Immunogenic compounds

Abstract

The antigenic peptide has the structure: X1-X2-X3-X4-X5-X6-X7-P-X9-X 10 -X 11 -X 12 and is derived from amino acids 113-124 of α-synuclein. According to the structure: P is proline; X1 is L, K, A, or S, where L is leucine, K is lysine, A is alanine, and S is serine; X2 is E or S, where E and S are as defined above; X3 is D, E, K, N, A, or S, where N is asparagine, D is aspartic acid, and D, E, K, A, and S are as defined above; X4 is M, A, S, L, or K, where M is methionine, and A, S, L, and K are as defined above; X5 is P or A, as defined above; X6 is V, A, or S, where V is valine, and A and S are as defined above; X7 is D or S, as defined above; X9 is D or A, as defined above; 10 is N, S or A, wherein N, S and A are as defined above; X 11 is E, A or S, where E, A and S are as defined above; X 12 is present or absent, and if present, is A, K, V, S, or G, where G is glycine and A, K, V, and S are as defined above. The structure contains at least one mutation compared to the wild-type LEDMPVDPDNEA sequence. The peptide comprises X 12 wherein Y is tyrosine and E is as defined above. The peptides are conjugated to a suitable carrier and are useful in treating synucleinopathies.

Description

[Technical Field]

[0001] The present invention relates to immunogenic compounds and their use in the prevention and treatment of synucleinopathy, particularly Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). [Background technology]

[0002] Parkinson's disease (PD) is a synucleinopathy and the second most common neurodegenerative movement disorder. The prevalence of PD ranges from 100 to 200 / 100,000 in the general population, affecting approximately 1% of the population over 60 years of age, with an annual incidence of approximately 15 / 100,000. It is a chronic, progressive disorder defined by a combination of motor syndromes (bradykinesia, rigidity, resting tremor, and postural instability) and non-motor syndromes (various autonomic dysfunctions, paresthesia, and psychiatric abnormalities) that usually precede the motor syndromes. A prominent feature of this disease is the severe loss of dopaminergic neurons in the substantia nigra (SN), accompanied by the accumulation of fibrous protein inclusions called Lewy bodies (LBs), which are primarily composed of alpha-synuclein (aSyn). PD, DLB, and other LB diseases exhibit aSyn accumulation and redistribution in various brain regions and cell populations.

[0003] MSA is another very important synucleinopathy. MSA is a sporadic neurodegenerative disease characterized by symptoms of L-DOPA-resistant parkinsonism, cerebellar ataxia, and autonomic dysfunction. Patients suffer from multisystem neuronal loss affecting various brain regions, including the striatum, substantia nigra, cerebellum, pons, inferior olivary gland, and spinal cord. MSA is characterized by aSyn-positive glial cytoplasm (GCI) and rare neuronal inclusions throughout the central nervous system. These inclusions are associated with striatonigral degeneration, olivopontocerebellar atrophy, and the involvement of autonomic nuclei in the medulla and spinal cord. The importance of GCI to the pathogenesis of MSA is generally recognized and has been clearly demonstrated by a recent analysis of a transgenic mouse model that analyzes the effects of aSyn overexpression in oligodendroglia. In tg mice overexpressing human aSyn, both GCI-like aggregates and biochemical markers of MSA were observed.

[0004] DLB is the second most common neurodegenerative dementia in Western societies, after Alzheimer's disease (AD). It accounts for 4–7% of clinically diagnosed dementias, and it is estimated that the same number of cases miss the correct clinical diagnosis. Diagnosing DLB is challenging because the disease represents an “intermediate” stage between AD and PD, exhibiting overlapping features of both entities. Four clinical consensus criteria that must exist to diagnose “possible DLB” are fluctuations in cognition and attention, recurrent hallucinations, REM sleep behavior disorder, and spontaneous Parkinsonian motor signs, which occur later in the disease than the other criteria. These are supported by a variety of additional clinical criteria that may occur but do not necessarily occur, including transient episodes of syncope or unresponsiveness, apathy, anxiety, depression, psychotic episodes, and neuroleptic hypersensitivity. Symptoms are not uniform among patients.

[0005] The pathology of DLB ​​is characterized by protein inclusions called Lewy bodies (LBs), which are primarily composed of α-synuclein (aSyn) and involved in the loss of neuronal function and structure. However, in DLB, LBs are found diffusely distributed throughout the cortex, whereas in PD, they are mainly found in dopaminergic neurons in the substantia nigra. DLB LBs are less clearly defined, less eosinophilic, and less fibrous than PD LBs. Furthermore, the brains of DLB ​​patients show amyloid plaques, mainly containing carboxylate-extended amyloid beta (Abeta), such as Abeta1-42. Cortical amyloid deposition is associated with lower temporal lobe perfusion and a tendency toward hippocampal atrophy.

[0006] aSyn is a 14kD native monomer protein typically located at presynaptic terminals bound to the membrane or cytosol of synaptic vesicles. Its native function is not well understood, but it may be involved in synaptic transmission. During pathogenesis, misfolding and aggregation of aSyn occurs in the central nervous system (CNS) and peripheral nervous system, possibly as a result of post-translational modifications, particularly C-terminal protease cleavage (Dufty 2007, Bassil 2016). Aggregation leads to the formation of various aSyn species, including oligomers, protofibrils, and fibrils, which are associated with the pathogenesis of LB disease. Fibrous forms of aSyn are primarily detected in LBs located in the nerve cell body (Kosaka et al., 1990; Dickson et al., 1989). Aggregates of aSyn can also be detected in astroglial cells (Braak 2007).

[0007] Not only fibrils, but also various oligomeric aSyn species were detected in the brains of diseased individuals. In contrast to filamentous aSyn, oligomeric aggregates are most likely located at neuronal projections and presynaptic terminals that can damage synapses, and therefore oligomeric aSyn are thought to be responsible for cytotoxicity.

[0008] Monomer aSyn has been shown to form various types of aggregates with different appearances, conformations, cytotoxicity, and chemical properties under a variety of in vitro conditions. Depending on the monomer conformation and general tolerance conditions, different types of aggregates can arise and have different structural characteristics. When seeded, different aSyn strains (e.g., "fibril" or "ribbon") imprint conformations on receptor cells, generating aggregates of the same strain in a process called "conformation template." When they are injected into rat brains, these types of aggregates exhibit different characteristics with respect to inclusion body formation and the generation of behavioral and neurotoxic phenotypes in vivo. Different types of aSyn aggregates are thought to expose different polypeptide chains due to their different conformations. These differently exposed surfaces allow for different sets of intramolecular interactions. Thus, the conformation of a particular aSyn strain determines characteristics such as tendency toward seeding and preference for specific cell types. Experimental data are beginning to emerge that demonstrate the diverse properties of aSyn strains extracted from PD and MSA materials; analysis of pathological brain material from patients with PD or MSA has demonstrated different properties of transmissible aSyn aggregates.

[0009] Currently available treatments only address the symptoms, and therapies that can correct the underlying neurodegeneration are still under development. Here, we present an aSyn-specific active immunotherapy (SAIT) approach that primarily targets the oligomeric and neurotoxic forms of aSyn, which may have the potential to hinder the progression of synucleinopathy.

[0010] Vaccination with previously developed aSyn-targeting AFFITOPE® peptides PD01 and PD03 has demonstrated efficacy in various animal models of aSyn aggregation disorders, resulting in reduction of aSyn pathology, preservation of neuroinflammation, and improvement of behavioral disorders (Mandler et al. 2014; WO 2009 / 103105 A1, WO 2011 / 020133 A1, WO 2017 / 076873 A1). These peptides have been found to be safe and well-tolerated vaccines capable of inducing target-specific antibodies in humans.

[0011] WO 2005 / 108423 A1 discloses peptides that confer environmental stress tolerance, particularly those derived from aSyn, β-synuclein (bSyn), or γ-synuclein (GSyn); these may result in reduced denaturation and / or increased solubility of the fusion partner protein when present in a fusion protein with a fusion partner protein. WO 2018 / 151821 A1 discloses antibodies against aSyn useful for the diagnosis, treatment, and prevention of neurodegenerative diseases. These antibodies are to be produced by immunization with native aSyn or the allele mutant A53T and should preferentially bind to pre-formed fibrils (PFFs). WO 2005 / 013889 A2 discloses fragments of native aSyn useful for the treatment or prevention of LB disease (LBD), or for providing monoclonal antibodies for the treatment or prevention of LBD. [Overview of the project] [Problems that the invention aims to solve]

[0012] The object of the present invention is to provide a drug for preventing and treating synucleinopathy based on a vaccine. A further object is to provide a vaccine peptide suitable for use in humans. [Means for solving the problem]

[0013] Furthermore, the present invention may provide an improved immunogenic peptide that is improved in terms of immunogenicity, inducing larger amounts of aSyn-specific antibodies in the periphery and in the brain. It is also desirable to increase the target binding of the induced antibodies through an oligoclonal antibody response (prolonged epitope).

[0014] Therefore, the present invention has a structure: X1-X2-X3-X4-X5-X6-X7-P-X9-X 10 -X 11 -X 12 The present invention provides an antigenic peptide that contains, essentially consists of, or comprises the same. Here, P is proline; X1 is L, K, A, or S, where L is leucine, K is lysine, A is alanine, and S is serine; X2 is either E or S, where E is glutamic acid and S is as defined above; X3 is D, E, K, N, A, or S, where N is asparagine, D is aspartic acid, and E, K, A, and S are as defined above; X4 is M, A, S, L, or K, where M is methionine, and A, S, L, and K are as defined above; X5 is P or A as defined above; X6 is V, A, or S, where V is valine, and A and S are as defined above; X7 is D or S with the same meaning as above; X9 is D or A as defined above; X 10 is N, S, or A, where N, S, and A are synonymous with those stated above; X 11 is E, A, or S, where E, A, and S are synonymous with those stated above; X 12is either present or absent, and when present, is A, K, V, S, or G, where G is glycine, and A, K, V, and S are as defined above; However, X1-X2-X3-X4-X5-X6-X7-P-X9-X 10 -X 11 -X 12 is not L-E-D-M-P-V-D-P-D-N-E-A, and contains 1 to 5 amino acid differences compared to the amino acid sequence: L-E-D-M-P-V-D-P-D-N-E-A, and the peptide does not contain the dipeptide: Y-E immediately after X 12 , where Y is tyrosine and E is as defined above. Thus, generally, single-letter amino acid codes are used herein.

[0015] The antigenic peptide may contain 1 to 5 amino acid differences (i.e., 1, 2, 3, 4, or 5 differences), or 1 to 4 amino acid differences compared to the amino acid sequence: L-E-D-M-P-V-D-P-D-N-E-A. The differences are generally amino acid substitutions (X 12 may be deleted depending on the options set at each position). It is preferred that there are 1 to 3 amino acid differences, most preferably 2 amino acid differences, from the amino acid sequence: L-E-D-M-P-V-D-P-D-N-E-A (amino acids 113 to 124 of the wild-type α-synuclein sequence). These differences can be selected from any of the amino acids X1-X 12 , except for X8 which is proline. In some embodiments, the antigenic peptide contains amino acid differences at one or more positions selected from X1, X3, X4, and X 12 . Preferably, the antigenic peptide contains 2 amino acid differences at positions selected from X1, X3, X4, and X 12 compared to the amino acid sequence L-E-D-M-P-V-D-P-D-N-E-A.

[0016] The antigen peptides of the present invention retain the ability to generate aSyn-specific antibodies when used as immunogens. Furthermore, the antigen peptides of the present invention are more immunogenic than the corresponding wild-type aSyn peptides (including the dodecamer: LEDMPVDPDNEA, p9524 (SEQ ID NO: 4)) with respect to the generation of aSyn-specific antibodies, as shown in comparative experiments. They are also more immunogenic than other aSyn peptides from the C-terminal region of aSyn, which have been shown to be less immunogenic than p9524; see Tables 1 and 2 below (e.g., p4456 (SEQ ID NO: 1) and p4572 (SEQ ID NO: 2)). These properties can be tested by those skilled in the art by typically testing the peptides in the form of a conjugate of the type described in detail herein (e.g., a peptide conjugated to CRM197 as a protein carrier), according to the teachings herein. The peptides are administered to suitable experimental animals, particularly mice, and samples (e.g., blood) are collected at appropriate intervals after administration (see Examples for specific details). The aSyn antibody titer can be determined, for example, by ELISA, as described herein.

