited by (SEQ ID NO: 113) reacted more strongly with α-Syn β-sheet fibrils and α-Syn β-sheet oligomers than with β-sheet monomers. 126-135 (SEQ ID NO: 112) and α-Syn 111-132 Anti-α-Syn antibodies elicited by (SEQ ID NO: 113) inhibited β-Syn or the amyloidogenic protein Aβ 1-42 and showed no detectable reactivity to different species of Tau441 (i.e., α-helical monomers, β-sheet monomers, β-sheet oligomers, and β-sheet fibrils) (Figures 8A, 8B, and 8C). This finding supports the conclusion that α-Syn 126-135 (SEQ ID NO: 112) and α-Syn 111-132 These results suggest that the anti-α-Syn antibodies induced by (SEQ ID NO: 113) have specificity for β-sheet monomers, β-sheet oligomers, and β-sheet fibrillar forms of α-Syn.

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

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

[0207] Pre-immunization, α-Syn 126-135 (SEQ ID NO: 112) or α-Syn 111-132 Another immunohistopathological study using anti-α-Syn antibodies elicited by (SEQ ID NO: 113) and a 1:1 combination of both anti-α-Syn antibodies was performed to test their reactivity with human Parkinson's disease brains. Histological sections (BioChain) from three regions (i.e., cerebellum, corpus callosum, and thalamus) were assayed. The results showed that α-Syn 126-135 (SEQ ID NO: 112) or α-Syn 111-132Anti-α-Syn antibodies elicited by (SEQ ID NO: 113) and a 1:1 combination of both anti-α-Syn antibodies showed positive reactivity (indicated by arrows) in PD brain sections in all three regions, compared with negative reactivity in healthy brain sections (Figures 10B and 10C). Quantification of reactivity to α-Syn aggregates in PD brain sections was performed by counting positive staining under a microscope. Results showed that α-Syn aggregates were significantly more abundant than those in healthy brain sections. 126-135 (SEQ ID NO: 112) or α-Syn 111-132 The anti-α-Syn antibodies elicited by (SEQ ID NO: 113) and a 1:1 combination of both anti-α-Syn antibodies were shown to be strongly positive in PD brain sections compared to healthy human brain sections. Of the three different anti-α-Syn antibodies assayed, α-Syn 111-132 Antibodies elicited by (SEQ ID NO: 113) had the strongest immunoreactivity to α-Syn aggregates in PD brain sections.

[0208] Example 15 Demonstration of the efficacy of α-Syn peptide immunogen constructs and their formulations in animal models a. Immunization and collection of blood / brain tissue A Parkinson's disease (PD) mouse model was established as described in Example 4. MPP + Two weeks after injection or seven weeks after inoculation of fibrillar α-Syn, mice were immunized with the adjuvant group (immunized with the adjuvant and solvent used in the preparation of the composition (ISA 51 VG, CpG3, 0.2% TWEEN®-80)) plus the UBITh1-bound α-Syn. 111-132 (SEQ ID NO: 113) Peptide, UBITh1-binding α-Syn 126-135 The mice were randomly divided into three groups, each containing a 40 μg dose of the 40 μg (SEQ ID NO: 112) peptide, and a combination of both peptides. Intramuscular (IM) immunization was administered three times at 3-week intervals. The administration and blood sampling schedule was performed according to Table 13.

[0209] At each time point, 200 μL of blood was collected via facial vein sampling. Blood dripping from the punctured submandibular vein was collected into microtubes and centrifuged at 300 rpm for 10 minutes to prepare serum. After sacrifice, brain tissue samples were collected for Western blotting.

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

[0211] Formulated α-Syn 111-132 (SEQ ID NO: 113), formulated α-Syn 126-135 (SEQ ID NO: 112), or a combination of both peptide immunogen constructs, were administered to two PD mouse models, respectively. + In the induction model (Fig. 11A) or fibrillar α-Syn challenge model (Fig. 11B), anti-α-Syn antibody optical density (OD) values ​​exceeded 3.0 after the second immunization and remained elevated until the end of the study, 15 and 19 weeks after the initial immunization, whereas adjuvanted animals did not elicit a measurable anti-α-Syn immune response.

