Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0007—Nervous system antigens; Prions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to immunogenic compounds and their use in the prevention and treatment of synucleopathies , especially of Parkinson ' s disease (PD) , Dementia with Lewy bodies ( DLB ) and Mul- tiple System Atrophy (MSA) .
• PD Parkinson ' s disease
• DLB Dementia with Lewy bodies
• MSA Mul- tiple System Atrophy
• PD is a synucleinopathy and the second most common neuro- degenerative movement disease .
• PD prevalence ranges between 100 and 200/ 100 , 000 in the general population, and af fects approxi- mately 1 % of the population above the age of 60 with an annual incidence of about 15/ 100 , 000 .
• It is a chronic progressive disor- der, defined by a combination of motoric syndromes (bradykinesia, rigidity, resting tremor and postural instability) and by non- motoric syndromes ( a variety of autonomic dys functions , sensory abnormalities , and psychiatric abnormalities ) that usually precede motoric syndromes .
• the hallmark of the disease is a profound loss of dopaminergic neurons in the substantia nigra ( SN) , accompanied by the accumulation of filamentous protein inclusions , termed Lewy Bodies ( LB ) , which are predominantly composed of alpha-synuclein ( aSyn) .
• PD, DLB and other LB diseases show accumulation and re- distribution of aSyn in various brains regions and cellular popu- lations .
• MSA is another very important synucleinopathy .
• MSA is a spo- radic neurodegenerative disorder that is characterised by symptoms of L-DOPA-resistant parkinsonism, cerebellar ataxia, and dysauto- nomia . Patients suf fer from multisystem neuronal loss af fecting various brain areas including striatum, substantia nigra, cere- bellum, pons , as well as the inferior olives and the spinal cord .
• MSA is characteri zed by aSyn-positive glial cytoplasmic ( GCI ) and rare neuronal inclusions throughout the central nervous system .
• GCI glial cytoplasmic
• GCI s for the patho- genesis of MSA is generally acknowledged and underscored by recent analysis of transgenic mouse models analysing the ef fect of aSyn overexpression in oligodendroglia . In tg mice overexpressing human aSyn both GCI-like aggregates and biochemical markers of MSA were observed .
• DLB is the second most common type of neurodegenerative de- mentias in western society after Al zheimer ' s disease (AD) . It makes up for 4-7 % of clinically diagnosed dementia, with the same number of cases predicted to escape correct clinical diagnosis . Diagnosis of DLB is challenging, as the disease represents an " in-between" of AD and PD and shows overlapping features of both entities .
• the four clinical consensus criteria, of which two must be present to diagnose "probable DLB" are fluctuation in cognition and atten- tion, recurrent visual hallucinations , REM sleep behaviour disor- der and spontaneous parkinsonian motor signs , which occur later in the disease than the other criteria .
• Symptoms are not uni form among pa- tients .
• DLB pathology is characteri zed by proteinaceous inclusions termed Lewy Bodies ( LB ) , predominantly composed of alpha synuclein ( aSyn) that has a role in the loss of function and structure of the neurons .
• LB Lewy Bodies
• aSyn alpha synuclein
• the LB are found distributed di f- fusely throughout the cortices , while in PD, they are found pre- dominantly in the dopaminergic neurons of the Substantia Nigra .
• the LB in DLB are less well demarcated, less eosinophilic and les s filamentous than those of PD .
• amyloid plaques con- taining mainly carboxy-terminally elongated forms of amyloid beta (Abeta ) such as Abetal-42 can be found in the brains of DLB pa- tients .
• Cortical amyloid deposition is associated with lower tem- poral lobe perfusion and a trend to hippocampal atrophy .
• aSyn is a 14 kD naturally monomeric protein that is normally located to presynaptic terminals either bound to membranes of the synaptic vesicles or in the cytosol . Its natural function remains poorly understood and is likely involved in the synaptic trans- mission .
• aSyn During pathogenesis mis folding and aggregation of aSyn occurs in the central nervous system ( CNS ) and the peripheral nervous system, possibly as a consequence of posttranslational modi fication, including among others C-terminal protease cleavage (Wy 2007 , Bassil 2016 ) . Aggregation leads to the generation of di f ferent aSyn species that have been associated with the patho- genesis of LB diseases , such as oligomers , protofibrils , and fi- brils . The fibrillar forms of aSyn are detected mostly in LBs which are located in neuronal cell body (Kosaka et al . , 1990 , Dickson et al , 1989 ) . Aggregates of aSyn can be also detected in astroglial cells (Braak 2007) .
• oligomeric aSyn has been attributed to cellular cyto- toxicity.
• aSyn can form different types of aggregates with different appearances, conformations, cytotox- icities and chemical properties under different in vitro condi- tions.
• different types of aggregates can develop, possessing different structural characteristics.
• distinct aSyn-strains impress (e.g. "fibrils” or "rib- bons") their conformation upon the receiving cell and generate aggregates of the same strain in a process termed "conformational templating".
• SAIT aSyn Specific Active ImmunoTherapy
• Vaccination with PD01 and PD03 has proven efficacy in various animal models of aSyn aggregation disorders, reducing aSyn pathology, preserva- tion of neuroinflammation, as well as amelioration of behavior deficits (Mandler et al. 2014; WO 2009/103105 A 1, WO 2011/020133 A 1, WO 2017 / 076873 A 1) .
• These peptides turned out to be safe and well-tolerated vaccines which are able to induce target-speci fic antibodies in humans .
• WO 2005/ 108423 A 1 discloses peptides conferring environmental stress resistance derived i . a . from aSyn, beta-synuclein (bSyn) , or gamma-synuclein ( GSyn) , which - i f present in a fusion protein with a fusion partner protein - may provide decreased denaturation and/or increased solubility to this fusion partner protein .
• WO 2018 / 151821 A 1 discloses antibodies to aSyn which are useful for diagnosing, treating and preventing neurodegenerative diseases .
• WO 2005/ 013889 A2 discloses fragments of native aSyn which are useful for the treatment or prophylaxis of a LB disease ( LBD) or to provide monoclonal antibodies for the treatment or prophylaxis of LBD .
• the invention may provide improved immunogenic pep- tides which are improved with respect to their immunogenicity, which induce higher amounts of aSyn-speci f ic antibodies in the periphery, and which induce higher amounts of aSyn-speci f ic anti- bodies in the brain .
• the present invention provides an antigenic pep- tide comprising, consisting essentially of or consisting of the structure :
• P is proline
• X 1 is L, K, A or S , wherein L is leucine , K is lysine , A is alanine and S is serine ;
• X 2 is E or S , wherein E is glutamic acid and S is as defined above ;
• X 3 is D, E , K, N, A or S , wherein N is asparagine , D is aspartic acid and E , K, A and S are as defined above ;
• X 4 is M, A, S, L or K, wherein M is methionine and A, S, L and K are as defined above;
• X5 is P or A as defined above;
• X 6 is V, A or S, wherein V is valine and A and S are as defined above ;
• X 7 is D or S as defined above;
• X 9 is D or A as defined above;
• X 10 is N, S or A, wherein N, S and A are as defined above;
• X 11 is E, A or S, wherein E, A and S are as defined above;
• X 12 is present or not and, if present, is A, K, V, S, or G wherein
• G is glycine and A, K, V and S are as defined above, with the proviso that X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -P-X 9 -X 10 -X 11 -X 12 is not L- E-D-M-P-V-D-P-D-N-E-A and which comprises between 1 and 5 amino acid differences compared with the amino acid sequence L-E-D-M-P- V-D-P-D-N-E-A and wherein the peptide does not comprise the di- peptide Y-E immediately following X 12 , wherein Y is tyrosine and E is as defined above.
• single letter amino acid code is generally used herein.
• the antigenic peptide may comprise between 1 and 5 amino acid differences (i.e. 1, 2, 3, 4 or 5 differences) or between 1 and 4 amino acid differences compared with the amino acid sequence L-E- D-M-P-V-D-P-D-N-E-A.
• the differences are generally amino acid substitutions (according to the options set out for each position, with the exception that X 12 may be deleted) .
• the antigenic peptide comprises amino acid differ- ences compared with the amino acid sequence L-E-D-M-P-V-D-P-D-N- E-A at one or more positions selected from X 1 , X 3 , X 4 and X 12 .
• the antigenic peptide comprises two amino acid differences compared with the amino acid sequence L-E-D-M-P-V-D-P-D-N-E-A at positions selected from X 1 , X 3 , X 4 and X 12 •
• the antigenic peptides of the invention retain their ability to generate aSyn-specif ic antibodies when employed as an immuno- gen. Moreover, the antigenic peptides of the invention are more immunogenic than the corresponding wild type aSyn peptide (com- prising the 12-mer L-E-D-M-P-V-D-P-D-N-E-A, p9524 (SEQ ID NO: 4) ) in terms of generating aSyn-specif ic antibodies, as demonstrated in the comparative experiments herein.
• aSyn antibody titer determination may be per- formed for example by ELISA, as described herein.
• the antigenic peptides of the invention typically do not com- prise further alpha synuclein amino acid residues after X 12 . In particular they do not comprise the dipeptide Y-E immediately fol- lowing X 12 . As described herein peptides including the amino acids Y 125 and E 126 are predicted by in silico analyses to bind with high affinity to different allelic variants of MHCI and thus be poten- tial cytotoxic T cell epitopes (www.syfpeithi.de) . The antigenic peptides may, however, comprise a limited number of further N terminal amino acid residues. Thus, the antigenic peptides may comprise, consist essentially of or consist of the structure
• Xa is present or not and, if present, is G, wherein G is glycine;
• Xb is G, wherein G is as defined above;
• X 1 -X 12 are as defined above.