[0017] The antigen peptide of the present invention is typically X 12 It does not contain any further α-synuclein amino acid residues after it. In particular, they do not contain X 12 It does not contain the dipeptide YE immediately following it, as described herein. 125 and E 126 Peptides containing MHCI are predicted by computer analysis to bind with high affinity to different allele variants of MHCI and are therefore predicted to be potential cytotoxic T cell epitopes (www.syfpeithi.de). However, antigen peptides may contain a limited number of further N-terminal amino acid residues. Therefore, antigen peptides have a structure: Xa-Xb-X1-X2-X3-X4-X5-X6-X7-P-X9-X 10 -X 11 -X 12 It includes, essentially consists of, or can consist of. Here, Xa is either present or absent; if present, it is G, where G is glycine; Xb is G, where G is the same as above; and X1-X 12 This is equivalent to the above.

[0018] Therefore, the antigen peptide of the present invention is typically 11 to 20 amino acids in length, preferably 12 to 14 amino acids in length (i.e., 12, 13, or 14 amino acids). The antigen peptide is particularly preferably 12 or 14 amino acids in length. The antigen peptide of the present invention elicits an antibody response in the absence of a T cell response. Therefore, the antigen peptide of the present invention itself typically does not contain a T cell epitope, particularly a cytotoxic T cell epitope.

[0019] As further described herein, the antigen peptides of the present invention are typically used in the form of immunogenic compounds conjugated to a carrier. To facilitate the binding process, the antigen peptides of the present invention may further include an amino acid that acts as a binding site. Typically, this amino acid is a terminal amino acid, preferably located at the N-terminus. In preferred embodiments, the antigen peptide further includes a terminal cysteine ​​residue, preferably an N-terminal cysteine ​​residue.

[0020] In some embodiments, X1 is L, S, or K, X2 is E or S, X3 is S, D, E, A, K, or N, X4 is M, X 10 is N and / or X 12 X is A, S, K or V, preferably X1 is L or K, X2 is E, X3 is S, D, E, K or A, X4 is M, X 10 is N and / or X 12 is A, S or K, and in particular X1 is L or K, X3 is D, K or S, X 12This is A. In these embodiments, it is preferable that the other positions of the peptide are wild-type α-synuclein amino acids.

[0021] In some embodiments, X1 is A, S, or K. In some embodiments, X2 is S. In some embodiments, X3 is A, S, E, K, or N. In some embodiments, X4 is A, S, L, or K. In some embodiments, X5 is A. In some embodiments, X6 is A or S. In some embodiments, X7 is S. In some embodiments, X9 is A. In some embodiments, X 10 is A or S, preferably S. In some embodiments, X 11 is A or S. In some embodiments, X 12 This is S, V, G, or K. According to these embodiments, it is preferable that the antigen peptide has up to one, two, or three additional mutations compared to the natural LEDMPVDPDNEA sequence. In some embodiments, the antigen peptide does not have any additional mutations compared to the natural LEDMPVDPDNEA sequence.

[0022] In some embodiments, the antigen peptide is AEDMPVPDPDNEA, KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, SEKMPVDPDNEA LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES, LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, LEKMPVDPDNES, LENMPVDPDNEA, KESMPVDPDNEK, and KEDMPVDPDNEA, preferably selected from the group consisting of SEDMPVDPDNEA, SEKMPVDPDNEA, LEEMPVDPDNEA, LEKMPVDPDNEK, LESMPVDPDNEA, LESMPVDPDNES, KESMPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNES, LEKMPVDPDNEA, and LESMPVDPDNES, in particular from the group consisting of LEKMPVDPDNEA, KESMPVDPDNEK, KESMPVDPDNEA, and KEDMPVDPDNEA. In preferred embodiments, the antigen peptide comprises, essentially consists of, or comprises the amino acid sequences KESMPVDPDNEA, GKESMPVDPDNEA, GGKESMPVDPDNEA, or CGGKESMPVDPDNEA.

[0023] The antigen peptide of the present invention is typically used in the form of an immunogenic compound conjugated to a carrier. The carrier functions as a source of T cell epitopes to enhance the immune response to the immunogenic peptide. Therefore, the present invention further provides an immunogenic compound comprising the antigen peptide of the present invention and a carrier containing T cell epitopes conjugated to the antigen peptide. Structure: carrier-X1-X2-X3-X4-PVDPDX 10 -EX 12 An immunogenic compound having the following characteristics is provided. Here, the support is a polypeptide support covalently bonded to X1, preferably the support molecule is peptide:X1-X2-X3-X4-PVDPDX 10 -EX 12 It includes a linker portion that is covalently bonded to it; D is aspartic acid, E is glutamic acid, P is proline, and V is valine; X1 is L, K, A, or S, where L is leucine, K is lysine, A is alanine, and S is serine; X2 is either E or S, where E and S are synonymous with those stated above; X3 is D, E, K, N, A, or S, where N is asparagine, and D, E, K, A, and S are as defined above; X4 is M, A, S, L, or K, where M is methionine, and A, S, L, and K are as defined above; X 10 is N, S, or A, where N, S, and A are synonymous with those stated above; X 12 is either present or absent, and if present, is A, K, V, S, or G, where G is glycine, and A, K, V, and S are synonymous with the foregoing; However, X1-E-X3-X4-PVDPDX 10 -EX 12 It is not LEDMPVDPDNEA.

[0024] The compounds of the present invention contain peptides (antigen peptides) capable of inducing a potent anti-aSyn antibody response. The induced antibodies exhibit high cross-reactivity with human aSyn, although these peptides have sequences different from the natural sequence (LEDMPVDPDNEA). A superior immune response (i.e., targeting the same natural structure) is achieved with the peptides of the present invention compared to the response with the natural sequence, and a highly effective and appropriate antibody response can be induced in vaccinated individuals. Antibodies induced by vaccination with the compounds of the present invention bind with high selectivity and specificity to aggregated toxic aSyn species and Lewy bodies in pathological human brain tissue.

[0025] It has been previously demonstrated that immunogenic peptides containing a non-natural amino acid sequence of aSyn can be provided to induce a specific immune response to aSyn with improved cross-reactivity to bSyn (WO 2009 / 103105 A1, WO 2011 / 020133 A1). Surprisingly, it has been demonstrated that improved peptides containing a non-natural aSyn amino acid sequence can be generated that, in addition to inducing a specific immune response to aSyn, exhibit increased immunogenicity and can induce antibodies with higher cross-reactivity than the peptides described in WO 2009 / 103105 A1 and WO 2011 / 020133 A1 (see examples below).

[0026] The preferred immunogenic compounds according to the present invention include the preferred peptides of the present invention, where X1 is L, S, or K, X2 is E or S, X3 is S, D, E, A, K, or N, X4 is M, and X 10 is N and / or X 12 is A, S, K, or V. Other preferred immunogenic compounds according to the present invention include preferred peptides, where X1 is L, S, or K, X2 is E, X3 is S, D, E, K, or A, X4 is M, and X 10 is N and / or X 12 is A, S, or K. Another preferred embodiment is a peptide according to the present invention, where X1 is L or K, X3 is D, K, or S, and X 12 A is the answer.

[0027] In a preferred embodiment, a carrier containing a T cell epitope is bound to an antigen peptide via a linker. Thus, the antigen peptide of the present invention bound to a linker is also provided. As will be readily apparent to those skilled in the art, any suitable linker can be used. The linker may be a chemical linker or a peptide (amino acid-based) linker. The linker may contain a reactive functional group that enables crosslinking of the antigen peptide antigen to the carrier via a suitable chemical reaction. Thus, the linker may contain two reactive groups. The first typically binds to the carrier (protein) via a reactive amino acid side chain, such as by reaction with a primary amine (e.g., on a lysine residue). Thus, typically an amide bond is formed. The second, again typically, binds to the antigen peptide via a reactive amino acid side chain, such as a sulfhydryl group (e.g., on a cysteine ​​residue). Thus, typically a thioether bond is formed. Thus, a preferred linker is a heterobifunctional linker, particularly containing an amine reactive group such as N-hydroxysuccinimide (NHS) ester and a sulfhydryl reactive group such as maleimide. Therefore, the linker may contain sGMBS (=sulfoGMBS)-(maleimidobutyryloxy)sulfosuccinimide ester, GMBS-maleimidobutyryloxysuccinimide ester, succinimidyl 3-(bromoacetamide)propionate (SBAP), or succinimidyl 6-(N-maleimido)-n-hexanoate (MHS).Other linkers that may be used include sEMCS (=sulfoEMCS)-N-(ε-maleimidocaproyloxy)sulfosuccinimide ester, MBS-m-maleimidobenzoyl-N-hydroxysuccinimide ester, sMBS (=sulfoMBS)-(m-maleimidobenzoyl-N-hydroxy)sulfosuccinimide ester, iodoacetamide-(PEG)2-maleimide (=N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)-3-(2-( Examples include 2-iodoacetamide)ethoxy)propanamide) and iodoacetamide-(PEG)-tri(maleimide)(=3,3'-((2-((3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-3-oxopropoxy)methyl)-2-(2-iodoacetamide)propan-1,3-diyl)bis(oxy))bis(N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)propanamide)). The linker can bind the antigen peptide to a specific amino acid residue or its side chain contained within the carrier (protein). Thus, in some embodiments, the antigen peptide is bound to a lysine residue contained within the carrier (protein) (via a primary amine group). Binding via a histidine residue is also possible. It should be noted that the antigen peptide may be provided in association with the remaining larger peptide molecule, which is not derived from the amino acid sequence of α-synuclein, in order to provide a linker or to facilitate binding. For example, the peptide may contain additional residues, such as one or more cysteine ​​residues, with or without a spacer such as polyenglycol (PEG), to facilitate binding to a carrier (protein). These additional residues are typically found at the N and / or C terminus of the antigen peptide, preferably at the N terminus.In this context, the term “essentially consisting of” may mean that the antigen peptide of the present invention comprises 11 to 20, preferably 12 to 14, consecutive amino acids derived from the α-synuclein sequence (amino acids 111-124 or 113-124, subject to at least one (and up to four) mutations as defined herein), but may also include a limited number of additional residues, such as additional cysteine ​​residues, to facilitate binding to a carrier protein, with or without spacers such as PEG or amino acid-based spacers.

[0028] According to preferred embodiments of the present invention, the linker portion of the immunogenic compound preferably comprises at least one cysteine ​​and / or glycine amino acid residue, linked to the polypeptide carrier portion by a chemical linker. Providing an amino acid linker at the N-terminus of the peptide (e.g., a dodecamer) of the present invention offers many advantages for coupling larger compounds (as carriers) and for inducing a stronger immune response; however, such linkers, particularly amino acid linkers, are not essential to the present invention.

[0029] Preferred amino acid linkers include glycine and cysteine ​​(or combinations thereof such as CG-, CGG-, CCG-, GC-, GGC-, GCC-, GG-, GGG-), as well as isoleucine, alanine, valine, leucine, serine, glutamic acid, aspartic acid, lysine, asparagine, and glutamine. Amino acid linkers may also consist of more than one amino acid residue, such as two, three, four, or five amino acid residues. In particular, when the amino acid linker is longer than five amino acid residues, and when the antigen peptide is directly bound to the carrier protein via peptide bonds, it is preferable to exclude certain amino acid residues within the peptide (amino acid-based) linker due to stability / folding issues. Therefore, if the amino acid linker (or amino acid sequence extending from X1 to the N-terminus) is longer than 5 amino acid residues, it is preferable that the linker does not contain an amino acid selected from the group consisting of proline, arginine, or histidine within the N-terminus of the region (of the linker) relative to X1, i.e., within the first 5 amino acids of the amino acid linker molecule starting from the amino acid that is N-terminus relative to X1.

[0030] Peptide linkers preferred for use in the present invention are those that do not form T cell epitopes. This can be evaluated using known methods, including computer-based methods, such as referring to the SYFPEITHI database of MHC ligands and peptide motifs (http: / / www.syfpeithi.de / ).

[0031] Alternatively, or in addition to an amino acid linker, the carrier preferably includes a chemical linking group (or a chemical component resulting from a chemical linking process) in addition to the polypeptide carrier. Preferred linking groups can be obtained using heterobifunctional compounds, such as chemical linkers such as GMBS or sulfo-GMBS. All chemical linkers known and used in the art, particularly those used to manufacture products administered to human subjects, may be used to provide the linkage of the carrier to the peptide of the present invention. Particularly preferred chemical linkers are those that link to the antigen peptide and carrier of the present invention via a non-peptide bond. Such non-peptide bond linkages are particularly advantageous with respect to their immunogenicity, stabilization, and / or manufacturing properties. Preferably, the linker portion is formed by NHS-poly(ethylene oxide) (PEO) (e.g., NHS-PEO4-maleimide) or other compounds used in biochemical techniques.