[0212] In the fibrillar α-Syn inoculation model, α-Syn 111-132 The construct was α-Syn 126-135 Although the construct elicited a stronger immune response than the MPP construct (Fig. 11B), + Note that no differences in immunogenicity were observed in the induction model (Figure 11A).

[0213] C. Decreased serum α-Syn levels The α-Syn levels in pooled serum from animals in each group were assayed using an ELISA kit (SEB222Mu, USCN) capable of detecting both alpha-helical and β-sheet α-Syn, as described in Example 3 .

[0214] The α-Syn quantitative ELISA was performed to test whether the anti-α-Syn antibody response in the immunized groups was associated with reduced amounts of peripheral α-Syn compared to untreated animals. + In both the induction model (Fig. 12A) and the fibrillar α-Syn inoculation model (Fig. 12B), α-Syn 126-135 (SEQ ID NO: 112), α-Syn 111-132 Immunization with either the α-Syn peptide construct (SEQ ID NO: 113), or a combination of these constructs demonstrated decreased optical density (OD) values ​​of α-Syn levels compared to adjuvanted animals. The results suggested that the development of an anti-α-Syn antibody response upon immunization with the α-Syn peptide immunogen construct resulted in a corresponding decrease in the amount of α-Syn in the peripheral circulation.

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

[0216] MPP + In the induction model, α-Syn 111-132 Animals immunized with the peptide immunogen constructs showed a decrease in the oligomeric α-Syn fraction (Fig. 13A). Similarly, in mice inoculated with fibrillar α-Syn, Western blots using lysates of the substantia nigra and striatum ipsilateral to fibrillar α-Syn inoculation (Figs. 14A and 14D) and striatum contralateral to fibrillar α-Syn inoculation (Fig. 14F) demonstrated that the 2- to 3-fold increase in oligomeric α-Syn levels seen in adjuvant control mice was significantly greater than that observed with formulated α-Syn. 111-132 (SEQ ID NO: 113) and α-Syn 126-135 (SEQ ID NO: 112) construct, the expression of IL-16 was reduced after treatment with the construct. Quantification of the Western blotting results is shown in Figures 13B, 14B, 14C, 14D, and 14G.

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

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

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

[0220] An average of five crossings was analyzed for each mouse. Because fibrillary α-Syn inoculation was performed in the right hemisphere, left hindlimb rearing time was considered the reference parameter, with running time alone.

[0221] In the fibrillar α-Syn inoculation model, α-Syn 126-135 (SEQ ID NO: 112) or α-Syn 111-132 After treatment with a composition containing (SEQ ID NO: 113), significant differences were observed in measurements of left hindlimb standing time (Figure 15A). Meanwhile, in both the fibrotic α-Syn inoculation model and the MPP+ challenge model, α-Syn 111-132 After treatment with a composition containing (SEQ ID NO: 113), significant differences in measured transit times were observed (Figures 15B and 15C). 126-135 (SEQ ID NO: 112) or formulated α-Syn 111-132 (SEQ ID NO: 113) and improved motor function in two PD mouse models.

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

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

[0224] Dr. Ronald Melki was a collaborator on this study. In the lab, they generated different types of α-Syn aggregates: (a) fibrils—long, twisted, zipped-together chains of α-Syn protein; (b) ribbons—wider, flatter structures; and (c) α-Syn oligomers (O550), dopamine-stabilized (ODA), and glutaraldehyde-stabilized (OGA) oligomers.

