TWI676635B - Peptide immunogens from the c-terminal end of alpha-synuclein protein and formulations thereof for treatment of synucleinopathies - Google Patents

Abstract

The invention relates to an α-synuclein (α-Syn) peptide immunogen structure, a composition containing the structure, an antibody induced by the structure, and a method for manufacturing and using the structure and the composition. The α-Syn peptide immunogen structure contains the B-cell epitope of α-Syn, which is connected to the heterologous T helper cell (Th) epitope directly or through a selective heterolinker. The B-cell epitope of the peptide immunogen structure contains about 10 to about 25 amino acids of α-Syn, corresponding to the glycine (G111) to about 135 of the full-length α-Syn Sequence of aspartic acid (D135) at three positions. The α-Syn peptide immunogen structure stimulates the production of highly specific antibodies that are β-sheets with α-Syn, which are monomers, oligomers, and fibrils, rather than the natural α-helix α- Syn cross-reactivity provides a therapeutic immune response to individuals at risk for synucleinopathy.

Description

Alpha-synuclein C-terminal peptide immunogen and its formula for treating synuclein diseases

The invention relates to a peptide immunogen at the C-terminus of α-synuclein (α-Syn) and its formula for treating synuclein diseases.

Synuclein (see website: en.wikipedia.org/wiki/Synuclein) is a common soluble protein family in vertebrates, which is mainly expressed in neural tissues and certain tumors. The synuclein family includes three known proteins: α-synuclein (see website: en.wikipedia.org/wiki/Alpha-synuclein) and β-synuclein (see website: en.wikipedia.org/ wiki / Beta-synuclein), and gamma-synuclein. All synucleins typically have a highly conserved α-helical lipid-binding region, which is similar to the A2 lipid-binding domain of exchangeable apolipoproteins. Although the existing data speculate that synuclein may be involved in membrane stability and / or turnover, it has not been shown to be related to normal cell function.

Full-length α-synuclein (α-Syn) has 140 amine groups Acidic protein (Accession No. NP_000336), encoded by the SNCA gene. At least 3 isoforms of α-Syn can be generated by alternative splicing. The main form is full-length protein. The other homologous protein is α-Syn-126, which lacks residues 41-54 and no exon 3; α-Syn-112 lacks residues 103-130 and no exon 5 ( exon 5).

The primary structure of α-Syn is usually divided into 3 different regions: (1) amino acids 1-60: bipolar N-terminal region consisting of 4 repeating 11-amino acids, which includes the KTKEGV consensus sequence, Has an alpha-helical structure, similar to apolipoprotein binding domains; (2) 61-95 amino acids: a central hydrophobic region, which includes non-amyloid-beta (NAC) regions, and is associated with protein aggregation; and (3) Amino acids 96-140: highly acidic and proline-rich regions, which have no obvious structural tendency. The α-Syn fragments of the 35 amino acids in the NAC region are present with Aβ in the non-amyloid-rich portion. NAC is a fragment of its precursor protein NACP, and a full-length human synuclein homologue was found in Torpedo californica , and is referred to herein as human α-Syn.

Α-Syn purified using high-resolution ion mobility mass spectrometry (IMS-MS) and HPLC showed that α-Syn had self-proteolytic, and various small molecular weight fragments were produced after cultivation. The full-length 14.46 kDa protein produces many smaller fragments, including a 12.16 kDa fragment (14-133 amino acids) and a 10.44 kDa fragment (40-140 amino acids) with C- and N-terminal truncations, and a 7.27 kDa fragment (amino acids 72-140). The 7.27kDa fragment contains multiple NAC regions, and the aggregation rate is significantly faster than the full-length α-Syn. These self-proteolysates α-Syn aggregates play an intermediate or cofactor role.

α-Syn is an extra component of the brain, which accounts for 1% of all proteins in the cytoplasm of the brain and glial cells. α-Syn is widely expressed in neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus and cerebellum. α-Syn is also highly expressed in hematopoietic cells, including B cells, T cells and NK cells, as well as monocytes and platelets. In the brain, α-Syn is mainly found in neurons (neurons) at the front of the synapse. In these structures, α-Syn interacts with phospholipids and proteins. The front of the synapse releases chemical information called neurotransmitters, such as dopamine from synaptic vesicles. The release of neurotransmitters can transmit messages between neurons and is important for normal brain function, including cognition.

Α-Syn in solution is a protein with no stable configuration, so it does not have a single stable 3D structure. It has been shown that α-Syn and tubulin react significantly with each other, and α-Syn may have the activity of a tubulin-related protein such as tau. α-Syn is generally considered to be a non-structurally soluble protein. Non-mutated α-Syn forms a stable folded tetramer that resists aggregation. However, α-Syn aggregates to form insoluble fibers in the pathological state of Lewy bodies. This condition is known as synuclein disease (see webpage: en.wikipedia.org/wiki/Synucleinopathies).

Synuclein diseases are a different group of neurodegenerative diseases that share common pathological characteristics: In neuropathological examination, lesions characterized by insoluble α-Syn abnormal aggregates are present in the neuron and glial cell injury population. The most common synuclein diseases include Lewy body disease (LBDs) such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), and road disease. Degenerative Dementia (DLB), as well as multiple systemic degeneration (MSA) or type 1 degenerative neurological degeneration of the brain. Current treatments for these diseases include symptomatic treatments, such as L-dopa, anticholinergic drugs, and monoamine oxide alcohol inhibitors. However, at present, all treatment opportunities can only relieve symptoms and cannot achieve sustained disease improvement for patients.

LBD is a progressive neurodegenerative disease characterized by tremor, stiffness, bradykinesia, and loss of dopaminergic neurons in the brain. Symptoms of DLB and PDD include cognitive impairment. In Western countries, more than 2% of the population over the age of 60 will develop the typical symptoms of PD / LBD. It shows that genetic susceptibility and environmental factors seem to be related to the development of the disease. Patients with this disease develop intracellular inclusions in the cortex and cortical areas of the brain, especially in areas with high levels of dopamine neurons or neuronal protrusions, known as Lewy bodies (LBs). In LBD, α-Syn accumulated in Lewy bodies affects the brain. In addition, point mutations and α-Syn gene recombination or proliferation have been shown to be associated with rare familial Parkinson's syndrome.

Multiple system degeneration (MSA) is a sporadic aging neurodegenerative disease characterized by L-DOPA-resistant Parkinson's syndrome, cerebellar dyskinesia, and autonomic disorders. Various brain regions in patients with multisystem neuron damage, including striatum, substantia nigra, cerebellum, pontine, and inferior olive nucleus and spinal cord are affected. MSA is characterized by alpha-Syn positive glial cytoplasm (GCI) and rare neuronal debris throughout the central nervous system.

Other rare diseases, such as various neurite disorders, also have α-Syn lesions, and α-Syn is the main structure of Lewy body fibers. Usually, Leu body contains tau protein; however, α-Syn and tau constitute two different intermediate filament subunits in the same inclusion body. Alpha-Syn lesions are also found in sporadic and familial Alzheimer's disease.

The mechanism of α-Syn accumulation is unclear. The α-Syn monomer does not fold in solution, but can bind to the membrane in an α-helical form. Unfolded monomers can first aggregate into small oligomers, which can be stabilized by β-sheet-like interactions to form higher molecular weight insoluble fibers. The presence of α-Syn maintains a balance with unstructured, α-helix and β-sheet-rich mixtures. Mutations or buffer conditions are known to enhance aggregation and greatly increase the population of beta conformers, so it may be related to pathogenic aggregation. There is evidence that structured intermediates are rich in beta structures, which are aggregate precursors that eventually form the Louis 4 body.

Many physiological factors can alter the formation of α-Syn leading to the formation of aggregates, including (1) phosphorylation by one or more enzymes, (2) cleavage by proteases such as calpain, and (3) nitric oxide during inflammation (NO) or other active nitrogen substances for nitration. Other proteolytic substances such as ER-high martensite transport, synaptic vesicles, mitochondria, and lysosomes are the target cells of α-Syn toxicity caused by aggregation.

The strategy for treating synuclein diseases is to use compounds that inhibit α-Syn aggregation. The small molecule cuminaldehyde has been shown to inhibit fibrosis of α-Syn. In addition to small molecule therapy, literature has revealed that α-Syn aggregation is associated with immunotherapy (Lee JS and Lee S-J, 2016). However, the literature indicates that there are still many potential problems in developing α-Syn immunotherapy, including (1) potential interference with the normal physiological functions of α-Syn; (2) anti- Difficulties in delivering body drugs to the brain parenchyma; and (3) efficacy of immunotherapy.

So far, the development of site-specific peptide immunogens and their preparations to treat patients with synuclein diseases has not been satisfied.

references

"Alpha-synuclein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Alpha-synuclein & oldid = 781366541 (accessed May 30, 2017).

"Synucleinopathies," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Synucleinopathies & oldid = 686287116 (accessed May 30, 2017).

"Beta-synuclein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Beta-synuclein & oldid = 763171134 (accessed May 30, 2017).

"Synucleinopathies," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Synucleinopathies & oldid = 686287116 (accessed May 30, 2017).

LEE, J.S., et al., "Mechanism of Anti-α-Synuclein Immunotherapy", J Mov Disord .; 9 (1): 14-19 (2016)

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)

WANG, C., et al. “Versatile Structures of α-Synuclein”, Front Mol Neurosci. 9:48 (2016)

The invention relates to the structure of an α-synuclein (α-Syn) peptide immunogen. The present invention also relates to a composition containing the peptide immunogen structure, a method for manufacturing and using the peptide immunogen, and antibodies produced by the peptide immunogen.

The peptide immunogen structure of the present invention contains a B-cell epitope, which is directly or via a selective heterologous linker connected to a heterologous T helper cell (Th). The B-cell epitope in the peptide immunogen structure contains 10 to 25 amino acid residues at the C-terminus of α-Syn, which is about the 111th amino acid in the full-length α-Syn (SEQ ID NO: 1). From glycine (G111) to about 135th aspartic acid (D135). The heterologous Th epitope of the peptide immunogen structure is derived in part from the amino acid sequence of the pathogen protein. When the B-cell epitope and Th epitope portion of the peptide immunogen structure are administered to the host, they can work together to stimulate the production of antibodies. This antibody can specifically recognize and bind to the α-Syn B-cell epitope.

In certain embodiments, the α-Syn peptide immunogen structure includes: (a) a B-cell epitope comprising 10 to 25 amino acids, which are about G111 to about D135 in SEQ ID NO: 1; (b) T-assisted antigen determination Mapping, selected from the group consisting of SEQ ID NOs: 70-98; and (c) selective heterologous linkers, selected from Lys-, Gly-, Lys-Lys-Lys-, (α , ε-N) Lys and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), in which the B cell epitope is covalently linked directly or through a selective heterologous linker T helper epitope. In a specific embodiment, the α-Syn peptide immunogen structure includes a group selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

The invention further relates to a composition containing a peptide immune structure of the present invention, including a pharmaceutical composition. The pharmaceutical composition of the present invention is capable of inducing an immune response and stimulating the host to produce antibodies against the pharmaceutical composition of the present invention. The composition of the invention may contain a mixture of one or more peptide immune structures. In certain embodiments, this composition contains the peptide immune structure of the present invention and other additional ingredients, including carriers, adjuvants, buffers, and other appropriate agents. In a specific embodiment, the composition contains a peptide immune structure, which is in the form of a stable immune stimulating complex, and optionally supplements the CpG oligomer as an adjuvant.

In some embodiments, the composition includes an α-Syn peptide immunogen structure containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, 115-147 . In a specific embodiment, the composition is a pharmaceutical composition, including an α-Syn peptide immunogen structure, which contains a group selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. Amino acid sequence and a pharmaceutically acceptable carrier or adjuvant.

The invention further relates to antibodies produced by immunizing a host with a peptide immunogen structure. The antibody of the present invention specifically recognizes and binds to the α-Syn B cell epitope portion of the peptide immunogen structure. The α-Syn antibodies of the present invention have an unexpectedly high degree of interaction with β-sheets of monomers, oligomers, and fibrous α-Syn. Based on its unique characteristics and properties, the antibodies of the present invention can provide an immunotherapy approach to target, identify and treat synuclein diseases.

In a specific embodiment, the antibody or epitope-binding fragment thereof can specifically bind to the B-cell epitope of the α-Syn peptide immunogen structure, which is selected from SEQ ID NOs: 107, 108, 111-113 , 115-147.

The invention further relates to methods for making and using the peptide immune structures, antibodies and compositions of the invention. The method of the present invention can provide a peptide immune structure with low manufacturing cost and high quality control, and a composition containing the same to inhibit and treat synuclein diseases.

The invention further includes a method for treating and / or inhibiting a synuclein disease, including using the peptide immune structure of the invention and / or an antibody against the peptide immune structure. In certain embodiments, a method of treating and / or inhibiting a synuclein disease comprises administering to a host a composition comprising a peptide antigen structure of the invention. In a specific embodiment, the composition used in this method comprises a peptide immunogen structure of the present invention and a negatively charged oligonucleotide, such as a CpG oligomer, which forms a stable immunostimulatory complex form through electrostatic binding. This complex further includes mineral salts or mineral oils that are selectively used as adjuvants for administration to patients suffering from synuclein diseases. The invention The method includes the administration mode, dosage form and route of administering the peptide immunogen structure to a host suffering from a synuclein disease or at risk.

In various embodiments, the invention includes methods of using antibodies elicited by the α-Syn peptide immunogen structure and / or the α-Syn peptide immunogen structure. In particular embodiments, the invention includes methods for generating antibodies, inhibiting α-Syn aggregation, reducing α-Syn aggregation, and identifying α-Syn aggregation of different sizes. Various methods include administering a pharmaceutically effective amount of an alpha-Syn peptide immunogen to a desired host.

Figure 1 shows the degree of in vitro α-Syn peptide immunogen accumulation in the presence of an α-Syn C-terminal antibody (Sample 1-4) or a vector control group (Sample 5). In particular, in order to α-Syn _111-132 (Sample 1); α-Syn _121-135 (Sample 2); α-Syn _123-135 (Sample 3); α-Syn _126-135 (Sample 4) of the induced Aggregation of α-Syn in the presence of an anti-α-Syn antibody or the vector control group (Sample 5). The degree of α-Syn aggregation was measured by thioflavin-T (ThT) staining. Samples 1-4 were normalized with the sample 5 vector control group. Error bars represent the SEM (mean standard error) of each replicate.

Figure 2 shows the degree of deaggregation of pre-formed in vitro α-Syn aggregates after 3 days of culture in the presence of α-Syn C-terminal antibody (Sample 1-3) or pre-immune serum control group (Sample 4). In particular, α-Syn preformed aggregates in α-Syn _111-132 (sample 1), α-Syn _126-135 (Sample 2) induce the anti -α-Syn antibody to α-Syn _111-132 And α-Syn _126-135- induced antibody combinations; or the pre-immune serum control group (sample 4) was cultured. The degree of α-Syn aggregation was measured by thioflavin-T (ThT) staining. Samples 1-3 were normalized with the sample 4 vector control group. Error bars represent the SEM (mean standard error) of each replicate.

Figure 3 shows that PC12 cells overexpressing α-Syn were cultured with nerve growth factor (NGF) in the presence of α-Syn C-terminal antibodies (Sample 1-4) or the vector control group (Sample 5). Α-Syn Degree of aggregation and α-Syn deaggregation. In particular, PC12 cells were treated with α-Syn _111-132 (sample 1), α-Syn _121-135 (sample 2), α-Syn _123-135 (sample 3), and α-Syn _126-135 (sample 4). The induced anti-α-Syn antibody; or the vector control group (sample 5) was cultured. Samples 1-4 were normalized with the sample 5 vector control group. Error bars represent SD (standard deviation) of each replicate.

Figure 4 shows the degree of α-Syn-induced release of TNF-α and IL-6 by cell culture under α-Syn C-terminal antibody (samples 1-4) or vector control group (sample 5). In particular, microglia cells were treated with α-Syn _111-132 (Sample 1), α-Syn _121-135 (Sample 2), α-Syn _123-135 (Sample 3), and α-Syn _126-135. (Sample 4) The induced anti-α-Syn antibody; or the vector control group (Sample 5) was cultured. Samples 1-4 were normalized with the sample 5 vector control group. Error bars represent SD (standard deviation) of each replicate.

Figures 5A-5C show the effect of anti-α-Syn antibodies on NGF-induced PC12 cells induced by preformed α-Syn aggregates in an in vitro neurodegeneration model. Figure 5A is an evaluation of NGF treatment (solid black line); NGF treatment with exogenous preformed α-Syn aggregates (short dashed lines); NGF treatment with pre-immune serum (light solid line); and Nerve process length of PC12 cells treated with NGF and exogenous pre-formed α-Syn aggregation and pre-immune serum (long dashed line). Figure 5B is an evaluation of NGF-treated and with a carrier (solid black line); NGF-treated and with exogenous preformed α-Syn aggregates (short dashed lines); NGF-treated and with α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibody (light solid line); and NGF-treated and exogenous preformed α-Syn aggregates and affected by α-Syn _111-132 (SEQ ID NO: 113) Induced anti-α-Syn antibody (dashed line) PC12 cell neuron neurite length. Figure 5C is an evaluation of NGF-treated and with a carrier (black solid line); NGF-treated and with exogenous pre-formed α-Syn aggregates (long dashed lines); NGF-treated and with α-Syn _126-135 (SEQ ID NO: 112) induced anti-α-Syn antibody (light solid line); and NGF-treated and exogenous preformed α-Syn aggregates and affected by α-Syn _126-135 (SEQ ID NO: 112) Neuronal process length of PC12 cells induced by anti-α-Syn antibody (long dashed line).

Figures 6A-6B show the effect of anti-α-Syn antibodies on cell number and neuronal process length in an in vitro neurodegeneration model of PC12 cells in vitro with overexpression of α-Syn using NGF-induced neuronal differentiation. Cell control group (Sample 1); α-Syn _101-132 (Sample 2), α-Syn _111-132 (Sample 3), α-Syn _121-135 (Sample 4), α-Syn _123-135 (sample 5), α-Syn _126-135 (sample 6) -α-Syn-induced anti-antibody, or by α-Syn _{111-132 126-135} -α-Syn-induced anti-antibody combinations and α-Syn (sample 7); or pre-immune serum control group (sample 8). Figure 6A is an assessment of the protective ability of each sample to restore the number of PC12 cells. Fig. 6B is an assessment of the neuronal process length of cells treated with each sample. Samples 1-8 were normalized with NGF-induced neuronal differentiation of wild-type overexpressing α-Syn PC12 cells. A t-test was used to test for significance (p-values less than 0.05 represent statistically significant and are indicated by *).

Figures 7A-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 the results of a western blot analysis with commercially available anti-α-Syn antibodies, Syn211 (lane 1), pre-immune serum control group (lane 2), and anti-α-Syn induced by Syn _111-132 . Antibody (lane 3), anti-α-Syn antibody induced by Syn _111-135 (lane 4), anti-α-Syn antibody induced by Syn _121-135 (lane 5), anti-α- induced by Syn _{123-135 Comparison of} Syn antibody (lane 6) and anti-α-Syn antibody (lane 7) induced with Syn _126-135 . Figure 7B is a bar graph showing the relative ability of each antibody to bind to various sizes of α-Syn molecular complexes, including monomers, dimers, trimers, tetramers, and oligomers. The chemical cold light signal of western ink dot method is shown in Fig. 7A, and after quantification, it is represented by the histogram of Fig. 7B.

Sections 8A-8C are the results of dot blot analysis, which show that antibodies against the C-terminus of α-Syn will only recognize and bind to different types of α-Syn (i.e., α-helical monomers, β-sheets). Monomers, β-sheet oligomers and β-sheet fibers) without binding to other amyloids of the same species (ie Aβ1-42 and Tau441). Figure 8A is a control group sample. Antibodies purified from guinea pig pre-immune sera will not bind to any protein. FIG. 8B shows that the anti-α-Syn antibody induced by Syn _111-132 (SEQ ID NO: 113) recognizes and binds different types of α-Syn, Aβ1-42 and Tau441 proteins. Figure 8C shows that anti-α-Syn antibodies induced by Syn _126-135 (SEQ ID NO: 112) recognize and bind to different types of α-Syn, Aβ1-42 and Tau441 proteins.

Figure 9 is a table summarizing the binding affinities of antibodies against the α-Syn C-terminus to the extracellular α-Syn of various PC12 cells, and measuring the positive signals in immunocytochemical (ICC) analysis. In particular, by _{α-Syn 111-132, α-Syn} 121-135, α-Syn 126-135 or preimmune serum control group induced the anti-α-Syn antibody PC12 cells to parent NGF treatment, the negative control PC12 cells Wild-type α-Syn overexpresses PC12 cells, and A53T mutant α-Syn overexpresses relative binding affinity in PC12 cells.

Figures 10A-10C show that antibodies directed against the C-terminus of α-Syn only bind to α-Syn in PD brain sections and not to healthy brain sections. FIG. 10A shows that the α-Syn antibody and pre-immune antibody induced by the α-Syn peptide immunogen structure did not detect an immune response in normal human brain tissue. FIG. 10B shows the immune response of antibodies against α-Syn aggregation in PD thalamus sections, as indicated by arrows. Figure 10C is a table showing the immune response to the α-Syn C-terminus and the pre-immune control group antibodies to α-Syn in PD and healthy brain sections. The number of positive staining was counted by microscopic observation.

Figures 11A-11B show 3 adjuvants (open circles) or alpha-Syn _111-132 (open squares), α-Syn _126-135 (closed circles), or α-Syn _111-132 and α- The amount of anti-α-Syn IgG in the PD mouse model following the peptide immunogen of the combination (solid square) of Syn _126-135 . Figure 11A shows the extent of IgG in the MPP + mouse model. Figure 11B shows the amount of IgG in a mouse model inoculated with alpha-Syn fibers.

Figures 12A-12B show PD mouse models receiving peptide immunogens containing pure adjuvant (open circles), α-Syn _111-132 (open squares), α-Syn _126-135 (closed circles), or α after -Syn _111-132 and α-Syn _126-135 (solid squares) immunizations composition 3, α-Syn amount of peripheral circulation. Figure 12A shows the amount of α-Syn in the MPP + induced mouse model. Figure 12B shows the amount of α-Syn in a mouse model inoculated with α-Syn fibers.

Figures 13A-13B show an untreated healthy mouse model (lane 1) or a PD mouse model (lane 2-3) receiving 3 times of simple adjuvant (lane 2) or containing an α-Syn _111-132 peptide immunogen Amount of α-Syn oligomers from brain samples. Untreated Balb / c mice were healthy mouse models, and MPP + induced mouse models were PD mouse models. Figure 13A is the result of Western blot method, which shows the amount of α-Syn oligomer and GAPDH as a protein loading control in the sample. FIG. 13B compares the amount of α-Syn oligomers shown in 13A Western blot method, normalizes the amount of protein with GAPDH, and normalizes the amount of lysate in untreated healthy mouse models to 1.00.

Figures 14A-14G show an untreated healthy mouse model (lane 1) or PD mouse model (lane 2-4) that received a simple adjuvant (lane 2) or contained α-Syn _111-132 (lane 3) or α -Syn _126-135 (lane 4) after peptide immunogen, the amount of α-Syn oligomers and the amount of tyrosine hydroxylase in brain tissue. Untreated FVB mice were healthy mouse models, and α-Syn fiber mouse models were PD mouse models. Figure 14A is the result of Western blot analysis, which shows the amount of α-Syn oligomer, tyrosine hydroxylase, and GAPDH as a protein loading control in the ipsilateral nigra solute. Figure 14B shows the amount of α-Syn oligomers compared to the Western blot method shown in Figure 14A to normalize the amount of protein by GAPDH. After normalizing the amount of protein with GAPDH, FIG. 14B shows the relative amount of α-Syn oligomers in the Western blot method compared to FIG. 14A. After normalizing the amount of protein with GAPDH, Fig. 14C shows the relative amount of tyrosine hydroxylase protein in the Western blot method compared to Fig. 14A. Figure 14D is a Western blot method, which shows the amount of α-Syn oligomers in the ipsilateral striatum, with GAPDH as the loading control. After normalizing the amount of protein with GAPDH, Fig. 14E shows the relative amount of α-Syn oligomers in the Western blot method comparing Fig. 14C. Fig. 14F is a Western blot method, which shows the amount of α-Syn oligomers in the ipsilateral striatum, with GAPDH as the loading control. After normalizing the amount of protein with GAPDH, Figure 14G shows the relative amount of α-Syn oligomers in the Western blot method compared to Figure 14E.