[0032] Particularly preferred immunogenic compounds according to the present invention are KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES, LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, KEDMPVDPDNEA, LENMPVDPDNEA, KESMPVDPDNEK, and KEDMPVDPDNEA, preferably SEDMPVDPDNEA, LEEMPVDPDNEA, LESMPVDPDNEA, KESMPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNEA, and LESMPVDPDNES, and especially preferred peptides X1-X2-X3-X4-PVDPDX selected from the group consisting of LEKMPVDPDNEA, KESMPVDPDNEA, and KEDMPVDPDNEA. 10 -EX 12 Includes.

[0033] The carrier functions as a source of T cell epitopes and therefore typically contains multiple T cell epitopes. The T cell epitopes are preferably universal cellular epitopes. A “universal” cellular epitope means an epitope specific to T cells present in the majority of the human population. The “universal” ability of a T cell epitope to activate T cells is a result of at least two complementary properties: i) affinity for binding to the HLA groove, meaning the strength of binding; and ii) the ability to indiscriminately bind to different HLA haplotypes, meaning the ability to cover a highly diverse human population with respect to differences in HLA molecule expression. Universal T cell epitopes can bind to the majority of MHC class II alleles present in the human population. Therefore, T cell epitopes in a carrier may be able to stimulate a CD4 T cell response. Therefore, T cell epitopes in a carrier may be able to stimulate a helper T cell response that enhances (antigen peptide-specific) antibody production by B cells.

[0034] According to preferred embodiments, the immunogenic compound according to the present invention comprises a pharmaceutically acceptable polypeptide carrier molecule, a carrier protein. The carrier protein may be selected from the group consisting of diphtheria toxin (DT) and its variants, particularly CRM197 (cross-reactant 197), keyhole limpet hemocyanin (KLH), tetanus toxoid, thermolabile enterotoxin (LT), cholera toxin (CT), tetanus toxoid (TT), mutagen toxin, albumin-binding protein, bovine serum albumin, and synthetically derived fusion peptides (e.g., Tet or PADRE) containing multiple T cell epitopes. Further carrier proteins that can be used include Pseudomonas exotoxin A (EPA), Haemophilus influenzae protein D (HiD), or meningococcal outer membrane protein conjugate (OMPC). CRM197 is a particularly preferred carrier protein.

[0035] Non-polypeptide carriers may also be included in the immunogenic compounds of the present invention. Examples include poly(lactic acid-co-glycolic acid) nanoparticles (PLG nanoparticles), poloxamer particles, virus-like particles, and dendrimers. Further carriers may include nanoparticles or liposomes.

[0036] The immunogenic compounds and antigenic peptides of the present invention are preferably used in therapeutic and prophylactic methods for treating human patients, particularly in the treatment or prevention of synucleinopathy. Preferred synucleinopathy for the treatment or prevention of synucleinopathy is Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and Lewy body dementia (DLB), as well as multiple system atrophy (MSA) or neurodegenerative type I with iron deposition in the brain (NBIA type I).

[0037] In another aspect of the present invention, the present invention relates to a pharmaceutical formulation comprising an immunogenic compound or antigenic peptide and a pharmaceutically acceptable excipient (sometimes interchangeably called a carrier). The term “excipient” encompasses any component other than the immunogenic compound present in the final formulation for administration. The pharmaceutical formulation is preferably intended for use as a vaccine in the treatment or prevention of synucleinopathy selected from the group consisting of synucleinopathy, preferably Lewy body disease (LBD), particularly Parkinson’s disease (PD), Parkinson’s disease with dementia (PDD) and Lewy body dementia (DLB), and multiple system atrophy (MSA) or neurodegenerative type I with iron deposition in the brain (NBIA type I).

[0038] The pharmaceutical formulation according to the present invention is preferably formulated as a vaccine. The pharmaceutical formulation, preferably a vaccine, contains an adjuvant, preferably aluminum phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligos, IC31, LPS, monophosphoryl hexaacyl lipid A ((MPLA)), polyphosphazene and aluminum hydroxide, or mixtures thereof, particularly aluminum hydroxide as an adjuvant; It may be formulated with an adjuvant selected from the group comprising the following. The purpose of the adjuvant is to increase or stimulate the immune response of the target. In some embodiments, at least one adjuvant forms part of the carrier. Other adjuvants that can be used according to the present invention include aluminum-containing adjuvants, in particular aluminum hydroxide (Alum), and imidazoquinolineamines and / or CpGs (synthetic oligodeoxynucleotides (ODNs) containing an unmethylated CpG motif), such as reximod (R-848) in particular. The adjuvant may be a Toll-like receptor (TLR) agonist.

[0039] Typically, a pharmaceutical formulation according to the present invention, particularly when formulated as a vaccine, contains 0.1 ng to 10 mg, preferably 10 ng to 1 mg, particularly 1 μg to 500 μg, of the immunogenic compound according to the present invention (or the peptide according to the present invention conjugated to an optional carrier), or alternatively, for example, 100 fmol to 10 μmol, preferably 10 pmol to 1 μmol, particularly 1 nmol to 500 nmol. In some embodiments, the amount of peptide may be 100 pmol to 100 nmol. In this specification, amounts indicate the peptide component of the composition.

[0040] Typically, pharmaceutical formulations, particularly vaccines, may contain excipients, such as buffers and stabilizers. Preferably, such excipients, pharmaceutically acceptable excipients such as water, buffers, and / or stabilizers, are present in amounts of 1 to 99% (by weight), more preferably 5 to 80% (by weight), and particularly 10 to 70% (by weight). Preferably, the pharmaceutical formulations according to the present invention are formulated as liposomes, wirosoms, iscoms, cocreates, or emulsions.

[0041] The antigenic peptides, immunogenic compounds, and pharmaceutical formulations of the present invention may be administered according to any appropriate schedule. They may be administered according to a prime-boost vaccination strategy. A prime-boost vaccination strategy involves multiple vaccinations, which are intended to enhance the effect of the vaccine. Generally, the same vaccine composition is administered each time; this is a so-called homologous prime-boost vaccination plan. Possible administration plans for the initial priming phase of a pharmaceutical formulation, in addition to other schemes already proposed for other vaccines, include 2-5 treatments, particularly 3-4, every two weeks up to a maximum of 4 months, with the first priming vaccine administration (at 1-5 months), followed by a booster or maintenance vaccination 3-12 months or several years later.

[0042] In absolute terms, it is preferable to use an amount of at least 10 μg, preferably at least 50 μg, of the peptide (as antigen) of the present invention. In this regard, it is important to note that "(peptide as antigen) μg" as referred to in the present invention means the amount of antigen peptide in the dose and does not include the carrier or linker portion of the vaccine conjugate (immunogenic compound, if present). Therefore, a preferred amount of antigen is at least 5 nmol, preferably at least 25 nmol.

[0043] Preferably, pharmaceutical formulations containing immunogenic compounds or antigenic peptides are formulated for parenteral administration. In certain embodiments, the formulations are formulated for subcutaneous, intradermal, or intramuscular administration. Intravenous administration may also be used.

[0044] The vaccination strategy of the present invention preferably follows a conventional vaccination strategy. In a preferred embodiment, an autoantigen vaccination strategy according to WO 2017 / 076873 A1 is used, i.e., inducing a primary immune response in the patient, followed by a booster or maintenance dose in the patient.

[0045] Preferably, booster / maintenance vaccinations are administered after the primary immunization response has been completed, i.e., when the antibody titer induced by the primary vaccination (induced by one, two, three, four, or more vaccine doses during the primary immunization response) has fallen below a certain level (e.g., above a predetermined threshold level in an assay suitable for testing a large number of samples), or when it has fallen to at least 30%, preferably less than 50%, and especially less than 80%, of the maximum antibody level present during the primary vaccination process. To maintain such high levels, it may be beneficial to administer booster / maintenance injections every 3 to 12 months after initial (primary) immunization.

[0046] Preferably, the amount of antigen in the booster / maintenance dose is at least 20%, preferably at least 50%, more preferably at least 100%, and particularly at least 200%, which is greater than the amount used in the dose for the primary immune response. In certain embodiments, the amount of antigen in the booster / maintenance dose is at least 300%, preferably at least 400%, more preferably at least 500%, and particularly at least 600%, which is greater than the amount used in the dose for the primary immune response. However, in some embodiments, the same composition is administered each time. Therefore, in some embodiments, the amount of antigen administered is the same each time (within manufacturing tolerances).

[0047] In a more preferred embodiment, the booster / maintenance dose is repeated after some time, for example, after 1, 2, 3, 5, or 10 years. Preferably, the second and subsequent booster / maintenance doses are administered in the same or similar manner as the first booster / maintenance dose, i.e., with an increased amount of antigen than the initial dose, or using the same amount of antigen.

[0048] The administration routes according to the present invention are typically the same as those used for current vaccinations. Therefore, preferred administration of the immunogenic compounds or antigenic peptides, or pharmaceutical formulations, according to the present invention is parenteral administration, such as subcutaneous, intradermal, or intramuscular. However, the immunogenic compounds or antigenic peptides, or pharmaceutical formulations of the present invention, can be administered to a target by any suitable route of administration. As those skilled in the art will recognize, such compositions (preferably vaccine compositions) can be administered via topical, oral, rectal, nasal, or parenteral (such as intravenous, intradermal, subcutaneous, or intramuscular) routes. Furthermore, the compositions can be incorporated into a sustained-release matrix, such as a biodegradable polymer, which is implanted near or very close to the site where delivery is desired. However, in preferred embodiments, the compositions are administered intramuscularly or subcutaneously.

[0049] According to a preferred embodiment of the present invention, an immunogenic compound or antigenic peptide, or a pharmaceutical formulation, is administered, preferably in the form of a vaccine, together with an adjuvant, preferably aluminum oxyhydroxide. According to this most preferred embodiment, the present invention relates to the use of European Pharmacopoeia grade (aluminum oxyhydroxide, monograph 1664), and more specifically, to a product (2% Alhydrogel) manufactured by Brenntag Biosector that has been tested for EP compliance. Three types of Alhydrogel are available: Alhydrogel 1.3%, Alhydrogel 2%, and Alhydrogel "85". Alhydrogel 2% was selected as the international standard formulation of aluminum hydroxide gel. The pharmaceutical formulations according to the present invention are aseptically formulated in a suitable buffer, preferably isotonic phosphate buffer (1 mM to 100 mM), in an alhydrogel (given as an Al2O3 equivalent) at a concentration of preferably 1.0 mg / ml or higher; this criterion (Al as an Al2O3 equivalent) is commonly used in the present invention; therefore, all doses and amounts referred to in this application, as far as aluminum oxyhydroxide is concerned, mean the equivalent (of aluminum oxyhydroxide (alhydrogel)), more preferably at a concentration of 1.5 mg / ml or higher, and more preferably at a concentration of 2.0 mg / ml or higher, of alhydrogel (given as an Al2O3 equivalent). The amount of aluminum salt in the alhydrogel is given as an Al2O3 equivalent according to the strength specified by the manufacturer (i.e., 2% alhydrogel corresponds to 2% Al2O3, i.e., 20 mg / mL). These concentrations can be directly converted to their respective aluminum concentrations using their respective molecular weights (20 mg / mL Al2O3 (Mw 101,96) corresponds to 10.6 mg / mL aluminum (molecular weight 26,98)).

[0050] The carrier of the present invention optionally has a linker connected to the antigen peptide of the present invention (X1 to X 12 It may be any suitable and pharmaceutically acceptable carrier portion having a linker for binding with (including).

[0051] According to a preferred embodiment of the present invention, the antigen peptide (which may be a dodecamer peptide) is bound to at least one pharmaceutically acceptable polypeptide carrier, preferably CRM197 (cross-reactive substance 197), KLH (keyhole limpet hemocyanin) (KLH), tetanus toxoid, albumin-binding protein, bovine serum albumin, or a synthetically derived fusion peptide comprising multiple T cell epitopes. Further carriers, or carrier or linker portions within a carrier, include dendrimers (MAP; Biol. Chem. 358: 581), peptide linkers (or flanking regions), and adjuvant substances or mixtures thereof as described in Singh et al., Nat. Biotech. 17 (1999), 1075-1081 (in particular those listed in Table 1 of that document), and O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727-735 (in particular the endogenous immunoenhancing compounds and delivery systems described therein) and others. Conjugation chemistry in this context (e.g., heterobifunctional compounds such as GMBS, and, of course, others as described in "Bioconjugate Techniques," Greg T. Hermanson) can be selected from reactions known to those skilled in the art.

