Cyclic peptides

Abstract

The present invention relates to cyclized peptides based on amino acids 1 to 14 of amyloid β. The cyclic peptides are useful for inducing immune responses for the treatment of neurodegenerative diseases such as Alzheimer's disease and as vaccines.

Description

[Technical Field]

[0001] The present invention relates to cyclized peptides and their use as vaccines for the prevention and treatment of neurodegenerative diseases, such as Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive pulmonary degenerative disease characterized by the presence of extracellular deposits composed of amyloid beta (Aβ) protein. The main variants of Aβ protein are full-length Aβ (SEQ ID NO: 18) and Aβ (SEQ ID NO: 19), N-truncated pyroglutamate Aβ (SEQ ID NO: 3-42) and Aβ (SEQ ID NO: 4-42) (SEQ ID NO: 21).

[0003] Amyloid-β protein tends to aggregate and form amyloid fibrils. Amyloid fibrils are large, insoluble polymers of Aβ found in senile plaques and are a major contributor to the neuronal loss and dementia typical of Alzheimer's disease. However, evidence is also growing regarding the role of soluble Aβ oligomers, rather than Aβ precipitated in plaques, in the progression of Alzheimer's disease. Soluble oligomers have a non-fibrillar structure, are stable in aqueous solution, and remain soluble even after high-speed centrifugation. While Aβ plaques have been shown to correlate poorly with clinical symptoms in AD patients, soluble oligomers have been suggested to be good predictors of synaptic loss (Non-Patent Document 1), neurofibrillary tangles (Non-Patent Document 2), and clinical phenotype (Non-Patent Document 3). Furthermore, memory impairment and pathological changes in many AD mouse models occur well before the onset of plaque deposition (Non-Patent Document 4). In particular, Aβ trimers and tetramers are known to be the most harmful Aβ peptides at the onset of AD pathology. As such, low molecular weight (LMW) oligomers of Aβ have been viewed as targets for the treatment of amyloid-β-related diseases such as AD.

[0004] Antibodies targeting low molecular weight oligomers have been developed with the aim of neutralizing these oligomers. Passive immunization has been demonstrated with the antibody 9D5, which detects low molecular weight (LMW) AβpE3-42 (Non-Patent Document 5 and Patent Document 1). The murine anti-amyloid beta (Aβ) antibody NT4X-167, first generated against the Aβ4-40 amyloid peptide, has been reported to specifically bind to the N-truncated amyloid peptides AβpE3-42 and Aβ4-42, but not to the amyloid peptide Aβ1-42 (Non-Patent Document 6). Passive immunization using NT4X-167 has been shown to be therapeutically beneficial in Alzheimer's mouse models (Non-Patent Document 7 and Patent Document 2). Regarding clinical applications, a humanized version of NT4X has also been developed that is useful, for example, in the treatment of Alzheimer's disease (AD) (Patent Document 3).

[0005] Active immunization approaches for Alzheimer's disease have also been proposed, such as those described in U.S. Patent No. 6,277,629, which discloses Aβ-derived peptides, including cyclic peptides formed through head-to-tail cyclization. The use of linear peptides based on different regions of Aβ has also been proposed, such as those described in U.S. Patent No. 6,277,629. U.S. Patent No. 6,277,629 describes an active immunization approach targeting the N-terminal epitope of Aβ, using linear peptides based on Aβ as part of an immunogenic construct. However, these approaches do not specifically generate antibodies against low molecular weight oligomers.

[0006] Therefore, compounds that can be used for active immunization would be useful in the treatment of Alzheimer's disease, particularly in treatments that target early stages of AD progression. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2011 / 151076 [Patent Document 2] International Publication No. 2013 / 167681 [Patent Document 3] International Publication No. 2020 / 070225 [Patent Document 4] International Publication No. 2006 / 0609718 [Patent Document 5] International Publication No. 2014 / 143087 [Non-Patent Document]