Figures 15A-15C show CatWalk ^TM XT analysis of healthy mice (lane 1-2) injected with saline (lane 1) or adjuvant (lane 2), or α-Syn ₁₂₆ containing adjuvant (lane 3), respectively. _-135 (lane 4) or α-Syn _111-132 (lane 5) peptide immunogen immunized PD mouse model (lanes 3-5) for motor function. Significance analysis was performed by t test (p-values less than 0.05 were defined as statistically significant differences and indicated by an asterisk "*"). Fig. 15A is an analysis of the left hind limb of treated mice, in which untreated FVB mice were used as healthy mouse models and α-Syn fiber immunized mice were used as PD mouse models. FIG. 15B is an analysis of the running period of the treated mice, in which untreated FVB mice were used as healthy mouse models, and α-Syn fiber-immunized mice were used as PD mouse models. Figure 15C is the analysis of the running time of treated mice, in which untreated Balb / c mice were used as healthy mouse models, and MPP + induced mice were used as PD mouse models.

Figure 16A shows that PD-021514 antibody (α-Syn _85-140 , wpi 08) can recognize α-Syn fibers with the highest affinity. Good binding with ribbons and fiber-91 was observed. Poor binding to oligomers and fiber-65. Poor binding with α-Syn monomers and fibers lacking 30 C-terminal amino acid residues (Fib-110). Figure 16B shows that PD-021522 (α-Syn _85-140 , wpi 13) can bind to all species / oligomers, but not to monomers. The detection signal does not have a significant concentration-dependent increase. The antibody can bind to fibers lacking the C-terminal 30 amino acid residues (Fib-110). Therefore, the epitope is not in this region. Figure 16C shows that PD-100806 (α-Syn _126-135 , wpi 09) can be combined with all species, and has the highest affinity with ribbon α-Syn. Low binding efficiency to natural α-Syn oligomers. It hardly binds to glutaraldehyde, dopamine crosslinked oligomer, and monomer α-syn. Since the antibody does not bind to a fiber lacking 30 C-terminal amino acid residues (Fib-110), the antibody may be directed to the 30 C-terminal amino acid residues of α-syn. Figure 16D shows that the commercially available antibody Syn-1 (clone 42, BD bioscience) can bind to all α-Syn species and oligomers except for glutaraldehyde cross-linking. It can also be combined with the monomer α-Syn. Its epitope spans between 91 and 96/99 residues. Similarly, it can bind to fibers lacking 30 C-terminal amino acid residues (Fib-110). Figure 16E shows that PRX002 antibody has slightly better affinity for fibrous α-Syn compared to α-Syn monomer. Figure 16F shows the background of the antibodies produced by guinea pigs. Figure 16G shows the background of the control antibody Syn-1. Figure 16H shows the background control group of PRX002 antibody.

Figures 17A-17D show the specificity analysis of α-Syn in the basal nucleus of patients with Lewy body (DLB) dementia by IHC analysis of UNS antibodies. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine the putamen (Figure 17A) and inner capsule (Figure 17B) with a total area of 7.5 mm ² And island leaves (Figure 17C). Figure 17D shows representative microscopic images obtained from immunostaining of the putamen with each antibody. UNS antibodies can detect the putamen (F (3,7) = 1.550, p = 0.284, ANOVA), the inner capsule (F (3,7) = 1.356, p = 0.332, ANOVA), and the insular cortex (F (3 8) = 2.050, p = 0.195, ANOVA) a higher area percentage of α-Syn aggregates. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

Figures 18A-18D are specific analysis of IHC analysis of UNS antibodies on basal nucleus α-Syn of patients with Parkinson's disease (PD). The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine the putamen (Figure 18A) and inner capsule (Figure 18B) with a total area of 7.5 mm ² And island leaves (Figure 18C). Figure 18D shows a representative microscopic image obtained by immunostaining the putamen. UNS antibodies can detect the putamen (F (3,18) = 4.152, p = 0.047, ANOVA), the inner capsule (F (3,8) = 1.995, p = 0.1934, ANOVA) and the insular cortex (F (3 8) = 0.4044, p = 0.754, ANOVA) a higher area percentage of α-Syn aggregates. Compared with NCL-L-ASYN, PD100806 can detect a significantly higher proportion of α-Syn area. (p = 0.023, PD100806 vs NCL-L-ASYN; n = 3). P <0.05 (*); P <0.01 (**); P <0.001 (***). Dunnett's test was performed after One-way ANOVA (One-way ANOVA). Values are expressed as mean + SD (error bar).

Figures 19A-19B are specific analysis of the basal nucleus α-Syn of patients with multiple system degenerative disease (MSA) by IHC analysis of UNS antibodies. Average area percentages of each antibody (PD062220, PD062205, PD100806 and NCL-L-ASYN) α-Syn stained aggregates, MSA patients to determine the total area of 7.5mm ² 3 putamen (second FIG. 19A) and internal capsule ( (Figure 19B). No pathological features were detected in the insular cortex of MSA patients, and therefore were not quantified. UNS antibody can detect α- in the higher area percentage in the putamen (F (3,8) = 1.56, p = 0.273, ANOVA) and inner capsule (F (3,8) = 1.126, p = 0.395, ANOVA). Syn aggregates. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

Figures 20A-20E are specific analysis of the basal nucleus α-Syn of patients with different synuclein diseases by IHC analysis of UNS antibodies. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine PD (Figure 20A), DLB (Figure 20B), and MSA (Figure 20C) patients. Substantia nigra with a total area of 7.5 mm ² . Compare the percentage of stained area of various antibodies with the diagnostic antibody NCL-L-ASYN. UNS antibodies can detect MSA (F (3,8) = 0.830, p = 0.51, ANOVA), DLB (F (3,7) = 2.493, p = 0.144, ANOVA) and PD (F (3,7) = 0.189, p = 0.900, ANOVA). The percentage of α-Syn aggregates in the substantia nigra was higher. Figures 20D (MSA) and 20E show representative microscope images of each antibody immunostaining. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

Figures 21A-21F show IHC analysis of the specificity of the UNS antibody for α-Syn in the white and gray matter of the temporal lobe cortex of patients with different synuclein diseases. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine PD (Figure 21A and 21D), DLB (Figure 21B and 21E), and MSA (Figure 21C and 21F) Cortical gray matter and cortical white matter with a total area of 7.5 mm ² . The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. P <0.05 (*); P <0.01 (**); P <0.001 (***). Dunnett's test was performed after One-way ANOVA (One-way ANOVA). Values are expressed as mean + SD (error bar).

Figures 22A-22C are specific analysis of α-Syn in cerebellum of patients with different synuclein by IHC analysis of UNS antibodies. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine patients with PD (Figure 22A), DLB (Figure 22B), and MSA (Figure 22C). Cerebellum with a total area of 7.5 mm ² . UNS antibody can detect MSA (F (3,8) = 0.929, p = 0.469, ANOVA); DLB (F (3,6) = 1.426, p = 0.325, ANOVA) and PD (F (3,6) = 2.509, p = 0.157, ANOVA) The area percentage of α-Syn aggregates was higher in patients. The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

Figures 23A-23B are representative images of brain substantia nigra (Figure 23A) and putamen (Figure 23B) of non-patient control groups with each antibody immunostaining. UNS antibody did not detect any α-Syn pathology, which was consistent with the results of the diagnostic antibody NCL-L-ASYN.

Figures 24A-24D are IHC analysis of α-Syn specific analysis of LBs in basal nucleus leaves of DLB or PD patients by UNS antibodies. The average area percentage of each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) -positive LBs was used to determine the total leaf area of 7.5 mm ² in patients with PD (Figure 24A ) and DLB (Figure 24B ). The area percentage of LB is the total α-Syn ratio detected by each antibody. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine an island with a total area of 7.5 mm ² in patients with PD (Figure 24A) and DLB (Figure 24B). leaf. The percentage area of LBs is the ratio of total α-Syn detected by each antibody. UNS antibody can detect LBs (or higher proportion) in island leaves of patients with DLB (F (3,7) = 0.836, p = 0.516, ANOVA) and PD (F (3,4) = 0.913, p = 0.510, ANOVA) LNs) has a lower area percentage. The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. Figures 24C (PD) and 24D (DLB) show representative microscope images of each antibody immunostaining. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

Figures 25A-25D are specific analysis of gray matter in temporal lobe cortex of patients with DLB or PD by UNS antibody analysis by IHC. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine the gray area of 7.5 mm ² in the total area of patients with PD (Figure 25A) and DLB (Figure 25B). . The area percentage of LBs is the proportion of total α-synuclein detected by each antibody. UNS antibody can detect LBs (or higher proportion of LNs) in gray matter of patients with PD (F (2,3) = 1.983, p = 0.282, ANOVA) and DLB (F (3,7) = 1.906, p = 0.217, ANOVA) ) Has a lower area percentage. The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. Figures 25C (PD) and 25D (DLB) show representative microscope images of each antibody immunostaining. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

Figures 26A-26B are representative images of brain substantia nigra in patients with DLB (Figure 26A) and PD (Figure 26B) immunostained with UNS antibodies and NCL-L-ASYN. Compared with NCL-L-ASYN, UNS antibodies have a higher degree of detection of LN.

Figures 27A-27C show cell-specific aggregation of α-Syn. The largest projections of basal nucleus and human midbrain α-Syn aggregates in patients with PD (figure 27A), DLB (figure 27B) and MSA (figure 27C) covered conjugate focus images. Α-Syn (PD062205, red) in neurons (HuD, green) aggregated in patients with PD and DLB, but not MSA. Mark α-Syn (PD062205) and HuD in black and white images, and provide color images on request. Scale bar: 10 μM.

Figures 28A-28C show cell-specific aggregation of α-Syn. The largest projections of α-Syn aggregates in patients with PD (Figure 28A), DLB (Figure 28B), and MSA (Figure 28C) covered conjugate focus images. Oligodendrocyte (Olig2, green) α-Syn (PD062205, red) aggregates exist in patients with MSA, not patients with PD and DLB. Mark α-Syn (PD062205) and Olig2 in black and white images, and provide color images on request. Scale bar: 10 μM.

The invention relates to the structure of an α-synuclein (α-Syn) peptide immunogen. The invention further relates to a composition containing a peptide immunogen structure, a method for manufacturing and using the peptide immunogen structure, and an antibody generated by the peptide immunogen structure.

The peptide immunogen structure of the present invention contains a B-cell epitope derived from α-Syn, which is connected to a heterologous T helper cell (Th) epitope directly or through a selective heterologous linker. The B cell epitope of the peptide immunogen structure contains about 10 to about 25 amino acids derived from the C-terminal region of α-Syn, which corresponds to about 111th of the full-length α-Syn (SEQ ID NO: 1) Sequence of glycine (G111) to about 135th aspartic acid (D135). The heterologous Th epitope of the peptide immunogen structure is derived in part from the amino acid sequence of the pathogenic protein. When the B-cell epitope and Th epitope of the peptide immunogen structure are administered to a single body, they can work together to stimulate antibodies that specifically recognize and bind to the α-Syn B-cell epitope.

The invention further relates to a composition containing a peptide immunogen structure, including a pharmaceutical composition. This pharmaceutical composition can stimulate the immune response and produce antibodies against the peptide immunogen structure in one body. This composition may contain A mixture of one or more peptide immunogen structures. In certain embodiments, this composition contains the peptide immunogen structure of the present invention and additional ingredients, including carriers, adjuvants, buffers, and other suitable reagents. In a specific embodiment, the composition contains the peptide immunogen structure of the present invention and a CpG oligomer to form a stable immunostimulatory complex, which selectively supplements an adjuvant.

The invention is even more concerned with antibodies produced by immunizing a peptide immunogen structure of the invention. The antibody of the present invention specifically recognizes and binds an epitope portion of an α-Syn B cell of a peptide immunogen structure. The α-Syn antibodies of the present invention have an unexpectedly high cross-reactivity to β-sheets of monomers, oligomers or α-Syn fibers. Based on its unique characteristics and properties, the antibodies of the present invention can provide an immunotherapeutic method for calibrating, identifying, and treating synuclein diseases.

The invention further relates to methods for making and using the peptide immunogen structure, antibodies, and compositions of the invention. The method of the present invention provides low-cost manufacturing quality control of the peptide immunogen structure and a composition containing the peptide immunogen structure, which can be used for inhibiting and treating disorders.

The present invention further includes a method for treating and / or inhibiting a synuclein disease using the peptide immunogen structure of the present invention and / or an antibody against the peptide immunogen structure. In certain embodiments, a method of treating and / or inhibiting a synuclein disease comprises administering to a subject a composition comprising a peptide immunogen structure of the invention. In a specific embodiment, the composition used in this method contains the peptide immunogen structure of the present invention and a negatively charged oligonucleotide, such as a CpG oligomer, through electrostatic coupling to form a stable immunostimulatory complex This complex is more selectively supplemented with mineral salts or oils as an adjuvant to patients with synuclein diseases. The method of the present invention also includes the administration design, dosage form and route to administer the peptide immunogen structure to an individual suffering from or having a synuclein disease.

The section headings in the description are for organizational purposes only and are not intended to limit the subject matter described. All or part of the cited documents may be incorporated into the description of the invention for any purpose in its entirety.

Unless otherwise specified, those skilled in the art can generally understand the same meanings of all technical and scientific terms used in the description of the present invention. Unless otherwise specified, the singular terms "a" and "the" include plural designations. Likewise, the term "or" includes "and" unless the context clearly dictates otherwise. Therefore, "including A or B" means including A or B, or A and B. It should be further understood that all amino acid sizes for polypeptides are similar to all molecular weights or molecular weight values and are disclosed in the description of the present invention. Although methods and materials similar or identical to those described in the description of the invention can also be used in the tests and methods of the present invention, suitable methods and materials are disclosed below. All documents, patent applications, patents, and other references in the description of the invention may be incorporated into the description of the invention. In the event of conflict, the description of the invention, including terminology, will be explained. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

α-Syn peptide immunogen structure

The invention provides a peptide immunogen structure containing a B-cell epitope derived from α-Syn, which is covalently linked to a heterologous T helper cell (Th) epitope directly or through a selective heterolinker. .

The “α-Syn peptide immunogen structure” in the present invention means that a peptide contains (a) a B-cell epitope with about 10 to 25 amino acids derived from the C-terminus of α-Syn, which corresponds to The sequence of glycine (G111) to aspartic acid (D135) at about the 111th position of the full-length α-Syn (SEQ ID NO: 1); (b) a heterologous Th epitope; (c) A selective heterologous linker.

In a specific embodiment, the peptide immunogen structure can be represented by the formula: (Th) _m- (A) _n- (α-Syn C-terminal fragment) -X or (α-Syn C-terminal fragment)-(A ) _n- (Th) _m -X where Th is a heterologous T helper epitope; A is a heterologous linker; (α-Syn C-terminal fragment) is an α-Syn C-terminal The fragment has B-cell epitopes of about 10 to 25 amino acids; X is α-COOH or α-CONH ₂ of a monoamino acid; M is 1 to about 4; and N is 0 to about 10.

The various components of the α-Syn peptide immunogen structure are shown below.

a. α-Syn and α-Syn C-terminal fragments

The “α-Syn”, “alpha-synuclein”, “α-synuclein” and the like in the present invention are derived from any manifestation (a) The full-length α-Syn protein and / or (b) a fragment of α-Syn. α-Syn has extreme conformational diversity, adapts to different conditions in membrane binding, cytoplasm, and amyloid aggregation, and has multiple functions. In certain embodiments, the alpha-Syn protein is from a human. In a specific embodiment, the full-length human α-Syn protein has a 140 amino acid (Accession No. NP_000336) (SEQ ID NO: 1).

The "C-terminal region" or "C-terminus" of α-Syn according to the present invention refers to any amino acid sequence derived from the C-terminus of α-Syn. In a specific embodiment, the C-terminal region or C-terminus of α-Syn is an amino acid sequence having 96-140 residues, or a fragment thereof. The C-terminal region of α-Syn is rich in proline and negatively charged, which is a characteristic common in essentially random proteins that maintains solubility. Due to low hydrophobicity and high net negative charge, the C-terminal region of α-Syn usually exists as a random ring structure. In extracellular studies, it has been shown that a decrease in pH to neutralize negative charges can promote the aggregation of α-Syn.

The “α-Syn C-terminal fragment” or “B-cell epitope derived from the α-Syn C-terminal” in the present invention refers to a part of the full-length α-Syn sequence, which includes about 10 to 25 -A C-terminal amino acid of Syn, which corresponds to the sequence of glycine (G111) to aspartic acid (D135) at about the 111th position of the full-length α-Syn. The α-Syn C-terminal fragment may also be α-Syn G111-D135 peptide and fragments thereof. The various α-Syn C-terminal fragments of the present invention are represented by corresponding amino acid positions in the full-length α-Syn (SEQ ID NO: 1).

The α-Syn C-terminal fragment amino acid sequence used in the α-Syn peptide immune structure is selected according to a design principle. These principles include the use of an α-Syn peptide sequence: (i) because β-Syn can bind to α-Syn and prevent it from aggregating, it has no obvious sequence identity with β-synuclein (β-Syn) Derived to avoid the production of antibodies that will cross-react with β-Syn; (ii) it does not have the auto-T helper epitope in α-Syn to avoid autologous T cell activation, as previously used against Aβ _{1 -42} of AN1792 vaccine in clinical trials to treat Alzheimer's disease, which can cause brain inflammation and acute meningococcal (meningococcal encephalitis); (iii) it is a region in α-Syn, which is easily affected by natural structures Conformation change effect; (iv) It is non-immunogenic because it is a self-molecule; (v) It can provide immunogenicity through a protein carrier or an effective T helper epitope; (vi) When it is immune When it is native and given to one individual: (a) stimulate high-valence antibodies against α-Syn peptide sequence (B cell epitope), and the antibody will not target protein carriers or effective T helper epitopes; (b) ) Stimulate high potency antibodies, which can react with denatured β-sheets of monomers, oligomers, or α-Syn fibers This antibody can prevent the formation of α-Syn aggregation, decompose any α-Syn aggregation, and remove toxic α-Syn oligomers, aggregations and / or fibers to reduce or avoid the α-Syn aggregation load in the brain; (c) It does not stimulate antibodies that react with natural α-Syn, because natural α-Syn is a major cellular protein widely distributed in tissues, which can provide high safety.

Taking these design principles into consideration, the C-terminal region of α-Syn can be selected as the target of peptide immunogen design. In addition, based on the structural characteristics, compared to other α-Syn regions, they are more easily regulated by antibodies or other physical factors, so the C-terminal region of α-Syn was selected.

Various peptide sequences derived from α-Syn are evaluated to determine and select multiple α-Syn peptides that meet design principles, as further described in the Examples. In particular, a sequence that conforms to the design principle has about 10 to 25 amino acids, which correspond to glycine (G111) to aspartic acid (D135) at about the 111th position of the full-length α-Syn.

In certain embodiments, the C-terminal fragment of α-Syn is an α-Syn G111-D135 peptide (SEQ ID NO: 12) of 25 amino acids. In other embodiments, the C-terminal fragment of α-Syn is about 10 consecutive amino acids containing α-Syn G111-D135 peptide (SEQ ID NO: 12). In a specific embodiment, the C-terminal fragment of α-Syn contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the α-Syn G111-D135 peptide (SEQ ID NO: 12). , 20, 21, 22, 23, 24, or 25 consecutive amino acids. In a specific embodiment, the C-terminal fragment of α-Syn has an amino acid sequence, such as SEQ ID NOs: 12-15, 17 or 49-64 as described in Table 1.

The α-Syn C-terminal fragment of the present invention also includes immunogenic functional analogs or homologues of α-Syn G111-D135 peptides and fragments thereof. The functional immune analogs or homologues of G111-D135 peptides and fragments thereof include various variants that retain substantially the same immunogenicity as the original peptide. Immunogenic functional analogues may have a conservative substitution at an amino acid position; Changes in overall charge; covalent attachment of other units; or addition, insertion or deletion of amino acids and / or any combination thereof.

Conservative substitutions are replaced by other amino acids with similar chemical properties. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids Includes 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.

Immunogenic functional analogs include amino acid sequences with conservative substitutions, additions, deletions, or insertions of 1 to about 4 amino acids, which can stimulate an immune response that cross-reacts with the α-Syn G111-D135 peptide. Conservative substitutions, additions, deletions or insertions can be done with natural or unnatural amino acids. Unnatural amino acids include, but are not limited to, ε-N lysine, ß-alanine, ornithine, n-leucine, n-valine, hydroxyproline, thyroxine, γ-amine Acid, homoseric acid, citrulamic acid, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-Aminohexanoic acid), hydroxyproline, thiopropionic acid (MPA), 3-nitro-tyrosine , Pyroglutamic acid and similar. Natural amino acids include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamic acid, glycine, histidine, isoleucine, leucine , Lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

In one embodiment, the functional immune analogue of a specific peptide contains the same amino acid sequence as the original peptide, and is further included in α-Syn G111-D135 peptide and its fragment have 3 lysine (Lys-Lys-Lys) added to the amine end. In this embodiment, the fusion of the three lysines to the original peptide will change the overall charge of the original peptide, but will not change the function of the original peptide.

In a particular embodiment, the functional analogue of the α-Syn C-terminal fragment has at least 50% similarity to the original amine sequence. In other embodiments, the functional similarity has at least 80% similarity to the original amine. In other embodiments, the functional similarity has at least 85% similarity to the original amine. In other embodiments, the functional similarity has at least 90% or at least 95% similarity to the original amine.

b. Heterologous T helper epitopes (Th epitopes)

The invention provides a peptide immunogen structure containing a B-cell epitope derived from α-Syn, which is covalently linked to a heterologous T helper cell (Th) epitope directly or through a selective heterolinker.

The heterologous Th epitope in the structure of the α-Syn peptide immunogen can promote the immunogenicity of the C-terminal fragment of α-Syn, and promote the specificity of optimized B-cell epitopes (that is, α-Syn C -Terminal fragment) production of high-valence antibodies.

The term "heterologous" in the present invention refers to an amino acid sequence derived from a part of a non-wild-type α-Syn sequence or a homologous sequence thereof. Therefore, the heterologous Th epitope is a Th epitope derived from an unnatural α-Syn (ie, the Th epitope is not autologous α-Syn) amino acid sequence. Because the Th epitope and α-Syn are heterologous, when the heterologous Th is covalently linked to the C-terminal fragment of α-Syn, the natural amino acid of α-Syn does not extend to the N-terminus or C- end.

The heterologous Th epitope of the present invention may be any Th epitope, which does not contain the amino acid sequence of natural α-Syn. The Th epitope may have an amino acid sequence derived from any species (e.g., human, pig, cow, dog, rat, mouse, guinea pig, etc.). Th epitopes can also have promiscuous binding regions for second-class MHC molecules of multiple species. In a specific embodiment, the Th epitope includes a plurality of mixed second-type MHC binding regions to obtain maximum T helper cell activity, and to initiate and regulate the immune response. The Th epitope is preferably immunosilent itself, that is, antibodies that are induced by the α-Syn peptide immune structure are rarely directed against the Th epitope, so it can have a very concentrated focus on the α-Syn peptide immunostructure Target B-cell epitope immune response.

The epitope of the present invention includes, but is not limited to, amino acid sequences derived from foreign pathogenic bacteria, as shown in Table 2 (SEQ ID NOs: 70-98). In addition, the Th epitope includes a combination of optimized artificial Th epitopes and artificial Th epitopes (eg, SEQ ID NOs: 71 and 78-84). Heterologous Th epitope peptides appear as a combined sequence (eg, SEQ ID NOs: 79-82), a mixed amine contained at specific positions in the peptide block in the homology variable residues of a specific peptide Acid residues. During the synthesis process, it is possible to synthesize a combination of peptides by adding a design-protected amino acid mixture in a procedure to replace a specific amino acid at a specific position. This combination of heterologous Th epitope sets has broad Th epitope coverage for species with different genetic backgrounds. Representative heterologous Th epitope peptide combination sequences include SEQ ID NOs: 79-82, as shown in Table 2. The epitope peptides of the present invention can provide broad response and immunogenicity in genetically diverse animals and patients.

The α-Syn peptide immunostructure containing Th epitope can be generated simultaneously with the α-Syn C-terminal fragment in a single solid phase peptide synthesis. Th epitopes further include immune analogs of Th epitopes. Immune Th analogs include immune stimulating analogs, cross-reactive analogs, and any fragments of the above-mentioned epitopes of Th, which are sufficient to promote or stimulate the immune response against the α-Syn C-terminal fragment.