[0052] Furthermore, vaccine compositions comprising immunogenic compounds or antigenic peptides according to the present invention may be formulated together with adjuvants, preferably low-solubility aluminum compositions, particularly aluminum hydroxide. Naturally, adjuvants such as MF59, aluminum phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligos, IC31, LPS, MPLA (including MPL), polyphosphazenes, emulsions (e.g., Freund's, SAF), liposomes, virosomes, ISCOM, COCREATE, PLG microparticles, poloxamer particles, virus-like particles, thermolabile enterotoxins (LT), cholera toxin (CT), diphtheria toxin (DT), tetanus toxoid (TT), mutagens (e.g., LTK63, LTR72), microparticles, liposomes and / or polymerized liposomes may also be used.

[0053] The peptide or polypeptide of the present invention is preferably conjugated to a carrier or adjuvant via a linker that is NHS-poly(ethylene oxide) (PEO) (e.g., NHS-PEO4-maleimide). Alternatives are as described above.

[0054] The carrier preferably comprises a toxoid protein. The toxoid protein may be a naturally occurring toxoid protein or a recombinant variant thereof used in pharmaceutical compositions. The toxin can be inactivated, for example, by treatment with formaldehyde, glutaraldehyde, UDP-dialdehyde, peroxides, oxygen, or by mutation (e.g., using recombinant methods). Mutant diphtheria toxins with reduced toxicity can also be produced using recombinant methods.

[0055] DT is a diphtheria toxin cross-reactant (DT-CRM) or diphtheria toxoid. DT-CRMs represent mutant diphtheria toxins that no longer possess sufficient ADP-ribosyl due to mutation or chemical modification, for example. Non-limiting examples of DT-CRMs include DT-CRM30, DT-CRM45, DT-CRM176, DT-CRM197, and DT-CRM228. Diphtheria toxoids are formaldehyde-inactivated diphtheria toxins. DTs are commercially available or can be prepared by methods known in the art, such as recombinant DNA technology.

[0056] CRM197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin that retains the immunological characteristics of wild-type diphtheria toxin. CRM197 differs from wild-type diphtheria toxin in one base of a structural gene that results in a single amino acid substitution from glutamate to glycine. CRM197 is typically isolated from cultures of Corynebacterium diphtheria strain C7(P197) grown in casamino acid and yeast extract-based media. CRM197 can be purified by ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography. Alternatively, CRM197 can be prepared by recombinant DNA. CRM197 is a Hibtiter TM It is used in the design of complex carbohydrate vaccines such as Menveo® or Prevnar®.

[0057] Tetanus toxoids are manufactured and used worldwide for mass vaccination against tetanus (or trismus) caused by Clostridium tetani. Tetanus toxoids are used alone or in combination with diphtheria and / or pertussis vaccines. The parent protein, tetanus toxin, is typically obtained from a culture of Clostridium tetani. Tetanus toxin is a protein of approximately 150 kDa and consists of two subunits (approximately 100 kDa and 50 kDa) linked by a sulfide bond. The toxin is typically detoxified with formaldehyde and can be purified from the culture filtrate using known methods such as ammonium sulfate precipitation or chromatography. Tetanus toxin can also be inactivated by recombinant genetic means. Tetanus toxoids are also used as carrier proteins in other vaccines, including pneumococcal conjugate vaccines. Mixed carriers can also be used, for example, a pneumococcal conjugate vaccine combined with CRM197, a serotype 3 vaccine combined with a tetanus toxoid carrier, or a serotype 3 vaccine conjugated with diphtheria toxoid.

[0058] The peptides of the present invention are variants of the natural human aSyn sequence, i.e., AFFITOPE® or VARIOTOPE® (peptides containing sequence mutations compared to the original native aSyn sequence, but exhibiting similar (same or improved) immunological properties, i.e., peptides capable of inducing an immune response equivalent to or better than that obtained with the native aSyn sequence). At the same time, AFFITOPE® is designed not to induce a cytotoxic response or a helper T cell response, and the first is CD8 having a linear sequence fragment of the immunizing peptide. +The first step is to evade cytotoxic attacks on tissues reachable by T cells, and the second is to evade responses to target-derived peptides independently of the vaccine, thereby avoiding the generation of a perpetually renewed and uncontrolled immune response. This AFFITOPE® or VARIOTOPE® technology is designed to (i) break down resistance to self-proteins, (ii) generate a high-titer antibody response against the peptide portion of the vaccine that cross-reacts with the natural target protein epitope, and (iii) avoid inducing an autoimmune response.

[0059] Therefore, in the process of the present invention, the length of the peptide is preferably 12aa(X1 to X 12 The antigen peptides of the present invention are typically 11 to 20 amino acids long, preferably 12 to 14 amino acids long. The antigen peptides are particularly preferably 12 or 14 amino acids long. These typically contain 1 to 4 amino acid mutations, preferably 2 or 3, compared to the natural alpha-synuclein sequence. However, it should be noted that amino acid elongations not based on (or identical to) the alpha-synuclein sequence may be permissible, particularly at the N-terminus of the peptide. Such additional amino acids may, for example, form part of a linker. Linkers (amino acids, or peptide, linker, etc.) may be present that covalently bond the peptide to other molecular parts, such as a carrier. The linker may contain additional amino acids, preferably amino acids with uncharged side chains, such as glycine. Linkers can be attached to any position of the immunogenic peptide, as long as the immunogenicity of the peptide is not significantly impaired. For example, the linker may be at the N-terminus or C-terminus aa(X1 or X 12Linking can occur via a ) or within the peptide. However, when the peptide of the present invention is applied as an immunogenic composition, it is preferable to link the peptide of the present invention to the carrier via the N-terminus of peptide (X1). When the peptide of the present invention is used for other purposes, such as a preparation portion (e.g., in the process of antibody purification) or a diagnostic probe (e.g., for detecting antibodies in a human sample), the linker and carrier can be more diverse, including linking the peptide of the present invention to a surface, i.e., a solid surface.

[0060] Therefore, the peptides of the present invention can also be used in a variety of assays and kits, particularly immunological assays and kits. Accordingly, the peptides of the present invention are particularly preferably part of another peptide or polypeptide, for example, they can be fused or bound to an enzyme used as a reporter in an immunological assay. Such reporter enzymes include, for example, a fluorescent moiety such as green fluorescent protein (GFP), a phosphatase such as alkaline phosphatase, or an oxidase / reductase such as horseradish peroxidase.

[0061] In a particular embodiment, the present invention has a structure: X1-X2-X3-X4-PVDPDX 10 -EX 12 Regarding antigen peptides having, Here, D is aspartic acid, E is glutamic acid, P is proline, and V is valine; X1 is L, K, A, or S, where L is leucine, K is lysine, A is alanine, and S is serine; X2 is either E or S, where E and S are synonymous with those stated above; X3 is D, E, K, N, A, or S, where N is asparagine, and D, E, K, A, and S are as defined above; X4 is M, A, S, L, or K, where M is methionine, and A, S, L, and K are as defined above; X 10 is N, S, or A, where N, S, and A are synonymous with those stated above; X 12 is either present or absent, and if present, is A, K, V, S, or G, where G is glycine, and A, K, V, and S are synonymous with the foregoing; However, X1-X2-X3-X4-PVDPDX 10 -EX 12 It is not LEDMPVDPDNEA.

[0062] In case of doubt, the term “having structure” should be understood as “consisting of” a given amino acid residues (i.e., excluding further amino acid residues such as the C-terminus of the antigen peptide). Minor modifications such as amidation, esterification, formylation, acetylation, and other chemical substitutions of the free C-terminus (or N-terminus) of the peptide or its side chain are not excluded, but the absence of such modifications is preferred. Such minor modifications fall within the scope of the term “essentially consisting of” as used herein. These 11-mer or 12-mer peptides according to the present invention ((the (antigen) peptides of the present invention); X1 to X 12 These compositions (etc.) can be provided as pharmaceutical compositions suitable for intended use for the prevention and / or treatment of synucleinopathy, and in particular, preferably in combination with a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be administered to patients in need in an amount effective to achieve preventive and / or therapeutic effects.

[0063] In one embodiment, the peptide according to the present invention is AEDMPVPDPDNEA, LEAMPVDPDNEA, LEDAPVDPDNEA, LEDMAVDPDNEA, LEDMPADPDNEA, LEDMPVDPANEA, LEDMPVDPDNAA, SEDMPVDPDNEA, LSDMPVDPDNEA, LESMPVDPDNEA, LEDSPVDPDNEA, LEDMPSDPDNEA, LEDMPVSPDNEA, LEDMPVDPDSEA, LEDMPVDPDNSA, LEDMPVDPDNES, LEEMPVDPDNEA, LEKMPVDPDNEA, LENMPVDPDNEA, LEDKPVDPDNEA, LEDMPVDPDNEV, LEDMP VDPDNEG, LEDMPVDPDNEK, LESMPVDPDNES, SESMPVDPDNEA, LESSPVDPDNEA, SEDMPVDPDNES, LEDSPVDPDNES, SEDSPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNES, SEKMPVDPDNEA, LEKMPVDPDNEK, KESMPVDPDNEA, KESMPVDPDNEK, preferably selected from the group consisting of KEDMPVDPDNEA, LEKMPVDPDNEA, LESMPVDPDNES, LEKMPVDPDNES, SEKMPVDPDNEA, LEKMPVDPDNEK, KESMPVDPDNEA, KESMPVDPDNEK.

[0064] Preferably, the peptide according to the present invention is selected from the group consisting of KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES, LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, KEDMPVDPDNEA, LENMPVDPDNEA, KESMPVDPDNEK, and KEDMPVDPDNEA, preferably SEDMPVDPDNEA, LEEMPVDPDNEA, LESMPVDPDNEA, KESMPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNEA, and LESMPVDPDNES, in particular LEKMPVDPDNEA, KESMPVDPDNEA, and KEDMPVDPDNEA.

[0065] According to another aspect, the present invention also relates to the structure: Linker-X1-X2-X3-X4-PVDPDX 10 -EX 12 Regarding antigen peptides having an amino acid linker, Here, X1, X2, X3, X4, X 10 , and X 12 This is equivalent to the above, where the amino acid linker contains 1 to 5 amino acid residues.

[0066] According to a preferred embodiment, the peptide having an amino acid linker according to the present invention comprises a linker, wherein the amino acid residue in the linker is selected from the group consisting of glycine, cysteine, isoleucine, alanine, valine, leucine, serine, glutamic acid, aspartic acid, lysine, asparagine, glutamine and combinations thereof, preferably, wherein the linker is selected from the group consisting of C-, G-, CG-, CGG-, CCG-, GC-, GGC-, GCC-, GG-, and GGG-; in particular, having an amino acid linker The peptides are from the following group: GGKESMPVDPDNEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GGCKESMPVDPDNEA, CCGKESMPVDPDNEA, CCGCKESMPVDPDNEA, CCCKESMPVDPDNEA, CCKESMPVDPDNEA, CGKESMPVDPDNEA, GCKESMPVDPDNEA, CKESMPVDPDNEA, GGKESMPVDPDNEK, GGKEDMPVDPDNEA GGKESMPVDPDNEA, GGKEDMPVDPDNEA, CLESMPVDPDNEA, CLESMPVDPDNES, CSEDMPVDPDNEA, CLEEMPVDPDNEA, CSESMPVDPDNEA, CLEDMPVDPDNEA, CLEAMPVD PDNEA, CLEDMPVDPDNEA, CLEDMPVDPDNEV, CGGKESMPVDPDNEA, CLEKMPVDPDNEA, CLSDMPVDPDNEA, CLEKMPVDPDNEA, CKEDMPVDPDNEA, CLENMPVDPDNEA, CGG KESMPVDPDNEK, and CGGKEDMPVDPDNEA, in particular selected from GGKESMPVDPDNEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GGCKESMPVDPDNEA, CCGKESMPVDPDNEA, CGCK-ESMPVDPDNEA, CCCKESMPVDPDNEA, CCKESMPVDPDNEA, CGKESMPVDPDNEA, GCKESMPVDPDNEA, and CKESMPVDPDNEA.

[0067] Preferably, according to all relevant aspects of the present invention, (a)X 12 There is a further amino acid residue X 12 It does not exist at the C-terminus; or (b)X 12 It is not present, and the further amino acid residue is X 11 It is not present at the C-terminus.

[0068] According to these particularly preferred embodiments, the antigen peptides of the present invention do not have further elongation at the C-terminus (except for the presence of some modifications that can stabilize the compound or peptide, such as amidation). In any case, amino acid elongation should not be present at the C-terminus, specifically at Tyr125 and Glu126, which represent the native amino acid sequence of aSyn, that can bind with high affinity to various allele variants of MHCI and thus may be a potential cytotoxic T cell epitope. Therefore, no Y amino acid residue or YE dipeptide chain should be present at the C-terminus of the peptide, where Y is tyrosine and E is as defined above.