[0008] [Non-Patent Document 1] Lue LF et al, Am J Pathol 1999, 155:853-862 [Non-Patent Document 2] McLean CA, et al, Ann Neurol 1999, 46:860-866 [Non-Patent Document 3] Snowdon DA: Aging and Alzheimer's disease: lessons from the Nun Study. Gerontologist 1997, 37:150-156 [Non-Patent Document 4] Bayer TA and, Wirths O. Front Aging Neurosci 2010, 2:1-10 [Non-Patent Document 5] Wirths et al. (2010) J. Biol. Chem. 285, 41517-41524 [Non-Patent Document 6] Antonios et al Acta Neuropathol. Commun. (2013) 6 1 56 [Non-Patent Document 7] Antonios et al Scientific Reports 5 17338; 2015 [Summary of the Invention]

[0009] The present invention generally relates to certain cyclic peptides based on amino acid residues 1 to 14 of the amyloid beta (Aβ) protein, which preferably specifically bind to antibodies that specifically bind to low molecular weight oligomers of the Aβ protein.

[0010] Thus, in a first aspect, the present invention provides a compound of formula (I) (SEQ ID NO: 1): X1X2X3FX4HDSGX5X6X7X8H (I) A cyclic peptide comprising an amino acid sequence having the sequence of During the ceremony, X1 is absent or any amino acid; X2 is alanine or cysteine; X3 is glutamic acid or cysteine, X4 is arginine or cysteine, X5 is tyrosine or cysteine, X6 is glutamic acid or cysteine, X7 is valine or cysteine; X8 is histidine or cysteine, The present invention relates to a cyclic peptide, wherein only one of X1, X2, X3, and X4 is a cysteine, and only one of X5, X6, X7, and X8 is a cysteine, and the peptide is cyclized through the cysteine ​​residues at positions 1, 2, 3, or 5 and 10, 11, 12, or 13. Preferably, X1 is present, more preferably, X1 is proline, aspartic acid, or cysteine, more preferably, cysteine ​​or aspartic acid. Preferably, there are at least 7 amino acids between any two cysteine ​​residues present in the sequence, and more preferably, there are 7 to 11, even more preferably, 8 or 11, amino acid residues between any two cysteine ​​residues present in the sequence.

[0011] In one embodiment, the present invention relates to a cyclic peptide comprising the sequence of formula (I) as described above, wherein the peptide does not contain cysteine ​​residues at both positions 5 and 12 or at both positions 3 and 10.

[0012] In one embodiment, X1 is absent or any amino acid; a) X1 is cysteine, X2 is alanine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is valine, and X8 is cysteine, or b) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is cysteine, and X8 is histidine, or c) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is cysteine, and X8 is histidine, or d) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is cysteine, X6 is glutamic acid, X7 is valine, and X8 is histidine, or e) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is valine, and X8 is cysteine, or f) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is cysteine, X7 is valine, and X8 is histidine, or g) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is tyrosine, X6 is cysteine, X7 is valine, and X8 is histidine, or h) X2 is alanine, X3 is glutamic acid, X4 is cysteine, X5 is tyrosine, X6 is glutamic acid, X7 is cysteine, and X8 is histidine, or i) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is cysteine, X6 is glutamic acid, X7 is arginine, and X8 is histidine, or j) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is valine, and X8 is cysteine; The peptide is cyclized via two cysteine ​​residues.

[0013] In one embodiment, the cyclic peptide has the formula (II) (SEQ ID NO: 2): X1ACFRHDSGYECHH (II) or a variant thereof, wherein the peptide is cyclized via the cysteine ​​residues located at positions 3 and 12, and X1 is as defined above. Preferably, X1 is aspartic acid.

[0014] In further embodiments, the cyclic peptide comprises: a) CAEFRHDSGYEVCH (SEQ ID NO: 14), in which the peptide is cyclized through the cysteine ​​residues located at positions 1 and 13; b) DACFRHDSGYECHH (SEQ ID NO: 4), in which the peptide is cyclized through the cysteine ​​residues located at positions 3 and 12; c) DCEFRHSGYECHH (SEQ ID NO: 5), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 12; d) DCEFRHDSGCEVHH (SEQ ID NO: 10), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 10; e) DCEFRHDSGYEVCH (SEQ ID NO: 12), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 13; f) DCEFRHDSGYCVHH (SEQ ID NO: 9), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 11; g) DACFRHDSGYCVHH (SEQ ID NO: 8), in which the peptide is cyclized through the cysteine ​​residues located at positions 3 and 11; h) DAEFCHDSGYECHH (SEQ ID NO: 7), in which the peptide is cyclized through the cysteine ​​residues located at positions 5 and 12; i) DACFRHDSGCEVHH (SEQ ID NO: 11), in which the peptide is cyclized through the cysteine ​​residues located at positions 3 and 10, and j) DACFRHDSGYEVCH (SEQ ID NO: 13), wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 13; The amino acid sequence may be selected from the following or a variant thereof.