• the antigenic peptides of the invention are thus typically 11-20 amino acids in length, preferably 12-14 amino acids in length (i.e. 12, 13 or 14 amino acids in length) . It is particularly preferred that the antigenic peptides are 12 or 14 amino acids in length.
• the antigenic peptides of the invention produce an anti- body response in the absence of a T-cell response.
• the an- tigenic peptides of the invention themselves do not typically con- tain T-cell epitopes, in particular cytotoxic T-cell epitopes.
• the antigenic peptides of the invention are typically employed in the form of immunogenic com- pounds in which they are conjugated to a carrier.
• the antigenic peptides of the invention may further comprise an amino acid that acts as a conjugation site.
• this amino acid is a terminal amino acid and is preferably positioned at the N terminus.
• the antigenic peptide further comprises a terminal cysteine residue, preferably an N-terminal cysteine residue.
• X 1 is L, S, or K
• X 2 is E or S
• X 3 is S, D, E, A, K or N
• X 4 is M
• X 10 is N
• X 12 is A, S, K or V, preferably wherein X 4 is L or K
• X 2 is E
• X 3 is S, D, E, K or A
• X 4 is M
• X 10 is N
• X 12 is A, S or K, especially wherein X 4 is L or K
• X 3 is D, K or S and X 12 is A.
• the other positions in the peptide are preferably wild type alpha synuclein amino acids .
• X 1 is A, S or K.
• X 2 is S.
• X 3 is A, S, E, K or N.
• X 4 is A, S, L or K.
• X 5 is A.
• X 6 is A or S.
• X 7 is S.
• X 9 is A.
• X 10 is A or S, preferably S.
• X 11 is A or S.
• X 12 is S, V, G or K.
• the antigenic peptide is selected from the group consisting of AEDMPVDPDNEA, KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES, SEDMPVDPDNEA, SEKMPVDPDNEA LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES , LEAMPVDPDNEA, LEDMPVDPDNEK, LED- MPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, LEKMPVDPDNES, LENMPVDPDNEA, KESMPVDPDNEK and KEDMPVDPDNEA, pref- erably SEDMPVDPDNEA, SEKMPVDPDNEA, LEEMPVDPDNEA, LEKMPVDPDNEK, LESMPVDPDNEA, LESMPVDPDNES, KESMPVDPDNEA, KEDMPV
• the antigenic peptide comprises , consists essentially of or consists of the amino acid sequence KESMPVDPDNEA, GKESMPVDPDNEA, GGKESMPVDPDNEA or CGGKESMPVDPDNEA.
• the antigenic peptides of the invention are typically employed in the form of immunogenic compounds in which they are conj ugated to a carrier .
• the carrier acts as a source of T-cell epitopes to improve the immune response to the immunogenic peptides .
• the invention further provides an immunogenic compound comprising an antigenic peptide of the invention and a carrier comprising T- cell epitopes attached to the antigenic peptide .
• Carrier is a polypeptide carrier covalently coupled to X 1 ; pref- erably comprising a linker moiety which covalently links a carrier molecule to the peptide X 1 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 ;
• D is aspartic acid
• E is glutamic acid
• P is proline
• V is valine ;
• X 1 is L, K, A or S , wherein L is leucine , K is lysine , A is alanine and S is serine ;
• X 2 is E or S , wherein E and S are as defined above ;
• X 3 is D, E , K, N, A or S , wherein N is asparagine and D, E , K, A and S are as defined above ;
• X 4 is M, A, S , L or K, wherein M i s methionine and A, S , L and K are as defined above ;
• X 10 is N, S or A, wherein N, S and A are as defined above ;
• X 12 is present or not and, i f present , is A, K, V, S , or G, wherein G is glycine and A, K, V and S are as defined above , with the proviso that X 1 -E-X 3 -X 4 - P-V-D- P-D-X 10 -E-X 12 is not L-E-D- M-P-V-D-P-D-N-E-A.
• the present compounds contain peptides which are able to elicit a strong anti-aSyn antibody response ("antigenic pep- tides" ) , i . e . that the induced antibodies show high cross-reac- tivity with human aSyn, although these peptides have a sequence which is di f ferent from the native sequence ( L-E-D-M-P-V-D-P-D-N- E-A) .
• Immune responses superior to the response with the native sequence i . e . targeting the same native structures ) was achieved with the peptides according to the present invention and a highly ef fective and suitable antibody response can be induced in vac- cinated individuals .
• the antibodies induced by the vaccination with the compounds according to the present invention bind to the aggregated toxic aSyn species and Lewy bodies in pathological human brain tissue with high selectivity and speci ficity .
• immunogenic peptides with non-native amino acid sequences of aSyn can be provided to elicit speci fic immune responses against aSyn which is improved with re- spect to cross-reactivity to bSyn (WO 2009/ 103105 A 1, WO 2011 / 020133 A 1) .
• Preferred immunogenic compounds according to the present in- vention comprise a preferred peptide of the present invention, wherein X 1 is L , S , or K, X 2 is E or S , X 3 i s S , D, E , A, K or N, X 4 is M, X 10 is N, and/or X 12 is A, S , K or V .
• Other preferred immunogenic compounds according to the present invention comprise a preferred peptide , wherein X 1 is L, S or K, X 2 is E , X 3 is S , D, E , K or A, X 4 is M, X 10 is N, and/or X 12 is A, S or K .
• Another preferred embodiment is a peptide according to the present inven- tion, wherein X 1 is L or K, X 3 is D, K or S and X 12 is A.
• the carrier comprising T-cell epitopes is attached to the antigenic peptide via a linker .
• a linker may be any suitable linker, as would be readily appreciated by one skilled in the art .
• the linker may be a chemical linker or a peptide ( amino acid based) linker .
• a linker may contain reactive functional groups to enable cross-linking of the antigenic peptide antigen to the carrier through a suitable chemical reaction .
• the linker may thus comprise two reactive groups .
• the first attaches to the carrier (protein) , typically via a reactive amino acid side chain, such as through reaction with a primary amine ( e . g . on a lysine residue ) .
• a reactive amino acid side chain such as through reaction with a primary amine ( e . g . on a lysine residue ) .
• a primary amine e. g . on a lysine residue
• an amide bond is formed .
• the second attaches to the antigenic peptide, again typically via a reactive amino acid side chain, such as with a sulfhydryl group (e.g. on a cysteine residue) .
• a thioether bond is formed.
• linkers are therefore heterobifunctional linkers, in particular those that contain an amine-reactive group, such as a N-hydroxysuccinimide (NHS) ester, and a sulfhydryl reactive group, such as a maleimide.
• the linker may attach the an- tigenic peptide to specific amino acid residues, or side chains thereof, contained within the carrier (protein) .
• the antigenic peptide is conjugated to lysine resi- dues (via the primary amine group) contained within the carrier (protein) . Conjugation via histidine residues is also envisaged.
• the antigenic peptide may be provided in the context of a larger peptide molecule, the remainder of which is not derived from the alpha synuclein amino acid sequence in order to provide a linker, or to facilitate linkage.
• the peptide can include additional residues, such as one or more cysteine residues with or without a spacer, such as polyethylene glycol (PEG) , to facilitate attachment to the carrier (protein) .
• additional residues are typically found at the N and/or C terminus of the antigenic peptide, preferably at the N terminus of the antigenic peptide.
• the term "consists essen- tially of” may mean that the antigenic peptide of the invention includes the 11-20, preferably 12 to 14 contiguous amino acids derived from the alpha synuclein sequence (amino acids 111-124 or 113-124, subject to at least one (and up to four) mutation as defined herein) but can include a limited number of additional residues, such as an additional cysteine residue, to facilitate attachment to the carrier protein with or without a spacer, such as PEG or an amino acid based spacer.
• the linker moiety of the immunogenic compound comprises at least one cysteine and/or glycine amino acid residue, preferably coupled with a chemical linker to the polypeptide carrier moiety.
• Providing amino acid linkers at the N-terminal end of the (e.g. 12mer) pep- tides of the present invention offers many benefits for the cou- pling of larger compounds (as carriers) and for the elicitation of strong (er) immune responses; however, such linkers, especially amino acid linkers are not mandatory for the present invention.
• Preferred amino acid linkers are glycine and cysteine (or combinations thereof, such as CG-, CGG-, CCG-, GC-, GGC-, GCC-, GG-, GGG-, etc.) ) as well as isoleucine, alanine, valine, leucine, serine, glutamic acid, aspartic acid, lysine, asparagine, gluta- mine, etc..
• the amino acid linker may also consist of more than 1 amino acid residue, for example of 2, 3, 4, or 5 amino acid resi- dues.
• the linker does not contain amino acids selected from the group consisting of proline, arginine, or histidine in the (linker) re- gion N-terminal to X 1 , i.e. within the first five amino acids of the amino acid linker molecule starting from the amino acid being N-terminal to X 1 .
• Preferred peptide linkers for use in the invention are those that do not form a T-cell epitope. This can be assessed using known methods, including in silico methods, such as by consulting the SYFPEITHI database of MHC ligands and peptide motifs (http://www.syfpeithi.de/) .