[0069] The peptides and compounds of the present invention can be synthesized by chemical synthesis methods well known in the art, either as isolated peptides or as part of another polypeptide. Alternatively, the peptides and compounds may be produced by a peptide-producing microorganism of the present invention, subsequently isolated, and further purified as necessary. The peptides and compounds can be produced by microorganisms such as bacteria, yeasts, or fungi, eukaryotic cells such as mammalian or insect cells, or recombinant viral vectors such as adenoviruses, poxviruses, herpesviruses, Semryki forest virus, baculoviruses, bacteriophages, Sindbis virus, or Sendai virus. Suitable bacteria for producing peptides and compounds include Escherichia coli, Bacillus subtilis, or any other bacteria capable of expressing peptides such as peptide mimotopes. Suitable yeast species for expressing peptides and compounds include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida, Pichia pastris, or other yeasts capable of expressing peptides. Corresponding methods are well known in the art. Furthermore, methods for isolating and purifying recombinant peptides and compounds are well known in the art, and examples include gel filtration, affinity chromatography, and ion exchange chromatography.

[0070] To facilitate the isolation of peptides and compounds, fusion polypeptides can be created in which a dodecamer peptide is translationally fused (covalently bonded) to a heterologous polypeptide that enables isolation by affinity chromatography. Typical heterologous polypeptides include His-Tag (e.g., His6;6 histidine residue) and GST-Tag (glutathione-S-transferase). Fusion polypeptides not only facilitate the purification of peptides and compounds but can also prevent degradation during purification. If it is desirable to remove the heterologous polypeptide after purification, the fusion polypeptide can include a cleavage site, for example, at the binding site between the peptide mimotope and the heterologous polypeptide. The cleavage site consists of an amino acid sequence that is cleaved by an enzyme (e.g., a protease) specific to the amino acid sequence at that site.

[0071] The antigenic peptides, immunogenic compounds, or pharmaceutical formulations of the present invention are intended for therapeutic use (i.e., as agents). More specifically, the present invention provides the antigenic peptides, immunogenic compounds, or pharmaceutical formulations of the present invention for use in the treatment or prevention of synucleinopathy. The present invention also provides the use of the antigenic peptides, immunogenic compounds, or pharmaceutical formulations of the present invention for the manufacture of agents for the treatment or prevention of synucleinopathy. The present invention also provides a method for the treatment or prevention of synucleinopathy, comprising administering the antigenic peptides, immunogenic compounds, or pharmaceutical formulations of the present invention to a subject in need thereof. In certain embodiments according to all of these aspects, synucleinopathy is selected from the group consisting of Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and Lewy body dementia (DLB), and multiple system atrophy (MSA) or neurodegenerative type I with iron deposition in the brain (NBIA type I). In some embodiments of these aspects, the term synucleinopathy (or α-synucleinopathy) is used to describe diseases in which α-synuclein aggregates are detected, including primary synucleinopathy and associated conditions. Primary synucleinopathy includes Parkinson's disease (sporadic, familial with α-synuclein mutations, familial with non-α-synuclein mutations, pure autonomic dysfunction and Lewy body dysphagia), Lewy body disease (LBD; including Lewy body dementia (DLB)) ("pure" Lewy body dementia), Parkinsonian dementia (PDD), or diffuse Lewy body disease, and multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy).Furthermore, a-syn lesions can be detected as an associated condition in the following diseases: sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, inclusion body myositis, traumatic brain injury, chronic traumatic encephalopathy, Boxer dementia, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia with parkinsonism associated with chromosome 17 and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt syndrome. Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, amyotrophic lateral sclerosis (sporadic, familial, and Guam's ALS-dementia complex), axonal dystrophy, type I neurodegenerative disease with iron deposition in the brain (Harrelforden-Spatz syndrome), prion disease, Gerstmann-Straussler-Scheinker disease, ataxia with telangiectasia, Mage syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease, and other lysosomal storage disorders (including Kfor-Rakeb syndrome and Sanfilippo syndrome), or REM sleep behavior disorder.

[0072] According to all these embodiments, the target of treatment is a mammalian target, preferably human. The antigenic peptide, immunogenic compound, or pharmaceutical formulation of the present invention is administered in an amount effective to treat or prevent synucleinopathy. Prevention is preferably achieved using the vaccine composition of the present invention. Prevention includes delaying the onset and / or severity of the disease (compared to no administration) and completely preventing the disease. Treatment includes improving one or more symptoms of the disease, preventing or delaying the progression of the disease (compared to no administration) and completely curing the disease. Appropriate dosages and routes of administration are described herein, and variations may be determined empirically by a clinician.

[0073] The present invention may be further defined by the following numbered clauses: 1. Structure: Carrier-X1-X2-X3-X4-PVDPDX 10 -EX12 It is an immunogenic compound that has the following properties: Here, the support is a polypeptide support covalently bonded to X1, preferably the support molecule is peptide:X1-X2-X3-X4-PVDPDX 10 -EX 12 It includes a linker portion that is covalently bonded to it; D is aspartic acid, E is glutamic acid, P is proline, and V is valine; X1 is L, K, A, or S, where L is leucine, K is lysine, A is alanine, and S is serine; X2 is either E or S, where E and S are synonymous with those stated above; X3 is D, E, K, N, A, or S, where N is asparagine, and D, E, K, A, and S are as defined above; X4 is M, A, S, L, or K, where M is methionine, and A, S, L, and K are as defined above; X 10 is N, S, or A, where N, S, and A are synonymous with those stated above; X 12 is either present or absent, and if present, is A, K, V, S, or G, where G is glycine, and A, K, V, and S are synonymous with the foregoing; However, X1-E-X3-X4-PVDPDX 10 -EX 12 LEDMPVDPDNEA is not an immunogenic compound.

[0074] 2. X1 is L, S, or K, X2 is E or S, X3 is S, D, E, A, K, or N, X4 is M, X 10 However, N, and / or X 12 X is A, S, K, or V, preferably X1 is L or K, X2 is E, X3 is S, D, E, K, or A, X4 is M, X 10 However, N is and / or X12 However, X1 is A, S, or K, and in particular X1 is L or K, and X3 is D, K, or S, X 12 However, A is an immunogenic compound as described in item 1.

[0075] 3. The immunogenic compound according to claim 1 or 2, wherein the linker portion preferably comprises at least one cysteine ​​and / or glycine amino acid residue bonded to the polypeptide carrier portion by a chemical linker, and in particular the linker portion is formed by NHS-poly(ethylene oxide) (PEO) (for example, by NHS-PEO4-maleimide).

[0076] 4. Peptide: X1-X2-X3-X4-PVDPDX 10 -EX 12 The immunogenic compound described in any one of claims 1 to 3, selected from the group consisting of KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES, LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, LENMPVDPDNEA, KESMPVDPDNEK, and KEDMPVDPDNEA, preferably SEDMPVDPDNEA, LEEMPVDPDNEA, LESMPVDPDNEA, KESMPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNEA, and LESMPVDPDNES, particularly LEKMPVDPDNEA, KESMPVDPDNEA, and KEDMPVDPDNEA.

[0077] 5. Peptide: X1-X2-X3-X4-PVDPDX 10 -EX 12 However, the immunogenic compound is KESMPVDPDNEA, as described in any one of items 1 to 4.

[0078] 6. An immunogenic compound according to any one of items 1 to 5, wherein the polypeptide carrier portion is a pharmaceutically acceptable carrier molecule selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, thermolabile enterotoxin (LT), cholera toxin (CT), tetanus toxoid (TT), diphtheria toxin (DT) and its variants, in particular CRM197, tetanus toxoid (TT), mutagen, albumin-binding protein, and bovine serum albumin.

[0079] 7. For use in the treatment or prevention of synucleinopathy, preferably selected from the group consisting of Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and Lewy body dementia (DLB), and multiple system atrophy (MSA) or neurodegenerative type I with cerebral iron deposition (NBIA type I), an immunogenic compound according to any one of claims 1 to 6.

[0080] 8. A pharmaceutical formulation comprising a compound described in any one of claims 1 to 7 and a pharmaceutically acceptable carrier for use as a vaccine in the treatment or prevention of synucleinopathy selected from the group consisting of synucleinopathy, preferably Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and Lewy body dementia (DLB), and multiple system atrophy (MSA) or neurodegenerative type I with cerebral iron deposition (NBIA type I).

[0081] 9. The pharmaceutical formulation according to item 8, which is formulated as a vaccine, preferably with an adjuvant selected from the group consisting of MF59 aluminum phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligos, IC31, LPS, MPL, polyphosphazenes, and aluminum hydroxide, or mixtures thereof; in particular with aluminum hydroxide as the adjuvant.

[0082] 10. A pharmaceutical preparation according to item 8 or 9, comprising any one of items 1 to 10 in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, and particularly 100 ng to 100 μg.

[0083] 11. A pharmaceutical formulation prepared for subcutaneous, intradermal or intramuscular administration; and / or a pharmaceutical formulation prepared as a liposome, wirosome, iscom, cocreate, or emulsion, according to any one of paragraphs 8 to 10.

[0084] 12. Structure: X1-X2-X3-X4-PVDPDX 10 -EX 12 An antigen peptide having, Here, D is aspartic acid, E is glutamic acid, P is proline, V is valine; (C-) is cysteine, which is either present or absent; X1 is L, K, A, or S, where L is leucine, K is lysine, A is alanine, and S is serine; X2 is either E or S, where E and S are synonymous with those stated above; X3 is D, E, K, N, A, or S, where N is asparagine, and D, E, K, A, and S are as defined above; X4 is M, A, S, L, or K, where M is methionine, and A, S, L, and K are as defined above; X 10 is N, S, or A, where N, S, and A are synonymous with those stated above; X 12 is either present or absent, and if present, is A, K, V, S, or G, where G is glycine, and A, K, V, and S are synonymous with the foregoing; However, X1-E-X3-X4-PVDPDX 10 -EX 12 It is not LEDMPVDPDNEA; Antigenic peptide.

[0085] 13. KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES, LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, LENMPVDPDNEA, KESMPVDPDNEK and KEDMPVDPDNEA, preferably SEDMPVDPDNEA, LEEMPVDPDNEA, LESMPVDPDNEA, KESMPVDPDNEA, KEDMPVDPDNEA, LEKMPVDPDNEA and LESMPVDPDNES, particularly LEKMPVDPDNEA, KESMPVDPDNEA and KEDMPVDPDNEA The peptide according to item 12, selected from the group consisting of.

[0086] 14. Structure: Linker-X1-X2-X3-X4-P-V-D-P-D-X 10 -E-X 12 An antigenic peptide having an amino acid linker, having where X1, X2, X3, X4, X 10 , and X 12 are as defined in item 13, and where the amino acid linker contains 1 to 5 amino acid residues, the antigenic peptide.

[0087] 15. The amino acid residues in the linker are selected from the group consisting of glycine, cysteine, isoleucine, alanine, valine, leucine, serine, glutamic acid, aspartic acid, lysine, asparagine, glutamine, and combinations thereof, preferably, the linker is selected from the group consisting of C-, G-, CG-, CGG-, CCG-, GC-, GGC-, GCC-, GG-, and GGG-; in particular, the peptide having an amino acid linker is from the following group: GGKESMPVDPD NEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GGCKESMPVDPDNEA, CCGKESMPVDPDNEA, CCGKESMPVDP DNEA, CCCKESMPVDPDNEA, CCKESMPVDPDNEA, CGKESMPVDPDNEA, GCKESMPVDPDNEA, CKESMPVDPDNEA, GGKESMPVDPDNEA, GGKESMPVDPDNEA GGKESMPVDPDNEK, CLESMPVDPDNEA, CLESMPVDPDNES, CSEDMPVDPDNEA, CLEEMPVDPDNEA, CSESMPVDPDNEA, CLEDMPVDPDNES, CLEAMPVDPDNEA, CLEDMPVDPDNEK, CLEDMPVDPDNEV, CGGKESMPVDPDNEA, CLEKMPVDPDNEK, CLSDMPVDPDNEA, CLEKMPVDPDNEA, CKEDMPVDPDNEA, CLEENMPVDPDNEA, CGGKESMPVDPDNEK, and CGGK EDMPVDPDNEA, in particular, antigen peptides having the amino acid linkers described in item 14, selected from GGKESMPVDPDNEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GGGKESMPVDPDNEA, CCGKESMPVDPDNEA, CGCK-ESMPVDPDNEA, CCCKESMPVDPDNEA, CCKESMPVDPDNEA, CGKESMPVDPDNEA, GCKESMPVDPDNEA, and CKESMPVDPDNEA.