Functional immune analogs of the Th epitope peptide are also effective and are part of the present invention. Functional Thyroid Epidemic Th analogs include conservative substitutions, additions, deletions, and insertions of 1 to 5 amino acids in the Th epitope, which do not substantially alter the Th-stimulating function of the Th epitope. Conservative substitutions, additions, and deletions of the α-Syn C-terminal fragments described above are accomplished using natural or unnatural amino acids. Table 2 shows functional analogs of other Th epitopes. In particular, SEQ ID NOs: 71 and 78 of MvF1 and MvF2 Th are analogs of SEQ ID NOs: 81 and 83 of MvF4 and MvF5, and the difference is that the N- and C-termini have two amino acid deletions (SEQ ID NOs: 71 and 78) or increased (SEQ ID NOs: 81 and 83). The differences in the sequences of these two series of analogs will not affect the function of the Th epitope in the sequence. Therefore, functional immunogen Th analogs include a variety of measles virus fusion proteins MvF1-4 Ths (SEQ ID NOs: 71, 78, 79, 81, and 83) and hepatitis surface protein HBsAg 1-3 Ths (SEQ ID NOs). : 80, 82, and 84).

The Th epitope in the α-Syn peptide immunogen structure is Covalently linked to the N- or C-terminus of the α-Syn C-terminal peptide. In certain embodiments, the Th epitope is covalently linked to the N-terminus of the α-Syn C-terminal peptide. In other embodiments, the Th epitope is covalently linked to the C-terminus of the α-Syn C-terminal peptide. In a specific embodiment, more than one Th epitope is covalently linked to an α-Syn C-terminal fragment. When more than one Th epitope is covalently linked to the α-Syn C-terminal fragment, each Th epitope has the same amino acid sequence or a different amino acid sequence. In addition, when more than one Th epitope is covalently linked to the α-Syn C-terminal fragment, the Th epitopes can be arranged in any manner. For example, a Th epitope may be continuously linked to the N-terminus of an α-Syn C-terminal fragment, or a C-terminus of an α-Syn C-terminal fragment may be continuously linked; or a Th epitope may be continuously linked to an α-Syn C-terminal fragment. The N-terminus of the terminal fragment, while another Th epitope can continuously link the C-terminus of the α-Syn C-terminal fragment. The arrangement of Th epitopes at the C-terminal fragment of α-Syn is not limited.

In certain embodiments, the Th epitope is directly covalently linked to the α-Syn C-terminal fragment. In other embodiments, the Th epitope is covalently linked to the α-Syn C-terminal fragment via a linker.

c. Heterologous linkers

The α-Syn peptide immunogen structure optionally comprises a heterologous linker, which covalently links the B-cell epitope of the α-Syn and the heterologous T helper cell (Th) epitope.

As mentioned above, the "heterologous" of the present invention refers to an amino acid sequence derived from a part of a non-wild-type α-Syn sequence or a homologous sequence thereof. Therefore, when heterologous linkers covalently bind α-Syn B cells At the epitope position, because the linker is heterologous to the α-Syn sequence, the natural amino acid sequence of the α-Syn does not extend to the N-terminus or C-terminus.

A linker is any molecule or compound that can link two amino acids and / or peptides. Linkers can vary in length or polarity depending on the application. The linker may be linked through an amine group or a carboxyl group, but may be other functional groups. The linker includes a compound, a natural amino acid, or a non-natural amino acid.

The linker can provide a variety of structural features of the α-Syn peptide immunogen structure. Structurally, the linker provides a physical separation of the Th epitope from the B-cell epitope of the α-Syn C-terminal fragment. The physical separation of this linker can destroy the artificial secondary structure produced by linking Th epitopes to B cell epitopes. In addition, the physical separation of epitopes through the linker can eliminate the interference of the reaction between Th epitopes and / or B cell epitopes. In addition, the linker can generate or modify the secondary structure of the peptide immunogen structure. For example, the linker may have a variable hinge to facilitate the separation of the Th epitope and the B epitope. The variable hinge may also allow more effective responses between the peptide immunogen and appropriate Th cells and B cells to promote Th epitope and B cell epitope immune responses. The sequence encoding the variable hinge may be an immunoglobulin heavy chain hinge region, which is generally rich in proline. A variable hinge that can be used as a linker may be a Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 148) sequence, where Xaa is any amino acid, preferably aspartic acid.

The linker may also provide functional features of the α-Syn peptide immunogen structure. For example, a linker can change the overall charge of the α-Syn peptide immunogen structure, which can affect the solubility of the peptide immunogen structure. Also, change The overall charge of the α-Syn peptide immunogen structure can affect the ability of the peptide immunogen structure to bind to other compounds and reagents. As will be described later, the α-Syn peptide immunogen structure may be a stable immunostimulatory complex and a highly charged oligonucleotide, such as a CpG oligomer, through electrostatic binding. The overall charge of the α-Syn peptide immunogen structure is important for the formation of stable immunostimulatory complexes.

Compounds that can be used as linkers include, but are not limited to, (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminohexanoic 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-tetraoxo Heteropenta-decanoic acid (mini-PEG3), trioxadecane-succinic acid (Ttds), 12-oxo-dodecanoic acid, Fmoc-5-amino-3-oxovaleric acid (O1Pen) Its analog.

Natural amino acids include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamine, glutamine, glycine, histamine, isoleucine, leucine Amino acids, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Unnatural amino acids include, but are not limited to, ε-N lysine, ß-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-aminobutyric acid Acid, serine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-Aminohexanoic acid), hydroxyproline, mercaptopropionic acid, 3-nitro-tyrosine, pyroglutamic acid and the like analog.

The linker in the structure of the α-Syn peptide immunogen can be covalently linked to the Th epitope and the N- or C-terminus of the α-Syn C-terminal peptide. In some In some embodiments, the linker is covalently linked to the C-terminus of the Th epitope and the N-terminus of the α-Syn C-terminal peptide. In other embodiments, the linker is covalently linked to the C-terminus of the C-terminal peptide and the N-terminus of the Th epitope. In particular embodiments, for example, when there is more than one Th epitope in the peptide immunogen structure, more than one linker may be used. When more than one linker is used, each linker may be the same or different. Furthermore, when the peptide has more than one Th epitope, the Th epitope can be separated by a linker, and this linker can be the same as or different from the linker used to separate the Th epitope and the B cell epitope. The arrangement of the linker with the Th epitope or the α-Syn C-terminal peptide fragment is not particularly limited.

In a particular embodiment, the heterologous linker is a natural amino acid or a non-natural amino acid. In other embodiments, the linker includes more than one natural or non-natural amino acid. In a particular embodiment, the linker is Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

d. Specific examples of α-Syn peptide immunogens

The structure of α-Syn peptide immunogen can have the following structure: (Th) m- (A) n- (α-Syn C-terminal fragment) -X or (α-Syn C-terminal fragment)-(A) n -(Th) mX

Where Th is a heterologous T helper epitope; A is a heterologous linker; (α-Syn C-terminal fragment) is an α-Syn C-terminal fragment with about 10 to 25 amines B-cell epitopes of amino acids; X is α-COOH or α-CONH ₂ of a monoamino acid; M is 1 to about 4; and N is 0 to about 10.

In certain embodiments, the heterologous Th epitope in the α-Syn peptide immunogen structure has an amino acid sequence selected from SEQ ID NOs: 70-98 or a combination thereof, as shown in Figure 2 As shown. In a particular embodiment, the Th epitope has an amino acid sequence selected from SEQ ID NOs: 78-84. In a specific embodiment, the α-Syn peptide immunogen structure contains more than one Th epitope.

In certain embodiments, the selective heterologous linker may be selected from Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys, ε-N-Lys-Lys-Lys- Any of Lys (SEQ ID NO: 148) and combinations thereof. In a specific embodiment, the heterologous linker is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

In certain embodiments, the α-SynC-terminal fragment has about 10 to 25 amino acids at the α-SynC-terminus, which is glycine (G111) to about 111th amino acid in the full-length α-Syn 135 amino acids aspartic acid (D135). In a specific embodiment, the α-SynC-terminal fragment has an amino acid sequence of SEQ ID NOs: 12-15, 17 or 49-64, as shown in Table 1.

In certain embodiments, the α-Syn peptide immunogen structure has an amino acid sequence selected from any one of SEQ ID NOs: 107-108, 111-113, and 115-147, as shown in Table 3. Show. In a specific embodiment, α-Syn The peptide immunogen structure has an amino acid sequence selected from any one of SEQ ID NOs: 107-108 and 111-113.

Composition

The invention further discloses a composition including the aforementioned α-Syn peptide immunogen structure.

a. Peptide composition

The composition containing the α-Syn peptide immunogen structure is liquid or solid. The liquid composition may include water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structural or functional properties of the α-Syn peptide immunogen structure. The peptide composition may contain one or more α-Syn peptide immunogen structures.

b. Pharmaceutical composition

The present invention also relates to a pharmaceutical composition containing an α-Syn peptide immunogen structure.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier and / or other additives. Therefore, the pharmaceutical composition may contain a pharmaceutically effective amount of an α-Syn peptide immunogen structure and a pharmaceutically acceptable carrier, adjuvant and / or excipient, such as a diluent, an additive, a stabilizer, a preservative, Solubilizers, buffers and the like.

The pharmaceutical composition may contain one or more adjuvants, which can accelerate, prolong, or promote the immune response to the structure of the α-Syn peptide immunogen without the specific immunogen effect itself. Adjuvants used in pharmaceutical compositions may include liposomes, saponins, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA 35, ISA 206, ISA50V, ISA51, ISA 720 and other adjuvants and emulsifiers.

In certain embodiments, the pharmaceutical composition includes Montanide ^™ ISA 51 (an oil adjuvant composition of a w / o emulsifier composed of vegetable oil and mannitol oleate), Tween® 80 (polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), CpG oligo and / or combinations thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (w / o / w) emulsifier with Emulsigen or Emulsigen D as an adjuvant.

The pharmaceutical composition may be an immediate release or a sustained release formulation. In addition, pharmaceutical compositions can stimulate systemic or local mucosal immunity through capture of immunogens and co-administration of microparticles. Those skilled in the art can easily understand this delivery system.

The pharmaceutical composition may be an injection solution or a liquid solution or suspension. Before injection, a liquid carrier containing the α-Syn peptide immunogen structure is prepared. The pharmaceutical composition can be administered in an appropriate mode, for example, i.d., i.v., i.p., i.m., nasal, oral, subcutaneous, and the like. In certain embodiments, the pharmaceutical composition may be prepared for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions can also be prepared to be suitable for other modes of administration, including oral and intranasal administration.

Pharmaceutical compositions can also be prepared in a suitable dosage unit form. In certain embodiments, the pharmaceutical composition contains from about 0.5 μg to about 1 mg of alpha-Syn peptide immunogen structure per kilogram of body weight. Regardless of whether the patient is human or animal, prevention or treatment, the effective dose of the pharmaceutical composition can be changed according to many different factors, including the means of administration, the target site, the physiological state of the patient, and other administered drugs. Usually, the patient is a human, but it can also be Non-human animals, including transgenic animals. When multiple doses are administered, the pharmaceutical composition can be conveniently divided into appropriate unit doses. In the field of treatment, a dose can be given according to the age, weight and health of the individual.

In certain embodiments, the pharmaceutical composition includes more than one α-Syn peptide immunogen structure. A pharmaceutical composition containing more than one α-Syn peptide immunogen structure can synergistically increase the immune efficiency of the structure. A pharmaceutical composition containing more than one α-Syn peptide immunogen structure has a broad Class II MHC coverage, and therefore can provide a better immune response to the α-Syn peptide immunogen structure.

In certain embodiments, the pharmaceutical composition contains an α-Syn peptide immunogen structure selected from SEQ ID NOs: 107-108, 111-113, 115-147, and homologues, analogs, and / or combination. In a particular embodiment, the pharmaceutical composition contains an α-Syn peptide immunogen structure selected from SEQ ID NOs: 107-108, 111-113, and any combination thereof.

The pharmaceutical composition containing the α-Syn peptide immunogen structure can stimulate the host's immune response and produce antibodies.

c. immune stimulating complex

The present invention also relates to a pharmaceutical composition containing an α-Syn peptide immunogen structure and a CpG oligonucleotide immunoregulatory complex. The immunoregulatory complex specifically acts as an adjuvant and peptide immunogen stabilizer. The immunoregulatory complex is a particle that can effectively present an α-Syn peptide immunogen to the immune system of a cell to generate an immune response. The immunomodulatory complex may be a suspension for parenteral administration. Immune regulatory complexes can also be w / o emulsions, as a combination of suspensions with inorganic salts or in situ gelling polymers. The alpha-Syn peptide immunogen is efficiently delivered to cells of the host's immune system after parenteral administration. The immune regulatory complex can generate an immune response against β-sheet α-Syn (eg, Figures 8A, 8B, and 8C of Example 13) to achieve a protective / therapeutic effect.

The stable immunoregulatory complex can be electrostatically combined with an α-Syn peptide immunogen structure and an anion molecule, an oligonucleotide, a polynucleotide, or a combination thereof. Stable immune regulatory complexes can be combined with a pharmaceutical composition as an immune delivery system.

In certain embodiments, the alpha-Syn peptide immunogen structure contains a cationic moiety that is positively charged at pH 5.0 to 8.0. The cationic portion of the α-Syn peptide immunogen structure or mixture, the charge of each lysine (K), arginine (R) or histidine (H) is +1, and each aspartate (D ) Or glutamic acid (E) has a charge of -1 and other amino acids have a charge of 0, and the net charge is calculated in this manner. The addition of the charges within the α-Syn peptide immunogen structure is expressed as the average net charge. A stable peptide immunogen has a cation with an average net positive charge +1. Preferred peptide immunogens have an average net positive charge greater than +2. In certain embodiments, the cation of the α-Syn peptide immunogen structure is a heterologous linker. In certain embodiments, when the linker sequence is (α, ε-N) Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), the structure of the α-Syn peptide immunogen The cation has a +4 charge.

The "anionic molecule" as used herein refers to any molecule that is positively charged at a pH of 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge of an oligomer or polymer can be calculated using a charge of -1 per phosphodiester or thiophosphate group. Suitable anionic oligonucleotides are single-stranded DNA molecules with 8 to 64 nucleotide bases, with 1 to 10 repeating CpG units. Preferred CpG immunostimulatory single-stranded DNA molecules contain 18 to 48 nucleotide bases and have 3 to 8 repeating CpG units.

A better anionic oligonucleotide is shown by the following formula: 5 'X1CGX2 3', where C and G are unmethylated; and X1 is selected from A (adenine), G (guanine), and T (thymine) X2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide is represented by the formula: 5 '(X3) ₂ CG (X4) ₂ 3', where C and G are unmethylated; and X3 is selected from A, T or G, and X4 C or T.

The final immune stimulating complex is generally 1-50 μm particles, and has the function of many factors, including electrochemical measurement and molecular weight of reactants. The granular immune stimulating complex has the advantages of adjuvanting and enhancing the specific immune response in vivo. Therefore, the stabilized immune stimulating complex can stably prepare a pharmaceutical composition by water-in-oil emulsification, an inorganic salt suspension, and a polymer gel.

antibody

The present invention further provides antibodies that are stimulated with the structure of the α-Syn peptide immunogen.

The C-terminal fragment of α-Syn contains about 10 to about 25 amino acids of α-Syn, which is the glycine (G111) to about 135 positions of the full-length α-Syn (D135) The sequence is non-immunogenic or weakly immunogenic. However, α-Syn peptides including an α-Syn C-terminal fragment, a heterologous Th epitope, and a selective heterologous linker When given to the host, the immunogen structure can stimulate the immune response and produce antibodies. α-Syn peptide immunogen structure can surpass α-Syn itself, and promote the generation of specific antibodies that can recognize non-linear, conformational epitopes.

Surprisingly, the α-Syn peptide immunogen structure does not produce natural α-helical binding to the α-Syn monomer in its native form. However, antibodies generated by the α-Syn peptide immunogen structure recognize and bind α-Syn denatured β-sheets in the form of monomers, oligomers, and fibers. In addition, the antibodies produced by the α-Syn peptide immunogen structure will not bind to other amyloid-like structures (i.e., Aβ1-42 and Tau441). Therefore, the special design of the α-Syn peptide immunogen structure (including the α-Syn C-terminal fragment, the heterologous Th epitope, and the selective heterolinker) has changed the conformation of the multifunctional α-Syn C-terminal fragment, To have a β-sheet-like conformation.

In many functional tests, extensive comparisons have been made of sera obtained from animals immunized with an α-Syn peptide immunogen structure. These comparisons demonstrate that antibodies can specifically bind β-sheet α-Syn of monomers and oligomers in NGF-treated PC12 cells without binding to other amyloids (see Example 9).

Antibodies induced by the α-Syn peptide immunogen structure can inhibit the aggregation of α-Syn (anti-aggregation activity) and de-aggregate pre-formed α-Syn aggregates (cleavage activity). In addition, surprisingly, antibodies can reduce the production of TNF-α and IL6 induced by microglial cells, and these antibodies can effectively reduce α-Syn aggregates or fiber-mediated microglia activity. In α-Syn overexpressing cells, this antibody can also reduce neurodegeneration caused by exogenous α-Syn aggregates and endogenous α-Syn aggregates. Into. In addition, this antibody recognizes and specifically binds pathological α-Syn oligomer aggregates or fibers, but does not react with non-pathological α-Syn. In particular, the antibodies can react with Lewy bodies in brain slices from patients with Parkinson's disease who have alpha synuclein-related diseases, but do not react with normal human tissues.

It is also surprising that two mouse models of Parkinson's disease (MPP + induced mouse model and mouse model inoculated with α-Syn fiber) can (a) produce a composition containing the α-Syn peptide immunogen structure Antibodies that are highly cross-reactive with β-sheet α-Syn, (b) decrease α-Syn serum, (c) decrease brain α-Syn oligomers, and (d) decrease neuropathology to restore motor function.

Immune response produced by animals immunized with the α-Syn peptide peptide immunogen structure of the present invention confirms that this structure can produce antibodies specific to specific positions, which can interact with mutant β-sheet α in the form of monomers, oligomers and fibers -Syn reaction and does not react with the random coil structure of the C-terminus of α-Syn in its natural form.

In vitro Functional test

In vitro functional analysis was performed using antibodies produced by the α-Syn peptide immunogen structure. This functional analysis includes, but is not limited to: (a) inhibition of in vitro recombinant α-Syn aggregates; and decomposition of recombinant α-Syn aggregates (see Example 8); (b) inhibition of in vitro cellular α-Syn aggregates And break down intracellular α-Syn aggregates (see Example 9); (c) reduce the secretion of microglia TNF-α and IL6 (see Example 10); (d) reduce the amount of exogenous α-Syn aggregates Induced neurodegeneration (see Example 11); (e) Reduced neurodegeneration in α-Syn overexpressing cells (see Example 12); (f) Demonstrated the smallness of Parkinson's disease induced by inoculation of α-Sy fibers and MPP + The murine model had a decrease in the α-Syn content in the serum, a decrease in the α-Syn oligomer content in the brain, a decrease in neurodegeneration, and a recovery in motor activity (see Example 15).

method

a. Method for preparing α-Syn peptide immunogen

The α-Syn peptide immunogen structure of the present invention can be prepared by chemical synthesis methods known to those skilled in the art (see Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WHFreeman & Co., New York, NY, 1992, p. 77). The α-Syn peptide immunogen structure can be synthesized using t-Boc or F-moc-protected α-NH ₂ branched-chain amino acids using automated Merrifield solid-phase synthesis technology, for example, Applied Biosystems Peptide Synthesizer Model 430A or 431. Alternative amino acids are used for coupling at specific variable positions to prepare an α-Syn peptide immunogen structure comprising a Th peptide library.

After the assembly of the α-Syn peptide immunogen structure is completed, the processing resin cuts the peptide from the resin and unblocks the amino acid branch chain. Free peptides can be purified by HPLC using amino acids Analysis or sequencing for biochemical analysis. Methods for the purification and analysis of peptides are well known to those skilled in the art.

The quality of the peptides produced by this chemical procedure can be controlled and determined to ensure the reproducibility of the structure, immunogenicity and yield of the α-Syn peptide. A detailed description of the structure of the α-Syn peptide immunogen produced by the solid-phase peptide synthesis method is shown in Example 1.

The range of structural variability that retains the expected immune activity is much greater than the structural variability of small molecule drugs or biologically derived drugs. Therefore, peptide analogs, intentionally designed or a mixture of deleted sequence by-products due to errors in the synthetic process (having similar chromatographic and immunological properties), are usually as effective as the purified peptide preparations. As long as there are identifiable QC procedures to monitor the process and product evaluation of specific designed analog and undesired analog mixtures, to ensure the reproducibility and efficacy of these peptides.

The α-Syn peptide immunogen structure can also be prepared using recombinant DNA technology, including nucleotide molecules, vectors, and / or host cells. Therefore, a nucleotide molecule encoding a nuclear α-Syn peptide immunogen structure and a functional analogue of the immunogen also belongs to the present invention. Likewise, vectors (including expression vectors) containing nucleotide molecules and host cells containing vectors also form part of the present invention.

Other embodiments also include methods for preparing the α-Syn peptide immunogen structure and immunogenic functional analogs of the α-Syn G111-D135 fragment derived from the peptide immunogen structure. For example, the method can include culturing a peptide containing an alpha-Syn peptide Cells expressing vectors of epidemic structure and / or their immunogenic functional analogue nucleotides. Longer synthetic peptide immunogens can be synthesized using conventional recombinant DNA techniques. This technique is documented in the well-known standard operating manual. In order to construct a gene encoding the peptide of the present invention, the amino acid sequence is reverse-translated to obtain a nucleotide sequence encoding the amino acid sequence, preferably having the best codon for the host. Then, if necessary, genes can usually be synthesized by synthesizing oligonucleotides encoding peptides and any regulatory elements. This synthetic gene is inserted into a suitable vector and transfected into a host cell. The peptide is then expressed under conditions suitable for the performance system and host. The peptide was purified and analyzed by standard methods.

b. Method for preparing immune complex

Various embodiments of the present invention also include methods of preparing an immunostimulatory complex comprising an α-Syn peptide immunogen structure and a CpG oligonucleotide (ODN) molecule. The stable immune stimulating complex (ISC) is derived from the cationic portion of the α-Syn peptide immunogen structure and the polyanionic CpG ODN molecule. Self-assembly systems can be completed using electrostatic neutralization of charge. The molar charge ratio of the cationic portion of the α-Syn peptide immunogen structure to the anionic oligomer determines the degree of binding. The non-covalent electrostatic binding of α-Syn peptide immunogen structure to CpG ODN is a completely repeatable process. The peptide / CpG ODN immune stimulating complex complex aggregates, which can promote "professional" antigen-presenting cells (APC) presented to the immune system, thereby enhancing the immunogenicity of the complex. This composite can easily control quality during the manufacturing process. Peptide / CpG ISC is well tolerated in vivo . This novel particle system including CpG ODN and α-Syn G111-D135 fragment peptide immunogen structure has the advantage of mitosis in generalized B cells associated with CpG ODN, but promotes the balance of Th-1 / Th-2 response.

In the pharmaceutical composition of the present invention, CpG ODN is 100% bound to the immunogen during the electrostatic neutralization of opposite charges, and forms nano-sized particles. This granular form can significantly reduce the doses used with traditional CpG adjuvants, reduce possible adverse innate immune responses, and facilitate selective immune procedures including antigen-presenting cells (APC). Therefore, this formulation offers possible advantages for an innovative concept and the stimulation of an immune response through alternative mechanisms.

c. Method for preparing pharmaceutical composition

Various embodiments also include pharmaceutical compositions containing an alpha-Syn peptide immunogenic construct. In certain embodiments, pharmaceutical compositions use water-in-oil emulsification and suspensions of mineral salts.

In order to make the pharmaceutical composition usable in most ethnic groups, and to suppress the aggregation of α-Syn is also one of the management purposes, safety has become another important factor for consideration. Although many formulations use water-in-oil emulsions in humans in clinical trials, alum is still the main adjuvant used in formulations based on safety. Therefore, alum or its mineral salt aluminum phosphate (ADJUPHOS) is often used as an adjuvant for clinical use.

d. Method of using pharmaceutical composition

The present invention further includes a method for using a pharmaceutical composition containing an α-Syn peptide immunogen structure.