• the Carrier preferably comprises - besides the polypeptide carrier also chemical linking groups (or chemical components resulting from chemical linking processes) .
• Preferred linking groups may be obtained with the aid of chemical linkers , such as heterobi func- tional compounds , such as GMBS or sul fo-GMBS .
• All chemical linkers known and used in the art especially those which are used for producing products that are administered to human individuals may be used for providing linkage of the carrier to the peptide of the present invention .
• a speci fically preferred chemical linker is a chemical linker which couples via a non-peptide bond to the anti- genic peptide of the invention and to the carrier .
• linker moiety is formed by NHS-poly ( ethylene oxide ) ( PEO) ( e . g . by NHS-PEO 4 -maleimide ) or other compounds used in biochemical technology .
• Speci fically preferred immunogenic compounds according to the present invention comprise a preferred peptide X 1 -X 2 -X 3 -X 4 -P-V-D- P-D-X 10 -E-X 12 which is selected from the group consisting of KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES , SEDMPVDPDNEA, LEEMPVDPDNEA, SESMPVDPDNEA, LEDMPVDPDNES , LEAMPVDPDNEA, LED- MPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, KEDMPVDPDNEA, LENMPVDPDNEA, KESMPVDPDNEK and KED- MPVDPDNEA, preferably SEDMPVDPDNEA, LEEMPVDPDNEA, LESMPVDPDNEA, KESMPVDPDNEA,
• the carrier acts as a source of T-cell epitopes and thus typically comprises multiple T-cel l epitopes .
• the T-cell epitopes are preferably universal T-cell epitopes .
• universal T-cell epitope is meant an epitope that i s speci fic to T-cells that are present in the maj ority of the human population .
• the "universal" ability of a T-cell epitope to activate T-cells is the result of at least two complementary properties : i ) af finity of binding to the HLA groove , meaning the strength of the binding, as well as ii ) its capacity to bind di f ferent HLA haplotypes in a promiscuous manner, meaning the ability to cover very diverse human popula- tions , with regards to the di f ferences in the expression of HLA molecules .
• the universal T-cell epitopes may bind to a maj ority of MHC class I I alleles present in the human population .
• the T-cell epitopes of the carrier may thus be capable of stimulating a CD4 T-cell response .
• the T-cell epitopes of the carrier may thus be capable of stimulating a helper T-cell response that enhances ( an- tigenic peptide speci fic ) antibody production by B-cells .
• 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 diph- theria toxin (DT) and variants thereof, especially CRM197 (cross- reacting material 197) , Keyhole Limpet Hemocyanin (KLH) , tetanus toxoid, heat-labile enterotoxin (LT) , cholera toxin (CT) , tetanus toxoids (TT) , mutant toxins, albumin-binding proteins, bovine se- rum albumin, and synthetically derived fusion peptides containing multiple T cell epitopes (e.g. Tet or PADRE) .
• KLH Keyhole Limpet Hemocyanin
• LT heat-labile enterotoxin
• CT cholera toxin
• TT tetanus toxoids
• mutant toxins albumin-binding proteins
• bovine se- rum albumin and synthetically derived fusion peptides containing multiple T cell epitopes
• carrier protein that may be employed include pseudomonas exotoxin A (EPA) , Haemophilus influenzae protein D (HiD) or meningococcal outer mem- brane protein complex (OMPC) .
• EPA pseudomonas exotoxin A
• HiD Haemophilus influenzae protein D
• OMPC meningococcal outer mem- brane protein complex
• CRM197 is a particularly preferred carrier protein.
• Non-polypeptidic carriers may also be included in the immu- nogenic compounds of the present invention.
• examples include poly ( lactic-co-glycolic acid) microparticles (PLG microparti- cles) , poloxamer particles, virus-like particles, and dendrimers.
• Further carriers may comprise nanoparticles or liposomes.
• the immunogenic compounds and antigenic peptides of the present invention are preferably used for thera-plastic and prophylactic methods for the treatment of human pa- tients, especially for use in the treatment or prevention of synu- cleopathies.
• Preferred synucleopathies to be treated or prevented are Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Parkinson's Disease with Dementia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Multiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I) •
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Parkinson's Disease with Dementia
• DLB Dementia with Lewy Bodies
• MSA Multiple System Atrophy
• NBIA Type I Neurodegeneration with Brain Iron Accumulation type I
• the present invention relates to a pharmaceutical preparation compris- ing an immunogenic compound or antigenic peptide according to the present invention and a pharmaceutically acceptable excipient (which may be referred to interchangeably as a carrier) .
• excipient encompasses any component apart from the immunogenic compound that is present in the final formulation for administra- tion.
• the pharmaceutical preparation is preferably for use as a vaccine in the treatment or prevention of a synucleinopathy, pref- erably a synucleinopathy selected from the group consisting of Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Parkinson's Disease with Dementia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Multiple System Atrophy (MSA) or Neuro- degeneration with Brain Iron Accumulation type I (NBIA Type I) .
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Parkinson's Disease with Dementia
• DLB Dementia with Lewy Bodies
• MSA Multiple System Atrophy
• NBIA Type I Neuro- degeneration with Brain Iron Accumulation type I
• the pharmaceutical preparation according to the present in- vention is preferably formulated as a vaccine.
• the pharmaceutical preparation may be formulated with an ad- juvant, preferably with an adjuvant selected from the group con- sisting of MF59 aluminium phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF) , saponins (e.g., QS21) , MDP deriva- tives, CpG oligos, IC31, LPS, monophosphoryl lipid A ( (MPLA) which term encompasses MPLA-derivatives such as Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D- (6-acyl) PHAD®) , PHAD® (Phos- phorylated HexaAcyl Disaccharide ) or MPL) , , polyphosphazenes, and aluminium hydroxide, or mixtures
• MPLA
• the adjuvant (s) is to increase or stimulate the immune response in the subject.
• the at least one adjuvant forms part of the carrier.
• Other adjuvants that may be employed according to the invention include aluminium containing adjuvants, in particular aluminium hydroxide (Alum) , an imidazoquinolinamine such as Resiquimod (R- 848) and/or CpG (synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs) amongst others.
• the adjuvant may be a Toll-like receptor (TLR) agonist.
• a pharmaceutical preparation according to the pre- sent invention contains the immunogenic compound according to the present invention (or the peptide of the present invention, optionally coupled to an alternative carrier) in an amount of from 0.1 ng to 10 mg, pref- erably 10 ng to 1 mg, in particular 1 ⁇ g to 500 ⁇ g, or, alterna- tively, e.g. 100 fmol to 10 ⁇ mol, preferably 10 pmol to 1 ⁇ mol, in particular 1 nmol to 500 nmol.
• the amount of the peptide may be 100 pmol to 100 nmol in some embodiments.
• the amounts herein refer to the peptide component of the composition.
• the pharmaceutical preparation may also contain auxiliary substances as excipients, e.g. buffers, stabilizers etc.
• auxiliary substances e.g. a pharmaceutically acceptable excipient, such as water, buffer and/or stabilisers, are contained in an amount of 1 to 99 % (weight) , more preferred 5 to 80% (weight) , especially 10 to 70 % (weight) .
• the pharmaceutical preparation according to the present invention is formulated as liposomes , virosomes , iscoms , cochleates , emulsions .
• the antigenic peptides , immunogenic compounds and pharmaceu- tical preparations of the invention may be administered according to any suitable schedule . They may be administered according to a prime-boost vaccination strategy . Prime-boost vaccination strategies involve multiple immuni zations . They aim to improve the ef fectiveness of the vaccine . Generally, the same vaccine compo- sition is administered each time ; a so-called homologous prime- boost vaccination regimen .
• Possible administration regimes for the initial priming phase of the pharmaceutical preparation include a biweekly up to four- monthly treatment ; 2 to 5 , especially 3 to 4 , initial priming vaccine administrations ( in 1 to 5 months ) , fol- lowed by booster or maintenance vaccinations 3 to 12 months there- after or even years thereafter are preferred - besides other re- gimes already suggested for other vaccines .
• the " ⁇ g peptide ( as antigen) " referred to in the present invention refers to the amount of antigenic peptide in the dose and does not include the carrier or linker part of the vaccine conj ugate ( immunogenic compound, i f present ) .
• preferred amounts of antigen are at least 5 nmol , preferably at least 25 nmo 1 .
• the pharmaceutical preparation comprising the im- munogenic compound or the antigenic peptide is formulated for par- enteral administration .
• the preparation is formulated for subcutaneous , intradermal or intramuscular ad- ministration .
• Intravenous administration may also be employed .
• Vaccination strategy for the present invention preferably follows usual vaccination strategies .
• the vaccination strategy for sel f-antigens according to WO 2017 / 076873 A 1 i . e . to elicit a primary immune response in a pa- tient and then to perform on the patient booster or maintenance administrations .
• the booster/maintenance vaccination is adminis- tered at a point in time when the primary immune response has already passed, i . e . when the antibody titers elicited with a primary vaccination ( elicited by one , two , three , four or more vaccine administration ( s ) within the course of the primary immune response elicitation) have dropped beyond certain levels ( e . g . beyond a given threshold level of an assay suitable for testing high numbers of samples ) or have at least gone under 30% , prefer- ably under 50% , especially under 80% , of the maximum antibody level being present in the course of the primary vaccination, so as to obtain a high titer of antibodies over the whole treatment period .