[0015] Preferably, the cyclic peptide is a) DACFRHDSGYECHH (SEQ ID NO: 4), in which the peptide is cyclized through the cysteine ​​residues located at positions 3 and 12; b) DCEFRHSGYECHH (SEQ ID NO: 5), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 12; c) DCEFRHDSGCEVHH (SEQ ID NO: 10), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 10; d) DCEFRHDSGYEVCH (SEQ ID NO: 12), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 13; e) DCEFRHDSGYCVHH (SEQ ID NO: 9), in which the peptide is cyclized through the cysteine ​​residues located at positions 2 and 11; f) DACFRHDSGYCVHH (SEQ ID NO: 8), in which the peptide is cyclized through the cysteine ​​residues located at positions 3 and 11, and g) DACFRHDSGYEVCH (SEQ ID NO: 13), in which the peptide is cyclized through the cysteine ​​residues located at positions 3 and 13; The amino acid sequence may be selected from the following or a variant thereof.

[0016] In one embodiment, the cyclic peptide comprises the amino acid sequence CAEFRHDSGYEVCH (SEQ ID NO: 14) or a variant thereof, wherein the peptide is cyclized through the cysteine ​​residues located at positions 1 and 13.

[0017] In one embodiment, the cyclic peptide is cyclized through two cysteine ​​residues. Preferably, the peptide is cyclized through a bridge having the formula -SS- or -S-CH2-S- between the two cysteine ​​residues. More preferably, the peptide is cyclized through a bridge having the formula -S-CH2-S- between the two cysteine ​​residues.

[0018] Preferably, the cyclic peptide comprises the amino acid sequence CAEFRHDSGYEVCH (SEQ ID NO: 14) or a variant thereof, and the peptide is cyclized through the cysteine ​​residues located at positions 1 and 13. More preferably, the cyclic peptide comprises the amino acid sequence CAEFRHDSGYEVCH or a variant thereof, and the peptide is cyclized through a bridge having the formula -S-CH2-S- between the two cysteine ​​residues at positions 1 and 13.

[0019] Preferably, the cyclic peptide comprises the amino acid sequence DACFRHDSGYECHH (SEQ ID NO: 4) or a variant thereof, wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 12. More preferably, the cyclic peptide comprises the amino acid sequence DACFRHDSGYECHH or a variant thereof, wherein the peptide is cyclized through a bridge having the formula -S-CH-S- between the two cysteine ​​residues at positions 3 and 12.

[0020] Preferably, the cyclic peptide comprises the amino acid sequence DACFRHDSGYEVCH (SEQ ID NO: 13) or a variant thereof, and the peptide is cyclized through the cysteine ​​residues located at positions 3 and 13. More preferably, the cyclic peptide comprises the amino acid sequence DACFRHDSGYEVCH or a variant thereof, and the peptide is cyclized through a bridge having the formula -S-CH-S- between the two cysteine ​​residues at positions 3 and 13.

[0021] In a further embodiment, the cyclic peptide consists of the amino acid sequence CAEFRHDSGYEVCH (SEQ ID NO: 14) or a variant thereof, and the peptide is cyclized between two cysteine ​​residues at positions 1 and 13 via a bridge having the formula -S-CH2-S-.

[0022] In a further embodiment, the cyclic peptide consists of the amino acid sequence DACFRHDSGYECHH (SEQ ID NO: 4) or a variant thereof, and the peptide is cyclized through a bridge having the formula -S-CH2-S- between the two cysteine ​​residues at positions 3 and 12.

[0023] In one embodiment, the cyclic peptide consists of the amino acid sequence DACFRHDSGYEVCH (SEQ ID NO: 13) or a variant thereof, and the peptide is cyclized through a bridge having the formula -S-CH2-S- between two cysteine ​​residues at positions 3 and 13.