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

[0226] Methods and Materials a. Assembly of α-Syn into fibrils and ribbons For fibril formation, soluble WT α-Syn was incubated in Buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl) at 37°C with continuous shaking in an Eppendorf Thermomixer set at 600 rpm. Assembly was continuously monitored in the presence of Thioflavin T (15 μM) in a 1 × 1 cm cuvette under stirring (100 rpm) using a magnetic stir bar (6 × 3 mm) using a Cary Eclipse spectrofluorometer (Varian Inc., Palo Alto, CA, USA) with an excitation wavelength set at 440 nm, emission wavelengths set at 440 and 480 nm, and an averaging time of 1 s. For ribbon formation, WT α-Syn was dialyzed against 1,000x buffer B (5 mM Tris-HCl pH 7.5) at 4°C for 16 hours and then incubated at 37°C with continuous shaking in an Eppendorf thermomixer set at 600 rpm. Assembly was monitored by measuring scattered light at 440 nm. Alternatively, the amount of protein remaining in the supernatant after sedimentation at 35,000 x g was determined by measuring absorbance at 280 nm on a Hewlett Packard 8453 diode array spectrophotometer. The nature of the oligomeric species was assessed using a Jeol 1400 (Jeol Ltd.) TEM by adsorption of samples to carbon-coated 200-mesh grids and negative staining with 1% uranyl acetate. Images were recorded with a Gatan Orius CCD camera (Gatan). The Congo Red binding ability of α-Syn assemblies was assessed as follows: α-Syn fibrils and ribbons were incubated with 100 μM Congo Red (Sigma-Aldrich, St. Louis, MO, USA) in 20 mM Tris buffer (pH 7.5) for 1 h. The polymers were then precipitated at 25,000 g for 30 min at 20 °C in a TL100 tabletop Beckman ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA). The pellets were washed four times with an equal volume of water. After resuspension of the pellets, aliquots were placed on cover slips and either imaged immediately or allowed to dry.Samples were observed in bright field and cross-polarized light by polarized light microscopy using a Leica (MZ12.5) microscope (Leica Microsystems, Ltd., Heerbrugg, Switzerland) equipped with cross-polarizers.

[0227] b. Determination of α-Syn fibril and ribbon concentrations Length heterogeneity of α-Syn fibrils and ribbons was reduced by sonication for 20 minutes on ice in 2 ml Eppendorf tubes in a VialTweeter equipped with an ultrasonic processor UIS250v (250 W, 2.4 kHz, Hielscher Ultrasonic, Teltow, Germany) set at 75% amplitude and 0.5-second pulses. The sedimentation velocities of α-Syn fibrils and ribbons were measured. The sedimentation boundaries were analyzed with Sedfit software using least-squares boundary modeling (1s-g*(s)) that best fits heterogeneous mixtures of large particles. This resulted in a distribution of particles with sedimentation coefficients ranging from 50 to 150 S for α-Syn ribbons and 100 to 1,000 S for α-Syn fibrils, centered around species with sedimentation coefficients of approximately 90 S and 375 S for α-Syn ribbons and fibrils, respectively. For α-Syn ribbons, this corresponds to a particle with a molecular weight of approximately 11,500 kDa, composed of, for example, approximately 800 α-Syn molecules (12,000 kDa / 14.5 kDa), and for α-Syn fibrils, this corresponds to a particle with a molecular weight of approximately 102,000 kDa, composed of, for example, approximately 7,000 α-Syn molecules (10,2000 kDa / 14.5 kDa). Therefore, assuming that 100% of the α-Syn assembles into ribbons or fibrils at steady state, since 100% of the protein is found in the pellet fraction upon centrifugation of the sample, at a working concentration of 20 μM, the concentrations of total α-Syn ribbon and fibril particles are 20 μM / ≈800 = ≈0.02 μM and 20 μM / ≈7,000 = ≈0.003 μM for α-Syn ribbons and fibrils, respectively.

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

[0229] Using a slot blot filtration apparatus, increasing amounts of fibrillar, oligomeric, or monomeric α-Syn, ranging from 20 pg to 200 ng, were spotted onto nitrocellulose filters. The filters were then blocked with nonfat milk and incubated with PRX002 or Syn1 antibodies, or the test GP antibodies of this disclosure, at the indicated dilutions. After extensive washing, secondary anti-human or anti-guinea pig IgG-HRP was used to detect the primary antibody binding profile. A secondary antibody-only control was also tested. Super Signal ECL (Pierce #34096) was applied to the blots, which were then imaged using a BioRad imager (Chemidoc MP Imaging System / BioRad imagelab software). Exposure times and dynamic ranges are shown in Figures 16A-16H. In this series of measurements, human brain homogenates from DLB cases were spotted onto the membranes.