• administration of booster/maintenance inj ections every 3 to 12 months after the initial (primary) im- muni zation may be beneficial .
• the amount of antigen in the dose for the booster/maintenance administration is at least 20% , preferably at least 50% , more preferred at least 100% , especially at least 200% , higher than the amount used in the dose for the administration for the primary immune response .
• the amount of antigen in the dose for the booster/maintenance administration is at least 300% , preferably at least 400% , more preferred at least 500% , especially at least 600% , higher than the amount used in the dose for the administration for the primary immune response .
• the same composition is administered on each occasion .
• the amount of antigen that is administered is the same on each occasion (within manufacturing tolerances ) .
• the booster/maintenance administration is repeated after some time , for example after one , two , three , five or ten years .
• the second or further booster/maintenance are performed in the same or similar manner than the first booster/maintenance admin- istration, i . e . with the increased amount of antigen compared to the dose of the primary vaccination, or with the same amount of antigen .
• Administration routes according to the present invention are usually the same routes as for current vaccinations . Therefore , preferred administration of the immunogenic compound or antigenic peptide , or pharmaceutical preparation, according to the present invention is parenteral , such as subcutaneous , intradermal or in- tramuscular administration . However, the immunogenic compound or antigenic peptide , or pharmaceutical preparation of the invention may be administered to the subj ect by any appropriate route of administration. As the skilled person would be aware, such compo- sitions (preferably vaccine compositions) may be administered by topical, oral, rectal, nasal or parenteral (such as intravenous, intradermal, subcutaneous, or intramuscular) routes.
• compositions may be incorporated into sustained release ma- trices such as biodegradable polymers, the polymers being im- planted in the vicinity of, or in close proximity to, where de- livery is desired.
• sustained release ma- trices such as biodegradable polymers
• the polymers being im- planted in the vicinity of, or in close proximity to, where de- livery is desired.
• the compo- sitions are administered intramuscularly or subcutaneously.
• the immunogenic compound or antigenic peptide, or pharmaceutical preparation is administered together with an adjuvant, preferably aluminium oxyhydroxide.
• an adjuvant preferably aluminium oxyhydroxide.
• the current invention relates to the use of European Pharmacopoeial grade (Aluminium- oxyhydroxide, monograph 1664) , more specifically to the product manufactured by Brenntag Biosector (2% Alhydrogel) tested towards EP compliance. Alhydrogel is available in three varieties: Alhy- drogel 1.3%; Alhydrogel 2% and Alhydrogel "85". Alhydrogel 2% was elected as the International Standard Preparation for aluminium hydroxide gels.
• the pharmaceutical preparation according to the present invention is aseptically formulated into a suitable buffer, preferably an isotonic phosphate buffer (1 mM to 100 mM) , preferably at a concentration of ⁇ 1.0 mg/ml Alhydrogel (given as AI 2 O 3 equivalent; this metric (Al as " AI 2 O 3 equivalent”) is used generally for the present invention; accordingly, all doses and amounts referred to in the present application, as far they are relating to aluminium oxyhydroxide refer to AI 2 O 3 equivalents (of aluminium oxyhydroxide (Alhydrogel) ) , even more preferably at a concentration of ⁇ 1.5 mg/ml Alhydrogel (given as AI 2 O 3 equiva- lent) , most preferable at a concentration of > 2.0 mg/ml Alhydrogel (given as AI 2 O 3 equivalent) .
• a suitable buffer preferably an isotonic phosphate buffer (1 mM to 100 mM) , preferably at a concentration
• the amount of aluminium salt for Alhy- drogel is given as AI 2 O 3 equivalent in line with the strength as stated by the manufacturer (i.e. 2% Alhydrogel equates to 2% AI 2 O 3 , i.e. 20 mg/mL) .
• This concentration is directly convertible into the respective concentration of aluminium by using the re- spective molecular masses (20 mg/mL AI 2 O 3 (Mw 101,96) corresponds to 10.6 mg/mL aluminium (molecular mass 26,98) ) .
• the carrier of the present invention may be any suitable and pharmaceutically acceptable carrier moiety, optionally with a linker to couple the linker with the antigenic peptide of the invention (comprising X 1 to X 12 ) .
• the antigenic peptide (which may be a 12mer peptide) of the present invention is coupled to a at least one pharmaceutically acceptable polypeptide carrier, preferably CRM197 (Cross reactive material 197) , KLH (Keyhole Limpet Hemocyanin) , tetanus toxoid, albumin- binding protein, bovine serum albumin, or synthetical fusion pep- tides containing multiple T cell epitopes.
• a pharmaceutically acceptable polypeptide carrier preferably CRM197 (Cross reactive material 197) , KLH (Keyhole Limpet Hemocyanin) , tetanus toxoid, albumin- binding protein, bovine serum albumin, or synthetical fusion pep- tides containing multiple T cell epitopes.
• Carriers or carrier or linker moieties within the Carrier include a dendrimer (MAP; Biol. Chem. 358: 581) , peptide linkers (or flanking regions) as well as the adju
• a vaccine composition comprising an immunogenic compound or antigenic peptide according to the present invention may be formulated with an adjuvant, preferably a low soluble alu- minium composition, in particular aluminium hydroxide.
• adjuvants like MF59 aluminium phosphate, calcium phosphate, cytokines (e.g., IL-2, IL-12, GM-CSF) , saponins (e.g., QS21) , MDP derivatives, CpG oligos, IC31, LPS, MPLA (which includes MPL) , polyphosphazenes, emulsions (e.g., Freund's, SAF) , liposomes, virosomes, iscoms, cochleates, PLG microparticles, poloxamer par- ticles, virus-like particles, heat-labile enterotoxin (LT) , chol- era toxin (CT) , diphtheria toxin
• LT
• the peptide or polypeptide of the present invention is pref- erably bound to the carrier or adjuvant via a linker, which is NHS-poly (ethylene oxide) (PEG) (e.g. NHS-PEO 4 -maleimide ) .
• linker which is NHS-poly (ethylene oxide) (PEG) (e.g. NHS-PEO 4 -maleimide ) .
• PEG poly (ethylene oxide)
• the carrier preferably comprises a toxoid protein.
• the toxoid protein may be a naturally occurring toxoid protein or a recombi- nant variant thereof used in pharmaceutical compositions.
• Toxins can be inactivated, for example, by treatment with formaldehyde, glutaraldehyde, UDP-dialdehyde, peroxide, oxygen or by mutation (e.g., using recombinant methods) .
• Mutant diphtheria toxins with reduced toxicity can also be produced using recombinant methods.
• DT diphtheria toxin cross-reacting materials (DT-CRM) or diphtheria toxoids.
• An DT-CRM refers to a mutant diphtheria toxin, e.g., by mutation or by chemical modification, such that it no longer possesses sufficient ADP-ribosyl.
• Non-limiting examples of DT-CRM include DT-CRM30, DT-CRM45, DT-CRM176, DT-CRM197 and DT- CRM228.
• a diphtheria toxoid is a f ormaldehyde-inactivated diph- theria toxin.
• DT is commercially available from or can be prepared by methods known in the art, such as recombinant DNA technology.
• CRM197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin that retains the immunologic properties of the wild type diphtheria toxin.
• CRM197 differs from the wild type diphtheria toxin at a single base in the structural gene, which gives rise to a single amino acid substitution from glutamic acid to glycine.
• CRM197 is typically isolated from cultures of Corynebacterium diphtheria strain C7 (Pl 97) grown on casamino acids and yeast extract-based medium.
• CRM197 may be purified through ultra-fil- tration, ammonium sulfate precipitation, and ion-exchange chroma- tography.
• CRM197 can be prepared recombinantly .
• CRM197 has been used in the design of glycoconjugate vaccines such as HibtiterTM, Menveo®, or Prevnar®.
• Tetanus toxoid is prepared and used worldwide for large-scale immunization against tetanus (or lockjaw) caused by Clostridium tetani . Tetanus toxoid is also used both singly and in combination with diphtheria and/or pertussis vaccines.
• the parent protein, tetanus toxin is generally obtained in cultures of Clostridium tetani. Tetanus toxin is a protein of about 150 kDa and consists of two subunits (about 100 kDa and about 50 kDa) linked by a sulfide bond.
• the toxin is typically detoxified with formaldehyde and can be purified from culture filtrates using known methods, such as ammonium sulfate precipitation, or chromatography tech- niques. Tetanus toxin may also be inactivated by recombinant ge- netic means. Tetanus toxoid has also been used as a carrier protein in other vaccines, including pneumococcal conjugate vaccines. Also mixed carrier can be used, e.g. pneumococcal conjugate vaccine in combination with CRM197, serotype 3 in combination with tetanus toxoid carrier, serotype 3 conjugated to diphtheria toxoid.
• the peptides of the present invention are variants of the native human aSyn sequence, i.e. an AFFITOPE ® or VARIOTOPE ® (being a peptide that contains sequence variations compared with the original native aSyn sequence, but which shows similar (the same or improved) immunization characteristics, i.e. is able to elicit an immune response that is similar or higher than the immune re- sponse that is obtained with the native aSyn sequence) .