[0024] A further aspect of the present invention relates to a cyclic peptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence CAEFRHDSGYEVCH (SEQ ID NO: 14) or a variant thereof, wherein the peptide comprises cysteine ​​residues at positions 1 and 13 and a phenylalanine residue at position 4, and the peptide is cyclized through the cysteine ​​residues at positions 1 and 13.

[0025] In a further embodiment of the invention, the cyclic peptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence DACFRHDSGYECHH (SEQ ID NO: 4) or a variant thereof, wherein the peptide comprises cysteine ​​residues at positions 3 and 12 and a phenylalanine residue at position 4, and wherein the peptide is cyclized through the cysteine ​​residues at positions 3 and 12.

[0026] In a further embodiment of the invention, the cyclic peptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence DACFRHDSGYEVCH (SEQ ID NO: 13) or a variant thereof, wherein the peptide comprises cysteine ​​residues at positions 3 and 13 and a phenylalanine residue at position 4, and wherein the peptide is cyclized through the cysteine ​​residues at positions 3 and 13.

[0027] In a further embodiment of the invention, the cyclic peptide comprises the amino acid sequence DAEFRHDSGYEVHH (SEQ ID NO: 3) or a variant thereof, wherein one of the amino acid residues at positions 1, 2, 3 or 5 is substituted with a cysteine ​​residue and one of the amino acid residues at positions 10, 11, 12 or 13 is substituted with a cysteine ​​residue such that the peptide contains two cysteine ​​residues and the peptide is cyclized between the two cysteine ​​residues.

[0028] Preferably, in one embodiment, the cyclic peptide comprises the amino acid sequence DAEFRHDSGYEVHH (SEQ ID NO: 3) or a variant thereof, in which the amino acid residue at position 1 is substituted with a cysteine ​​residue and one of the amino acid residues at positions 10, 11, 12, or 13 is substituted with a cysteine ​​residue. Preferably, the amino acid residue at position 13 is substituted with a cysteine ​​residue.

[0029] Alternatively, in one embodiment, the cyclic peptide comprises the amino acid sequence DAEFRHDSGYEVHH (SEQ ID NO: 3) or a variant thereof, in which one of the amino acid residues at positions 2, 3, or 5 is substituted with a cysteine ​​residue, and one of the amino acid residues at positions 10, 11, 12, or 13 is substituted with a cysteine. Preferably, there are at least seven amino acids between any two cysteine ​​residues in the sequence, more preferably, there are between seven and ten amino acid residues, and even more preferably, there are eight or nine amino acid residues between any two cysteine ​​residues in the sequence. In one embodiment, the peptide does not contain cysteine ​​residues at positions 5 and 12 or both positions 3 and 10.

[0030] A further aspect of the present invention relates to pharmaceutical compositions comprising the above-described cyclic peptides and a pharmaceutically acceptable carrier. Preferably, the compositions further comprise an adjuvant. The compositions may be immunogenic compositions. In one embodiment, these compositions may be vaccine compositions.

[0031] Aspects of the present invention also relate to cyclic peptides for use as pharmaceuticals. One embodiment relates to a method for treating a neurodegenerative disease, comprising administering to an individual in need thereof a cyclic peptide or composition as described above. Preferably, the neurodegenerative disease is Alzheimer's disease.

[0032] A further embodiment of the present invention relates to a method of inducing an immune response in a subject, comprising administering to the subject a cyclic peptide or composition as described above, i.e. a cyclic peptide adopting the hairpin structure of amyloid-β or a composition comprising said peptide. Preferably, the immune response generates antibodies against amyloid-β, more preferably the amyloid-β is in the form of low molecular weight amyloid-β oligomers, and the method induces an immune response against low molecular weight amyloid-β oligomers.

[0033] One embodiment of the present invention relates to a cyclic peptide as described above for use in treating a neurodegenerative disease, preferably Alzheimer's disease.

[0034] A further embodiment of the present invention relates to a cyclic peptide as described above for use in inducing an immune response in a subject, preferably the immune response generates antibodies against amyloid beta, more preferably the amyloid beta is in the form of low molecular weight amyloid beta oligomers, and the use is to induce an immune response against low molecular weight amyloid beta oligomers.