In certain embodiments, a pharmaceutical composition containing an α-Syn peptide immunogen structure can be used for:

(a) inhibiting the aggregation of α-Syn in the host;

(b) promote the breakdown of pre-formed α-Syn aggregates in the host;

(c) reduce the secretion of TNF-α and IL6 by microglia in the host;

(d) Reduce neurodegeneration in the host induced by external α-Syn aggregates

(e) reducing neurodegeneration in α-Syn overexpressing cells;

(f) reducing serum α-Syn content in the host;

(g) reducing the serum α-Syn content in the host brain;

(h) Reduce neuropathy and restore motor function in the host; and similar.

The method includes administering a pharmaceutical composition to a desired individual, wherein the medical composition includes a medically effective amount of an α-Syn peptide immunogen structure.

Specific embodiment

The present invention includes, but is not limited to, the following specific examples: (1) an α-synuclein (α-Syn) peptide immunogen structure, including: a B-cell epitope, which includes about 10 to 25 sources The amino acid sequence from the C-terminus of α-Syn, which corresponds to the sequence of glycine (G111) to the 135th position of aspartic acid (D135) of the full-length α-Syn; SEQ ID NO: 1; The T helper epitope includes an amino acid sequence selected from SEQ ID NOs: 70-98; and a selective heterolinker selected from monoamino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), in which the B cell epitope is covalently directly or through selective heterologous The linker connects the T helper epitope.

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

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

(4) The α-Syn peptide immunogen structure according to (1), wherein the selective heterolinker is (α, ε-N) Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

(5) The α-Syn peptide immunogen structure according to (1), wherein the T helper epitope is covalently linked to the amine end of the B cell epitope.

(6) The α-Syn peptide immunogen structure according to (1), wherein the T helper epitope is covalently connected to the amine base of the B cell epitope through a selective heterolinker.

(7) The α-Syn peptide immunogen structure according to (1), including the following structures (Th) m- (A) n- (α-Syn C-terminal fragment) -X or (α-Syn C-terminal fragment )-(A) n- (Th) mX where Th is the T helper epitope; A is a heterologous linker; (α-Syn C-terminal fragment) is the B cell epitope; X is a monoamino acid Α-COOH or α-CONH ₂ ; m is from about 1 to about 4; and n is from 1 to about 10.

(8) The α-Syn peptide immunogen structure according to (1), including a monoamino acid sequence, selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

(9) The α-Syn peptide immunogen structure according to (1), comprising a monoamino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, and 111-113.

(10) A composition comprising the α-Syn peptide immunogen structure of (1).

(11) A composition comprising more than one α-Syn peptide immunogen structure according to (1).

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

(13) A pharmaceutical composition comprising the α-Syn peptide immunogen structure of (1) and a pharmaceutically acceptable carrier and / or adjuvant.

(14) The pharmaceutical composition according to (13), wherein a. The α-Syn peptide immunogen structure is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; and b. The adjuvant is a mineral salt selected from the group consisting of Al (OH) ₃ or AlPO ₄ .

(15) The pharmaceutical composition according to (13), wherein a. The α-Syn peptide immunogen structure is selected from SEQ ID NOs: 107, 108, A group consisting of 111-113, and 115-147; and b. The α-Syn peptide immunogen structure and a CpG oligonucleotide (ODN) to form a stable immunostimulatory complex.

(16) An isolated antibody or epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogen structure of (1).

(17) The isolated antibody or epitope-binding fragment thereof according to (16), which binds to an α-Syn peptide immunogen structure.

(18) An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogen structure of (9).

(19) A composition comprising the isolated antibody of (16) or an epitope-binding fragment thereof.

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

(21) The composition of (20), comprising a. An isolated antibody or an epitope-binding fragment thereof that specifically binds to a B-cell epitope of SEQ ID NO: 112; and b. Specifically binds to SEQ ID NO : 113 B-cell epitope isolated antibody or combination of epitope-binding fragments thereof.

(22) An antibody for recognizing α-Syn in a host, comprising administering to the host a composition comprising the α-Syn peptide immunogen structure of (1) and a carrier and / or adjuvant.

(23) A method of inhibiting α-Syn aggregation in an animal, comprising administering the A pharmaceutically effective amount of (1) α-Syn peptide immunogen structure.

(24) A method for reducing α-Syn aggregation in an animal, comprising administering to the animal a pharmaceutically effective amount of the α-Syn peptide immunogen structure of (1).

(25) An α-Syn aggregate of different sizes in a biological sample, including: a. Contacting the biological sample with the antibody or an epitope-binding fragment of (16) under conditions that are capable of being contacted; b. The amount of the antibody or epitope-binding fragment that binds to the α-Syn aggregate in the biological sample.

The following examples provide detailed implementations.

[Example]

Example 1. Synthesis of α-synuclein peptide and preparation of its formulation

a. Synthesis of α-Syn C-terminal fragment

This example discloses a synthetic method for developing an α-Syn C-terminal fragment of an α-Syn peptide immunogen structure. It can be used for small-scale synthesis for serological testing, laboratory tests and field research, as well as large-scale (kilogram) synthesis for industrial / commercial production of pharmaceutical compositions.

In order to screen and select the best peptide constructs for effective α-Syn peptide immunogen structure, a large library of α-Syn related antigenic peptides with a length of about 10 to 40 amino acid sequences was designed.

Use representative full-length α-Syn (SEQ ID NO: 1) and β-Syn (SEQ ID No: 2), α-Syn fragments, such as α-Syn _111-132 , α-Syn _126-135, etc. 10-mer The peptides identified epitopes in various serum tests, as shown in Table 1 (SEQ ID NOs: 1 and 3 to 69). The selected α-Syn fragment was synthetically linked to measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza, tetanus, and Epstein-Barr virus (EBV) ) Specially designed helper T cell (Th) epitopes to make an α-Syn peptide immunogen structure, as shown in Table 2 (SEQ ID NOs: 70-98). Th epitope can use a single sequence (SEQ ID NOs: 70-78 and 83-98) or a combined sequence library (SEQ ID NOs: 79-82) to promote the immunogenicity of each α-Syn peptide immunogen structure .

Representative α-Syn peptide immunogen structures selected from more than 100 peptide structures are shown in Table 3 (SEQ ID NOs: 99-147). All peptides used for immunogenicity research or detection and / or measurement of anti-α-Syn antibody-related serological tests are performed using F-moc chemistry with Applied BioSystems Models 430A, 431, and / or 433 peptide synthesizers.量 合成。 Volume synthesis. Each peptide is independently synthesized on a solid phase support, and its N-terminus has F-moc protection and three functional amino acid branch-chain protecting groups. The peptide was excised from the solid phase support and the branched protecting groups were removed with 90% trifluoroacetic acid (TFA). The synthetic peptide was analyzed by (MALDI-TOF) mass spectrometry to ensure that the amino acid sequence was correct. The synthesis results and concentration of each synthetic peptide can also be confirmed by reversed-phase HPLC (RP-HPLC). Despite strict control over synthetic procedures, including stepwise monitoring of coupling efficiency, peptide analogs can still be generated by unexpected events during extended cycles, including amino acid insertions, deletions, substitutions, and early termination. Therefore, peptide synthesis generally includes target peptides and various peptide analogs. Despite the inclusion of this unexpected peptide analog, this final peptide was synthesized Still suitable for immunological applications, including immunoassay (antibody capture antigen) and pharmaceutical composition (peptide immunogen). In general, peptide analogs, whether deliberately designed or mixed by-products generated during the synthesis process, as long as the manufacturing process and product evaluation procedures are monitored with rigorous QC procedures, the reproducibility and efficacy of the final product can be guaranteed. Usually the same effect as the desired peptide. Hundreds to several kilograms of large peptides can be synthesized using a customized automatic peptide synthesizer UBI2003 or similar, with a concentration between 15mmol and 50mmol. For the active ingredients in the final pharmaceutical composition of clinical trials, the α-Syn peptide structure can be purified by RP-HPLC at a low concentration gradient, and the characteristics of the peptide can be analyzed by MALDI-TOF mass spectrometry and amino acids, and Purity and consistency were analyzed by RP-HPLC.

b. Preparation of composition containing α-Syn peptide immunogen structure

Formulations using water-in-oil emulsions and inorganic salt suspensions. In order to make the pharmaceutical composition applicable to a wide range of ethnic groups, and prevention is also one of the purposes of administration, safety has become another important factor to consider. Despite the use of water-in-oil emulsions in many pharmaceutical compositions in human clinical practice, alum remains the main adjuvant for pharmaceutical compositions based on safety. Therefore, in clinical applications, alum or its mineral salt ADJUPHOS (aluminum phosphate) is often used as an adjuvant.

The specific formulations described below contain all types of α-Syn peptide immunogen structures. The relative immunogenicity of more than 100 α-Syn peptide immunogen structures to representative α-Syn peptides was evaluated in guinea pigs, and the application of different peptides selected from SEQ ID NOs: 1-153 was applied. 96-well plates were analyzed by ELISA for serum cross-reactivity between various homologous peptides.

α-Syn peptide immunogen structure (i) Prepared as a water-in-oil emulsion with Seppic Montanide ^TM ISA 51, which can be used in humans, or (ii) peptide structures with different concentrations and ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum) mixing. The α-Syn peptide immunogen structure is dissolved in water at a concentration of 20 to 800 μg / mL, and is mixed with Montanide ^TM ISA 51 (1: 1 volume ratio), or mineral salt or ALHYDROGEL (alum) (1: 1 volume ratio) Formulated as a water-in-oil emulsion. The composition was maintained at room temperature for about 30 minutes and mixed with a shaker for 10 to 15 seconds before immunization. Animals were immunized with the specific composition 2 to 3 times and given by intramuscular injection. Immunization was performed at week 0 and 3 weeks (boost) after the initial immunization (wpi), and the second boost was optionally performed at 5 or 6 wpi Immunity. The immunized animals were tested using the selected B epitope peptides to evaluate the immunogenicity of each α-Syn peptide immunogen structure in the formulation and its cross-reactivity with the relevant target peptide or protein. The α-Syn peptide immunogen structure, which may be immunogenic, will be initially screened in guinea pigs. It will be further tested in primates with specific dosing procedures in the form of water-in-oil emulsions, mineral salts and alum formulations.

Only the most promising α-Syn peptide immunogen structure is further evaluated for its immunogenicity, duration, toxicity, and efficacy according to GLP specifications before being prepared into a final preparation in preparation for patients with synucleinopathy In the clinical application of new drugs and clinical trials.

Example 2. Preparation of recombinant α- Syn protein

For the method of constructing the α-Syn gene into the pGEX-4T1 vector, refer to Neurotoxicology and teratology 2004, 26 (3): 397-406. The target sequence (SEQ ID NOs: 1) was inserted into the restriction endonucleation site between BamHI and XhoI in the pGEX-4T1 vector. This fragment was replicated by polymerase chain reaction (PCR) using KAPA HiFi DNA polymerase (Kapa Biosystems, Inc., Woburn, MA, USA). The primer sequence is as follows: forward primer 5'-cgggatccgatgtgtttatgaaaggtctgag-3 '(SEQ ID NO: 149); reverse primer 5'-ggaattccgatgtgtttatgaaaggtctgag-3' (SEQ ID NO: 150). The PCR conditions were as follows: heating at 94 ° C for 1 minute, heating at 94 ° C for 15 seconds for denaturation, bonding at 60 ° C for 30 seconds, and extension at 68 ° C for 2 minutes, 30 cycles, and 5 minutes at 68 ° C. The end reaction is performed. A Q5 Site-Directed Mutagenesis kit (New England BioLabs, Beverly, MA, USA) was used to complete the A53T α-Syn point mutation. The primer sequence of the mutant α-Syn is: forward primer 5'-tcatggtgtgaccaccgttgcag-3 '(SEQ ID NO: 151); reverse primer 5'-accacgccttctttggttttg-3' (SEQ ID NO: 152).

The pGEX-4T1 GST vector containing α-Syn was transfected into E. coli BL21 (DE3) for protein expression. E. coli was cultured in LB medium at 37 ° C, and β-d-1 thiogalactoside (IPTG) was added at a final concentration of 4 mM when the OD600 reached 0.8. After 4 hours of incubation, cells were collected by centrifugation at 5000xg for 20 minutes at 4 ° C. The collected cells were suspended in PBS, placed on ice for ultrasonic destruction, and centrifuged at 5000xg for 20 minutes at 4 ° C. The supernatant was placed in a glutathione agarose-4B column (GE Healthcare) and equilibrated with PBS. After washing 3 times with PBS, 1 mL of thrombin (20 U / mL in PBS) was added and left at 4 ° C overnight to release the GST of the fusion protein. The unlabeled α-Syn was washed out and thrombin was removed on a HiTrap Benzamidine FF column (GE Healthcare). The dialyzed α-Syn was immediately frozen at -80 ° C. The purified α-Syn has a molecular weight of 14 kDa MW. After separation by 10% SDS-PAGE, anti-α-Syn antibody (1: 2000, Millipore, targeting α-Syn _111-131 ) was used for Western _blot analysis.

Example 3. Serological analysis and reagents

In the following, serological assays and reagents for evaluating the functional immunogenicity of synthetic peptide structures and their preparations are disclosed.

a. Analysis of antibody specificity by peptide ELISA

The ELISA analysis used to evaluate immune serum samples in the following examples is described below. The holes of the 96-well plate were at a concentration of 2 μg / mL (unless otherwise specified), 100 μL of target peptide α-Syn fragments A85-A140, A91- dissolved in 10 mM NaHCO ₃ buffer (pH 9.5 (unless specified)) 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 win The peptides (SEQ ID NOs: 4-17) were reacted at 37 ° C for 1 hour to perform coating.

b. Evaluation of Th peptide response by Th peptide ELISA

The peptide-coated holes (SEQ ID Nos: 70-98) were reacted with 250 μL of 3% by weight gelatin in PBS at 37 ° C. for 1 hour to block non-specific protein binding sites, and then contained 0.05% by volume TWEEN® 20 is washed 3 times and dried. The serum to be tested was diluted 1:20 with PBS containing 20% by volume of goat serum, 1% by weight of gelatin and 0.05% by volume of TWEEN® 20 (unless otherwise specified). Add 100 μL of the diluted sample (such as serum and plasma) to each well and react at 37 ° C for 60 minutes. Next, wash the wells 6 times with TWEEN® 20 in 0.05% PBS to remove unbound antibodies. Horseradish peroxidase (HRP) -conjugated species (such as mouse, guinea pig, or human) specific goat anti-IgG is a tracking marker that can bind to the antibody / antigen complex in the hole. 100 μL of diluted peroxidase-labeled goat anti-IgG was added to each well, and reacted at 37 ° C. for 30 minutes. Wash the wells 6 times with PBS containing 0.05% TWEEN® 20 to remove unbound antibodies and 100 μL of 0.04% by weight of 3 ', 3', 5 ', 5'-tetramethylbenzidine (TMB) And 0.12% by volume sodium hydrogen citrate buffer for 15 minutes. This mixture can detect peroxidase by producing colored substances. The reaction was stopped by adding 100 μL of 1.0MH ₂ SO ₄ and the absorbance (A450) at 450 nm was detected. Measure the serum valence of 10-fold dilution (dilution ratio of 1: 100 to 1: 10,000) and express it as Log _10. Make A450 linear regression, and use linear regression and A450 threshold of 0.5 to calculate various α-Syn peptide immunity Antibody titer of the original immunized animal.

c. B-cell epitope 10-mer peptide ELISA to evaluate the specificity and epitope position of α-Syn fragment

Epitope mapping was used to determine the precise specificity of anti-α-Syn peptide antibodies in immunized hosts. In other words, a 96-well plate was coated with 0.5 μg / 0.1 mL of α-Syn 10-mer peptide (SEQ ID NOs: 18 to 69) per hole, and then 100 μL of a serum sample (1: 100 ratio to PBS dilution) was performed, and the antibody ELISA analysis step was performed. The corresponding α-Syn peptide (SEQ ID No: 99, 102, 108, 110, 112, 113) or a fragment thereof can also be used, without the linker and Th sequence, or with β-Syn (SEQ ID NO: 153) The peptide performs additional responses and specificity analysis to test the specificity of the B-cell epitope of the α-Syn peptide immunogen and the associated immune serum anti-α-Syn antibody.

d. Evaluation of immunogenicity

Pre-immune and immune serum samples were collected according to the immunization program, and heated at 56 ° C for 30 minutes to deactivate serum complement factors. After administering the pharmaceutical composition, a blood sample is obtained in accordance with the procedure, and the immunogenicity of a specific target location is analyzed. Serially diluted sera were analyzed and force valence was expressed as the inverse of Log ₁₀ . With high-valence B-cell antibody response that can be specific for the epitope in the target antigen, while maintaining the "helper T-cell epitope" used to enhance the B-cell response at a low or even negligible antibody reactivity, To assess the immunogenicity of a particular pharmaceutical composition.

e. Immunoassay of α-Syn content in mouse immune serum

Using sandwich ELISA (Cloud-clon, SEB222Mu) with anti-α-Syn antibody as capture antibody and biotin-labeled antibody as detection antibody, α-Syn in the serum of mice administered with α-Syn peptide immunogen was analyzed content. In other words, 100 ng / well of an antibody dissolved in a coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) was fixed in a 96-well plate and reacted at 4 ° C. overnight. 200 μL / well of analytical dilution (0.5% BSA, 0.05% TWEEN®-20, 0.02% ProClin 300 in PBS) was used to block the coated holes for 1 hour at room temperature. A standard curve (2-fold serial dilution, concentration 156 to 1250 ng / mL) was obtained using an analytical dilution containing purified recombinant α-Syn and 5% mouse serum. 50 μL of diluted serum (1:20) and standards were added to the coated wells. The reaction was carried out at room temperature for 1 hour. All holes were aerated and washed 6 times with 200 μL / well of washing buffer. The captured human α-Syn was reacted with 100 μL of a detection antibody solution (50 ng / ml of the biotin-labeled HP6029 in an analytical dilution) for 1 hour at room temperature. Next, streptavidin poly-HRP (1: 10000 dilution, Thermo Pierce) was reacted with the bound biotin-HP6029 for 1 hour. All holes were aerated, washed 6 times with 200 μL / well of washing buffer, and 100 μL / well of 1M H ₂ SO _{4 was added to} stop the reaction. A standard curve was prepared using SoftMax Pro software (Molecular Devices) and a 4-parameter logistic curve fit was generated to calculate the α-Syn concentration in all test samples.

f. Preparation of α-Syn aggregates and recombinant α-Syn

To prepare aggregated α-Syn, purified wild-type or A53T mutant α-Syn [dissolved in 100 μL PBS / KCl aggregation buffer (2.5 mM MgCl ₂ , 50 mM HEPES and 150 mM KCl in 1 x PBS, pH 7.4), [Concentration: 0.1 [mu] g / [mu] L] was placed in a 1.5 mL microcentrifuge tube and reacted for 7 days at 37 [deg.] C. using a thermostatic shaker. The obtained aggregated α-Syn was immediately frozen at -80 ° C for subsequent use.

g. Purification of anti-α-Syn antibodies

Guinea pigs were immunized with an α-Syn peptide immunogen structure containing different sequences (SEQ ID NOs: 99-121). Affinity columns (Thermo Scientific, Rockford) were used from serum collected 3 to 15 weeks after injection (WPI). Purified anti-α-Syn antibody. In other words, after equilibrating the buffer (0.1 M phosphoric acid and 0.15 M sodium chloride, pH 7.2), 400 μL of serum was added to a Nab Protein G Spin column, mixed up and down for 10 minutes, and centrifuged at 5,800 xg for 1 minute. To binding buffer (400 μ L) washed three times with the column. Next, wash buffer (400 μL, 0.1M glycine, pH 2.0) was added to the Spin column, and the antibodies were washed out by centrifugation at 5,800 xg for 1 minute. The washed out antibody was mixed with a neutralization buffer (400 μL, 0.1 M Tris pH 8.0), and fetal bovine serum (BSA) was used as a standard. The OD280 was detected using Nan-Drop to calculate the antibody concentration.

h. Specificity of anti-α-Syn antibodies of guinea pigs with different α-Syn peptide immunogen structural immunity against α-Syn molecules of different sizes

Western blot analysis was used to analyze the specificity of anti-α-Syn antibodies derived from guinea pigs immunized with different α-Syn peptide immunogen structures for α-Syn molecules of different sizes. 20 μM α-Syn was separated with 12% Tris-glycine SDS-PAGE and transfected onto nitrocellulose (NC) membranes before photo-induced cross-linking (PICUP) treatment. Derived from 1 μg / mL The nitrocellulose membrane was treated with anti-α-Syn antibody of guinea pig, and then reacted with donkey anti-guinea pig antibody (706-035-148, Jackson) conjugated with HRP. The chemical dots of the film were developed with a chemical cold light reagent Western Lightning ECL Pro (PerkinElmer). From the results, it is found that the molecular weight of the monomer α-Syn (Mw 14,460 Da) is about 14 kDa, and the dimer, trimer or oligomer thereof has a molecular weight several times that of the monomer α-Syn. As a positive control group, a commercially available Syn211 antibody (Abcam) capable of detecting various oligomers such as dimer, trimer or oligomer was used.

i. Analysis of different types of amyloids by circular staining method

The preparations of Aβ _1-42 , Tau and α-Syn α-helical monomers, β-sheet monomers, β-sheet oligomers, and fibrous β-sheets are shown below.

1. Aβ _1-42 α-helical monomer: Add 20 μg of Aβ _1-42 β-fold monomer (50 μL) to 20% trifluoroacetic acid and 20% hexafluoroisopropanol (10 μL), place in 4 It was reacted at 24 ° C for 24 hours to form an α-helical monomer.

2. Aβ _1-42 β-folded monomer: Place 60 μg of Aβ _1-42 in 120 μL of 5% TFA in 1xPBS, react at 37 ° C for 24 hours, and place in a 10 kDa filter column (Millipore) to obtain β -Folding monomer.

3. Aβ _1-42 β-folded oligomer: 120 μL 1xPBS containing 60 μg Aβ _1-42 was reacted at 37 ° C. for 3 days, placed on ice for ultrasonic shock, and placed in 10 and 30 kDa filter columns (Millipore ) To obtain β-sheet fibrous oligomers with a molecular weight of less than 35 kDa.

4. Aβ1-42 fibrous β-sheet: 120 μL 1xPBS containing 60 μg Aβ _1-42 was reacted at 37 ° C for 3 days, placed on ice for ultrasonic shock, and placed in a 30 kDa filter column (Millipore) to obtain fibers. Like β-sheet.

5. α-Syn α-helical monomer: 40 μg of freshly prepared α-Syn was dissolved in 100 μL of ice 1xPBS at 4 ° C. and immediately placed in a 10 kDa filter column (Millipore) to obtain α-helical monomer.

6. α-Syn β-folded monomer: 100 μL of PBS / KCl buffer containing 40 μg of α-Syn was reacted at 37 ° C. for 24 hours, and a β-folded monomer was obtained using a 10 kDa filter column (Millipore).

7. α-Syn β-folded monomer: 100 μL of 40 μg aggregated α-Syn PBS / KCl buffer solution was reacted at 37 ° C. for 8 days, placed on ice for ultrasonic shock, and placed in 30 kDa and 100 kDa filter tubes. Column (Millipore) to obtain β-sheet monomer.

8. α-Syn fibrous β-sheet: 100 μL of PBS / KCl buffer solution containing 40 μg aggregated α-Syn is reacted at 37 ° C. for 8 days, placed on ice for ultrasonic shock, and placed in 30 kDa and 100 kDa filter tubes. Column (Millipore) to obtain β-sheet monomer.

9. Tau441 α-helical monomer: 100 μL of 1 × PBS containing 60 μg of Tau at 4 ° C. was placed in a 100 kDa filter column (Millipore) to obtain α-helical monomer.

10. Tau441 β-fold monomer: 100 μL of 60 μg aggregated Tau was reacted with 10 unit / mL heparin 1x PBS buffer at 25 ° C for 48 hours, and placed in a 100kDa filter column (Millipore) at 4 ° C to obtain β-fold monomer.

10. Tau441 β-fold oligomer: 100 μL of 1 × PBS buffer containing 60 μg of aggregated Tau was reacted at 37 ° C. for 48 hours, and placed in 100 kDa and 300 kDa filter columns (Pall) to obtain β-fold oligomer.

11. Tau441 fibrous β-sheet: 100 μL of 60 μg aggregated Tau was reacted with 10 unit / mL heparin 1xPBS buffer at 37 ° C for 6 days, and placed in a 300kDa filter column (Pall) at 4 ° C to obtain fibrous β- fold.