• an AFFITOPE ® or VARIOTOPE ® being a peptide that contains sequence variations compared with the original native aSyn sequence, but which shows similar (the same or improved) immunization characteristics, i.e. is able to elicit an immune response that is similar or higher than the immune re- sponse that is obtained with the native aSyn sequence
• the AFFITOPES® are designed not to elicit cytotoxic, or helper T cell responses, the first to avoid cytotoxic attacks against any tissue reachable by CD8+ T cells bearing linear se- quence fragments of the immunizing peptide, the latter to avoid responses to target-derived peptides independently of the vaccine and thus the generation of a permanently renewed and uncontrolled immune response.
• This AFFITOPE ® or VARIOTOPE ® technology has been designed with the aim of (i) breaking tolerance against self- proteins, (ii) generating high titer antibody responses to the peptide moiety of the vaccine which cross-react with the native target protein epitope, (iii) and to not induce an autoimmune response .
• the length of the peptide has therefore been limited, preferably to 12 aa (X 1 to X 12 ) .
• the antigenic peptides of the invention are typically 11-20 amino acids in length, preferably 12-14 amino acids in length. It is particularly preferred that the antigenic peptides are 12 or 14 amino acids in length. They typically contain between 1 and 4, preferably 2 or 3, amino acid mutations compared to the native alpha synuclein sequence. Note, however, that amino acid exten- sions may be permitted that are not based on (or identical to) the alpha synuclein sequence, especially at the N terminus of the peptide. Such additional amino acids may form part of a linker for example.
• Linkers may be present which covalently link the peptide to other molecular moieties, such as the Carrier.
• the linker may comprise additional amino acids, preferably amino acids with uncharged side chains such as glycines.
• the linkers may be attached to all positions in the immunogenic peptide, as long as the immunogenic properties of the peptides are not significantly worsened. For example, linkers may be linked via the N-terminal or C-terminal aa (X 1 or X 12 ) ; also linking inside the peptide may be possible.
• linkers and carriers may be more diverse, including linking the peptides of the present invention to surfaces, i.e. solid surfaces.
• the peptides of the present invention may also be used in various assays and kits, in particular in immunological assays and kits. Therefore, it is particularly preferred that the peptides of the present invention may be part of another peptide or polypeptide, for example, they may be fused or conjugated with an enzyme which is used as a reporter in immunological assays.
• reporter enzymes include e.g. fluorescent moieties, such as a green fluorescent protein (GFP) , phosphatases, such as an alka- line phosphatase, or oxidases/reductases , such as a horseradish peroxidase .
• the present invention relates to an antigenic peptide having the structure
• D is aspartic acid
• E is glutamic acid
• P is proline
• V is valine ;
• X 1 is L, K, A or S, wherein L is leucine, K is lysine, A is alanine and S is serine;
• X 2 is E or S, wherein E and S are as defined above;
• X 3 is D, E, K, N, A or S, wherein N is asparagine and D, E, K, A and S are as defined above;
• X 4 is M, A, S, L or K, wherein M is methionine and A, S, L and K are as defined above;
• X 10 is N, S or A, wherein N, S and A are as defined above;
• X 12 is present or not and, if present, is A, K, V, S, or G wherein G is glycine and A, K, V and S are as defined above, with the proviso that X 1 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 is not L-E-D- M-P-V-D-P-D-N-E-A.
• the term "having the structure” is to be understood as “consisting of ( the structure ) " the amino acid res- idues given ( i . e . excluding further amino acid residues , e . g . at the C-terminus of the antigenic peptide ) .
• Minor modi fications such as amidation, esteri fication, formylation, acetylation, other chemical substitution etc . of a free C-terminal ( or N-terminal ) end of a peptide or its side chains are not excluded but it is preferred not to have such modi fications present .
• Such minor mod- i fications are within the scope of the term “consisting essentially of” as used herein .
• These l lmer or 12mer peptides according to the present invention can be provided in compositions suitable for the intended use for preventing and/or treating synu- cleinopathies , especially in pharmaceutical compositions , prefer- ably combined with a pharmaceutically acceptable carrier .
• Such pharmaceutical compositions can be administered to a patient in need thereof in an ef fective amount to achieve the preventive and/or therapeutic ef fect .
• the peptide according to the present in- vention is selected from the group consisting of AEDMPVDPDNEA,
• LEAMPVDPDNEA LEDAPVDPDNEA, LEDMAVDPDNEA, LEDMPADPDNEA
• LEDMPVDPANEA LEDMPVDPDNAA
• SEDMPVDPDNEA SEDMPVDPDNEA
• LEDMPVDPDSEA LEDMPVDPDNSA
• LEDMPVDPDNES LEDMPVDPDNES
• LEKMPVDPDNEA LEKMPVDPDNEA, LEKMPVDPDNEA, LEDKPVDPDNEA, LEDMPVDPDNEV
• LEDMPVDPDNEG LEDMPVDPDNEK
• LESMPVDPDNES SESMPVDPDNEA
• LESSPVDPDNEA SEDMPVDPDNES , LEDSPVDPDNES , SEDSPVDPDNEA, KED- MPVDPDNEA, LEKMPVDPDNES , SEKMPVDPDNEA, LEKMPVDPDNEK,
• KESMPVDPDNEA KESMPVDPDNEK
• KEDMPVDPDNEA KEDMPVDPDNEA
• LEKMPVDPDNEA LEKMPVDPDNEA
• LESMPVDPDNES LEKMPVDPDNES
• SEKMPVDPDNEA KEDMPVDPDNEA
• the peptide according to the present invention i s selected from the group consisting of KESMPVDPDNEA, LESMPVDPDNEA, LESMPVDPDNES , SEDMPVDPDNEA, LEEMPVDPDNEA, SESMPVDPDNEA, LED- MPVDPDNES , LEAMPVDPDNEA, LEDMPVDPDNEK, LEDMPVDPDNEV, LEKMPVDPDNEK, LSDMPVDPDNEA, LEKMPVDPDNEA, KEDMPVDPDNEA, LENMPVDPDNEA, KESMPVDPDNEK and KEDMPVDPDNEA, preferably SED- MPVDPDNEA, LEEMPVDPDNEA, LESMPVDPDNEA, KESMPVDPDNEA, KED- MPVDPDNEA, LEKMPVDPDNEA and LESMPVDPDNES , especially LEKMPVDPDNEA, KESMPVD
• Linker-X 1 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 wherein X 1 , X 2 , X 3 , X 4 , X 10 , and X 12 are defined as above , and wherein the amino acid linker comprises between 1 and 5 amino acid resi- dues .
• the peptide with an amino acid linker comprises a linker, wherein the amino acid residue ( s ) in the linker is ( are ) selected from the group consisting of glycine , cysteine , isoleucine , ala- nine , valine , leucine , serine , glutamic acid, aspartic acid, ly- sine , asparagine , glutamine and combinations thereof , preferably wherein the linker is selected from the group C- , G- , CG- , CGG- , CCG- , GC- , GGC- , GCC- , GG- , and GGG- ; especially wherein the pep- tide with an amino acid linker is selected from the group GGKESMPVDPDNEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GCGKESMPVDPDNEA,
• CLEAMPVDPDNEA CLEDMPVDPDNEK, CLEDMPVDPDNEV, CGGKESMPVDPDNEA, CLEKMPVDPDNEK, CLSDMPVDPDNEA, CLEKMPVDPDNEA, CKEDMPVDPDNEA,
• CLENMPVDPDNEA, CGGKESMPVDPDNEK, and CGGKEDMPVDPDNEA especially GGKESMPVDPDNEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, CGCK- ESMPVDPDNEA, CGGKESMPVDPDNEA, GGKESMPVDPDNEA, GGKESMPVDPDNEA, GGKESMPVDPDNEA, and GKESMPVDPDNEA.
• X 12 is not present and no further amino acid residue is present C-terminally to X 11 .
• the an- tigenic peptide of the invention has no further extensions at the C-terminal end (except that minor modi fications may be present , e.g. which may stabilise the compound or peptide (e.g. amidation, etc.) ) .
• no amino acid extensions should (in contrast to the fusion proteins disclosed e.g. in WO 2005/108423 A 1) be present C-terminally which represent the native amino acid se- quence of aSyn, i.e. specifically Tyr 125 and GlU 126 , which could bind with high affinity to different allelic variants of MHCI and thus could be potential cytotoxic T cell epitopes.
• no Y amino acid residue or YE dipeptide chain should be present at the C-terminal end of the peptide, wherein Y is tyrosine and E is as defined above.
• the peptides and compounds of the present invention can be synthetically produced by chemical synthesis methods which are well known in the art, either as an isolated peptide or as a part of another polypeptide.
• the peptides and compounds can be produced in a microorganism which produces the peptide of the present invention which is then isolated and if desired, fur- ther purified.
• the peptides and compounds can be produced in mi- croorganisms such as bacteria, yeast or fungi, in eukaryote cells such as a mammalian or an insect cell, or in a recombinant virus vector such as adenovirus, poxvirus, herpesvirus, Semliki forest virus, baculovirus, bacteriophage, Sindbis virus or Sendai virus.
• mi- croorganisms such as bacteria, yeast or fungi
• eukaryote cells such as a mammalian or an insect cell
• a recombinant virus vector such as adenovirus, poxvirus, herpesvirus, Semliki forest virus, baculovirus, bacteriophage, Sindbis virus or Sendai virus.