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

[0231] Figures 16A-16H show that the reference antibody PRX002 recognizes fibrillar α-Syn with slightly better affinity when compared to monomeric α-Syn, while both antibodies recognize fibrillar α-Syn with slightly better affinity than the α-Syn of the present disclosure. 126-135 The peptide constructs PD-100806 and PD-021514 had much higher affinity for fibrillar α-Syn compared to monomeric α-Syn, indicating that both have preferential binding to fibrillar α-Syn. The affinity of PRX002 for oligomeric and fibrillar α-Syn was found to be similar. The Syn1 monoclonal antibody bound to fibrillar α-Syn as well as oligomeric and monomeric α-Syn, but with less distinct preference.

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

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

[0234] b human brain tissue For this study, μm-thick sections were obtained from the UCL Brain Bank. All samples were collected and prepared in accordance with National Research Ethics Service-approved procedures.

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

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

[0237] These brain regions are known to be affected by α-Syn aggregation to varying degrees and at different stages of disease progression in each disease type. Generally, the basal ganglia and midbrain are affected in the early stages of DLB, PD, and MSA, and have the highest aggregate burden. The temporal cortex and cerebellum are affected in the later stages of the disease, with cerebellar aggregates being almost absent in PD and DLB. A negative control (without primary antibody) was performed alongside each IHC protocol to confirm the absence of nonspecific binding of the secondary antibody. Paraffin-embedded slides were dewaxed in a 60°C oven for 15-20 minutes and then immersed in xylene I and II for 5 minutes each. The tissue was rehydrated in four dilutions of IMS, from 100% to 50%, for 5 minutes each. The tissue was washed three times for 5 minutes in 1x PBS, followed by a 3-minute incubation in 100% formic acid for antigen retrieval. The tissue was then thoroughly washed in 1x PBS before quenching endogenous peroxidase activity with 3% H2O2 for 10 minutes. After cooling, the tissues were washed three more times (5 min each) with 1x PBS and then microwaved in citrate buffer (15 mM Tris-sodium citrate, TWEEN, pH 6) at medium temperature for 25 min. Three racks of slides in three containers were included each time to ensure comparable microwave times per run. After cooling and washing three times (5 min) with 1x PBS, nonspecific binding sites were blocked with 15% normal goat serum. The tissues were incubated with primary antibody (1:100 in 0.1% TBS / t) overnight at 4°C. The tissues were washed three times for 5 min with 1x PBS and then incubated in biotinylated secondary antibody for 1 h (RT). ABC solution was prepared 30 min prior to its application. The tissues were washed three times for 5 min with 1x PBS and then incubated with ABC for 1 h at room temperature. VIP peroxidase substrate was prepared using the ImmPACT VIP Peroxidase Kit as detailed in the manufacturer's instructions. VIP peroxidase substrate was added for 7 min at RT and washed with dH2O. Before mounting in DPX, tissues were dehydrated in 50%, 70%, 95%, 100%, and 100% IMS, and xylene I and II for 2 min each. For double immunofluorescence staining, tissues were not quenched with 3% H2O2 before application of the primary antibody.After application of the first primary antibody and the corresponding secondary antibody, the tissue was blocked with 15% normal goat serum for 30 minutes and then incubated with the second primary and secondary antibodies as described above. After the final application of the fluorescently labeled secondary antibody, the tissue was incubated with 1% Sudan Black for 5 minutes to quench autofluorescence, washed with 0.1% TBS / T, and immediately mounted in Mowiol Cituflour. Fluorescently stained tissues were stored at 4°C until imaging.

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

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

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

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

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

[0243] e.α-Syn 126-135 The antibody detected a different pattern of α-Syn aggregates compared to NCL-L-ASYN The cell type and subcellular localization of α-Syn aggregates differ between different synucleinopathies. While MSA is characterized by glial cytoplasmic inclusions (GCIs), α-Syn aggregates occur within neuronal cell bodies (LBs) and neurites (LNs) in DLB and PD. Analysis of the percentage of stained area allowed for quantification of the total α-Syn aggregates detected by each antibody. However, this did not account for differences in the type or subcellular location of the aggregates detected. The distinct patterns of α-Syn aggregates within cell bodies and neurites in PD and DLB allowed us to quantify the relative sensitivity of the UNS antibodies disclosed herein to these different types of α-Syn aggregates.