[0088] 16. An immunogenic compound according to any one of items 1 to 7, or a peptide having an amino acid linker according to item 14 or 15, wherein the linker moiety or amino acid linker does not contain an amino acid selected from the group consisting of proline, arginine, or histidine.

[0089] 17. A Y amino acid residue or a YE dipeptide chain is not present at the C-terminus of the peptide, where Y is tyrosine and E is the same as above; preferably, (a)X 12 There is a further amino acid residue X 12 It does not exist at the C-terminus; or (b)X 12 It is not present, and the further amino acid residue is X 11 It does not exist at the C-terminus; An immunogenic compound or peptide having an amino acid linker as described in item 16.

[0090] 18. Use of an immunogenic compound as described in any one of claims 1-7, 16 and 17, or an antigenic peptide as described in claim 12 or 13, or a peptide having an amino acid linker as described in claims 14-17, for the manufacture of a therapeutic or prophylactic agent for synucleinopathy, preferably selected from the group consisting of Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and Lewy body dementia (DLB), and multiple system atrophy (MSA) or neurodegenerative type I with cerebral iron deposition (NBIA type I).

[0091] 19. A method for treating or preventing synucleinopathy, preferably Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) or neurodegeneration type I with brain iron deposition (NBIA type I), the method comprising administering to a human individual needing such treatment or prevention an effective amount of an immunogenic compound according to any one of claims 1 to 7, 16 and 17, or an antigenic peptide according to claim 12 or 13, or a peptide having an amino acid linker according to claims 14 to 17.

[0092] Abbreviations used herein: aa Amino acid ab Antibody aSyn α-Synuclein BSA Bovine serum albumin bSyn β-Synuclein CNS Central nervous system DLB Dementia with Lewy bodies DHA Docosahexaenoic acid (purified) EC 50 Half maximal effect concentration ELISA Enzyme-linked immunosorbent assay Fc Flow cell FELASA Federation of European Laboratory Animal Science Associations h Hour HBS HEPES buffered saline HNE 4-Hydroxy-2-nonenal HPLC High performance liquid chromatography IC 50 Half maximal inhibitory concentration IHC Immunohistochemistry IQR Interquartile range IR Immune response kDa Kilodalton KLH Keyhole limpet hemocyanin LB Lewy body mAb Monoclonal antibody MSA Multiple system atrophy OD Optical density OD max / 2 Optical density at half maximum PD (Parkinson's disease) RT room temperature RU reaction unit SAIT-specific active immunotherapy sc subcutaneous SEM standard error SN substantia nigra SPR Surface Plasmon Resonance vs vs wt wild type

[0093] The present invention will be further illustrated by the following embodiments and drawings, but will not be limited thereto. [Brief explanation of the drawing]

[0094] [Figure 1] Schematic timeline of the experiment. Injections are represented by arrows, and blood collection by droplets. PP: Pre-treatment plasma, EP: Final plasma. Pn: Plasma n. Wn: nth week of the experiment. [Figure 2] Comparison of aSyn cross-reactive antibodies induced by p4456 and p4572, or by corresponding native aSyn epitopes of different lengths. (A) Antibody concentrations against the aSyn protein induced from the final plasma of all individual mice are shown. Bars represent the mean values ​​with SEM. Extreme outliers (greater than IQR × 3) have been removed from "Graph (B) Relative position of injected peptides along the amino acid sequence of the native aSyn sequence". p9524 is used as a scaffold for further development of AFFITOPE® and is highlighted in gray. [Figure 3] Immunogenicity of peptides (delivered as peptide-carrier protein conjugates) having a single alanine (A) or serine (B) exchange with aSyn sequences aa113-124. Group median concentrations of anti-aSyn antibodies in final plasma derived from individual immunized mice were determined and set relative to the concentration of anti-aSyn antibodies induced by vaccination with p9524, set as 100%. The sequences of the injected peptides are shown in Tables 3 and 4. [Figure 4] Immunogenicity of peptides (delivered as peptide-carrier protein conjugates) having aSyn sequences aa113-124 with single amino acid exchanges at positions 1 or 3 (A) and 4 or 12 (B). The group median concentration of anti-aSyn antibody in final plasma derived from individual immunized mice was determined and set in relation to the concentration of anti-aSyn antibody induced by vaccination with p9524, set as 100%. The sequences of the injected peptides are shown in Tables 7 and 8. [Figure 5] Immunogenicity of peptides having the aSyn sequence aa113-124 with double serine and other amino acid exchanges. The group median concentration of anti-aSyn antibody in the final plasma derived from individual immunized mice was determined and set in relation to the concentration of anti-aSyn antibody induced by vaccination with p9524, set as 100%. The sequences of the injected peptides are shown in Table 11. [Figure 6] Immunogenicity to aSyn induced by the aSyn sequence aa113-124 and the N-terminal extension sequence. The group median concentration of anti-aSyn antibody in the final plasma derived from individual immunized mice was determined and set in relation to the concentration of anti-aSyn antibody induced by vaccination with p10074, set as 100%. The sequences of the injected peptides are shown in Table 13. [Figure 7] Immunogenicity to aSyn was determined by the aSyn sequence aa113-124, the N-terminal extension sequence, and the aSyn sequence 115-121, induced by p4456. The group median concentration of anti-aSyn antibody in the final plasma derived from individual immunized mice was determined and set in relation to the concentration of anti-aSyn antibody induced by vaccination with p4456, set as 100%. The sequences of the injected peptides are shown in Table 13. [Figure 8]Immunogenicity of Syn target sequences p10033 and p10118 and their C-terminal truncate sequences. (A) The group median concentration of anti-aSyn antibody in final plasma derived from individual immunized mice was determined and set in relation to the concentration of anti-aSyn antibody induced by vaccination with p10033, set as 100%. (B) The group median concentration of anti-aSyn antibody in final plasma derived from individual immunized mice was determined and set in relation to the concentration of anti-aSyn antibody induced by vaccination with p10118, set as 100%. The sequences of the injected peptides are shown in Table 16. [Figure 9] IHC staining of postmortem human DLB brain with AFFITOPE® candidate-inducing antibodies. The antibodies used in each panel are induced by the peptides shown at the top of each panel. Underlined letters indicate amino acids that differ from the native sequence. The size bars indicate 50 μM in the main image and 10 μM in the small box in the lower right corner, focusing on a single Lewy body (LB). [Figure 10] Preferential binding of toxic oligomeric aSyn species to monomeric species (BiaCore data). Sensorograms of stable binding of AFFITOPE® candidate-inducing antibodies and monoclonal antibodies LB509 and 28A7 to oligomeric (red curve) or monomeric aSyn (green curve) species. The blue line represents negative control (binding to HBS buffer only). X-axis: execution time (seconds), y-axis: relative binding reaction units. [Figure 11] AFFITOPE® Candidate - A competitive ELISA demonstrating concentration-dependent inhibition of the binding of specific antibodies to aSyn monomers, oligomers, and fibers. AFFITOPE® Candidate - Purified antibodies from immunized mice were pre-incubated with increasing amounts of different aSyn species, and then tested for binding to plate-bound aSyn oligomers. Inhibition by monomeric aSyn is shown by the red curve, inhibition by fibrous aSyn by the blue curve, and inhibition by oligomer by the green curve. X-axis: Decimal logarithm of aSyn species concentration (ng / ml), Y-axis: OD405 values ​​measured for each aSyn species and concentration. [Examples]

[0095] Identification of Syn-AFFITOPE (registered trademark) Given the potential toxicity of various aSyn aggregates, therapeutic approaches to synucleinopathy may include reducing intracellular and extracellular aSyn levels or accumulations. The fact that oligomeric aSyn tends to be secreted into the intercellular space and can migrate like prions from one affected neuron or oligodendrocyte to adjacent neurons or glial cells (e.g., in the case of MSA) (Lee et al., 2008, Lashuel et al., 2013, Bengoa-Vergniory et al., 2017, Bernis et al., 2015) suggests that long-term targeting of aSyn transmission, AFFIRIS-specific active immunotherapy ( s pecific a ctive i mmuno t This opens the way to therapeutic approaches such as herapy (SAIT). AFFITOPE® PD01 and PD03 [short synthetic peptides that mimic distinct regions of aSyn] have been tested as compounds for SAIT in Phase I clinical trials in PD [PD01A (NCT01568099; Volc et al., 2020) and PD03A (NCT02267434)] and in patients with multiple system atrophy (MSA) [PD01 and PD03 (NCT02270489)]. These drugs have been shown to be well-tolerated and induce aSyn-specific antibodies that preferentially target oligomeric aSyn.

[0096] In the course of this invention, a second-generation aSyn-targeting AFFITOPE® was developed that induces higher titers and cross-reactivity to aSyn proteins than those observed with PD01 and PD03, respectively. Furthermore, a focus of selection was the ability of the induced antibody to distinguish between aggregated toxic aSyn species (oligomeric aSyn) and monomeric aSyn protein. Thus, although the antibody induced by AFFITOPE® is thought to bind to monomeric aSyn species that are excessively present in the periphery to a small extent, it is preferable that binding occurs to toxic oligomers and aSyn species that are present in small amounts in the CNS and periphery.

[0097] To identify highly potential AFFITOPE®, aSyn epitope aa 113-124 The target species was identified. Clearly defined selection criteria were applied, including high immunogenicity, high cross-reactivity to aSyn natural epitopes, and binding of inducing antibodies with high selectivity for oligomeric and fibrous toxic aSyn species (oligomeric binding > fibrous binding).

[0098] The selection strategy consisted of several steps: (i) epitope discovery within the C-terminus of aSyn target protein to identify a suitable epitope with high immunogenicity; (ii) alanine scan to detect a location along a native target sequence that can be exchanged to enhance immunogenicity and cross-reactivity; (iii) serine scan to detect a location along a native target sequence that can be exchanged to enhance immunogenicity and cross-reactivity; (iv) biserine exchange along a native target sequence; (v) non-Ala or Ser amino acid exchange that can improve immunogenicity and cross-reactivity to aSyn; and (vi) removal of one C-terminal amino acid from two selected AFFITOPE® sequences.

[0099] Immunization schedule for all in vivo experiments In all experiments using BALB / c described herein, mice were injected with AFFITOPE® or a natural aSyn epitope sequence (10 μg net peptide per injection) three times at bi-weekly intervals (Figure 1).

[0100] Discovery of an epitope in the C-terminus of aSyn target protein (aSyn-28 in vivo experiment) Epitope screening for highly immunogenic sequences along the C-terminal region of the aSyn protein is being performed. For this purpose, amino acid stretches of varying lengths, ranging from 7 to 13 amino acids, were used in immunogenicity studies of wt BALB / c mice (Table 1, Figure 1). In parallel, AFFITOPE®, previously selected by AFFiRiS p4456 and p4572, was included for comparison. Plasma was collected from all individual animals two weeks after the third immunization and subsequently analyzed by ELISA to determine the titers of the injected peptide and aSyn protein, as well as the concentration of aSyn reaction antibodies. 113-124 The peptide p9524 corresponding to the sequence induced the highest levels of aSyn-specific antibody (Figure 2, Table 2). Based on these findings, this sequence was selected as a natural target sequence for further AFFITOPE® candidate selection. Two other peptides, p9964 and p9556, which also induced high anti-aSyn titers, were excluded from further development. Peptide p9964 contains amino acid Y 125 and E 126 The peptide fragment derived from p9964, including p9964, binds with high affinity to different allele variants of MHCI by computer analysis and is predicted to be a potential cytotoxic T cell epitope (www.syfpeithi.de). Peptide p9556 contains the calpain cleavage site aSynL, which may be relevant to pathology. 113 / E 114 Because it did not cover the necessary parameters, it was not selected for further development. Table 2 summarizes the titers for aSyn found in AFFITOPE®-induced mouse plasma.

[0101] Table 1: Experiment aSyn-28 setup. The table shows the drug, the sequence of the drug peptide, and the corresponding sequence number. [Table 1]

[0102] Table 2: Key results including aSyn-28, titer, induced antibody concentration, and cross-reactivity. All parameters were evaluated in one mouse, and values ​​are median. Plasma concentrations of anti-aSyn antibodies from immunized mice were extrapolated from a reference curve generated with mAb LB509. [Table 2]

[0103] aSyn 113-124 Alanine scan (aSyn-30) and serine scan (aSyn-31) along epitopes As the next step, natural aSyn 113-124 Each amino acid position in the sequence was replaced with either alanine or serine to identify substitutable positions for maintaining or enhancing immunogenicity (Tables 3 and 4). BALB / c mice were each injected with AFFITOPE® three times. Two weeks after the third and final injection, plasma was collected from individual mice and analyzed by ELISA to determine AFFITOPE®-inducible titer and aSyn antibody concentration. Alanine scanning revealed that aSyn 113-124 It was revealed that the Leu (p9988) at position 1 and the Asp (p9990) at position 3 along the aa(p9524) sequence are not essential, and conversely, replacing them with Ala increases cross-reactivity to aSyn (Figure 3A). Serine scan, i.e., aSyn 113-124 The exchange of naturally occurring amino acids with serine along the sequence (Figure 3B) revealed higher cross-reactivity to aSyn due to the exchange of serine at positions 1 (p9999), 2 (p10000), 3 (p10001), and 12 (p10010). Tables 5 and 6 summarize the induced titers of aSyn in immunized mouse plasma relative to the titers induced by the WT sequence.