• Suitable bacteria for producing the peptides and compounds include E.coli, B.subtilis or any other bacterium that is capable of ex- pressing peptides such as the peptide mimoto
• Suitable yeast types for expressing the peptides and compounds include Saccharo- myces cerevisiae, Schizosaccharomyces pombe, Candida, Pichia pas- toris or any other yeast capable of expressing peptides. Corre- sponding methods are well known in the art. Also methods for iso- lating and purifying recombinantly produced peptides and compounds are well known in the art and include e.g. gel filtration, affinity chromatography, ion exchange chromatography etc.
• a fu- sion polypeptide may be made wherein the 12mer peptide is trans- lationally fused (covalently linked) to a heterologous polypeptide which enables isolation by affinity chromatography.
• Typical het- erologous polypeptides are His-Tag (e.g. His 6 ; 6 histidine resi- dues) , GST-Tag (Glutathione-S-transferase) etc.
• His-Tag e.g. His 6 ; 6 histidine resi- dues
• GST-Tag Glutathione-S-transferase
• the fusion polypeptide may comprise a cleavage site e.g. at the junction between the peptide mimotope and the heterologous polypeptide.
• the cleavage site consists of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site (e.g. proteases) .
• the antigenic peptide of the invention, the immunogenic com- pound of the invention or the pharmaceutical preparation of the invention are for use in therapy (i.e. as a medicament) . More specifically, the invention provides the antigenic peptide of the invention, the immunogenic compound of the invention or the phar- maceutical preparation of the invention are for use in the treat- ment or prevention of a synucleinopathy . The invention also pro- vides for use of the antigenic peptide of the invention, the im- munogenic compound of the invention or the pharmaceutical prepa- ration of the invention, for the manufacture of a medicament for the treatment or prevention of a synucleinopathy.
• the invention also provides a method for the treatment or prevention of a synu- cleinopathy comprising administering the antigenic peptide of the invention, the immunogenic compound of the invention or the phar- maceutical preparation of the invention to a subject in need thereof.
• the synucleinopathy is selected from the group consisting of Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Park- inson's Disease with Dementia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Multiple System Atrophy (MSA) or Neurodegenera- tion with Brain Iron Accumulation type I (NBIA Type I) .
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Park- inson's Disease with Dementia
• DLB Dementia with Lewy Bodies
• MSA Multiple System Atrophy
• NBIA Type I Neurodegenera- tion with Brain Iron Accumulation type I
• synucleinopathies or alpha-synucleinopathies
• synucleinopathies is used to describe diseases where a- synuclein aggregates are detected and comprises primary synucle- inopathies and concomitant pathology.
• Parkinson's disease sporadic, familial with alpha-synu- clein mutations, familial with mutations other than alpha-synu- clein, pure autonomic failure and Lewy body dysphagia
• LBD Lewy Body dementia
• DLB dementia with Lewy bodies
• PPD Parkinson's disease dementia
• Dif- fuse Lewy Body Disease multiple system atrophy (Shy-Drager syn- drome, striatonigral degeneration and olivopontocerebellar atro- phy) .
• a-syn lesions can be detected as concomitant pathology in the following diseases: sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzhei- mer's disease with PS-1, PS-2 or other mutations, familial British dementia, inclusion-body myositis, traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Frontotemporal dementia with Parkinsonism linked to chromosome 17 and Niemann-Pick type C1 dis- ease) , Down syndrome, Creutzfeldt- Jakob disease, Huntington's dis- ease, motor neuron disease, amyotrophic lateral sclerosis (spo- radic, familial and ALS-dementia complex of Guam) , neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type 1 (Hallervorden-Spatz syndrome
• the subject that is treated is a mammalian subject, preferably a human.
• the antigenic peptide of the invention, the immunogenic compound of the invention or the pharmaceutical preparation of the invention is administered at an amount effective to treat or prevent a synucleinopathy .
• Prevention is preferred using vaccine compositions of the invention.
• Pre- vention encompasses delaying the onset and/or severity of disease (as compared to the situation without administration) as well as complete prevention of disease.
• Treatment encompasses ameliorat- ing one or more symptoms of disease, preventing or delaying disease progression (as compared to the situation without administration) as well as complete treatment of disease. Suitable dosages and administration routes are described herein and variations may be determined empirically by a clinical practitioner.
• Carrier-X 1 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 wherein Carrier is a polypeptide carrier covalently coupled to X 4 ; pref- erably comprising a linker moiety which covalently links a carrier molecule to the peptide X 4 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 ;
• D is aspartic acid
• E is glutamic acid
• P is proline
• V is valine ;
• X 1 is L, K, A or S, wherein L is leucine, K is lysine, A is alanine and S is serine;
• X 2 is E or S, wherein E and S are as defined above;
• X 3 is D, E, K, N, A or S, wherein N is asparagine and D, E, K, A and S are as defined above;
• X 4 is M, A, S, L or K, wherein M is methionine and A, S, L and K are as defined above;
• X 10 is N, S or A, wherein N, S and A are as defined above;
• X 12 is present or not and, if present, is A, K, V, S, or G, wherein G is glycine and A, K, V and S are as defined above, with the proviso that X 4 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 is not L-E-D- M-P-V-D-P-D-N-E-A.
• linker moiety comprises at least one cysteine and/or glycine amino acid residue, preferably coupled with a chemical linker to the polypeptide carrier moiety, especially wherein the linker moiety is formed by NHS-poly (ethylene oxide) (PEG) (e.g. by NHS-PEO 4 - maleimide) .
• PEG ethylene oxide
• the polypeptide carrier moiety is or comprises a pharma- ceutically acceptable carrier molecule selected from the group consisting of Keyhole Limpet Hemocyanin (KLH) , tetanus toxoid, heat-labile enterotoxin (LT) , cholera toxin (CT) , diphtheria toxin (DT) and variants thereof, especially CRM 197, tetanus toxoids (TT) , mutant toxins, albumin-binding proteins, and bovine serum albumin .
• KLH Keyhole Limpet Hemocyanin
• LT heat-labile enterotoxin
• CT cholera toxin
• DT diphtheria toxin
• variants thereof especially CRM 197, tetanus toxoids (TT) , mutant toxins, albumin-binding proteins, and bovine serum albumin .
• Immunogenic compound according to any one of clauses 1 to 6 for use in the treatment or prevention of synucleopathies pref- erably for use in the treatment or prevention of a synucleinopathy selected from the group consisting of Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Parkinson's Disease with De- mentia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Mul- tiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I) .
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Parkinson's Disease with De- mentia
• DLB Dementia with Lewy Bodies
• MSA Mul- tiple System Atrophy
• NBIA Type I Neurodegeneration with Brain Iron Accumulation type I
• a synucleinopathy preferably a synucleinopathy selected from the group consisting of Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Parkinson's Disease with Dementia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Multiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I) .
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Parkinson's Disease with Dementia
• DLB Dementia with Lewy Bodies
• MSA Multiple System Atrophy
• NBIA Type I Neurodegeneration with Brain Iron Accumulation type I
• composition according to clause 8 wherein the preparation is formulated as a vaccine with an adjuvant, preferably with an adjuvant selected from the group consisting of MF59 alu- minium 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 aluminium hydroxide, or mix- tures thereof; especially with aluminium hydroxide as adjuvant.
• an adjuvant selected from the group consisting of MF59 alu- minium phosphate, calcium phosphate, cytokines (e.g., IL-2, IL- 12, GM-CSF) , saponins (e.g., QS21) , MDP derivatives, CpG oligos, IC31, LPS, MPL, polyphosphazenes
• D is aspartic acid, E is glutamic acid, P is proline, and V is valine; (C-) is cysteine being present or not;
• X 1 is L, K, A or S, wherein L is leucine, K is lysine, A is alanine and S is serine;
• X 2 is E or S, wherein E and S are as defined above;
• X 3 is D, E, K, N, A or S, wherein N is asparagine and D, E, K, A and S are as defined above;
• X 4 is M, A, S, L or K, wherein M is methionine and A, S, L and K are as defined above;
• X 10 is N, S or A, wherein N, S and A are as defined above;
• X 12 is present or not and, if present, is A, K, V, S, or G wherein G is glycine and A, K, V and S are as defined above, with the proviso that X 1 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 is not L-E-D-
• Linker-X 1 -X 2 -X 3 -X 4 -P-V-D-P-D-X 10 -E-X 12 wherein X 1 , X 2 , X 3 , X 4 , X 1 , and X 12 are defined as in clause 13 , and wherein the amino acid linker comprises between 1 and 5 amino acid residues .
• CLSDMPVDPDNEA CLEKMPVDPDNEA, CKEDMPVDPDNEA, CLENMPVDPDNEA,
• CGGKESMPVDPDNEK CGGKESMPVDPDNEK
• CGGKEDMPVDPDNEA especially GGKESMPVDPDNEA, GKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, GCGKESMPVDPDNEA, GGGKESMPVDPDNEA, CGGKESMPVDPDNEA, CGCK- ESMPVDPDNEA, CGGKESMPVDPDNEA, GGKESMPVDPDNEA, GGKESMPVDPDNEA, GGKESMPVDPDNEA, and GKESMPVDPDNEA.
• Immunogenic compound according to any one of clauses 1 to 7 or peptide with an amino acid linker according to clause 14 or 15 , wherein the linker moiety or the amino acid linker does not contain amino acids selected from the group consisting of proline , argi- nine , or histidine .
• X 12 is not present and wherein no further amino acid residue is present C-terminally to X 11 .