[0244] To investigate this, we estimated the percentage of aggregates detected within cell bodies for each antibody in DLB and PD cases. Using FIJI software, aggregates within cell bodies were selected based on their size and circularity. The average percentage area of ​​cell body aggregates was then calculated as a percentage of total α-Syn detected, and the results are shown in Figures 24A-D and 25A-D. The difference in the percentage area of ​​total α-Syn and cell body α-Syn was attributed to axonal aggregates (LNs) of α-Syn based on qualitative analysis of the tissue. A decrease in the percentage of cell body α-Syn detection results in an increase in LN detection. This analysis was performed on the gray matter of the temporal and insular cortices, as these regions exhibited both LB- and LN-like pathology. Because LNs were very sparse and unevenly distributed throughout the putamen and capsule, these regions of the basal ganglia were not selected for this analysis. A similar correlation was observed in the substantia nigra of the midbrain (Figures 26A and 26B), where the UNS antibody of the present disclosure detected higher levels of LN compared to NCL-L-ASYN in DLB and PD, however, the complex morphology of LN and LB meant that they could not be reliably distinguished and quantified using the same method.

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

[0246] f. α-Syn aggregation is cell type specific α-Syn-containing aggregates are a characteristic pathogenic feature of synucleinopathies, including MSA, DLB, and PD. While α-Syn aggregates are the primary causative protein in synucleinopathies, the pattern of aggregation and the cell types prone to aggregate formation differ between specific disease subtypes. Clinical features of MSA, DLB, and PD explain the accumulation of α-Syn within the cell bodies and superficial neurites of neurons in both DLB and PD, whereas in MSA it is found primarily within glial cells and oligodendrocytes.

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

[0248] The results in Figures 27A-27C show that α-Syn detected by PD062205 co-localizes within neuronal cell bodies in the basal ganglia and midbrain (high pathology areas) of PD and DLB, but not MSA. Using a marker for oligodendrocytes (Olig2), Figures 28A-28C show that α-Syn aggregates within glial cells in MSA, but not in PD or DLB. These results support the notion that α-Syn is aggregated within glial cells in MSA, but not in PD or DLB. 126-135We show that the antibodies match the clinical features of these synucleinopathies, confirming the specificity of these antibodies for pathological aggregates of α-Syn.

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

[0250] b. Representative α-Syn 126-135 Antibodies derived from immunization of guinea pigs with peptide immunogen constructs are more sensitive than commercially available diagnostic antibodies in binding to α-Syn aggregates Disclosed α-Syn 126-135 To examine the relative antigenicity of the antibodies, we compared the α-Syn load detected by each antibody with that of a commercially available diagnostic antibody for synucleinopathy (NCL-L-ASYN). First, by examining the overall pattern of results shown in Figures 17A-D through 22A-C, we found that the α-Syn load was significantly higher than that of NCL-L-ASYN. 126-135 It can be seen that there is a significant increase in the mean percentage area of ​​α-Syn detected by the antibody. This trend is consistent across brain regions and disease types, and is consistent with the disclosed α-Syn 126-135 This suggests that the antibody is more sensitive or selective in binding to aggregated α-Syn than to NCL-L-ASYN. Although the sample size was relatively small in this study (n=3), clear trends are still evident in the data. 126-135 The specificity of the antibodies was confirmed in the same brain region of the unaffected control patient's brain. These results, shown in Figures 23A-B, demonstrate the complete absence of immunopositive staining with each antibody, including NCL-L-ASYN. These data support the disclosed α-Syn 126-135This indicates that the antibody is specific for the pathological form of α-Syn.