[0104] Table 3: Experiment aSyn-30 setup. The table shows the drug, the peptide sequence of the drug, and the corresponding sequence number for each different therapeutic group and application. [Table 3]

[0105] Table 4: Experiment aSyn-31 setup. The table shows the drug, the peptide sequence of the drug, and the corresponding sequence number for each different therapeutic group and application. [Table 4]

[0106] Table 5: aSyn-30, main results. Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the median obtained with p9524. [Table 5]

[0107] Table 6: aSyn-31, main results. Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the median obtained with p9524. [Table 6]

[0108] aSyn aa 113-124 Amino acid exchange at positions 1, 3, 4, and 12 along the epitope sequence (in vivo experiments with aSyn-32 and aSyn-33) Leu in 1st place, Asp in 3rd place, Met in 4th place, and Ala in 12th place are all natural aSyn 113-124Since swapping the sequence with Ala and / or Ser induces high aSyn-specific antibody concentrations (Figure 3), these positions are promising candidate modifications leading to the design of AFFITOPES®. As a next step, these positions were swapped with amino acids having different properties (e.g., amino acids with different charges or amino acids with different polarities) to define preferred or less preferred swaps in terms of immunogenicity and aSyn cross-reactivity (Tables 7 and 8). For comparison, a group injected with natural aSyn113-124 (p9524) was included. Plasma from immunized mice was analyzed by ELISA to measure antibody titers against aSyn compared to titers induced with the natural sequence (Figures 4A, B). Replacing both the 1st and 3rd positions with Lys induced higher levels of aSyn-specific antibodies (p10029 and p10033), while replacing with Trp dramatically reduced aSyn-specific antibody production (p10026 and p10031) (Figure 4A). Furthermore, replacing Asp with Glu at the 3rd position resulted in increased cross-reactivity to aSyn compared to the native aSyn sequence (Figure 4A). Met at the 4th position and Ala at the 12th position were replaced using a similar strategy to experimental aSyn-33 (Table 8), as shown for the 1st and 3rd positions in experimental aSyn-33. Here again, replacing the 12th position with Lys or Val (p10045 or p10042) resulted in higher anti-aSyn antibody titers, while replacing the 4th or 12th position with Trp reduced cross-reactivity to aSyn (Figure 4B). Tables 9 and 10 summarize the induced titers for aSyn present in the plasma of immunized mice, induced by WT sequences.

[0109] Table 7: Experiment aSyn-32 setup. The table shows the drug, the peptide sequence of the drug, and the corresponding sequence number for each drug in different therapeutic groups and applications. [Table 7]

[0110] Table 8: Experiment aSyn-33 setup. The table shows the drug, the peptide sequence of the drug, and the corresponding sequence number for each drug in different therapeutic groups and applications. [Table 8]

[0111] Table 9: aSyn-32, main results. Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the median obtained with p9524. [Table 9]

[0112] Table 10: aSyn-33, main results. Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the median obtained with p9524. [Table 10]

[0113] aSyn aa 113-124 Bi-serine exchange along the epitope sequence (in vivo experiment aSyn-37) aSyn 113-124 VARIOTOPES® (p9524) was designed with added serine exchanges along the natural sequence, and its immunogenicity was tested in wtBALB / c mice (Table 11, Figure 5). Two weeks after the final injection (the third injection), plasma was collected from individual mice and analyzed by ELISA to determine AFFITOPE®-induced titer and aSyn antibody concentration. The natural aSyn sequence was injected for direct comparison (Group 1). Double Ser exchanges at positions 3 and 12 (p10074) and positions 1 and 3 (p10075) further increased the titer of aSyn-specific antibodies compared to the natural target sequence (Figure 5). Double Serine exchanges at positions 1 and 4, and positions 4 and 12 did not increase immunogenicity against aSyn (Figure 5). Table 12 summarizes the induced titers against the WT sequence-induced titer against aSyn present in the plasma of immunized mice.

[0114] Table 11: Experiment aSyn-37 setup. The table shows the drug, the peptide sequence of the drug, and the corresponding sequence number for each drug in different therapeutic groups and applications. [Table 11]

[0115] Table 12: aSyn-37, main results. Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the median obtained with p9524. [Table 12]

[0116] aSyn aa 113-124 Amino acid exchange with Ser and Lys at positions 1, 3, and 12 along the epitope sequence (in vivo experiment aSyn-44) The exchange of Ser and Lys at positions 1, 3, and 12 of the natural aSyn113-124 sequence has been shown to be favorable for inducing high concentrations of anti-aSyn antibodies (Figures 3B, 4, and 5). Immunogenic studies presented herein have shown that aSyn 113-124 Combinations of serine and lysine exchanges along the epitope were tested in wtBALB / c mice (aSyn-44 experiment, Table 13). Additionally, linker amino acids were attached to different peptides. In parallel, the previously selected AFFITOPE® candidate p4456 was included in this experiment for direct comparison. Two weeks after the final injection in the third dose, plasma was collected from individual mice and analyzed by ELISA to determine AFFITOPE®-induced titers and aSyn antibody concentrations. The AFFITOPE® candidates tested in groups 2–7 were able to induce higher titers of anti-aSyn antibodies compared to the highly successful AFFITOPE® p10074 shown in Figure 5 (Figure 6). In particular, AFFITOPE® p10118 was found to induce the highest titer against aSyn. Table 14 summarizes the induced titers against p10074-induced titers against aSyn present in the plasma of immunized mice.

[0117] To directly compare the immunogenicity of newly selected AFFITOPE® sequences (groups 2-7) with the previously selected peptide p4456 in AFFiRiS, the group median concentrations of anti-aSyn antibodies induced by these AFFITOPE® candidates were set relative to the group median anti-aSyn antibody concentrations induced by p4456 (Figure 7). All novel AFFITOPE® candidates tested induced significantly higher aSyn-specific titers compared to p4456 (Figure 7). Table 15 summarizes the induced titers of aSyn in immunized mouse plasma relative to the titers induced by the AFFITOPE® sequence p4456.

[0118] Table 13: Experiment aSyn-44 setup. The table shows the drug, the peptide sequence of the drug, and the corresponding sequence number for each drug in different therapeutic groups and applications. [Table 13]

[0119] Table 14: aSyn-44, main results. Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the p10074 median set to 100%. [Table 14]

[0120] Table 15: Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the p10074 median set to 100%. [Table 15]

[0121] C-terminal cleavage by removing Ala at position 12 of two selected AFFITOPE® sequences, p10033 and p10118. The AFFITOPE® sequences p10033 and, in particular, p10118 have been shown to induce high concentrations of anti-aSyn antibodies (Figures 4A and 6, respectively). In the immunogenicity studies described herein, the C-terminal Ala at position X12 was removed from the AFFITOPE® sequences p10033 and p10118 to test whether C-terminal cleavage affects the immunogenicity and aSyn cross-reactivity of the designed peptides. wtBALB / c mice were independently immunized with either p10033, p10118, p10166, or p10167. Table 16 shows the peptide sequences. Two weeks after the third injection, plasma was collected from each mouse and analyzed by ELISA to determine AFFITOPE®-inducing titer and aSyn antibody reactivity. The tested cleaved AFFITOPE® candidates were able to induce lower anti-aSyn antibody titers than those generated using AFFITOPE® sequences incorporating Ala at the X12 position, but higher than those obtained using p4456 (Figure 8). Table 17 summarizes the induced titers against aSyn present in the plasma of immunized mice, induced by either AFFITOPE® sequence p10033(A) or AFFITOPE® sequence p10118(B).

[0122] Table 16: The table shows the respective drugs, their peptide sequences, and corresponding sequence numbers for different therapeutic groups and applications. [Table 16]

[0123] Table 17: Titer for aSyn was evaluated in one mouse, and the value represents the median relative to the median obtained with either p10033 or p10118. [Table 17]

[0124] Interception of antibodies induced by the selected AFFITOPE(registered trademark) To investigate whether antibodies induced by the AFFITOPE® candidate could detect asynchronous aSyn in situ, serum from wtBALB / c mice vaccinated with the AFFITOPE® candidate was tested by IHC staining on brain sections of human brain tissue derived from cadaveric PD / DLB patients. Lewy bodies are a pathological feature in the brains of PD / DLB patients and are primarily rich in pathogenic asynchronous forms of aSyn (Spillantini et al., 1997).

[0125] Plasma from AFFITOPE® candidate-treated mice detected Lewy bodies in cerebral cortical brain sections, similar to control anti-aSyn mAb 28A7 (Figure 9). Specificity was confirmed by the absence of staining after pre-absorption of serum with the corresponding peptide portion of the AFFITOPE® candidate (data not shown).

[0126] aSyn oligomer (toxic) vs. preferential binding to monomer aSyn species. Next, antibodies induced by different AFFITOPE® antibodies were tested for selective binding to oligomeric aSyn (low molecular weight, soluble aggregates of aSyn, mainly dimers and trimers) rather than monomer species using an SPR-based methodology (Figure 10). Equal volumes of AFFITOPE® candidate-inducing antibodies or monoclonal antibodies were first immobilized on chips coated with anti-mouse capture antibodies. Subsequently, monomer and oligomeric aSyn species were applied sequentially, and different binding (defined as RU) to aSyn monomer and oligomeric aSyn species was evaluated. As controls, two monoclonal antibodies, LB509 (Biolegend, San Diego, California) and 28A7 (AFFiRiS AG), were used. Immobilization of LB509 on the chip surface did not distinguish between monomer and oligomeric aSyn, resulting in equivalent RU for both aSyn species. The second control antibody, 28A7, induced against peptide p4456, distinguished between monomeric aSyn and oligomeric aSyn species. The AFFITOPE® candidate inducing antibody showed high selectivity for oligomeric aggregates of aSyn compared to the monomeric form of aSyn (Figure 10).

[0127] In addition to SPR (BiaCore) analysis, AFFITOPE®-inducing antibodies have been tested by inhibitory ELISA for preferential binding to aSyn filaments over aSyn monomer species. In these assays, a fixed amount of affinity-purified AFFITOPE®-inducing antibody was pre-incubated with titrations of monomer and filamentous aSyn, and then transferred to an ELISA plate coated with aSyn filaments (see M&M for details). Figure 11 shows the results for two representative AFFITOPE® candidates (p10033 and p10118). Very good competition was observed between aSyn oligomers and subsequent aSyn filaments, but very little competition was observed with aSyn monomer species (Figure 11).

[0128] mAb28A7, which is known to preferentially bind to the oligomeric and aggregated forms of aSyn, was used as a control (Figure 11C).

[0129] Overall, the binding data for AFFITOPE® candidate-specific antibodies provide clear evidence of the high selectivity of AFFITOPE® candidate-inducing antibodies against toxic oligomeric aggregates of aSyn, which are considered to be associated toxic species leading to cell death, in contrast to monomeric forms.

[0130] material and method mouse I purchased the BALB / c mouse from Janvier Elevages (Le Genest-Sainte-Isle, F).

[0131] The animals were kept and stored under the standard conditions described in the IMP application for authorization to engage in activities as breeder, supplier, and user. Each authorization was granted by the relevant authorities on May 13, 2013, in notification GZ:223633 / 2013 / 4.

[0132] In short, the mice were housed in TECNIPLAST Sealsafe NextIVC Blue Line cages (Milan, Italy), with groups of five mice per cage. The cages were equipped with nesting reinforced structures and small plastic houses for hiding and playing. The mice were 6-8 weeks old at the start of the experiment. They were given a standard diet and acidic water freely and lived in a 12-hour light-dark cycle.

[0133] All animal experiments are conducted in accordance with Austrian and European law and with the permission of the Vienna City Administration, municipal department 58.

[0134] Peptides and proteins The peptides used for immunization were purchased from EMC microcollections (Tübingen, Germany). CRM197 was purchased from Pfenex (San Diego, California).