• an immunogenic compound according to any one of clauses 1 to 7, 16 and 17 or an antigenic peptide according to clauses 12 or 13 or a peptide with an amino acid linker according to clauses 14 to 17 for the manufacture of a medicament for the treatment or prevention of synucleopathies, preferably for the treatment or prevention of a synucleinopathy selected from the group consisting of Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Parkinson's Disease with Dementia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Multiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I) •
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Parkinson's Disease with Dementia
• DLB Dementia with Lewy Bodies
• MSA Multiple System Atrophy
• NBIA Type I Neurodegeneration with Brain Iron Accumulation type I
• a method for the treatment or prevention of synucleopathies preferably for the treatment or prevention of a synucleinopathy selected from the group consisting of Lewy Body Disorders (LBDs) , especially Parkinson's Disease (PD) , Parkinson's Disease with De- mentia (PDD) and Dementia with Lewy Bodies (DLB) , as well as Mul- tiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I) , wherein an effective amount of an immunogenic compound according to any one of clauses 1 to 7, 16 and 17 or an antigenic peptide according to clauses 12 or 13 or a peptide with an amino acid linker according to clauses 14 to 17, is administered to a human individual in need of such treatment or prevention .
• LBDs Lewy Body Disorders
• PD Parkinson's Disease
• PPD Parkinson's Disease with De- mentia
• DLB Dementia with Lewy Bodies
• MSA Mul- tiple
• Figure 1 Schematic time-course of the experiments . Inj ec- tions are represented by arrows and blood sampling by drops .
• PP Pre-plasma
• EP End-plasma
• Pn Plasma n
• Wn Week n of the ex- periment .
• Figure 2 Comparison of aSyn cross-reacting antibodies in- prised by p4456 and p4572 or by the corresponding native aSyn epitope of different length.
• A Induced antibody concentrations against aSyn protein from end-plasma of all individual mice are presented. Columns represent mean values with SEM. Extreme outli- ers (beyond 3 x the IQR) have been removed from the graph
• B Relative position of the injected peptides along the amino-acid sequence of the native aSyn sequence. p9524 was used as scaffold for further development of AFFITOPE ® s and is highlighted in gray.
• Figure 3 Immunogenicity of peptides (delivered as peptide- carrier protein conjugates) with aSyn sequence aall3-124 with sin- gle alanine (A) or serine (B) exchanges.
• Group median concentra- tions of anti-aSyn antibodies in the end-plasma derived from in- dividual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p9524, which was set as 100 %. Sequences of the injected peptides are shown in Table 3 and 4.
• FIG. 4 Immunogenicity of peptides (delivered as peptide- carrier protein conjugates) with aSyn sequence aall3-124 with sin- gle amino acid exchanges at position 1 or 3 (A) and 4 or 12 (B) .
• Group median concentrations of anti-aSyn antibodies in the end- plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elic- ited by vaccination with p9524, which was set as 100 %. Sequences of the injected peptides are shown in Table 7 and 8.
• Figure 5 Immunogenicity to aSyn induced by aSyn sequence aan3-i24 with double serine and other amino acid exchanges.
• Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p9524, which was set as 100 %. Sequences of the injected peptides are shown in Table 11.
• Figure 6 Immunogenicity to aSyn induced by aSyn sequences aan3-i24 and N-terminally prolonged sequences.
• Group median concen- trations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with pl0074, which was set as 100 %. Sequences of the injected peptides are shown in Table 13.
• FIG. 7 Immunogenicity to aSyn induced by aSyn sequences aa 113 - 124 , N-terminally prolonged sequences, and by the aSyn sequence aa 115 - 121 p4456.
• Group median concentrations of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were de- termined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with p4456, which was set as 100 %. Sequences of the injected peptides are shown in Table 13.
• Figure 8 Immunogenicity of aSyn targeting sequences pl0033 and pl0118 and C-terminally truncated sequences thereof.
• A Group median immunogenicity of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccination with pl0033, which was set as 100 %.
• B Group median immunogenicity of anti-aSyn antibodies in the end-plasma derived from individual immunized mice were determined and set in relation to the concentration of anti-aSyn antibodies elicited by vaccina- tion with p10118, which was set as 100 %. Sequences of the injected peptides are shown in Table 16.
• Figure 9 IHC staining of post mortem human DLB brains by AFFITOPE ® candidate-induced antibodies.
• the antibodies used in each panel are induced by the peptide shown on top of the respective panel.
• Underlined letters indicate amino acids that are different from the native sequence. Size bars indicate 50 ⁇ M in the main pictures and 10 ⁇ M in the smaller box in the lower right corner focusing on single Lewy Bodies (LBs) .
• Figure 10 Preferential binding to the oligomeric and toxic aSyn species vs the monomeric form (BiaCore data) .
• the blue lines represent the negative control (binding to HBS buffer only) .
• X- axis running time (sec)
• y-axis relative binding response units.
• Figure 11 Competition ELISA showing concentration-dependent inhibition of the binding of AFFITOPE ® candidate-specific antibod- ies to aSyn monomers, oligomers, and filaments.
• Purified antibod- ies from AFFITOPE ® candidate-immunized mice were pre-incubated with increasing amounts of different aSyn-species and then tested for binding to plate bound aSyn-oligomers .
• Inhibition of binding by monomeric aSyn is shown in red curves, inhibition by fibrillar aSyn in blue curves, and inhibition by oligomers in green curves.
• X-axis decadic logarithm of aSyn-species concentration (ng/ml)
• y-axis OD 405 value measured with each aSyn species and concen- tration . Examples
• ther- apeutic approaches for synucleinopathies might involve reducing the levels or accumulation of intracellular and extracellular aSyn.
• oligomeric aSyn is prone to be secreted into the intercellular space and is able to move from one affected neuron or (e.g.
• oligodendrocyte to a neigh- boring neuron or neuroglial cell in a prion-like fashion opens an avenue for a therapeutic approach such as AFFIRIS specific active immunotherapy (SAIT) , that targets aSyn-transmission in a long-term manner.
• SAIT AFFIRIS specific active immunotherapy
• the AFFITOPE ® s PD01 and PD03 - short synthetic peptides mimicking defined regions of aSyn - have been tested as compounds for SAIT in clinical Phase I trials in PD - PD01A (NCT01568099; Volc et al., 2020) and PD03A (NCT02267434 ) - and in multiple system atrophy (MSA) patients (PD01 and PD03) (NCT02270489) .
• the agents were shown to be well tolerated and elicited aSyn-specif ic antibodies with a preference for oli- gomeric aSyn.
• aSyn-targeting AFFITOPE ® s were developed, which induced higher ti- ters and cross-reactivity to aSyn protein, than seen for PD01 and PD03, respectively. Furthermore, the focus of selection was also on the ability of the induced antibodies to discriminate between the aggregated and toxic aSyn species (oligomeric aSyn) and the monomeric aSyn protein.
• the binding of AFFITOPE ® -induced antibodies to the overrepresented monomeric aSyn species in the periphery is supposed to be of minor extent, however, the binding to the toxic oligomeric and underrepresented aSyn species in the CNS and the periphery would occur preferably.
• aSyn epitope aa 113 - 124 was targeted.
• Well-defined selection criteria were applied such as high immunogenicity, high cross-reactivity to aSyn native epitope, and binding of induced antibodies with high selectivity towards oligomeric and fibrillar toxic aSyn species (oligomer binding > fibril binding) .
• the selection strategy consisted of several steps: (i) epitope finding within the C-terminus of aSyn target protein in order to identi fy a highly immunogenic and suitable epitope , ( ii ) alanine scan in order to detect positions along the native target sequence that can be exchanged in order to enhance immunogenicity and cross- reactivity, ( iii ) serine scans in order to detect positions along the native target sequence that can be exchanged in order to en- hance immunogenicity and cross-reactivity, ( iv) double serine ex- changes along the native target sequence , (v) other than Ala or Ser amino acid exchanges able to improve immunogenicity and cross- reactivity towards aSyn, and (vi ) removal of one amino acid at the C-terminus from two selected AFFITOPE ® sequences .
• mice received 3 inj ections with AFFITOPE ® s or native aSyn epitope sequences ( 10 ⁇ g net peptide per inj ection) , in biweekly intervals ( Figure 1 ) .
• the peptide p9524 corresponding to aSyn aa 113- 124 sequence induced the highest amount of aSyn-speci f ic antibodies ( Figure 2 , Table 2 ) . Based on these findings this sequence was selected as native target sequence for further AFFI TOPE ® candidate selection . The two other peptides p9964 and p9556 which elicited also high anti-aSyn titers were excluded from further development .
• the peptide p9964 includes the amino acids Y 125 and E 126 and peptide fragments derived from p9964 are predicted by in sili co analyses to bind with high af finity to di f ferent allelic variants of MHCI and thus be poten- tial cytotoxic T cell epitopes (www . syfpeithi . de ) .
• the peptide p9556 was not chosen for further development because it does not cover the potentially pathology-related calpain cleavage site aSyn L 113 /E 114 .
• Table 2 summarizes the titers to aSyn found in AFFITOPE ® induced mouse plasmas.
• Table 1 Setup of experiments aSyn-28. The table shows the drug product, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.