This monomer and oligomer were confirmed by thioflavin-T (ThT, Sigma) fluorescence or polyacrylamide gel electrophoresis (PAGE). The amyloid concentration was determined using Nano-Drop and was based on the commercially available amyloid Aβ _1-42 . Monomers and oligomers were respectively inked onto PVDF membranes, Aβ _1-42 was 3 μg, α-Syn was 4 μg, and Tau was 7 μg. The PVDF membrane was purified with guinea pig anti-α-Syn antibody (1: 1000 dilution) as a primary antibody, and then hybridized with an anti-guinea pig HRP-conjugated secondary antibody (1: 5000; Vector Laboratories). The PVDF membrane was treated with a Luminata Western HRP substrate (Bio-Rad, Hercules, CA, USA), and the generated signal was detected with a ChemiDoc-It 810 digital imaging system (UVP Inc., Upland, CA, USA).

j. Specific α-Syn binding specificity in PC12 cells overexpressing α-Syn after treatment with nerve growth factor (NGF)

The anti-α-Syn antibodies of guinea pigs collected at 8 or 9 WPI were subjected to immunocytochemistry with parental PC12 cells treated with NGF, mock-controlled PC12 cells, and PC12 cells overexpressing α-Syn ( ICC) analysis to assess antibody affinity force. Nuclei were counterstained with DAPI (4 ', 6-diamidino-2-phenylindole). Photographs were taken with a fluorescence microscope, and the number of positively stained cells and the total number of cells were expressed as "<1%", "1-15%" with "-", "+", "++", and "+++", respectively. , "16-50%", "> 50%".

Example 4. Cells and animals used in immunogenicity and effectiveness tests

a. PC12 cells overexpressing α-Syn:

A cDNA encoding a full-length wild-type α-Syn or A53T mutant α-Syn was inserted into a pZD / XOL-L vector to construct a pZD / XOL-L-α-Syn plastid. According to the instruction manual, Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect plastids into PC12 cells. 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 reacted at room temperature for 25 minutes. After the medium was replaced with 1.5 mL of RMPI 1640 growth medium, 500 μL of the transfection mixture was directly added to each well, and reacted at 37 ° C. for 1 day. The transfection efficiency was confirmed by PCR and Western blot method.

b. Guinea pigs:

Immunogenicity tests were performed using Duncan-Hartley guinea pigs (300-350 g / BW) in mature, natural, adult males and females. There are at least 3 guinea pigs in each group. Involving Duncan-Hartley Guinea pigs (8-12 weeks old; Covance Research Laboratories, Denver, PA, USA) were tested and approved by IACUC for animal facilities and UBI.

c. Parkinson's mouse model inoculated with α-Syn fibers:

FVB female mice (body weight 25-30 g) were kept in a 12-hour light, 12-hour dark cycle, and were reared according to the instructions of AAALAC. The α-Syn peptide (5 mg / mL) was shaken at 37 ° C. in PBS / high KCl buffer containing 0.1% NaN ₃ for 7 days to prepare α-Syn fibers. Fibrosis was monitored by measuring ThT fluorescence, and fibrosis was confirmed when the signal exceeded the original α-Syn monomer by three times. Unilateral substantia nigra (anterior-posterior: -3.0mm; medial-lateral: -1.3mm; dorsal-abdominal: away from the acupoint and dura mater -4.7mm) and dorsal striatum in isoflurane anesthetized animals Aggregation of α-Syn was confirmed in front of the body (anterior-rear: + 0.2mm; medial-lateral: -2mm; dorsal-abdominal: acupoint and dura mater -3.2mm) using Western blot method.

d. MPP + -induced Parkinson's disease mouse model:

Balb / c female mice (18-20 g in weight) were maintained in a 12-hour light and 12-hour dark cycle and were reared according to AAALAC guidelines. MPP + iodine (Sigma, St. Luis, MO) was dissolved in physiological saline, and 10 μl of a solution containing 18 μg of MPP + iodine (0.8 mg / kg) was injected into the unilateral ventricle of the anesthetized animal. The three-dimensional coordinate of the injection site is the front pit -1.0mm, the side is 1.0mm, and the depth is 2.0mm.

Example 5. Pharmaceutical group containing multiple components of the α-Syn immunogen structure Design principles, screening, identification and optimization of compounds

a. Design History

Each α-Syn peptide immunogen structure or immunotherapy product has its own design focus and method based on specific disease mechanisms and target proteins. The target of the model design may include cellular proteins of the disease pathway or multiple protein infectious agents which may be involved from pathogens. The process from R & D to commercialization is very long and usually takes a dozen or decades to complete.

Once the target molecule is selected, extensive serological testing is required. The identification and distribution of B-cell and T-cell epitopes within the target molecule is important for the design of the α-Syn peptide immunogen structure. Once the target B-cell epitope is identified, serial lead immunogenicity studies in small animals can be performed to assess the functional characteristics of antibodies raised by the designed peptide drug composition. All studies were performed in multiple parallel groups, and serum was collected from immunized hosts for analysis. In the case of human pharmaceutical compositions, early immunogenicity studies of target species or non-human primates are conducted to further verify immunogenicity and design direction. The target peptides are then prepared from various mixtures, and when used in combination to prepare each formulation design, the subtle differences in the functional characteristics of the interactions between the peptide structures are evaluated. After further evaluation, the final peptide structure, the peptide composition and its preparation, and the physical parameters of the preparation are determined, which is the final product development process.

b. Design and verification of α-Syn-derived peptide immunogen structure for pharmaceutical composition, which can be used to treat patients with synucleinopathy

In order to generate the most effective peptide structure suitable for incorporation into pharmaceutical compositions, a large number of promiscuous T helper cells derived from various pathogens were further designed from the measles virus fusion (MVF) protein sequence or hepatitis B surface antigen (HBsAg) protein. The epitope or artificial helper T helper epitope was used for guinea pig immunogenicity test. Α-Syn _126-140 , α-Syn _121-140 , α-Syn _111-140 , α-Syn _101-140 , α-Syn _91-140 , shown in Table 3 (SEQ ID NOs: 99 to 121) 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 Yansheng Sheng peptide structure is connected through a variety of mixed εK and / or linker KKK T helper epitopes bits, for a representative experiment.

i) Selecting the C-terminal portion of α-Syn as a target for peptide immunogen design

α-Syn is an inherently abnormal protein. It consists of 140 amino acids and is divided into three regions. The N-terminal region (residues 1-60) is capable of forming a bipolar helical structure, which is a typical conformation of membrane characteristics and combinations. The central region containing residues 61-95 is known as the non-amyloid beta portion (NAC), which was first found in AD senile plaques. This region is highly inclined to form a conformation rich in β structure and has a high degree of aggregation. Different types of post-translational modifications in this region show different effects on regulating α-Syn aggregation. The C-terminal region with residues 96-140 is rich in proline and negatively charged residues, which is a common feature of inherently disordered proteins to maintain solubility. Because the C-terminal domain has low hydrophobicity and high net negative charge, it exhibits an irregularly curled structure. In vitro experiments have shown that α-Syn aggregation can be induced by lowering the pH and neutralizing negative charges. α-Syn has extreme conformational diversity, which corresponds to different conditions of membrane binding, cytoplasm, and amyloid aggregation and has multiple functions. After careful consideration, the non-selective curl and inherent disordered regions of the C-terminus are important for maintaining protein solubility, and were selected as targets for peptide immunogen design because this region is most susceptible to regulation by antibodies or other physical factors, and Non-N-terminal bipolar helix and central beta-rich region.

ii) Exclude the α-Syn B epitope and identify the auto-Th epitope

Preliminary immunogenicity analysis confirmed the presence of helper T cell epitope structural features at the C-terminus of α-Syn, α-Syn _126-140 (SEQ ID NO: 9), α- Syn _121-140 (SEQ ID NO: 8), α-Syn _111-140 (SEQ ID NO: 7) peptides are completely non-immunogenic, while α-Syn _101-140 (SEQ ID NO: 6), α- Syn _91-140 (SEQ ID NO: 5) and α-Syn _85-140 (SEQ ID NO: 4) peptides (Table 4) have a possible auto Th structure in the C-terminal sequence, which has a certain degree of Immunogenicity. This sequence is included in the B epitope. When boosting immunity, the activation of autologous T cells may induce brain inflammation, such as AN1792 Alzheimer's vaccine. Therefore, the present invention designs an α-Syn peptide immunogen structure having a B-cell epitope starting from a G111 amino acid residue.

iii) Arrange heterologous T helper epitopes and their inclusion in α-Syn Design in peptide immunogen structure restores and enhances the immunogenicity of selected α-Syn B epitope peptides

Table 2 lists a total of 29 heterologous Th epitopes (SEQ ID NOs: 70-98). Their relative properties have been tested in mice, rats, guinea pigs, baboons, macaques and other species, and they have been found to enhance B Cell epitope immunogenicity. As shown in Table 5, UBITh1 (SEQ ID NO: 83) and UBITh2 (SEQ ID NO: 84) T cell epitopes derived from the MvF protein can separate non-immunogenic α-Syn _101-140 (SEQ ID NO: 6) The peptide transition has strong to moderate immunogenicity. In an ELISA test, a long α-Syn peptide A91-A140 (SEQ ID NO: 5) was used to coat a 96-well plate, and it was found that when UBITh3 (SEQ ID NO: 81) was covalently linked to various C-terminal The α-Syn peptide (SEQ ID NOs: 4 to 9) has similar immune enhancing properties as shown in Table 6.

iv) Immunogenicity assessment of the C-terminal α-Syn peptide immunogen structure and its corresponding α-Syn and β-Syn antibody reactivity

The synuclein family includes three known proteins: α-Syn, β-Syn and γ-synuclein. All synucleins share a highly conserved alpha-helical lipid-binding region, which is similar to the A2 class lipid-binding domain of the exchangeable apolipoprotein. β-Syn is highly homologous to α-Syn. β-Syn is suggested as an inhibitor of α-Syn aggregation, which occurs in neurodegenerative diseases such as Parkinson's disease. Therefore, β-Syn can protect the central nervous system from the neurotoxic effects of α-Syn. Therefore, the resistance induced by the α-Syn peptide immunogen structure is preferred. Body, reacts with α-synuclein, rather than the corresponding aggregate-protective β-Syn. The six peptide immunogen structures were tested, all of which ended with C-terminal A140. All antibodies of these structural immune sera had significant cross-reactivity with the corresponding size of β-Syn, as shown in Table 6. When carefully studying the sequence homology between α-Syn and β-Syn (SEQ ID NOs: 1 and 2), it was found that the sequences corresponding to the 5 amino acids YEPEA at the C-terminus are the same between the two proteins of. Therefore, the present invention is designed to not contain the B epitope of this "YEPEA" 5 amino acid sequence.

A careful study of the sequence homology between α-Syn and β-Syn (SEQ ID NOs: 1 and 2) revealed that the five amino acid "YEPEA" sequences corresponding to the C-terminus were identical between the two proteins . Therefore, the design of the B epitope does not contain this "YEPEA" amino acid sequence. Based on the findings of the immunological study, the design of the B epitope was deleted from the YEPEA (Y136 to A140) fragment, as shown in Table 6. On the merged linker sequence, for example, the artificial T helper peptide UBITh1 (SEQ ID NO: 83) is inserted into the α-Syn peptide immunogen structure (SEQ ID NOs: 107- 114), as shown in Table 7, when the long α-Syn peptide K97-A140 (SEQ ID NO: 110) was evaluated, all were highly immunogenic. Immune serum does not react with β-Syn. Therefore, from the results of Tables 6 and 7, it can be seen that the design of the B epitope of the immunogen structure is limited to G111 to D135 of α-Syn and fragments thereof.

v) The antibodies induced by the α-Syn peptide immunogen structure only react with β-sheet monomers, oligomers or fibrils, and do not react with α-helical monomers

Although the present invention rationally designs the α-Syn peptide immunogen, the antibodies produced by the design of the α-Syn peptide immunogen with the B epitope of the G111 to D135 sequence or a fragment thereof will be single with β-sheet α-Syn Oligomers, oligomers, or fibrils have specific responses but do not react with β-folded Aβ _1-42 or Tau1-441, so ideal candidates for α-Syn peptide immunogen structure, such as representative The structure of the α-Syn peptide immunogen (SEQ ID Nos: 112 and 113) is shown in FIG. 8.

vi) Use of α-Syn-derived peptides with different promiscuous T helper epitopes with broad MHC coverage

When designing pharmaceutical compositions for treating patients with different genetic backgrounds, it is important to design the largest populations that cover different genetic backgrounds. Therefore, the co-immunogenicity of the α-Syn-derived peptide immunogen structure to the composition was investigated. Since promiscuous T-helper epitopes derived from MVF or HBsAg provide the strongest immunogenicity, peptide peptide combinations containing helper T-epitopes were designed for research. Compared to a single peptide structure, a mixture of two peptide immunogen structures with the same B epitope can trigger a powerful immune response.

Example 6. The α-Syn peptide immunogen structure elicits an antibody response against the epitope of the target B cell only

All carrier proteins (e.g. keyhole limpet hemocyanin (KLH) or other carrier proteins such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins) that are known to enhance the immune response against phase B epitope Chemically conjugated B-cell antigen-determining peptides, and more than 90% of the antibodies stimulated in the host are directed against carrier proteins. And less than 10% of the antibodies are directed against the target B cell epitope. Therefore, the specificity of the α-Syn peptide immunogen construct of the present invention was evaluated. Eight α-Syn peptide immunogen structures (SEQ ID NOs: 107 to 114) containing B cell epitopes of different lengths were linked to a heterologous T cell epitope UBITh1 (SEQ ID NO: 83) to evaluate immunogenicity. UBITh1 (T-helper peptide used to enhance B epitope immunity) was spread on well plates, and guinea pig immune serum was used to test cross-reactivity with UBITh1 peptide. Compared with the high immunogenicity of these structures for the target B epitope, as shown in Tables 6 and 7, the most potent antibodies against the B epitope, most of the immune serum will not react with the UBITh1 peptide, such as Table 8 shows.

In summary, a simple immunogen design that links the target B-cell epitope with a specially selected T helper epitope can generate an immune response specific to the α-Syn B-cell epitope. For the design of the pharmaceutical composition, it can lead to a more specific immune response and higher safety. Therefore, the α-Syn peptide immunogen structure of the present invention is highly specific and very effective for its target.

Example 7. Prediction of epitopes for precise and specific analysis of various α -synuclein peptide immunogen structures using immune serum (9 WPI)

In an accurate epitope prediction study (Table 9), in order to determine antibody binding sites for specific residues in the C-terminal region of α-Syn, There are 52 overlapping 10-mers (SEQ ID Nos: 18-69), which cover the α-Syn amino acid sequence (K80-A140). The positive control group used (97-135, SEQ ID No: 10) and (111-132, SEQ ID No: 17) two longer peptides. These 10-mer peptides and two longer peptides were spread on a 96-well plate as solid-phase immunoadsorbants. The mixed guinea pig antisera and the sample dilution buffer were diluted 1: 100, added to a well plate coated with 2.0 μg / mL 10-mer peptide, and reacted at 37 ° C. for 1 hour. After the wells were washed with a buffer solution, horseradish peroxidase conjugated protein A / G was added and reacted for 30 minutes. After washing with PBS, the reaction was detected by an ELISA plate measuring instrument at 450 nm, and each sample was analyzed twice. The binding of the antiserum to the long alpha-Syn peptide of the B epitope immunogen structure represents maximum binding.

As shown in Table 9, the six α-Syn peptide immunogen structures were combined [(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)] obtained from 9 wpi guinea pig immune serum, For accurate epitope prediction. These 6 B epitope fragments of different lengths completely cover the 97-140 sequence of the C-terminal region of α-synuclein. ELISA results showed that all six immune sera had strong responses to the representative α-Syn long peptide (97-135, SEQ ID No: 10). The epitope prediction study for 10-mer showed that it can cover the area around AA114 to 125 (114-123, 115-124, 116-125 of SEQ ID NOs: 52, 53 and 54 peptides) The immunogenic epitope has a high immunogenic region at the C-terminus and is represented by peptide 131-140 (SEQ ID NO: 69). Interestingly, most antibodies stimulated by immune sera derived from the C-terminal α-Syn peptide immunogen structure can recognize epitopes in a non-linear conformation, except for those located in the α- In addition to the epitope of the C-terminus of Syn, it can also cross-react with β-Syn protein.

The epitope mapping was found to be less than expected, but was similar to the structure of the α-Syn peptide immunogen derived from α-Syn 111-132 (SEQ ID NO: 113) and α-Syn 126-135 (SEQ ID NO: 112). Antibodies are completely related. The structure of the α-Syn peptide immunogen is derived from the irregular coiled region of α-Syn, which is linked to the heterologous Th epitope, resulting in a conformational structure similar to the degenerate β-fold of α-Syn, and does not Cross-reacts with α-Syn of natural α-helix.

Example 8. Antibodies induced by α- SYN peptide immunogen structure and its preparation: the effects of anti-aggregation and deaggregation on recombinant alpha synuclein

Anti-α-Syn antibodies purified from recombinant sera of immunized guinea pigs were used to evaluate the role of the α-Syn peptide immunogen structure in the in vitro anti-aggregation test and disaggregation test.

a. Inhibition of α-Syn aggregation

The detection of thiofavin T was used to quantify the degree of change in α-Syn aggregation, and the anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogens were initially screened for anti-aggregation ability, as shown in Example 3. 100 μM recombinant α-Syn prepared in PBS was placed in 5 μM 40 μL PBS / KCl buffer (2.5 mM MgCl ₂ , 50 mM HEPES and 150 mM KCl in 1 × PBS, pH 7.4), and reacted in a 384-well plate for 6 days to Induced aggregation. Anti-α-Syn antibodies purified at different concentrations (0.05, 0.5, or 5 μg / mL) and purified from anti-sera from guinea pigs with different α-Syn peptide immunogen structures were collected from different time points and added to the reaction mixture for evaluation. Effect of inhibiting α-Syn aggregation. After the reaction was completed, the degree of aggregation was analyzed using ThT. The degree of aggregation in the vehicle control was set to 100%, and the value obtained without α-Syn was 0%, and the results of each test were standardized.

As shown in Table 10, via the α-Syn 9 WPI collected above and _111-132, α-Syn _121-135 or 3 types of anti-α-Syn antibody α-Syn _126-135 induced aggregation for more α-Syn Has effect and concentration-dependent inhibition. Among all anti-α-Syn antibodies, 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) (collected at 9WPI), compared to the vehicle control (100%), it has about 40% inhibition of α-Syn aggregates (Figure 1).

b. Deaggregation of pre-formed α- Syn aggregates

From the above test, the anti-α-Syn antibody purified from the guinea pig antiserum immunized with the α-Syn peptide immunogen has the effect of inhibiting the aggregation of α-Syn. To further evaluate whether antibodies raised by the α-Syn peptide immunogen structure remained active in the disaggregated pre-formed α-Syn aggregates, anti-α-Syn purified from guinea pig antisera was used The antibodies were subjected to an in vitro disaggregation test.

Α-Syn was aggregated in 200 μL of 5 μM PBS / KCl buffer for 3 days. After centrifugation (13,000xg, 4 ° C, 30 minutes), α-Syn aggregates were collected and confirmed by ThT analysis. The pre-formed α-Syn aggregates were reacted in 100 μL of PBS / KCl buffer for 3 days. This PBS / KCl buffer contained or did not contain anti-α-Syn antibody purified from guinea pig antisera (5 μg / mL). After the reaction, aggregates were collected by centrifugation at 13,000 xg for 30 minutes at 4 ° C, and ThT quantitative analysis was performed as described in Example 3. Residual α-Syn aggregates after spontaneous decomposition in the vehicle control group were normalized to 100%.

Against 2 anti-α-Syn antibodies _elicited by α-Syn _111-152 (SEQ ID NO: 113) or α-Syn _126-135 (SEQ ID NO: 112), and α-Syn _111-132 (SEQ The anti-α-Syn antibody composition induced by ID NO: 113) and α-Syn _126-135 (SEQ ID NO: 112) was subjected to an in vitro depolymerization test. From the results, it can be seen that the anti-α-Syn antibodies elicited by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) were compared with the vehicle control group (100%). The de-aggregation effect on pre-formed α-Syn aggregates is about 50%, while other anti-α-Syn antibodies and purified antibodies in pre-immune animals do not have this effect (Figure 2).

Example 9. Anti-aggregation and deaggregation effects of α- SYN peptide immunogen structure and its preparation-induced antibodies: α- SYN overexpresses α- SYN aggregation kinetics in cells

Alpha-Syn aggregation is known to accelerate during neuronal differentiation. In order to evaluate the effect of α-Syn peptide immunogen structure on the inhibition of α-Syn aggregation or isolation of pre-formed α-Syn aggregates under cell conditions, NGF-treated neurons were used to differentiate PC12 cells with over-expressed α-Syn to perform anti- Aggregation test and disaggregation test to evaluate anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures.

a. Inhibition of α-Syn aggregation

Α-Syn over-expressed PC12 cells were added to poly-D-lysine-coated 96-well plates. Nerve growth factor (NGF) (100ng / mL) and different α-Syn peptide immunogen structures were used ( 0 or 0.5 μg / mL) treated with anti-α-Syn antibodies purified from guinea pigs for 4 days to confirm anti-aggregation activity.

The treated cells were lysed, 20 μg of cell lysate was separated by SDS-PAGE, and analyzed using α-Syn antibody (BD). The α-Syn signal detected in the high molecular weight region was quantified and normalized to a 100% vehicle control group. Referring to FIG. 3, compared with the amount of α-Syn aggregates in the vehicle control group, the ratio of α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), _Aggregation of 4 anti-α-Syn antibodies to α-Syn induced by α-Syn _123-135 (SEQ ID NO: 111) or α-Syn _126-135 (SEQ ID NO: 112) α-Syn peptide immunogen structure The inhibitory effect of the body is as high as 80-90%.

b. Deaggregation of pre-formed α-Syn aggregates

To demonstrate disaggregation on pre-formed α-Syn aggregates Set, PC12 cells overexpressed with α-Syn treated with NGF (100ng / mL) for 3 days, undergoing neuronal differentiation to cause aggregation of α-Syn, and then different α-Syn peptide immunogen structures (0 or 0.5 μg) / mL) treated with anti-α-Syn antibodies purified from guinea pigs for 4 days.

The treated cells were lysed, 20 μg of cell lysate was separated by SDS-PAGE, and analyzed using α-Syn antibody (BD). The α-Syn signal detected in the high molecular weight region was quantified and normalized to a 100% vehicle control group. Referring to FIG. 3, compared to the amount of α-Syn aggregates in the vehicle control group, the ratio of α-Syn _121-135 (SEQ ID NO: 107), α-Syn _123-135 (SEQ ID O: 111), or α-Syn _126-135 (SEQ ID NO: 112) The inhibitory effect of anti-α-Syn antibodies on α-Syn aggregates induced by α-Syn peptide immunogen structure is 50-60%, while α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibodies can reduce the amount of aggregated α-Syn by more than 90%.

Example 10. Antibodies Induced by the Structure of α- SYN Peptide Immunogen and Its Preparation: Effects on Reducing TNF- α and IL-6 Secretion

It is believed that damage to substantia nigra neurons releases aggregated α-Syn into substantia nigra, activates microglia to produce inflammatory mediators, and results in continuous and progressive substantia nigra degeneration in PD. In order to evaluate the effect of anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogen structures on reducing microglial activation, in the presence or absence of different anti-α-Syn antibodies, -Syn Ag aggregate-treated microglia releases inflammatory mediator TNF-α (tumor Amount of necrosis factor alpha) and IL-6 (interleukin-6).

Mouse BV2 cells or human SVG p12 cells were added to RPMI 1640 medium containing 1% FBS in an amount of 5,000 cells / well. The cells were treated with 1 μM α-Syn and treated at 37 ° C., 5% CO ₂ , and humid environment for 24 hours. After that, the culture medium was collected, centrifuged, and the supernatant was separated. The mouse IL-6 or human TNF-α mouse ELISA kit (Thermofisher) was used to analyze the concentration of IL-6 secreted by BV2 cells and the TNF-α secreted by SVG p12 cells in the supernatant, and this was repeated 3 times. Normalize the signal so that the vehicle control group is set to 100%.