[0135] Manufacturing of immunogenic products All immunogenic AFFITOPE® products used in the described experiments are conjugates of synthetic AFFITOPE® peptides to the carrier protein CRM197. The conjugation is a directed procedure using the amino group of the lysine residue side chain of CRM197 and the free thiol group of the amino(N)-terminal cysteine ​​of the peptide. To activate CRM197, an aqueous solution of CRM197 is prepared in 10 mM phosphate-buffered saline (PBS) and gently shaken with the bifunctional linker 4-maleimidobutyrate N-hydroxysuccinimide ester (GMBS). Unreacted GMBS is then removed by dialysis or ultrafiltration. The resulting activated CRM197 solution is then incubated with the AFFITOPE® peptide dissolved in phosphate buffer (pH 6.7). The free thiol group of the cysteine ​​in the peptide reacts with the maleimide group to form the final AFFITOPE®-CRM197 product.

[0136] Application of immunogens The vaccine is allowed to return to ambient temperature, vortexed, and 200 μl of insulin (Omnican(C) 50, B. Braun Melsungen AG, Melsungen, Germany) is applied subcutaneously (sc) to the flank of the mouse using a G30 gauge 20 syringe. Immunization is repeated three times at bi-weekly intervals.

[0137] Sample collection Blood samples were taken two weeks after each injection. Plasma was collected and stored at -20°C until analysis. Blood collection and slaughter of animals were performed using FELASA-approved procedures.

[0138] Monoclonal antibodies In this study, two monoclonal antibodies were used as controls: LB509 (Biolegend, California, US) and 28A7. LB509 is a commercially available purified anti-aSyn, 115-121 antibody. Mouse mAb 28A7(IgG1) is used against aSyn 115-121 AFFITOPE® PD01, which mimics an epitope, was generated in-house using mouse B-cell hybridomas (Mandler et al., 2014).

[0139] Sample collection Blood samples were taken approximately two weeks after each injection, and at least one day before the next injection. Plasma was collected and stored at -20°C until analysis. Blood collection and slaughter of animals were performed using FELASA-approved procedures.

[0140] Titer determination by ELISA The titers for immunized peptides and recombinant human aSyn protein were analyzed. The presence of AFFIOTPE®-inducing antibodies in the plasma of immunized mice was determined by ELISA. 96-well plates (Nunc-Maxisorp) were coated with either recombinant human aSyn (1 μg / ml) or injected peptide (BSA conjugate; 1 μM). Titers were determined by nonlinear regression analysis (4-parameter logistic fit function) using PRISM® 5.04 (GraphPad Inc, San Diego, California) to obtain EC2. 50 It was calculated as a value.

[0141] Immunohistochemistry (IHC) The identification of aSyn-positive inclusion bodies (Lewy bodies) was performed on postmortem brain sections from frontal cortical biopsies of DLB ​​patients (DLB patient, case number: X5631, Department of Neuroscience, UCSD, La Jolla, California).

[0142] Frontal cortical brain biopsy sections from DLB patients were stained using plasma from AFFITOPE® immunized mice. After tissue preparation (rehydration, deparaffinization, antigen recovery, and blocking), sections were incubated with diluted mouse plasma at room temperature for 2 hours or overnight at 4°C. Sections were incubated with undiluted Dako EnVision HRP-labeled polymer (Agilent, Santa Clara, California) at RT for 1 hour. Counterstaining with hematoxylin was performed for each IHC stain. After this step, the slides were dehydrated and mounted in Enteran (Sigma-Aldrich). Slides were scanned in bright-field mode using Panoramic (Mirax) Scanner 150 (Carl Zeiss MicroImaging GmbH).

[0143] Surface plasmon resonance (Biacore) analysis All experiments were performed on a Biacore T200 (GE Healthcare, Chicago, Illinois) using Biacore T200 Control Software 2.0.1. CM5 chips were immobilized with antibodies from a commercially available mouse antibody capture kit (GE-Healthcare) by amine bonding in flow cells (Fc) 1 and Fc2 of the chip, according to the manufacturer's instructions. Fc1 served as the reference flow cell. The immobilization levels of anti-mouse antibodies against both Fc cells were comparable, reaching approximately 11,000 RU for both Fc1 and Fc2.

[0144] To achieve equivalent levels of capture antibodies, the injection time for each specific antibody was tested before actual execution, and the injection time was adjusted accordingly. This resulted in equivalent capture levels for all tested antibodies, including nonspecific antibodies captured by Fc1 in each cycle.

[0145] The experimental setup for one cycle is as follows: 1. Capture of nonspecific antibodies in reference Fc1 2. Capture of aSyn-specific antibodies in Fc2 3. Inject the sample (oligomer aSyn, monomer aSyn, and buffer only) into Fc1 and Fc2. 4. Regeneration of Fc1 and Fc2 at low pH; only anti-mouse antibodies remain on the chip. 5. Chip surface prepared for the next cycle

[0146] Preparation of oligomer and monomer aSyn: The aSyn oligomer (SynAging, Vendévre-les-Nancy, France) was thawed and diluted to 5 μg / ml with HBS immediately before injection. To remove the high molecular weight fraction from the true (bona fide) monomer aSyn (rPeptide), aSyn was freshly dissolved, diluted to 5 μg / ml with HBS, and centrifuged at 14,000 xg for 10 minutes using a 50 kDa cutoff column (Amicon Ultra 0.5 ml). Two aSyn-specific antibodies, LB509 (BioLegend, San Diego, California) and 28A7 (AFFiRiS AG), were used as controls.

[0147] Affinity purification of AFFITOPE®-specific antibodies from immunoplasma Iodoacetyl magnetic beads (FG-106, Bioclone Inc., San Diego, California) were coupled with each peptide (HPLC purified) at room temperature for 1 hour, and any remaining excess free sites were blocked with cysteine ​​for a further 1 hour. After the blocking reaction, the AFFITOPE®-conjugated beads were incubated with 150 μl of plasma from mice immunized with the corresponding AFFITOPE® candidate (at room temperature for 2 hours). Next, the AFFITOPE®-specific antibody was eluted with Elution Buffer (Thermo Scientific). The eluent was then concentrated to a volume of 150 μl (equal to the input volume) in an ultracentrifugation (Millipore) tube (30 kDa).

[0148] Competitive ELISA Titration volumes of different aSyn species, monomers (rPeptide), and fibril-containing aggregated forms (Proteos Inc.) or oligomers (Crossbeta Bicosciences) at concentrations ranging from 100 to 0.05 μg / ml (corresponding to a range of 69 to 0.034 μM in relation to the monomer aSyn) were pre-incubated with antibodies pre-purified from terminal plasma of AFFITOPE® candidate immunized mice, and with the control anti-aSyn antibody 28A7. The added aSyn species compete for binding to plate-coated aSyn fibers (Proteos Inc.). IC 50 The values ​​were calculated as the concentration of monomer, oligomer, or fibrous aSyn required to quench half of the ELISA signal. 50 The values ​​were calculated using nonlinear regression analysis (4-parameter logistic fit function) with PRISM® 5.04 (GraphPad Inc., San Diego, California).

[0149] Table 18: Tested AFFITOPE® and original sequence peptides [Table 18] [Table 19]

[0150] References Dufty et al., Am J Pathol. 2007 May;170(5):1725-38. [PubMed: 17456777] Bassil et al., Proc Natl Acad Sci US A.2016 Aug 23; 113(34): 9593-8.doi: 10.1073 / pnas.1609291113 [PubMed: 27482103] Kosaka, J Neurol.1990; 237:197-204.[PubMed: 2196340] Dickson et al., Acta Neuropathol.1989; 78:572-584.[PubMed:2683563] Braak et al., Acta Neuropathol.2007; 114:231-241.[PubMed:17576580] Lee et al., Int J Biochem Cell Biol.2008; 40:1835-1849.[Pub-Med: 18291704] Bengoa-Vergniory et al., Acta Neuropathol.2017;134(6):819-838.doi: 10.1007 / s00401-017-1755-1.[PubMed: 28803412] Bernis et al., Acta Neuropathol Commun. 2015 Nov 26;3:75. doi: 10.1186 / s40478-015-0254-7. [PubMed: 26612754] Mandler et al., Acta Neuropathol.2014;127(6):861-79.doi: 10.1007 / s00401-014-1256-4.[PubMed: 24525765] Spillantini et al., Nature.1997 Aug 28;388(6645):839-40.(PubMed: 9278044] Lashuel, et al., Nat.Rev.Neurosci.14, 38-48.doi: 10.1038 / nrn3406 Volc, et al., Lancet Neurol. 19, 591-600. doi: 10.1016 / S1474-4422(20)30136-8

Claims

1. Antigenic peptides containing, essentially consisting of, or comprising the amino acid sequences KESMPVDPDNEA, GKESMPVDPDNEA, GGKESMPVDPDNEA, or CGGKESMPVDPDNEA.

2. An immunogenic compound comprising an antigen peptide as described in claim 1 and a carrier containing a T cell epitope bound to the antigen peptide.

3. The immunogenic compound according to claim 2, wherein a carrier containing a T cell epitope is bound to an antigen peptide via a linker.

4. The immunogenic compound according to claim 2 or 3, wherein a carrier containing a T cell epitope is bound to the N-terminus of the antigen peptide.

5. Carrier proteins, a. Keyhole limpet hemocyanin (KLH), tetanus toxoid, fever enterotoxin (LT), cholera toxin (CT), diphtheria toxin (DT) and its variants; or b. CRM197, tetanus toxoid (TT), mutagen, albumin-binding protein, and bovine serum albumin; or c. CRM197; An immunogenic compound according to claim 4, selected from the group consisting of any of the following.

6. A pharmaceutical formulation comprising the antigen peptide described in claim 1 or the immunogenic compound described in any one of claims 2 to 5 and a pharmaceutically acceptable excipient.

7. The pharmaceutical preparation according to claim 6, further comprising an adjuvant.

8. The adjuvant, a. MF59 aluminum phosphate, calcium phosphate, cytokines, IL-2, IL-12, GM-CSF, saponins, QS21, MDP derivatives, CpG oligos, IC31, LPS, MPLA, polyphosphazenes and aluminum hydroxide, or mixtures thereof; or b. Aluminum hydroxide; A pharmaceutical preparation according to claim 7, selected from the group consisting of any of the following.

9. The pharmaceutical preparation according to claim 7 or 8, wherein the antigen peptide is contained in an amount of 0.1 ng to 10 mg, 10 ng to 1 mg, or 100 ng to 100 μg.

10. A pharmaceutical formulation according to any one of claims 6 to 9, wherein the formulation is formulated for parenteral administration, subcutaneous administration, intradermal administration, intravenous administration or intramuscular administration.

11. A pharmaceutical preparation according to any one of claims 6 to 10 for treating or preventing synucleinopathy.

12. The pharmaceutical preparation according to claim 11, wherein the synucleinopathy is primary synucleinopathy or an associated medical condition.

13. The pharmaceutical formulation according to claim 12, wherein the primary synucleinopathy is selected from the group consisting of Parkinson's disease (sporadic, familial with α-synuclein mutations, familial with mutations other than α-synuclein, pure autonomic dysfunction and Lewy body dysphagia), Lewy body disease (LBD; including Lewy body dementia (DLB)) ("pure" Lewy body dementia), Parkinsonian dementia (PDD), or diffuse Lewy body disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy).

14. Associated conditions include sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, inclusion body myositis, traumatic brain injury, chronic traumatic encephalopathy, Boxer dementia, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia with parkinsonism associated with chromosome 17, and Niemann-Pick type C1 disease), Down syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron disease, and amyotrophic lateral sclerosis. A pharmaceutical formulation according to claim 12, selected from the group consisting of sclerosis (sporadic, familial, and Guam's ALS-dementia complex), axonal dystrophy, type I neurodegenerative disease with iron deposition in the brain (Harrelforden-Spatz syndrome), prion disease, Gerstmann-Straussler-Scheinker disease, telangiectatic ataxia, Mäge syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease, and other lysosomal storage disorders (including Kfor-Rakeb syndrome and Sanfilippo syndrome), or REM sleep behavior disorder.

15. The pharmaceutical formulation according to claim 11, wherein the synucleinopathies are selected from the group consisting of Lewy body disease (LBD), particularly Parkinson's disease (PD), Parkinson's disease with dementia (PDD), and Lewy body dementia (DLB), and multiple system atrophy (MSA) or neurodegenerative type I with iron deposition in the brain (NBIA type I).

16. A pharmaceutical preparation according to any one of claims 11 to 15 for treating a human patient.

17. Use of the antigen peptide according to claim 1, the immunogenic compound according to any one of claims 2 to 5, or the pharmaceutical formulation according to any one of claims 6 to 10 for the manufacture of a drug for the treatment or prevention of synucleinopathy.

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