• Table 2 aSyn-28, key results including titers, induced antibody concentration, and cross-reactivity. All parameters were evaluated in single mice and values refer to medians. The concentration of anti-aSyn antibodies in the plasma of immunized mice was extrapo- lated from a reference curve generated with the mAb LB509. Table 2
• each amino acid position of the native aSyn 113 _ 124 sequence was exchanged either by an alanine or a serine in order to identify substitutable positions in order to maintain or enhance immunogenicity (Tables 3 and 4) .
• BALB/c mice were injected three times with the respective AFFITOPE ® s and two weeks after the third and last injection, plasma of each individual mouse was collected and analyzed by ELISA in order to determine the AFFITOPE ® -induced titers and aSyn antibody concentrations.
• Table 3 Setup of experiment aSyn-30. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product, and the corresponding sequence ID numbers. Table 3
• Table 4 Setup of the experiment aSyn-31. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corre- sponding sequence ID numbers.
• Table 5 aSyn-30, key results.
• the titers to aSyn evaluated in single mice and values represent medians relative to the median obtained with p9524.
• Table 6 aSyn-31, key results.
• the titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.
• Table 7 Setup of experiment aSyn-32. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.
• Table 8 Setup of experiment aSyn-33. The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.
• Table 9 aSyn-32, key results.
• the titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.
• Table 10 aSyn-33, key results.
• the titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.
• VARIOTOPES® with additional serine exchanges along the aSynu 3 _ 124 native sequence were designed and tested for their immunogenicity in wt BALB/c mice (Table 11, Figure 5) .
• the na- tive aSyn sequence was injected for direct comparison purposes (Group 1) .
• Table 12 summarizes the induced titers relative to the titers induced by the WT sequence against aSyn present in the plasma of immunized mice.
• Table 11 Setup of experiment aSyn-37. The table shows the dif- ferent treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.
• Table 12 aSyn-37, key results.
• the titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p9524.
• Table 15 summarizes the induced titers relative to the titers induced by the AFFITOPE ® sequence p4456 against aSyn present in the plasma of immunized mice.
• Table 13 Setup of experiment aSyn-44. The table shows the dif- ferent treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers.
• Table 14 aSyn-44, key results.
• the titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with p10074 set to 100%.
• Table 25 The titers to aSynwere evaluated in single mice and values represent group medians relative to the group median ob- tained with p4456 set to 100%.
• AFFITOPE ® sequences p10033 and especially p10118 have been shown to induce high anti-aSyn antibody concentrations ( Figure 4A and Figure 6, respectively) .
• the C-terminal Ala at position X 1 2 was removed from AFFITOPE ® sequences p10033 and p10118 in order to test whether C- terminal truncation has an influence on the immunogenicity and aSyn cross-reactivity of designed peptides, wt BALB/c mice where immunized in independent experiments with either p10033, p10118, p10166 or p10167.
• Table 16 lists the peptide sequences.
• Table 17 summarizes the induced titers relative to the titers induced by either by the AFFITOPE ® sequence p10033 (A) or by the AFFITOPE ® sequence p10118 (B) against aSyn present in the plasma of immunized mice.
• Table 16 The table shows the different treatment groups, the respective drug product in use, the sequence of the peptide in the drug product and the corresponding sequence ID numbers. Table 16
• Table 17 The titers to aSyn were evaluated in single mice and values represent medians relative to the median obtained with ei- ther p 10033 or p 10118 .
• the second control antibody, 28A7 which was raised against the peptide p4456 , did discriminate between monomeric and oligo- meric aSyn species .
• the AFFI TOPE ® candidate-induced antibodies showed high selectivity for oligomeric aggregates of aSyn compared to the monomeric form of aSyn ( Figure 10 ) .
• AFFITOPE ® -induced Abs have been tested for their preferential binding to the aSyn fila- ments over the monomeric species of aSyn by inhibition ELISA.
• a constant amount of af finity-puri fied AFFITOPE ® - induced antibodies was preincubated with titrated amounts of mon- omeric and filamentous aSyn and then trans ferred to ELISA plates coated with aSyn filaments ( for details see M&M) .
• Figure 11 the results of two representative AFFITOPE ® candidates (p 10033 and p 10118 ) are shown . Very good competition was seen with aSyn oli- gomers followed by aSyn filaments , whereas the competition with the monomeric form of aSyn was only of minor extent ( Figure 11 ) .
• a mAb 28A7 that was known to preferably bind the oligomeric and aggregated forms of aSyn was used as control ( Figure 11C ) .
• AFFITOPE ® candidate-speci fic antibodies provide clear evidence of high selectivity of AFFITOPE ® candidate-induced antibodies to the toxic, oligomeric aggregates of aSyn, which are considered to be the relevant toxic species that lead to cell death, as opposed to the monomeric form .
• mice were purchased from Janvier Elevages ( Le Genest- Sainte- I sle , F) .
• the animals were housed and kept under standard conditions described in the application of the IMP to license its activity as breeders , suppliers and users .
• the respective permission was granted by the relevant authorities on May 13 th 2013 with the notification GZ : 223633/2013/4.
• mice were kept in TECNIPLAST Sealsafe NextIVC Blue Line - Cages (Milano, IT) a five mice. Cages were equipped with enrichment in the form of nesting material and little plastic houses for hiding/playing purposes. The age of the mice at the beginning of the experiment was between 6 and 8 weeks. They were provided with standard diet and acidified water ad libitum and were kept under a 12 hour light/dark cycle.
• Peptides used for immunization were purchased from EMC micro- collections (Tubingen, Germany) .
• CRM197 was purchased from Pfenex (San Diego, CA) .
• All immunogenic AFFITOPE ® -based products used in the described experiments are conjugates of the synthetic AFFITOPE ® peptides to the carrier protein CRM197.
• the conjugation is a directed procedure using the side chain amino groups of lysine residues in CRM197 and the free thiol group of the amino (N) -terminal cysteine in the peptide.
• the aqueous CRM197 solution is adjusted to 10 mM phosphate buffered saline (PBS) and is then gently shaken with the bifunctional linker 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) .
• PBS phosphate buffered saline
• GMBS 4-maleimidobutyric acid N-hydroxysuccinimide ester
• Vaccines are brought to ambient temperature, vortexed and applied subcutaneously (s.c.) in the flank of mice (200 ⁇ l) with an insulin 20 syringe with a G30-gauge (Omnican ⁇ 50, B. Braun Mel- sungen AG, Melsungen, Germany) . Immunization is repeated three times in biweekly intervals. Sample collection
• LB509 Biolegend, CA, US
• 28A7 is a commercially available purified anti-aSyn, 115-121 Ab.
• the mouse mAb 28A7 (IgGl) was generated in-house with mouse B cell hybridomas (Mandler et al., 2014) against the AFFITOPE ® PD01 which mimics the aSynn5-i2i epitope .
• Titers against the immunizing peptides and against the recom- binant human aSyn protein were analyzed.
• the presence of AFFIOTPE ® - induced antibodies in plasma of immunized mice was determined by ELISA.
• 96-well plates (Nunc-Maxisorp) were coated with either re- combinant human aSyn (1 ⁇ g/ml) or the injected peptide (BSA-con- jugate; 1 ⁇ M) .
• Titers were calculated as EC 50 _ values with PRISM® 5.04 (GraphPad Inc, San Diego, CA) by non-linear regression anal- ysis (four-parameter logistic fit function) .
• Plasma from AFFITOPE ® - immunized mice was used to stain sec- tions from frontal cortex brain biopsies from DLB patients. After tissue preparation (rehydration, deparaf f inization, antigen re- trieval, and blocking) , sections were incubated with diluted mouse plasma for 2 h at RT or overnight at 4 °C. Sections were incubated for 1 h at RT with undiluted Dako Envision HRP labelled polymer (Agilent, Santa Clara, CA) . For each IHC staining, counterstaining was done with Haematoxylin, and after this step, slides were de- hydrated and mounted in Entellan (Sigma-Aldrich) . Slides were scanned in the brightfield mode using a Panoramic (Mirax) Scanner 150 (Carl Zeiss MicroImaging GmbH) .
• aSyn oligomers (SynAging, Vandcewin-les-Nancy, ER) were thawed and diluted to 5 ⁇ g/ml in HBS shortly before injection.
• aSyn was freshly dissolved, diluted to 5 ⁇ g/ml in HBS, and cut-off centri- fuged for 10 min at 14.000 x g using 50 kDa cut-off columns (Amicon Ultra 0,5ml) .
• two aSyn-specif ic antibodies were used, LB509 (BioLegend, San Diego, CA) and 28A7 (AFFiRiS AG) .
• AFFITOPE ® specific antibodies out of im- mune plasmas lodoacetyl magnetic beads were coupled with the respective peptide (HPLC purified) for 1 h at RT and the remaining excess free sites were blocked with cysteine for an additional hour.
• AF- FITOPE ® -coupled beads were incubated with 150 pl plasma of mice immunized with the corresponding AFFITOPE ® candidate (for 2 h at RT) .
• the AFFITOPE ® -specif ic Abs were then eluted with Elution Buffer (Thermo Scientific) . Afterwards the eluents were concen- trated by Ultra Centrifugation (Millipore) tubes (30 kDa) to a volume of 150 ⁇ l (equal to the input volume) .
• IC 50 values were calculated as the concentration of either monomeric, oligomeric, or fibrilic aSyn which was needed to quench half of the ELISA signal. IC 50 values were calculated with PRISM® 5.04 (GraphPad Inc, San Diego, CA) by non-linear regression analysis (four-parameter logistic fit func- tion) .

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