Test results showed that the _{α-Syn 111-132 (SEQ ID NO} : 113): the induced antibody anti-α-Syn, can be reduced by 30 to 50% α-Syn and _{α-Syn 123-135 (111 SEQ ID} NO) Aggregate-induced TNF-α release from SVG p12 cells, and anti-α-Syn antibodies induced by α-Syn _123-135 (SEQ ID NO: 111) reduced IL-6 release from SVGP12 cells by approximately 30% ( (Figure 4). The results show that the anti-α-Syn antibodies induced by α-Syn _123-135 (SEQ ID NO: 111) can reduce the effects caused by α-Syn aggregates more effectively than other anti-α-Syn antibodies. Microglia activation.

Reduce the impact of exogenous [alpha] -synuclein-induced neurodegeneration of: 11. α -SYN peptide immunogen structure and formulation induced antibody Example

In order to evaluate the neuroprotective effect of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogens, An in vitro neurodegeneration model of PC12 cells differentiated with NGF-treated neurons with exogenous pre-formed α-Syn aggregates.

PC12 cells were treated with NGF (100ng / mL) for 6 days to induce neuronal differentiation. The morphology of neuronal differentiated cells was confirmed and analyzed using the InCell high-content image analysis system (GE Healthcare). Assess the growth of neurites that respond to neurotrophic effects and the number of neuronal differentiated cells. After normalization, the degree of neurite growth and the number of neuronal differentiated cells is shown as a percentage (mean ± SEM). The neurite lengths of PC12 cells treated with and without NGF were set to 100% and 0%, respectively.

Neuron-differentiated PC12 cells were added with exogenous pre-formed α-Syn aggregates to observe neurodegeneration. In the presence of pre-formed α-Syn aggregates, in neuron-differentiated PC12 cells, the length of neurites was shortened and the number of cells was reduced. This α-Syn aggregate-driven neurodegeneration is proportional to the amount of exogenous α-Syn aggregates added, and can be blocked by curcumin in a concentration-dependent manner, which is known to resist α-Syn aggregates The neurotoxicity has neuroprotective effects. Commercially available anti-α-Syn antibodies (BD bioscience), not antibodies purified by guinea pigs, can alleviate neurodegenerative lesions caused by α-Syn aggregates. This model was used as a screening platform to identify anti-α-Syn antibodies with concentration-dependent restoration of neuroprotective effects in neurite growth and neuron survival (Tables 11 and 12).

It can be seen from the results that more than half of the anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures restored the growth of neurites in a concentration-dependent manner (Table 11), and almost all anti- -α-Syn antibody protects neuron-differentiated PC12 cells from neuronal death triggered by α-Syn aggregates (Table 12). Putting two different parameters together, it was found that nearly one-third of anti-α-Syn antibodies can affect neurite length and cell survival against neurotoxicity of α-Syn aggregates. To observe and quantify _{α-Syn 111-132 (SEQ ID NO} : 113), α-Syn 126-135 (SEQ ID NO: 112) -α-Syn preimmune anti-antibodies and the guinea pig induced antibodies, neurite length for And the effect of cell number calibration using calcein AM (Life Technologies), a fluorescent living cell marker dye. Shown in PC12 cells differentiated by neurite-rich neurons, α-Syn _111-132 (SEQ ID NO: 113) (Fig. 5B) and α-Syn _126-135 (SEQ ID NO: 112) ( Fig. 5C) The induced antibodies have a protective effect on the shortening of neurites caused by α-Syn aggregates, but the guinea pigs do not have pre-immune antibodies (Fig. 5A).

Example 12. Antibodies Induced by the Structure of α- SYN Peptide Immunogens and Their Preparations: Effects of Reduced Neurodegenerative Lesions in α- SYN Overexpressing Cells

To evaluate the neuroprotective effect of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogens, wild-type PC12 cells overexpressing α-Syn and A53T mutations overexpressing α-Syn PC12 cells serve as a model of neurodegenerative lesions in vitro.

After reacting with NGF, the blank control cells (transfected with cytoplasmic vector) produced long neurite extensions and increased the number of cells. This result is similar to the parent wild-type PC12 cells, but it is over-expressed compared to α-Syn wild-type PC12. Cells and A53T mutant PC12 cells with over-expression of α-Syn did not form neurite outgrowth or increase the number of cells, confirming that neuropathy accompanied by α-Syn aggregation after NGF treatment. Western blot methods and ThT analysis were used to determine cell lysates of wild-type PC12 cells overexpressed by NGF-treated α-Syn to understand their characteristics. Western blotting results show that after NGF treatment, the over-represented α-Syn in the cell lysate is an oligomer. ThT analysis results show that the over-represented α-Syn in the cell lysate has a β-sheet structure (that is, enhanced ThT fluorescent signal). Compared with Western blot and ThT measurements of wild-type PC12 cells overexpressing α-Syn without NGF treatment, the overexpression of α-Syn in the differentiation of neurons induced by NGF changed from α-helix to β- Folding, which in turn causes neuropathy of the β-sheet α-Syn oligomer.

In addition, compared to wild-type α-Syn over-expressed PC12 cells, α-Syn over-expressing A53T mutations will lead to a stronger neurodegenerative response, reflecting a reduction in the length of neurites and a decrease in the number of cells treated with NGF. It was shown that A53T mutant α-Syn caused stronger neurodegenerative effects in wild-type α-Syn than in PC12 cells overexpressed by α-Syn.

From 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 IID NO: 112) induced anti-α-Syn antibody, and α-Syn _111-132 (SEQ ID NO: 113) and α-Syn _{126 -135} (SEQ IID NO: 112) -induced anti-α-Syn antibody composition can be measured using an in vitro neurodegeneration model of wild-type PC12 cells overexpressing α-Syn to assess the protective effect of antibodies on neurodegeneration in individuals . α-Syn over-expressed wild-type PC12 cells treated with NGF for 3 days caused neuronal differentiation, and then reacted with anti-α-Syn antibody (final concentration of 5 μg / mL) and NGF for 3 days. After the reaction, the cells were observed under a microscope. Compared with the vehicle control group, it showed that the reaction with the selected anti-α-Syn antibody could restore the neurite length and increase the number of cells. The detection value of parental PC12 cells treated with NGF for 6 days was standardized to 100% to quantify neurite length and cell number. From the results, compared with the vector control group, α-Syn _101-132 (SEQ ID NO: 114), α-Syn _111-132 (SEQ ID NO: 113), or α-Syn _123-135 (SEQ ID NO: 111) -induced anti-α-Syn antibodies, and anti-α- induced by α-Syn _111-132 (SEQ ID NO: 113) and α-Syn _126-135 (SEQ ID NO: 112) The Syn antibody composition has a significantly larger number of cells, while α-Syn _101-132 (SEQ ID NO: 114), α-Syn _111-132 (SEQ ID NO: 113), α-Syn _123-135 (SEQ ID NO: 111) or α-Syn _126-135 (SEQ ID NO: 112) induced anti-α-Syn antibody, and α-Syn _111-132 (SEQ ID NO: 115) and α-Syn _126-135 (SEQ ID NO: 114) The induced anti-α-Syn antibody composition has significantly longer neurites (Figures 6A and 6B).

Example 13. Antibodies induced by α- SYN peptide immunogen structure and its preparation: specificity for β -sheet oligomers and fibrous α- synuclein

In order to analyze the specificity of anti-α-Syn antibodies purified from anti-sera from guinea pigs with different α-Syn peptide immunogen structures, specific α-Syn molecular complexes of different sizes, including α-Syn, Aβ, and tau proteins, etc. A series of in vitro experiments were performed with different amyloids and α-Syn aggregated in PC12 cells overexpressed by α-Syn after NGF treatment.

a. Specificity for larger α- Syn molecular complexes

Western blotting tests were performed on α-Syn molecular complexes of different sizes using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures as primary antibodies. The results show that except for the smaller α-Syn monomers, all anti-α-Syn antibodies are complexed with larger α-Syn molecules, including dimers, trimers, tetramers, and oligomers. Strong response. Compared with commercially available anti-α-Syn antibodies, Syn211 (Abcam), α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), α-Syn _123- Anti-α-Syn antibodies induced by ₁₃₅ (SEQ ID NO: 111) and α-Syn _126-135 (SEQ ID NO: 112) have higher and larger α-Syn molecular complexes (including dimers, The signal ratios of polymers, tetramers, and oligomers) to smaller α-Syn monomers (Figures 7A and 7B) indicate that anti-α-Syn is specific for larger α-Syn molecular complexes.

b. Specificity of α- Syn in different amyloids

As described in Example 3, different amyloids (ie α-Syn, Aβ1-42) of different species (ie α-helical monomers, β-fold monomers, β-fold oligomers, and β-fold fibers) were prepared. And Tau441), using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures as primary antibodies, and performing the round staining method. The results show that anti-α-Syn antibodies elicited by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) specifically bind to all β-sheets (monomers). , Oligomers, and fibrous species) instead of α-Syn monomers (Figures 8A, 8B, and 8C). In addition, anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) are associated with fibrous β-sheet α-Syn and β- The reaction of folded α-Syn oligomers is stronger than the reaction with β-folded α-Syn monomers. In contrast, anti-α-Syn antibodies _elicited by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) do not differ from β-Syn or different species Reactions of amyloid-producing proteins Aβ _1-42 and Tau441 (α-helical monomers, β-fold monomers, β-fold oligomers, and β-fold fibers) (Figures 8A, 8B, and 8C). These findings show that anti-α-Syn antibodies against β-sheet monomers, β-sheet oligos induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) Polymers and β-sheet fibrous α-Syn are specific.

c. α- Syn treated with NGF overexpresses the binding specificity of α- Syn aggregated in PC12 cells

Anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogens were overexpressed in parental PC12 cells, blank control PC12 cells, α-Syn and wild-type PC12 cells and α-Syn. An A53T mutant PC12 cell was subjected to an immunocytochemical test (ICC) to evaluate the binding affinity of the antibody to aggregated α-Syn after NGF treatment, as described in Example 3. The quantitative results shown in FIG. 9, the _{α-Syn 111-132 (SEQ ID NO} : 113), α-Syn 121-135 (SEQ ID NO: 107) , or _{α-Syn 126-135 (SEQ ID NO} : 112) Induced anti-α-Syn antibodies have higher levels of α-Syn overexpressing wild-type PC12 cells and α-Syn overexpressing A53T mutant PC12 cells than parental PC12 cells or NGF-treated blank control PC12 cells. Greater reactivity. Since NGF treatment can induce over-expressing α-Syn aggregates, it was found that α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), or α-Syn _126-135 (SEQ ID NO: 112) The elicited anti-α-Syn antibody is specific for α-Syn aggregated in NGF-treated α-Syn overexpressing wild-type PC12 cells and α-Syn overexpressing A53T mutant PC12 cells.

Example 14. Immunohistochemical staining of the brain of Parkinson's patients to evaluate the tissue specificity of the α- SYN peptide immunogen structure and its formulation

Anti-α-Syn antibody _elicited with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) before immunization, and the two antibodies were mixed in a 1: 1 ratio The anti-α-Syn antibody combination is used to perform immunohistopathological tests in normal human tissues to monitor specific and adverse antibody self-response. Before administering an antibody to a guinea pig immunized with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) and a composition in which the two antibodies are mixed in a ratio of 1: 1, Human tissue sections (Pantomics) were deparaffinized with xylene, dehydrated in ethanol, then treated with 0.25% trypsin solution and 0.5% CaCl ₂ in PBS for 30 minutes, and reacted in a methanol solution containing 1% hydrogen peroxide to prevent Endogenous peroxidase activity was cut off, and then reacted with 10% Block Ace (Sigma) in PBS. Tissue sections were stained with 3-3'-diaminobenzidine (DAB), counterstained with hematoxylin, and observed under a microscope. Compared with the positive response of commercially available anti-α-Syn antibody (BD, 610708), guinea pigs were immunized with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) The composition of the antibody and the 1: 1 ratio of the two antibodies was negative to normal human tissues. This result is consistent with the results of guinea pig pre-immune antibodies (Figure 10A).

Pre-immune, α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibody and a 1: 1 mixture of the two antibodies Composition to test its reactivity with human Parkinson's disease brain tissue. Tissue sections from three regions (ie, cerebellum, corpus callosum, and thalamus) (BioChain) were measured. From the results, compared with negative reactions in healthy brain sections, anti-α-Syn induced by α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) The combination of the antibody and the 1: 1 ratio of the two antibodies was positive for all three regions of the PD brain (Figures 10B and 10C). Positive staining was counted under a microscope to quantify the reactivity to α-Syn aggregates in PD brain sections. Compared to healthy brain sections, α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibodies and a 1: 1 ratio 2 A combination of these antibodies had a significant positive response to PD brain sections. Of the three different anti-α-Syn antibodies tested, antibodies induced by α-Syn _111-132 (SEQ ID NO: 113) had the strongest immune response to α-Syn aggregates in PD brain sections.

Example 15. Proof of the efficacy of α- SYN peptide immunogen structure and its formulation in animal models

a. Immune and blood / brain tissue collection

For the establishment of a mouse model of Parkinson's disease (PD), refer to Example 4. Two weeks after MPP + injection or 7 weeks after α-Syn fiber inoculation, mice were randomly divided into three groups, including UBITh1-α-Syn _111-132 (SEQ ID NO: 113) peptide, UBITh1-α-Syn _{126 -135} (SEQ ID NO: 112) peptide, a combination of two peptides, and an adjuvant group (adjuvants and solvents used in the composition for immunization (ISA 51 VG, CpG3, 0.2% TWEEN®-80) ). Intramuscularly (IM) immunized 3 times, 3 weeks apart, a dose of 40 μ g. The injection and blood collection schedule is shown in Table 13.

At each time point, the extraction face 200 μ L of blood through venous sampling. Blood dripped from the submandibular vein was collected into a centrifuge tube and centrifuged at 300 rpm for 10 minutes to prepare serum. After the animals were sacrificed, brain tissue samples were collected for Western blot analysis.

b. Inoculation with α-Syn _111-132 (SEQ ID NO: 113) and / or α-Syn _126-135 (SEQ ID NO: 112) PD mouse model response of peptide immunogen structure

The mixed serum samples of each experimental group were diluted with PBST containing 1% BSA, and then added to an ELISA disk (Cloud-clone) coated with 200 μL of full-length α-Syn peptide and containing 0.1 M sodium bicarbonate (α-Syn concentration 4.4 μg / μL, pH 9.6). After reacting at room temperature for 2 hours, washing with PBST three times, adding 100 μL of HRP conjugated anti-mouse IgG antibody diluted with 1% BSA at a ratio of 1: 3000, and reacting for 2 hours at room temperature. Next, it was washed 3 times with PBST, and reacted with 100 μL of 3,3,5,5-tetramethylbenzidine (TMB) in the dark for 10 minutes. Add 100 μL of 2M H ₂ SO ₄ , react for 15 to 30 minutes, and measure the absorbance (OD) at 450 nm using SpectraMax i3x Multi-Mode Detection (Molecular Devices).

After the second immunization, _{α-Syn 111-132 (SEQ ID NO} : 113), α-Syn 126-135 (SEQ ID NO: 112) or a combination of two structures immunogenic peptides immunity induced by MPP + model ( (Fig. 11A) or fibrous α-Syn-model (Fig. 11B) The absorbance (OD) of the anti-α-Syn antibody produced is greater than 3.0 and remains until the end of the 15wpi or 19wpi experiment, and the adjuvant is injected Animals cannot detect an anti-α-Syn immune response.

It should be noted that in the α-Syn fiber model, the α-Syn _111-132 structure can induce a stronger immune response than the α-Syn _126-135 structure (Figure 11B), but it is not available in the MPP + model. Difference in originality (Figure 11A).

c. Decreasing the amount of α-Syn in serum

The amount of α-Syn in the serum of each experimental group was determined using an ELISA reagent kit (SEB222Mu, USCN) capable of detecting both α-helix and β-fold α-Syn described in Example 3.

The α-Syn quantitative ELISA analysis was used to determine whether the anti-α-Syn antibody response of the immunized group was related to a decrease in the amount of peripheral α-Syn compared to the non-immunized animals. The results showed that compared with animals injected with adjuvant, the MPP + induction model was immunized with α-Syn _126-135 (SEQ ID NO: 112), α-Syn _111-132 (SEQ ID NO: 113), or a combination of these two structures. (Figure 12A) or the fibrous α-Syn model (Figure 12B) can reduce the absorbance (OD) of α-Syn.

The results show that the structure of the α-Syn peptide immunogen can produce anti-α-Syn antibody response, and the amount of α-Syn in the peripheral circulation system is correspondingly reduced.

d. Reduced α-Syn oligomers in the brain

After the animals were sacrificed, brain tissue samples were collected for Western blot analysis. For MPP + -induced mice, the brain was removed and homogenized; for fibrous mice inoculated with α-Syn, the striatum and substantia nigra regions were first separated and homogenized. Lysis buffer (Amresco) and 1x protease inhibitor (Roche) were added to the homogenate to prepare a brain tissue lysate. 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel Electrophoresis) The lysate was separated, transfected onto a PVDF membrane, and reacted with 5% milk in PBS overnight. In order to detect the number of dopamine neurons, PVDF membrane was reacted with anti-tyrosine hydroxylase antibody (1: 1000 dilution, Abcam), and then reacted with goat anti-rabbit IgG (H + L) HRP conjugated secondary antibody (1: 5000 dilution, Jackson Immunoresearch) hybridization. To visualize the data, the Luminata Western HRP matrix was used, and the experimental results were processed using the ChemiDoc-It810 digital imaging system. The quantification of the α-Syn oligomer was normalized to the amount of GAPDH, and the lysate ratio of the undamaged tissue was set to 100% for comparison.

In the MPP ⁺ mouse model, α-Syn oligomers were shown to be reduced in animals immunized with α-Syn _111-132 peptide immunogen structurally (Figure 13A). Similarly, Western blot analysis was performed on lysates of nigra and ipsilateral striatum in mice inoculated with α-Syn fibers (Figures 14A and 14D), and Western blot analysis was performed on the contralateral striatum. analysis (of FIG. 14F), the results are shown by _{α-Syn 111-132 (SEQ ID NO} : 113) and _{α-Syn 126-135 (sEQ ID NO} : 112) after the configuration process, the adjuvant control mice α- The amount of Syn is increased by 2 to 3 times. The quantification of Western blot analysis results is shown in Figures 13B, 14B, 14C, 14D and 14G.

e. Reduction of neuropathology

The substantia nigra area of mice injected with α-Syn fibers was isolated and homogenized. Lysis buffer (Amresco) and 1x protease inhibitor (Roche) were added to the homogenate to prepare a tissue lysate. The lysate was separated by 10% SDS-PAGE (sodium lauryl sulfate-polyacrylamide gel electrophoresis), transfected onto a PVDF membrane, and reacted with 5% milk in PBS Overnight. In order to detect the amount of dopamine neurons, the PVDF membrane was reacted with anti-tyrosine hydroxylase antibody (1: 1000 dilution, Abcam), and then reacted with goat anti-rabbit IgG (H + L) HRP conjugated secondary antibody (1: 5000 dilution, Jackson Immunoresearch) hybridization. To visualize the data, a Luminata Western HRP matrix was used and the detected values were received with a ChemiDoc-It810 digital imaging system. GAPDH was used to normalize the amount of α-Syn oligomers, and the ratio of lysed tissue lysate was set to 100% for comparison. To visualize the data, the Luminata Western HRP matrix was used, and the experimental results were processed using the ChemiDoc-It810 digital imaging system. The expression of α-Syn is based on the amount of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and GAPDH is also used as a protein loading control.

The results demonstrated that the amount of tyrosine hydroxylase was restored to a level equivalent to that of normal animals without damage by the immunization with the α-Syn _111-132 structure (Figures 14C-14D), indicating that the α-Syn peptide immunogen structure was suitable for vaccination. The neurotoxicity caused by the aggregation of α-Syn mice is neuroprotective.

f. Recovery of activity

CatWalk ^TM XT (Noldus information Technology, Wageningen, Netherlands) is an image analysis system that can dynamically measure the various conditions of the footsteps based on position, pressure, and the surface area of each footstep. Prior to the experiment, all mice were trained in a consistent manner and ran at least three times a day. A successful run was defined as an animal running without interruption or hesitation, and rats that failed training were excluded. The average of 5 runs per mouse was analyzed. Since α-Syn fiber is inoculated in the right brain, the standing time of the left hind foot during running can be used as a reference parameter.

In the α-Syn fiber inoculation model, after inoculation with a composition containing α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), the standing time of the left hind limb was found There are significant differences (Figure 15A). In addition, after treatment with α-Syn _111-132 (SEQ ID NO: 113) (Figures 15B and 15C), there was a significant difference in running time between the α-Syn fiber inoculation model and the MPP + induction model. The results show that in these two PD models, the processing and movement of the α-Syn peptide immunogen structure of α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) Functionality is related.

Example 16. Reactivity of antibodies produced by α-syn peptide immunogen structure with different types of α-syn in neurodegenerative diseases

Alpha-Syn causes Parkinson's disease and other synuclein diseases. α-Syn protein can form different types of aggregates with different sizes and structures, and has different effects on cells. Therefore, various diseases are caused by one or more different types of aggregates. Different shapes of α-Syn aggregates may cause different damage patterns in the brain, and even different brain diseases. This study evaluates how antibodies produced by the α-Syn peptide immunogen structure interact with different α-Syns in neurodegenerative diseases.

This trial is in collaboration with Dr. Ronald Melki. Produce a variety of different α-Syn aggregates in the laboratory, including (a) fibrous-a Long, twisted, hinged α-Syn protein; (b) ribbon-like, a broader, flatter structure, and (c) α-Syn oligomer (O550), dopamine stable (ODA), and glutaraldehyde Stable (OGA) oligomers.

The relative affinities of antibodies produced by various α-Syn peptide immunogen structures of the present invention in guinea pigs were tested. Will be derived from PD-021514 (α-Syn _85-140 , wpi 08), PD-021522 (α-Syn _85-140 , wpi 13), PD-100806 (α-Syn _126-135 , wpi 09), PRX002 Representative samples of a commercially available monoclonal antibody Syn1 (clone 42) and different α-Syn peptide aggregates (fibrous, ribbons, fibers 65, fibers 91, fibers 110, fiber aggregation pathways) Fiber oligomers (O550), dopamine stabilized (ODA) and glutaraldehyde stabilized (OGA) oligomers, and control monomers were analyzed by filter paper binding.

Materials and Methods

a. α-Syn aggregated into fiber and ribbon

For fiber formation, soluble WT α-Syn was placed in Buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl) and shaken continuously at 600 rpm at 37 ° C. In the presence of Thioflavin T (15 μM), agitate (100 rpm) with a magnet (6 × 3 mm) and observe the aggregation using a Cary Eclipse spectrophotometer (Varian Inc., Palo Alto, CA, USA), where the excitation wavelength is 440 nm, The emission wavelength is 440nm and 480nm, and the average time is 1s. For band formation, WT α-Syn was dialyzed at 1,000 times the volume of buffer B (5 mM Tris-HCl pH 7.5) at 4 ° C. for 16 hours, and continuously shaken at 600 rpm at 37 ° C. Observe aggregation by analyzing 440nm scattered light Happening. In addition, after sedimentation at 35,000xg, the amount of protein in the supernatant was detected using a 280 nm absorbance of a Hewlett Packard 8453 diode array spectrophotometer. A Jeol 1400 (Jeol Ltd.) TEM was used to adsorb the sample onto a 200-mesh grid coated with carbon, and negative staining was performed with 1% uranium acetate. Images were recorded using a Gatan Orius CCD camera (Gatan). The ability of α-Syn to bind Congo red was evaluated in the following manner: Fibrous and ribbon-shaped α-Syn and 20 μM Congo red (Sigma-Aldrich, St Louis, MO, USA) dissolved in Tris buffer (pH7.5) ) Reaction for 1 hour. The polymer was then centrifuged at 25,000 g for 30 minutes at 20 ° C using a TL100 desktop Beckman ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA). Wash the pellet 4 times with an equal volume of water. Immediately after resuspending the particles, place an aliquot of the sample on a glass coverslip and photograph or dry it. A Leica (MZ12.5) microscope with a cross polarizer (Leica Microsystems, Ltd., Heerbrugg, Switzerland) was used to observe the bright field and cross-polarized samples through a polarizing microscope.

b. Determination of fibrous and ribbon α-Syn concentration

Ultrasound cell pulverizer UIS250v (250W, 2.4kHz; Hielscher Ultrasonic, Teltow, Germany) with an amplitude of 75% and a pulse of 0.5 seconds. Ultrasonic crushing on ice for 20 minutes to reduce fibrous and band α -Syn length. The sedimentation rates of fibrous and banded α-Syn were measured. The Sedfit software was used to analyze the settlement interface with the least square boundary model ls-g * (s). Settlement coefficient The particle size distribution of banded α-Syn from 50S to 150S and the particle size distribution of fibrous α-Syn from 100S to 1,000S are based on fibrous and banded α-Syn with deposition coefficients of ~ 90S and 375S, respectively. Center, with a molecular weight of about 11,500 kDa, for example, about 800 band-shaped α-Syn molecules (12,000 kDa / 14.5 kDa); about 102,000 kDa, for example, about 7,000 fibrous α-Syn molecules (10, 2000, kDa / 14.5kDa). Therefore, at an operating concentration of 20 μM, all banded and fibrous α-Syns are 20 μM / ~ 800 = ~ 0.02 μM, 20 μM / ~ 7,000 = ~ 0.003 μM, and 100% α-Syn is formed in a stable state. Banded or fibrous, 100% of the protein is present in the pellet after centrifugation.

c. Evaluation of the affinity of inclusion bodies for different fibrous and banded α-Syn

Using filter paper binding analysis and using antibodies as a control group, the affinity of the antibodies produced by the α-Syn peptide immunogen structure of the present invention to different α-Syn aggregates was evaluated. α-Syn aggregates (fibrous, ribbon-like, fiber 65, fiber 91, fiber 110, fiber oligomer (O550) on fiber aggregation pathway, dopamine-stabilized (ODA) and glutaraldehyde-stabilized (OGA) oligomerization Has been disclosed 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. This example includes the use of the control monomer α-Syn.