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

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

[0253] To investigate the use of the reagent as an immunotherapy for common synucleinopathies, we investigated the effects of α-Syn on α-Syn in different synucleinopathies with different neuropathologies. 126-135 It is important to assess the selectivity of the antibodies. Clinically confirmed cases of PD, DLB, and MSA were selected for this purpose. PD and DLB are the second most common forms of dementia and are primarily caused by α-Syn accumulation within neurons (LBs and LNs). In contrast to PD, amyloid beta and tau pathology are known to contribute to neurodegeneration in DLB2. A distinct pattern of α-Syn aggregation is seen in MSA, where aggregates form primarily within glial cells rather than neurons (Figures 27A-C and 28A-B). Furthermore, the progression of α-Syn pathology differs between disease types, with the midbrain and basal ganglia being common areas of early pathology. Examining the antigenicity of each antibody in brain regions affected at different stages of the disease will provide insight into which antibodies may be more effective in treating the early stages of the disease.

[0254] d.α-Syn 126-135 The antibodies (PD062220, PD062205, and PD100806) can specifically bind to pathological aggregates of α-Syn in human brain tissue from PD, DLB, and MSA (Figures 17A-D to 22A-C), without detecting any synuclein pathology in healthy controls (Figures 23A-B). Disclosed α-Syn 126-135 Detection of α-Syn by antibodies was achieved with the same cell type specificity as described in clinical neuropathology (Figures 27A-B and 28A-B). Importantly, the disclosed α-Syn 126-135 The antibodies did not exhibit equal antigenicity to all forms of human α-Syn.

[0255] The specificity of PD062205 and PD100806 was further validated by the ability of each antibody to detect a greater proportion of LN than NCL-L-ASYN in the basal ganglia (Figures 24A-D). This was also observed visually in the midbrain (Figures 26A-B). Taken together, the high percentage area of ​​α-Syn detected by PD062205 and PD100806 supports the disclosed α-Syn. 126-135 These results indicate that the additional α-Syn detected by the antibodies may be due in part to the increased specificity of these antibodies for LNs. These results are beneficial for immunotherapy, as LNs are the primary form of α-Syn aggregates in the basal ganglia in the early stages of disease. Other reagents for treating synucleinopathies in preclinical development do not provide IHC detection of LNs. Therefore, the disclosed peptide immunogen constructs and α-Syn generated from the peptide immunogen constructs are useful for immunotherapy. 126-135 Antibodies have unique properties and characteristics compared to other commercially available products.

[0256] In this study, we assessed the effects of the disclosed peptide immunogen constructs on α-Syn expression by measuring the average amount of α-Syn aggregates in affected brain regions. 126-135The sensitivity of the antibody was analyzed using IHC. The present study quantified the mean percentage area of ​​α-Syn in brain samples. 126-135 We demonstrate that the antibody is highly sensitive for detecting α-Syn early in the disease progression of MSA, DLB, and PD compared to commercially available antibodies.

[0257] The higher sensitivity found in this study is due to the higher specificity of the disclosed antibodies for LN compared with the diagnostic antibody NCL-L-ASYN. 126-135 These findings suggest that antibodies are likely to be the most effective candidates for investigation of antibody-assisted clearance of α-Syn aggregates in synucleinopathies.

[0258] [Table 1]

[0259] [Table 2]

[0260] [Table 3]

[0261] [Table 4]

[0262] [Table 5]

[0263] [Table 6]

[0264] [Table 7]

[0265]

Table 8

[0266]

Table 9

[0267]

Table 10

[0268]

Table 11

[0269]

Table 12

[0270]

Table 13

[0271]

Table 14

[0272]

Table 15

[0273] Table 16

[0274]

Table 17

[0275]

Table 18

Claims

1. 1. An alpha-synuclein (α-Syn) peptide immunogen construct comprising: a B-cell epitope having an amino acid sequence selected from any one of SEQ ID NOs: 13, 14, 15, and 17; A T helper cell epitope of the amino acid sequence of SEQ ID NO: 83; and a heterologous spacer comprising Lys-Lys-Lys- and / or (α,ε-N)Lys; The α-Syn peptide immunogen construct above, wherein said B cell epitope is covalently linked to said T helper cell epitope via said heterologous spacer.

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

3. 3. The α-Syn peptide immunogenic construct of claim 1 or 2, wherein the T helper cell epitope is covalently linked to the amino terminus of the B cell epitope via the heterologous spacer.