A 20 pg-200 ng fibrous, oligo or monomeric α-Syn was transferred to a nitrocellulose membrane using a transfer filter tank. The nitrocellulose membrane was sealed with skim milk powder, and reacted with diluted PRX002, Syn1 antibody or GP antibody tested in this example. After sufficient washing, the binding characteristics of the primary antibodies were analyzed using secondary anti-human or anti-guinea pig IgG-HRP. The secondary antibodies of the control group were also analyzed. Super Signal ECL (Pierce # 34096) was added and developed with BioRad imager (Chemidoc MP imaging system / BioRad imagelab software). Exposure time and its dynamics are shown in Figures 16A-16H . In the experimental group, the brain homogenate of DLB patients was transferred to the membrane.

d. Results

Filter paper binding analysis was used to compare the antibodies PD-021514 (α-Syn _85-140 , wpi 08), PD-021522 (α-Syn _85-140 , wpi 13), PD-100806 ( _{Affinity of} α-Syn _126-135 , wpi 09), PRX002, and commercially available antibody Syn1 (clone 42) to different α-Syn aggregates. The α-Syn aggregates used include fibrous, ribbon, fiber 65, fiber 91, fiber 110, fiber oligomers on the fiber aggregation pathway (O550), dopamine stabilization (ODA) and glutaraldehyde stabilization (OGA) Oligomers, and control monomers.

Of FIG. 16A-16H show compared to monomeric α-Syn, a control antibody having a slightly PRX002 good affinity for α-Syn, but may recognize α-Syn PD-100806 peptide structure of the present invention for PD- _126-135 wins 021514 antibody has a high affinity for α-Syn fibrous, and the two have a good binding to fibrous α-Syn. Syn1 monoclonal antibodies can bind fibrous α-Syn as well as oligomers and monomer α-Syn without much difference from each other.

Example 17. Immunohistochemical Study of Antibodies Formed by Alpha-SYN Peptide Immunogen in the Brain of Patients with Parkinson's Disease (PD), Multiple System Degeneration (MSA), and Lewy Body Dementia (DLB)

Immunohistochemical studies were performed using antibodies obtained by immunizing guinea pigs with the representative α-Syn _126-135 peptide immunogen structure of the present invention to analyze their ability to bind to α-Syn in brain slices of patients with α-synuclein disease . This research is in collaboration with Professor Roxana Carare. The ability of antibodies to bind to α-Syn protein in brain slices from PD, LBD, and MSA patients was analyzed. Healthy tissue was used as the negative control group. A commercially available monoclonal antibody NCL-L-ASYN for autopsy was used as a positive control group. This experiment provides _evidence that α-Syn _126-135 peptide immunogen structural antibodies have positive immunoreactivity to brain tissue sections from patients with PD, LBD, and MSA. Binding was specifically seen in the brains of patients with synuclein disease, but was not found in the brains of non-patients, and the binding of test antibodies was more pronounced compared to commercially available diagnostic antibodies.

Materials and Methods

a. Reagents and their suppliers

Antibodies obtained from a representative α-Syn _126-135 peptide immunized guinea pig were diluted at a ratio of 1: 100. PD062220-09-1-2-Syn, PD062205-09-1-2-Syn, PD100806-09-1-2-Syn antibodies were derived from United Brain Scientific Corporation (UNS), NCL-L-ASYN antibody (mouse single Strain antibody diluted 1: 100) HuD (EI) from Leica Biosystems, Santa Cruz Biotechnology, (mouse monoclonal antibody diluted 1: 100), Olig2 antibody (rabbit antibody diluted 1: 100) from In Millipore, Alexa Flour 594 antibody (goat anti-guinea pig antibody diluted 1: 200), Alexa Flour 488 antibody (goat anti-guinea pig antibody diluted 1: 200) and Alexa Flour 488 antibody (goat anti-guinea pig antibody 1: 200 (Proportional dilution) from Molecular Probes life technologies.

b. Human brain tissue

This example uses a slice from the UCL brain bank with a thickness of μm. All samples were collected and prepared in a manner approved by the National Research Ethics Services Committee.

Tissue obtained from subjects with typical α-Syn pathology including multiple system degenerative disease (MSA; n = 3), Lewy body dementia (DLB; n = 3), and Parkinson's disease (PD; n = 3) (Table 15). Subjects were diagnosed according to published criteria **.

c. Immunohistochemical analysis of human synuclein disease subjects

Immunohistochemical (IHC) analysis was performed on patients with three different synuclein diseases (MSA, DLB, and PD) to quantify the specificity of the three UNS antibodies to α-Syn aggregates. The specificity of UNS antibodies (PD062220, PD062205, and PD100806) and commercially available diagnostic antibodies (NCL-L-ASYN) for α-Syn aggregates was compared. Analysis of antibody specificity in 4 brain regions of each patient, (1) Putamen, Internal Capsule, and Insula Cortex; (2) Midbrain: Substantia nigra; (3) Temporal Cortex: Cortical Grey Matter; and (4) Cerebellum: Subcortical White Matter; Cerebellar White Matter .

It is known that these brain regions are affected by α-Syn aggregation to varying degrees and disease progression stages of each disease type. Generally, in the early stages of DLB, PD, and MSA, the basal ganglia and midbrain are affected and have the highest cumulative burden. The temporal lobe cortex and cerebellum are affected late in the disease, and there are very few cerebellar aggregates in PD and DLB. A negative control group was used in accordance with each IHC program (primary antibody was not used) to confirm non-specific binding of the secondary antibody. The paraffin-embedded glass slides were dewaxed in an oven at 60 ° C for 15-20 minutes, and immersed in xylene I & II for 5 minutes each. Tissues were rehydrated in 4-fold dilutions of 100% to 50% IMS for 5 minutes each. The tissues were washed 3 times in 1xPBS for 5 minutes each, and then reacted in 100% formic acid for 3 minutes for antigen retrieval. After thoroughly washing the tissue with 1xPBS, it was reacted with 3% H ₂ O ₂ for 10 minutes to remove endogenous peroxidase activity. The tissues were frozen and further washed 3 times (5 minutes each) with 1xPBS, and microwaved with citrate buffer (15 mM trisodium citrate, TWEEN, pH 6) at medium temperature for 25 minutes. Microwave, use 3 containers and 3 slicing racks each time. Sections were frozen and washed 3 times (5 minutes each) with 1xPBS, blocking non-specific binding sites with 15% goat serum. Tissues reacted with primary antibodies (diluted with 0.1% TBS / t at a ratio of 1: 100) at 4 ° C overnight. Tissues were washed 3 times in 1xPBS for 5 minutes each and reacted with a secondary antibody with biotin for 1 hour at room temperature. 30 minutes before use, freshly prepare ABC solution. The tissue was washed 3 times with 1xPBS, and after 5 minutes each time, it was reacted with the ABC solution at room temperature for 1 hour. ImmPACTVIP peroxidase kit was used to prepare VIP peroxidase substrate according to the instructions. VIP peroxidase substrate was added for 7 minutes at room temperature, and washed with dH ₂ O. Before placing the tissue in DPX, dehydrate it in IMS 50%, 70%, 95%, 100%, 100% and xylene I & II, respectively. For dual immunofluorescence staining, the tissues were not treated with 3% H ₂ O ₂ before the primary antibody was administered. After the first primary antibody and the same secondary antibody were administered, the tissue was blocked with 15% goat serum for 30 minutes, and reacted with the second primary and secondary antibodies. After giving the final fluorescently labeled secondary antibody, the tissue was reacted with 1% Sudan Black for 5 minutes to remove autofluorescence, washed with 0.1% TBS / T, and immediately placed in mowiol cituflour. The fluorescently stained tissue was placed at 4 ° C until image photography was completed.

d. Image analysis and statistics

The Olympus VS110 high-throughput virtual microscopy system or Olympus scanning microscopy virtual microscopy system was used to scan the sections at x20 objective magnification for analysis. Use Olympus VS software to capture 30 images (500 μm ² each) of the same position of each subject in each area from the scanned images (17A-17D, 18A-18D, 19A-19B, 20A -20E, 21A-21F, 22A-22C, 24A-24D and 25A-25D). An area with a total area of 7.5 mm ² in each brain was analyzed. ImageJ version Fiji windows-64 software was used to quantify the α-Syn immunoreactivity of each image.

To analyze the total amount of α-Syn detected by each antibody, the percentage of the total area of the image was used as the immunoreactivity. In order to analyze the differences in background staining, each brain region was selected to adjust the threshold of α-Syn positive immunoreactivity. For the analysis of each antibody and brain region, the average percentage of α-Syn positive aggregate coverage area was calculated.

In order to analyze the relative specificity of each antibody to LBs or LNs, Fiji software was used to quantify the immunoreactivity of LBs based on parameters of size and roundness to distinguish them from LNs (refer to sections 24A-24D, 25A-25D, and 26A Figure -26B). Brain regions with different morphologies of LBs and LNs were selected for analysis to avoid false positives, and the brain regions included the basal nucleus of the insular cortex and the gray matter of the temporal cortex. LB immunoreactivity is expressed as a percentage of total α-Syn immunoreactivity.

Statistical analysis was performed using GraphPad Prism v7.01 software and expressed as mean ± SD (unless otherwise stated). Results were analyzed by one-way analysis of variance (ANOVA) and post hoc analys were analyzed by Dunnett's correction. When p <0.05 (*), it is considered to have a significant difference. The number (n) refers to the number of samples used in each experiment.

Qualitative analysis of α-Syn in neurons or glial cells was performed using dual immunofluorescence staining as previously described. The glass was observed using a Leica SP8 laser scanning conjugate focal microscope. Get maximum overlapping images under x40 series objectives. The image includes a series of 2 colors in the z-direction Color overlay the image to show its relative position.

e. Take α-Syn _126-135 Antibodies detect different states of α-Syn aggregates and compare with NCL-L-ASYN antibodies

The cell type and subcellular location of α-Syn aggregates varies between different synuclein diseases. MSA is characterized by glial cytoplasmic inclusions (GCI), while in DLB and PD, α-Syn aggregation occurs in neuronal cell bodies (called Lewy bodies (LB)) and axonal processes (called Lewy processes (LN) )Inside. Analysis of the percentage of stained areas quantifies the total α-Syn aggregates detected by each antibody. However, this does not take into account differences in aggregate types or subcellular locations. In the case of PD and DLB, the appearance of the α-Syn aggregates in the cell body and neuronal processes is different, which can make the UNS antibody of the present invention relatively sensitive to the quantification of these different types of α-Syn aggregates.

To investigate this phenomenon, in the case of DLB and PD, the proportion of aggregates detected by each antibody in the cell was analyzed. Using FIJI software, aggregates within cells were selected based on their size and roundness. The area average percentage of cell body aggregates was calculated as the total α-Syn, and the results are shown in Nos. 24A-24D and 25A-25D. According to the qualitative analysis of tissue, the difference between the percentage of α-Syn area in the whole and the cell body was related to the axon aggregates (LNs) of α-Syn. A decrease in the cell body α-Syn ratio reflects an increase in LN. Since the temporal lobe cortex and the insular cortex both show pathologies similar to LB and LN, analysis was performed in the temporal lobe cortex and insular cortex. LNs are very few and unevenly distributed on the putamen and inner capsule, so the basal nuclei were not selected for analysis. In the substantia nigra A similar correlation was observed (Figures 26A and 26B), and the UNS antibody of the present invention detected a higher amount of LN than the NCL-L-ASYN antibodies in DLB and PD. However, due to the complex morphology of LNs and LBs, they cannot be reliably distinguished and quantified in the same way.

The results of Figures 24A-24D show that the total amount of α-Syn detected by each antibody was lower than that of NCL-L-ASYN. This indicates that the ratio of inclusions to LNs in the body is reduced, and a higher proportion of LNs is detected with UNS antibodies. Among the UNS antibodies, the PD062205 antibody detected the high proportion of LNs in the insular cortex in DLB and PD (Figures 17A-17D and 18A-18D). In contrast, compared to NCL-L-ASYN, all α-Syn _126-135 antibodies detected a high proportion of cell-body aggregates in the gray matter of the temporal lobe of patients with DLB and PD (p. 25A-25B).

f. α-Syn aggregates are cell type specific

Containing α-Syn aggregates are characteristic pathogenic markers of synuclein diseases, including MSA, DLB and PD. And α-Syn aggregation is the main pathogenic protein in synuclein diseases. Aggregation and cell appearance will affect the formation of aggregation, and it varies between specific disease subtypes. The clinical features of MSA, DLB, and PD show that α-Syn aggregates in the intracellular and neritic processes of neurons in DLB and PD. Aggregation of α-Syn was found in oligodendrocytes.

To determine the selectivity of the α-Syn _126-135 antibody for cell-specific α-Syn aggregates, dual immunofluorescence staining was performed using PD062205 with a marker against neurons (HuD) or oligodendrocytes (Olig2).

Figures 27A-27C show that PD062205 detected α-Syn in neurons of the basal nucleus and midbrain (high pathological region) in PD and DLB, but not MSA. Figures 28A-28C show that using oligodendrocyte (Olig2) labeling, α-Syn was found to aggregate in glial cells in MSA, but not in PD or DL. This result demonstrates that the α-Syn _126-135 antibodies are consistent with the clinical characteristics of synuclein diseases and that these antibodies are specific for α-Syn pathological aggregates.

result

a. Use a representative α-Syn _126-135 Quantitative analysis of antibodies produced by immunizing guinea pigs with peptide immunogen structure

In order to study the novel anti-α-Syn antibodies for immunotherapy, immunohistochemistry (IHC) was used to quantitatively analyze the relative specificity of α-Syn in human patients with three synuclein diseases (MSA, DLB and PD) with various antibodies.

b. Representative α-Syn _126-135 Antibodies derived from peptide immunogens structurally immunized guinea pigs are more sensitive to binding to α-Syn aggregates than commercially available diagnostic antibodies

In order to study the relative antigenicity of the α-Syn _126-135 antibody of the present invention, the amount of α-Syn detected by each antibody was compared with a commercially available synuclein disease diagnostic antibody (NCL-L-ASYN). First, by analyzing the overall results of the 17A-17D to 22A-22C plots, it can be found that compared with NCL-L-ASYN, the average percentage area of α-Syn detected by the α-Syn _126-135 antibody is significantly larger. This trend is consistent in each brain region and disease type, and shows that the α-Syn _126-135 antibody of the present invention is more sensitive or selective in binding to aggregated α-Syn than NCL-L-ASYN. Although the sample size of the present invention is relatively small (n = 3), the data still shows a clear trend. The specificity of the α-Syn _126-135 antibody of the present invention for α-Syn can be confirmed by comparing the same brain region in the non-patient and the patient's brain. The results in Figures 23A-23B show that each antibody, including NCL-L-ASYN, did not have any positive immunostaining. This data indicates that the alpha-Syn _126-135 antibodies of the invention are specific for the pathogenic form of alpha-Syn.

c. α-Syn _126-135 Antibodies can detect higher amounts of α-Syn, showing higher sensitivity and specificity than commercially available antibodies

The α-Syn _126-135 antibody of the present invention can detect a higher amount of α-Syn than NCL-L-ASYN, showing that the antibody of the present invention is more conducive to the application of immunotherapy to promote the elimination of α-Syn aggregates.

The first step in selecting an appropriate antibody as an immunotherapy agent is to determine the selectivity of the antibody for the target antigen (α-Syn) in human brain tissue with primary α-Syn pathology. Different synuclein diseases have different mechanisms of α-Syn aggregates, neuroanatomical patterns, and vulnerability of specific cell type aggregation.

In order to study the immunotherapy using agents as synuclein diseases in general, it is important to evaluate the selectivity of α-Syn _126-135 antibodies for α-Syn of different neuropathologies in different synuclein diseases. To this end, clinically confirmed cases of PD, DLB, and MSA were selected. PD and DLB are the second most common form of dementia, and are mainly caused by the accumulation of α-Syn in neurons (LB and LN). In contrast to PD, the pathologies of amyloid-beta and tau are known to cause neurodegeneration in DLB2. Different patterns of α-Syn aggregation can be seen in MSA, where aggregates are formed mainly in glial cells rather than neurons (Figures 27A-27C and 28A-28B). In addition, the progression of α-Syn pathology varies between different disease types, with early pathology common in the midbrain and basal nucleus regions. By analyzing the antigenicity of each antibody in the brain affected by different stages of the disease, it can be distinguished which antibody can be more effective in treating the early stages of the disease.

d. α-Syn _126-135 Antibodies (PD062220, PD062205, and PD100806) specifically bind PD, DLB, and MSA (Figures 17A-D to 22A-22C) human α-Syn pathological aggregates, while no synapses were observed in healthy controls Riboprotein Pathology (Figures 23A-23B)

Α-Syn was detected using the published α-Syn _126-135 antibody, and the specificity of the same cell type was described in clinical neuropathology (Figures 27A-27B and 28A-28B). Importantly, the α-Syn _126-135 antibodies of the invention do not have the same antigenicity to all forms of human α-Syn.

Further analysis of the specificity of PD062205 and PD100806 antibodies in each antibody's ability, a greater proportion of LN than LNL-L-ASYN was detected in the basal nucleus (Figures 24A-24D). The same phenomenon was observed in the midbrain (Figures 26A-26B). In summary, higher percentage areas of α-Syn can be detected by PD062205 and PD100806 antibodies. This result indicates that the additional α-Syn portion detected by the α-Syn _126-135 antibodies of the present invention can be attributed to the specificity of these antibodies for LN Sex increased. This result is helpful for immunotherapy, because in the early stages of the disease, LN is the main form of α-Syn aggregation in the basal ganglia. Other synucleinopathy treatment agents in the preclinical development phase do not provide detection of IHC for LN. Therefore, compared with other commercially available products, the peptide immunogen structure of the present invention has the same properties and characteristics as the α-Syn _126-135 antibody produced by this peptide immunogen structure.

The average amount of α-Syn aggregates in the affected brain region was measured, and the sensitivity of the α-Syn _126-135 antibody produced by the peptide immunogen structure of the present invention was analyzed by IHC. The invention quantifies the average percentage area of α-Syn in brain samples, and proves that compared to commercially available antibodies, α-Syn _126-135 antibodies are very sensitive to detection of α-Syn in the early stages of disease progression in MSA, DLB, and PD.

The higher sensitivity in the present invention can be attributed to the higher specificity of the antibody of the invention for LN compared to the diagnostic antibody NCL-L-ASYN. This result proves that the α-Syn _126-135 antibody of the present invention may be the most effective candidate for antibody-assisted clearance of α-Syn aggregates in synuclein diseases.

The technical features of the invention disclosed in all the specifications can be combined in any way. Each technical feature disclosed in the description can be replaced in other ways for the same, equivalent or similar purpose. Therefore, unless stated otherwise, all features disclosed herein are merely examples of a general series of equivalent or similar features.

As can be seen from the above, those skilled in the art can easily understand the necessary features of the present invention, and without departing from the spirit and scope, can make many changes and adjustments to the present invention to apply to different uses and conditions.

<110> United United Biomedical Corporation

<120> Alpha -synuclein C-terminal peptide immunogen and its formula for treating synuclein diseases

<130> UNS1001-PRO

<140> TBD

<141> 2017-06-16

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<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 95-104

<400> 33

<210> 34

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 96-105

<400> 34

<210> 35

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 97-106

<400> 35

<210> 36

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 98-107

<400> 36

<210> 37

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 99-108

<400> 37

<210> 38

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 100-109

<400> 38

<210> 39

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 101-110

<400> 39

<210> 40

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 102-111

<400> 40

<210> 41

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 103-112

<400> 41

<210> 42

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 104-113

<400> 42

<210> 43

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 105-114

<400> 43

<210> 44

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 106-115

<400> 44

<210> 45

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 107-116

<400> 45

<210> 46

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 108-117

<400> 46

<210> 47

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 109-118

<400> 47

<210> 48

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 110-119

<400> 48

<210> 49

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 111-120

<400> 49

<210> 50

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 112-121

<400> 50

<210> 51

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> alphal-Synuclein 113-122

<400> 51

<210> 52

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 114-123

<400> 52

<210> 53

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 115-124

<400> 53

<210> 54

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 116-125

<400> 54

<210> 55

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 117-126

<400> 55

<210> 56

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 118-127

<400> 56

<210> 57

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 119-128

<400> 57

<210> 58

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 120-129

<400> 58

<210> 59

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 121-130

<400> 59

<210> 60

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 122-131

<400> 60

<210> 61

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 123-132

<400> 61

<210> 62

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 124-133

<400> 62

<210> 63

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 125-134

<400> 63

<210> 64

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 126-135

<400> 64

<210> 65

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 127-136

<400> 65

<210> 66

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 128-137

<400> 66

<210> 67

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 129-138

<400> 67

<210> 68

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 130-139

<400> 68

<210> 69

<211> 10

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (10)

<223> Alpha -synuclein 131-140

<400> 69

<210> 70

<211> 17

<212> PRT

<213> Clostridium tetani

<220>

<221> Peptide

<222> (1) .. (17)

<223> Clostridium tetani 1 Th

<400> 70

<210> 71

<211> 15

<212> PRT

<213> Measles virus

<220>

<221> Peptide

<222> (1) .. (15)

<223> MvF1 Th

<400> 71

<210> 72

<211> 24

<212> PRT

<213> Bordetella pertussis

<220>

<221> Peptide

<222> (1) .. (24)

<223> Bordetella pertussis Th

<400> 72

<210> 73

<211> 17

<212> PRT

<213> Clostridium tetani

<220>

<221> Peptide

<222> (1) .. (17)

<223> Clostridium tetani 2 Th

<400> 73

<210> 74

<211> 23

<212> PRT

<213> diphtheria bacilli

<220>

<221> Peptide

<222> (1) .. (23)

<223> Diphtheria Th

<400> 74

<210> 75

<211> 21

<212> PRT

<213> Plasmodium falciparum

<220>

<221> Peptide

<222> (1) .. (21)

<223> Plasmodium falciparum Th

<400> 75

<210> 76

<211> 17

<212> PRT

<213> Schistosoma mansoni

<220>

<221> Peptide

<222> (1) .. (17)

<223> Schistosoma mansoni Th

<400> 76

<210> 77

<211> 25

<212> PRT

<213> Cholera Toxin

<220>

<221> Peptide

<222> (1) .. (25)

<223> Cholera Toxin Th

<400> 77

<210> 78

<211> 15

<212> PRT

<213> Measles virus

<220>

<221> Peptide

<222> (1) .. (15)

<223> MvF 2 Th

<400> 78

<210> 79

<211> 22

<212> PRT

<213> Measles virus

<220>

<221> Peptide

<222> (1) .. (22)

<223> KKKMvF 3 Th

<220>

<221> SITE

<222> (7) .. (7)

<223> S or T

<220>

<221> SITE

<222> (10) .. (10)

<223> K or R

<220>

<221> SITE

<222> (11) .. (11)

<223> G or T

<220>

<221> SITE

<222> (15) .. (15)

<223> H or T

<220>

<221> SITE

<222> (16) .. (16)