4. Formula: (Th) ｍ -(A) ｎ -(C-terminal fragment of α-Syn)-X; or Formula: (C-terminal fragment of α-Syn)-(A) ｎ - (Th) ｍ -X wherein Th is the T helper cell epitope; A is the heterologous spacer; (C-terminal fragment of α-Syn) is the B-cell epitope; X is α-COOH or α-CONH of an amino acid ２ and m is 1 to 4; n is 1 to 10. The α-Syn peptide immunogenic construct of any one of claims 1 to 3, comprising:

5. The α-Syn peptide immunogenic construct of claim 4, wherein m is 1 and n is 1.

6. The α-Syn peptide immunogenic construct of any one of claims 1 to 5, wherein the B-cell epitope is the amino acid sequence of SEQ ID NO:

14.

7. The α-Syn peptide immunogenic construct of any one of claims 1 to 5, wherein the B-cell epitope is the amino acid sequence of SEQ ID NO:

15.

8. The α-Syn peptide immunogenic construct of any one of claims 1 to 5, wherein the B-cell epitope is the amino acid sequence of SEQ ID NO:

17.

9. The α-Syn peptide immunogen construct of claim 1, which has or consists solely of an amino acid sequence selected from any one of SEQ ID NOs: 107, and 111 to 113.

10. 2. The α-Syn peptide immunogenic construct of claim 1, having or consisting solely of the amino acid sequence of SEQ ID NO:

112.

11. 2. The α-Syn peptide immunogenic construct of claim 1, having the amino acid sequence of SEQ ID NO:113 or consisting solely of the amino acid sequence of SEQ ID NO:

113.

12. A pharmaceutical composition comprising one or more α-Syn peptide immunogenic constructs according to any one of claims 1 to 11, further comprising a pharmaceutically acceptable delivery vehicle and / or adjuvant.

13. The pharmaceutical composition described in claim 12, having an α-Syn peptide immunogen construct of the amino acid sequence of SEQ ID NO:

112.

14. The pharmaceutical composition described in claim 12, having an α-Syn peptide immunogen construct of the amino acid sequence of SEQ ID NO:

113.

15. 13. The pharmaceutical composition of claim 12, comprising an α-Syn peptide immunogenic construct of the amino acid sequence of SEQ ID NO: 112 and an α-Syn peptide immunogenic construct of the amino acid sequence of SEQ ID NO:

113.

16. The adjuvant is Al(OH) ３ and AlPO ４ The pharmaceutical composition according to any one of claims 12 to 15, wherein the inorganic salt of aluminum is selected from the group consisting of:

17. The pharmaceutical composition of any one of claims 12 to 16, wherein the α-Syn peptide immunogenic construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

18. An α-Syn peptide immunogen construct described in any one of claims 1 to 11, or a pharmaceutical composition described in any one of claims 12 to 17, used to produce an antibody that recognizes α-Syn in a subject.

19. An α-Syn peptide immunogen construct described in any one of claims 1 to 11, or a pharmaceutical composition described in any one of claims 12 to 17, for use in inhibiting α-Syn aggregation in a subject.

20. An α-Syn peptide immunogen construct described in any one of claims 1 to 11, or a pharmaceutical composition described in any one of claims 12 to 17, for use in reducing the amount of α-Syn aggregates in a subject.

21. An α-Syn peptide immunogen construct described in any one of claims 1 to 11, or a pharmaceutical composition described in any one of claims 12 to 17, for use in treating synucleinopathy in a subject.

22. An α-Syn peptide immunogenic construct described in any one of claims 1 to 11, or a pharmaceutical composition described in any one of claims 12 to 17, for use in treating Parkinson's disease in a subject.

23. An α-Syn peptide immunogen construct described in claim 10 or 11 for treating Parkinson's disease in a subject.

24. An α-Syn peptide immunogen construct according to any one of claims 13 to 15 for use in treating Parkinson's disease in a subject, wherein the pharmaceutically acceptable delivery vehicle and / or adjuvant comprises an inorganic salt of aluminum selected from Al(OH)3 and AlPO4, and the α-Syn peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

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