<223> K or R

<220>

<221> SITE

<222> (19) .. (19)

<223> G or T

<400> 79

<210> 80

<211> 18

<212> PRT

<213> Hepatitis B virus

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg 1 Th

<220>

<221> SITE

<222> (1) .. (1)

<223> K or R

<220>

<221> SITE

<222> (2) .. (2)

<223> K or R

<220>

<221> SITE

<222> (3) .. (3)

<223> K or R

<220>

<221> SITE

<222> (4) .. (4)

<223> L or I or V or F

<220>

<221> SITE

<222> (5) .. (5)

<223> F or K or R

<220>

<221> SITE

<222> (6) .. (6)

<223> L or I or V or F

<220>

<221> SITE

<222> (7) .. (7)

<223> L or I or V or F

<220>

<221> SITE

<222> (9) .. (9)

<223> K or R

<220>

<221> SITE

<222> (10) .. (10)

<223> L or I or V or F

<220>

<221> SITE

<222> (11) .. (11)

<223> L or I or V or F

<220>

<221> SITE

<222> (13) .. (13)

<223> L or I or V or F

<220>

<221> SITE

<222> (15) .. (15)

<223> Q or L or I or V or F

<220>

<221> SITE

<222> (17) .. (17)

<223> L or I or V or F

<220>

<221> SITE

<222> (18) .. (18)

<223> D or R

<400> 80

<210> 81

<211> 19

<212> PRT

<213> Measles virus

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<400> 81

<210> 82

<211> 18

<212> PRT

<213> Hepatitis B virus

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg 2 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> I or F

<220>

<221> SITE

<222> (5) .. (5)

<223> I or F

<220>

<221> SITE

<222> (6) .. (6)

<223> T or L

<220>

<221> SITE

<222> (7) .. (7)

<223> I or L

<220>

<221> SITE

<222> (11) .. (11)

<223> I or L

<220>

<221> SITE

<222> (14) .. (14)

<223> P or I

<220>

<221> SITE

<222> (15) .. (15)

<223> Q or T

<220>

<221> SITE

<222> (16) .. (16)

<223> S or T

<220>

<221> SITE

<222> (17) .. (17)

<223> L or I

<400> 82

<210> 83

<211> 19

<212> PRT

<213> Measles virus

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 5 Th

<400> 83

<210> 84

<211> 18

<212> PRT

<213> Hepatitis B virus

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg 3 Th

<400> 84

<210> 85

<211> 11

<212> PRT

<213> Influenza virus

<220>

<221> Peptide

<222> (1) .. (11)

<223> Influenza Matrix protein 1_1 Th

<220>

<221> Peptide

<222> (1) .. (11)

<223> Influenza Matrix protein 1_1 Th

<400> 85

<210> 86

<211> 15

<212> PRT

<213> Influenza virus

<220>

<221> Peptide

<222> (1) .. (15)

<223> Influenza Matrix protein 1_2 Th

<400> 86

<210> 87

<211> 9

<212> PRT

<213> Influenza virus

<220>

<221> Peptide

<222> (1) .. (9)

<223> Influenza Non-structural protein 1 Th

<400> 87

<210> 88

<211> 19

<212> PRT

<213> Epstein-Barr virus

<220>

<221> Peptide

<222> (1) .. (19)

<223> EBV BHRF1 Th

<400> 88

<210> 89

<211> 15

<212> PRT

<213> Clostridium tetani

<220>

<221> Peptide

<222> (1) .. (15)

<223> Clostridium tetani TT1 Th

<400> 89

<210> 90

<211> 20

<212> PRT

<213> Epstein-Barr virus

<220>

<221> Peptide

<222> (1) .. (20)

<223> EBV EBNA-1 Th

<400> 90

<210> 91

<211> 21

<212> PRT

<213> Clostridium tetani

<220>

<221> Peptide

<222> (1) .. (21)

<223> Clostridium tetani TT2 Th

<400> 91

<210> 92

<211> 16

<212> PRT

<213> Clostridium tetani

<220>

<221> Peptide

<222> (1) .. (16)

<223> Clostridium tetani TT3 Th

<400> 92

<210> 93

<211> 16

<212> PRT

<213> Clostridium tetani

<220>

<221> Peptide

<222> (1) .. (16)

<223> Clostridium tetani TT4 Th

<400> 93

<210> 94

<211> 18

<212> PRT

<213> Epstein-Barr virus

<220>

<221> Peptide

<222> (1) .. (18)

<223> EBV CP Th

<400> 94

<210> 95

<211> 14

<212> PRT

<213> Human cytomegalovirus

<220>

<221> Peptide

<222> (1) .. (14)

<223> HCMV IE1 Th

<400> 95

<210> 96

<211> 15

<212> PRT

<213> Epstein-Barr virus

<220>

<221> Peptide

<222> (1) .. (15)

<223> EBV GP340 Th

<400> 96

<210> 97

<211> 13

<212> PRT

<213> Epstein-Barr virus

<220>

<221> Peptide

<222> (1) .. (13)

<223> EBV BPLF1 Th

<400> 97

<210> 98

<211> 11

<212> PRT

<213> Epstein-Barr virus

<220>

<221> Peptide

<222> (1) .. (11)

<223> EBV EBNA-2 Th

<400> 98

<210> 99

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (38)

<223> Alpha -synuclein 126-140

<400> 99

<210> 100

<211> 43

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (43)

<223> Alpha -synuclein 121-140

<400> 100

<210> 101

<211> 53

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (53)

<223> Alpha -synuclein 111-140

<400> 101

<210> 102

<211> 63

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (63)

<223> Alpha -synuclein 126-140

<400> 102

<210> 103

<211> 63

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (63)

<223> Alpha -synuclein 101-140

<400> 103

<210> 104

<211> 62

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg3 Th

<220>

<221> SITE

<222> (19) .. (19)

<223> ε -K

<220>

<221> Peptide

<222> (19) .. (22)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (23) .. (62)

<223> Alpha -synuclein 101-140

<400> 104

<210> 105

<211> 73

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (73)

<223> Alpha -synuclein 91-140

<400> 105

<210> 106

<211> 79

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (79)

<223> Alpha -synuclein 85-140

<400> 106

<210> 107

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (38)

<223> Alpha -synuclein 121-135

<400> 107

<210> 108

<211> 48

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (48)

<223> Alpha -synuclein 111-135

<400> 108

<210> 109

<211> 58

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (58)

<223> Alpha -synuclein 101-135

<400> 109

<210> 110

<211> 62

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (62)

<223> Alpha -synuclein 97-135

<400> 110

<210> 111

<211> 36

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK as a spacer

<220>

<221> Peptide

<222> (24) .. (36)

<223> Alpha -synuclein 123-135

<400> 111

<210> 112

<211> 33

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (33)

<223> Alpha -synuclein 126-135

<400> 112

<210> 113

<211> 45

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (45)

<223> Alpha -synuclein 111-132

<400> 113

<210> 114

<211> 55

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (55)

<223> Alpha -synuclein 101-132

<400> 114

<210> 115

<211> 45

<212> PRT

<213> Mus musculus

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (45)

<223> Mouse α -synuclein 111-132

<400> 115

<210> 116

<211> 33

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (33)

<223> Alpha -synuclein 126-135

<400> 116

<210> 117

<211> 45

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF 4 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> S or T

<220>

<221> SITE

<222> (7) .. (7)

<223> K or R

<220>

<221> SITE

<222> (8) .. (8)

<223> G or T

<220>

<221> SITE

<222> (12) .. (12)

<223> H or T

<220>

<221> SITE

<222> (13) .. (13)

<223> K or R

<220>

<221> SITE

<222> (20) .. (20)

<223> ε -K

<220>

<221> Peptide

<222> (20) .. (23)

<223> ε K-KKK is the linker

<220>

<221> Peptide

<222> (24) .. (45)

<223> Alpha -synuclein 111-132

<400> 117

<210> 118

<211> 30

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε K is a linker

<220>

<221> Peptide

<222> (21) .. (30)

<223> Alpha -synuclein 126-135

<400> 118

<210> 119

<211> 42

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> MvF5 Th

<220>

<221> SITE

<222> (20) .. (20)

<223> ε K is a linker

<220>

<221> Peptide

<222> (21) .. (42)

<223> Alpha -synuclein 111-132

<400> 119

<210> 120

<211> 29

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg3 Th

<220>

<221> SITE

<222> (19) .. (19)

<223> ε K is a linker

<220>

<221> Peptide

<222> (20) .. (29)

<223> Alpha -synuclein 126-135

<400> 120

<210> 121

<211> 41

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg3 Th

<220>

<221> SITE

<222> (19) .. (19)

<223> ε K is a linker

<220>

<221> Peptide

<222> (20) .. (41)

<223> Alpha -synuclein 111-132

<400> 121

<210> 122

<211> 40

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (17)

<223> Clostridium tetani 1 Th

<220>

<221> SITE

<222> (18) .. (18)

<223> ε K is a linker

<220>

<221> Peptide

<222> (19) .. (40)

<223> Alpha -synuclein 111-132

<400> 122

<210> 123

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (15)

<223> MvF1 Th

<220>

<221> SITE

<222> (16) .. (16)

<223> ε K is a linker

<220>

<221> Peptide

<222> (17) .. (38)

<223> Alpha -synuclein 111-132

<400> 123

<210> 124

<211> 47

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (24)

<223> Bordetella pertussis Th

<220>

<221> SITE

<222> (25) .. (25)

<223> ε K is a linker

<220>

<221> Peptide

<222> (26) .. (47)

<223> Alpha -synuclein 111-132

<400> 124

<210> 125

<211> 40

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (17)

<223> Clostridium tetani 2 Th

<220>

<221> SITE

<222> (18) .. (18)

<223> ε K is a linker

<220>

<221> Peptide

<222> (19) .. (40)

<223> Alpha -synuclein 111-132

<400> 125

<210> 126

<211> 46

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (23)

<223> Diphtheria Th

<220>

<221> SITE

<222> (24) .. (24)

<223> ε K is a linker

<220>

<221> Peptide

<222> (25) .. (46)

<223> Alpha -synuclein 111-132

<400> 126

<210> 127

<211> 44

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (21)

<223> Plasmodium falciparum Th

<220>

<221> SITE

<222> (22) .. (22)

<223> ε K is a linker

<220>

<221> Peptide

<222> (23) .. (44)

<223> Alpha -nucleolytic protein 111-132

<400> 127

<210> 128

<211> 40

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (17)

<223> Schistosoma mansoni Th

<220>

<221> SITE

<222> (18) .. (18)

<223> ε K is a linker

<220>

<221> Peptide

<222> (19) .. (40)

<223> Alpha -synuclein 111-132

<400> 128

<210> 129

<211> 48

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (25)

<223> Cholera toxin Th

<220>

<221> SITE

<222> (26) .. (26)

<223> ε K is a linker

<220>

<221> Peptide

<222> (27) .. (48)

<223> Alpha -synuclein 111-132

<400> 129

<210> 130

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (15)

<223> MvF2 Th

<220>

<221> SITE

<222> (16) .. (16)

<223> ε K is a linker

<220>

<221> Peptide

<222> (17) .. (38)

<223> Alpha -synuclein 111-132

<400> 130

<210> 131

<211> 45

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (22)

<223> KKKMvF3 Th

<220>

<221> SITE

<222> (7) .. (7)

<223> S or T

<220>

<221> SITE

<222> (10) .. (10)

<223> K or R

<220>

<221> SITE

<222> (11) .. (11)

<223> G or T

<220>

<221> SITE

<222> (15) .. (15)

<223> H or T

<220>

<221> SITE

<222> (16) .. (16)

<223> K or R

<220>

<221> SITE

<222> (19) .. (19)

<223> G or T

<220>

<221> SITE

<222> (23) .. (23)

<223> ε K is a linker

<220>

<221> Peptide

<222> (24) .. (45)

<223> Alpha -synuclein 111-132

<400> 131

<210> 132

<211> 41

<212> PRT

<213> Hepatitis B virus

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg 1 Th

<220>

<221> SITE

<222> (1) .. (1)

<223> K or R

<220>

<221> SITE

<222> (2) .. (2)

<223> K or R

<220>

<221> SITE

<222> (3) .. (3)

<223> K or R

<220>

<221> SITE

<222> (4) .. (4)

<223> L or I or V or F

<220>

<221> SITE

<222> (5) .. (5)

<223> F or K or R

<220>

<221> SITE

<222> (6) .. (6)

<223> L or I or V or F

<220>

<221> SITE

<222> (7) .. (7)

<223> L or I or V or F

<220>

<221> SITE

<222> (9) .. (9)

<223> K or R

<220>

<221> SITE

<222> (10) .. (10)

<223> L or I or V or F

<220>

<221> SITE

<222> (11) .. (11)

<223> L or I or V or F

<220>

<221> SITE

<222> (13) .. (13)

<223> L or I or V or F

<220>

<221> SITE

<222> (15) .. (15)

<223> Q or L or I or V or F

<220>

<221> SITE

<222> (17) .. (17)

<223> L or I or V or F

<220>

<221> SITE

<222> (18) .. (18)

<223> D or R

<220>

<221> SITE

<222> (19) .. (19)

<223> ε K is a linker

<220>

<221> Peptide

<222> (20) .. (41)

<223> Alpha -synuclein 111-132

<400> 132

<210> 133

<211> 41

<212> PRT

<213> Hepatitis B virus

<220>

<221> Peptide

<222> (1) .. (18)

<223> HBsAg 2 Th

<220>

<221> SITE

<222> (4) .. (4)

<223> I or F

<220>

<221> SITE

<222> (5) .. (5)

<223> I or F

<220>

<221> SITE

<222> (6) .. (6)

<223> T or L

<220>

<221> SITE

<222> (7) .. (7)

<223> I or L

<220>

<221> SITE

<222> (11) .. (11)

<223> I or L

<220>

<221> SITE

<222> (14) .. (14)

<223> P or I

<220>

<221> SITE

<222> (15) .. (15)

<223> Q or T

<220>

<221> SITE

<222> (16) .. (16)

<223> S or T

<220>

<221> SITE

<222> (17) .. (17)

<223> L or I

<220>

<221> SITE

<222> (19) .. (19)

<223> ε K is a linker

<220>

<221> Peptide

<222> (20) .. (41)

<223> Alpha -synuclein 111-132

<400> 133

<210> 134

<211> 34

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (11)

<223> Influenza MP1_1 Th

<220>

<221> SITE

<222> (12) .. (12)

<223> ε K is a linker

<220>

<221> Peptide

<222> (13) .. (34)

<223> Alpha -synuclein 111-132

<400> 134

<210> 135

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (15)

<223> Influenza MP1_2 Th

<220>

<221> SITE

<222> (16) .. (16)

<223> ε K is a linker

<220>

<221> Peptide

<222> (17) .. (38)

<223> Alpha -synuclein 111-132

<400> 135

<210> 136

<211> 32

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (9)

<223> Influenza NSP1 Th

<220>

<221> SITE

<222> (10) .. (10)

<223> ε K is a linker

<220>

<221> Peptide

<222> (11) .. (32)

<223> Alpha -synuclein 111-132

<400> 136

<210> 137

<211> 42

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (19)

<223> EBV BHRF1 Th

<220>

<221> Peptide

<222> (20) .. (20)

<223> ε K is a linker

<220>

<221> SITE

<222> (20) .. (20)

<223> ε K is a linker

<220>

<221> Peptide

<222> (21) .. (42)

<223> Alpha -synuclein 111-132

<400> 137

<210> 138

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (15)

<223> Clostridium tetani TT1 Th

<220>

<221> SITE

<222> (16) .. (16)

<223> ε K is a linker

<220>

<221> Peptide

<222> (17) .. (38)

<223> Alpha -synuclein 111-132

<400> 138

<210> 139

<211> 43

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (20)

<223> EBV EBNA-1 Th

<220>

<221> SITE

<222> (21) .. (21)

<223> ε K is a linker

<220>

<221> Peptide

<222> (22) .. (43)

<223> Alpha -synuclein 111-132

<400> 139

<210> 140

<211> 44

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (21)

<223> Clostridium tetani TT2 Th

<220>

<221> SITE

<222> (22) .. (22)

<223> ε K is a linker

<220>

<221> Peptide

<222> (23) .. (44)

<223> Alpha -synuclein 111-132

<400> 140

<210> 141

<211> 39

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (16)

<223> Clostridium tetani TT3 Th

<220>

<221> SITE

<222> (17) .. (17)

<223> ε K is a linker

<220>

<221> Peptide

<222> (18) .. (39)

<223> Alpha -synuclein 111-132

<400> 141

<210> 142

<211> 39

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (16)

<223> Clostridium tetani TT4 Th

<220>

<221> SITE

<222> (17) .. (17)

<223> ε K is a linker

<220>

<221> Peptide

<222> (18) .. (39)

<223> Alpha -synuclein 111-132

<400> 142

<210> 143

<211> 41

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (18)

<223> EBV CP Th

<220>

<221> SITE

<222> (19) .. (19)

<223> ε K is a linker

<220>

<221> Peptide

<222> (20) .. (41)

<223> Alpha -synuclein 111-132

<400> 143

<210> 144

<211> 37

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (14)

<223> HCMV IE1 Th

<220>

<221> SITE

<222> (15) .. (15)

<223> ε K is a linker

<220>

<221> Peptide

<222> (16) .. (37)

<223> Alpha -synuclein 111-132

<400> 144

<210> 145

<211> 38

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (15)

<223> EBV GP340 Th

<220>

<221> SITE

<222> (16) .. (16)

<223> ε K is a linker

<220>

<221> Peptide

<222> (17) .. (38)

<223> Alpha -synuclein 111-132

<400> 145

<210> 146

<211> 36

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (13)

<223> EBV BPLF1 Th

<220>

<221> SITE

<222> (14) .. (14)

<223> ε K is a linker

<220>

<221> Peptide

<222> (15) .. (36)

<223> Alpha -synuclein 111-132

<400> 146

<210> 147

<211> 34

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (11)

<223> EBV EBNA-2 Th

<220>

<221> SITE

<222> (12) .. (12)

<223> ε K is a linker

<220>

<221> Peptide

<222> (13) .. (34)

<223> Alpha -synuclein 111-132

<400> 147

<210> 148

<211> 4

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (4)

<223> ε K-KKK is the linker

<220>

<221> SITE

<222> (1) .. (1)

<223> ε -K

<400> 148

<210> 149

<211> 31

<212> DNA

<213> Homo sapiens

<220>

<221> Primer_binding position

<222> (1) .. (31)

<223> Forward primer sequence of α -synuclein

<400> 149

<210> 150

<211> 31

<212> DNA

<213> Homo sapiens

<220>

<221> Primer_binding position

<222> (1) .. (31)

<223> Alpha -synuclein reverse primer

<400> 150

<210> 151

<211> 23

<212> DNA

<213> Homo sapiens

<220>

<221> Primer_binding position

<222> (1) .. (23)

<223> Primer sequence of mutant α -synuclein

<400> 151

<210> 152

<211> 21

<212> DNA

<213> Homo sapiens

<220>

<221> Primer_binding position

<222> (1) .. (21)

<223> Reverse primer for mutant α -synuclein

<400> 152

<210> 153

<211> 32

<212> PRT

<213> Homo sapiens

<220>

<221> Peptide

<222> (1) .. (32)

<223> β -synuclein 103-134

<400> 153

Claims (25)

An α-synuclein (α-Syn) peptide immunogen structure, including: a B cell epitope, including 10 to 25 amino acids derived from the C-terminal fragment of α-Syn, which corresponds to SEQ ID NO: 1 Glycine (G111) from position 111 to 135 aspartic acid (D135); one T-helper epitope, including optional free SEQ ID NOs: 70-98 A group of amino acid sequences; and a selective heterologous linker selected from amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys, and A group consisting of ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), in which the B cell epitope is covalently linked to the T helper epitope directly or through a selective heterologous linker . The α-Syn peptide immunogen structure according to item 1 of the patent application range, wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17 and 49-63. The α-Syn peptide immunogen structure according to item 1 of the patent application scope, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 81, 83, and 84. The α-Syn peptide immunogen structure according to item 1 of the scope of the patent application, wherein the selective heterologous linker is (α, ε-N) Lys or ε-N-Lys-Lys-Lys-Lys ( (SEQ ID NO: 148). The α-Syn peptide immunogen structure according to item 1 of the scope of the patent application, wherein the T helper epitope is covalently linked to the N-terminus of the B cell epitope. The α-Syn peptide immunogen structure according to item 1 of the patent application scope, wherein the T helper epitope is covalently connected to the N-terminus of the B cell epitope through the selective heterologous linker. The α-Syn peptide immunogen structure according to item 1 of the patent application scope, including the following formula: (Th) _m- (A) _n- (α-Syn C-terminal fragment) -X or (α-Syn C -Terminal fragment)-(A) _n- (Th) _m -X where Th is the T helper epitope; A is the heterologous linker; (α-Syn C-terminal fragment) is the B cell epitope Position; X is α-COOH or α-CONH ₂ of an amino acid; m is 1, 2, 3, or 4; and n is 1, ₂ , 3, 4, 5, 6, 7, 8, 9, or 10 . The α-Syn peptide immunogen structure according to item 1 of the patent application scope includes a monoamino acid sequence, which can be selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. The α-Syn peptide immunogen structure described in item 1 of the patent application scope includes a monoamino acid sequence, which can be selected from the group consisting of SEQ ID NOs: 107, 108, and 111-113. A composition comprising the α-Syn peptide immunogen of the first patent application. A composition comprising more than one α-Syn peptide immunogen in the first patent application. The composition according to item 11 of the scope of patent application, wherein the α-Syn peptide immunogen structure has SEQ ID NOs: 112 and 113 amino acid sequences. A medicinal composition includes the α-Syn peptide immunogen structure and the pharmaceutically acceptable delivery carrier and / or adjuvant in the first patent scope of the application. The pharmaceutical composition according to item 13 of the patent application scope, wherein: a. The α-Syn peptide immunogen structure is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. group; and b. the mineral adjuvant is an aluminum salt, which lines the desired group consisting of Al (OH) ₃ or AlPO ₄ composed of. The pharmaceutical composition according to item 13 of the patent application scope, wherein a. The α-Syn peptide immunogen structure is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; And b. The α-Syn peptide immunogen structure is mixed with a CpG oligonucleotide (ODN) to form a stable immunostimulatory complex. An isolated antibody or epitope-binding fragment thereof specifically binds to the B-cell epitope of the α-Syn peptide immunogen structure of the first patent application. The isolated antibody or epitope-binding fragment thereof according to item 16 of the scope of the patent application, which binds to the α-Syn peptide immunogen structure. An isolated antibody or an epitope-binding fragment thereof specifically binds to a B-cell epitope of an α-Syn peptide immunogen structure of the patent application No. 9. A composition comprising an isolated antibody or an epitope-binding fragment thereof according to item 16 of the patent application. A composition comprising an isolated antibody or an epitope-binding fragment thereof according to item 18 of the patent application. The composition according to item 20 of the scope of patent application, including the following mixture: a. An isolated antibody or an epitope-binding fragment thereof specifically binding to the B-cell epitope of SEQ ID NO: 112; and b An isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of SEQ ID NO: 113. An application of the α-Syn peptide immunogen structure according to item 1 of the scope of patent application, which is a pharmaceutical composition for manufacturing an antibody that recognizes α-Syn in a host, and comprises administering the pharmaceutical composition to the host The pharmaceutical composition includes an alpha-Syn peptide immunogen and a delivery vehicle and / or an adjuvant in the first patent application. An application of the α-Syn peptide immunogen structure as described in item 1 of the scope of patent application, which is used for manufacturing a pharmaceutical composition that inhibits the aggregation of α-Syn in an animal, and comprises administering the pharmaceutical composition to the animal The medicinal composition includes a pharmaceutically effective amount of an α-Syn peptide immunogen in the first patent application. An application of the α-Syn peptide immunogen structure as described in item 1 of the scope of the patent application, which is used for manufacturing a pharmaceutical composition for reducing the amount of α-Syn aggregation in an animal, which comprises administering the pharmaceutical composition to the animal The pharmaceutical composition includes a pharmaceutically effective amount of an α-Syn peptide immunogen in the first patent application. A method for identifying α-Syn aggregates of different sizes in a biological sample, including: a. Exposing the biological sample to the scope of the patent application under conditions that allow antibodies or epitope-binding fragments thereof to bind α-Syn aggregates The antibody or epitope-binding fragment thereof according to item 16; and b. Detecting the amount of the antibody or epitope-binding fragment that binds to the α-Syn aggregate in the biological sample.