[0035] A further embodiment of the present invention relates to cyclic peptides, i.e. cyclic peptides adopting the hairpin structure of amyloid beta, for the manufacture of a medicament for treating a neurodegenerative disease, such as Alzheimer's disease, and / or for inducing an immune response that preferably produces antibodies against amyloid beta oligomers, preferably low molecular weight amyloid beta oligomers.

[0036] A further aspect of the present invention is a method for producing a cyclic peptide as described above, comprising the steps of: (a) synthesizing a linear peptide containing the sequence of such a peptide; (b) cyclizing the linear peptide through the cysteine ​​residue to obtain a cyclic peptide according to formula (I); The present invention relates to a method, comprising:

[0037] A further aspect of the present invention is a method for producing an antibody that recognizes low molecular weight oligomers of amyloid beta, comprising the steps of: (a) immunizing an animal with a cyclic peptide or a variant thereof as described above; (b) obtaining antibodies produced by immunization in step (a); The method may further comprise a step (c) comprising screening the antibody obtained in step (b) for recognizing low molecular weight oligomers of amyloid beta. Preferably, the antibody is also screened for its ability to not bind to, or not significantly bind to, Aβ1-42, Aβ1-40, and / or Aβ1-38. Preferably, the antibody is screened for its ability to specifically recognize low molecular weight oligomers of amyloid beta, preferably low molecular weight AβpE3-x and Aβ4-x, more preferably AβpE3-42 and Aβ4-42. Preferably, the method comprises immunizing an animal with a cyclic peptide having the sequence of SEQ ID NO: 4, 13, or 14.

[0038] Further aspects of the invention include antibodies obtainable by the above methods. The obtained antibodies may be used in compositions, such as vaccine compositions. The antibodies may be used in the treatment of Alzheimer's disease.

[0039] Other aspects and embodiments of the invention are described in more detail below. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows the structure of TAP01 Fab. [Figure 2] FIG. 1 shows the structure of TAP01-pE3-14 Fab. [Figure 3] FIG. 1 shows (a) the structure of pGlu3-14 and (b) the TAP01-pGlu3-14 amyloid peptide structure. [Figure 4] FIG. 1 shows a comparison of the structures of TAP01-pE3-14 and TAP01_01-pE3-14. [Figure 5] FIG. 1 shows the structure of the TAP01-1-14 cyclized peptide. [Figure 6] FIG. 1 shows a comparison of the structures of (A) 1-14 (cysteines 3,12) and (B) pGlu3-14 cyclic amyloid peptides. [Figure 7] FIG. 1 shows binding ELISA data for the binding of reference antibodies (bapineuzumab, solanezumab, BAN2401, ProBioDrug 6_1_6, ProBioDrug 24_2_3) and Tap01 to disulfide-bridged 1-14 cyclic peptide 3,12. [Figure 8] FIG. 1 shows binding ELISA data for binding of animal sera to disulfide-bridged 1-14 cyclic peptide 3,12. [Figure 9] FIG. 1 shows binding ELISA data for binding of animal sera to Aβ1-42 peptide. [Figure 10] FIG. 1 shows binding ELISA data for binding of animal sera to AβpE3-42 peptide. [Figure 11] FIG. 1 shows binding ELISA data for binding of animal sera to Aβ4-42 peptide. [Figure 12] FIG. 1 shows binding ELISA data for binding of animal sera to KLH antigen. [Figure 13] FIG. 1 shows binding ELISA data for binding of animal sera to thioacetal-bridged 1-14 cyclic peptide 3,12. [Figure 14] FIG. 1 shows binding ELISA data for binding of animal sera to Aβ1-42 peptide. [Figure 15] FIG. 1 shows binding ELISA data for binding of animal sera to AβpE3-42 peptide. [Figure 16] FIG. 1 shows binding ELISA data for binding of animal sera to Aβ4-42 peptide. [Figure 17] Immunostaining of AD mouse model brain sections with M2 antiserum: SXFAD is mostly Aβ1-42 and plaques, Tg4-42 is only Aβ4-42, and TBA42 is only pyroglutamate Aβ3-42. [Figure 18] Immunostaining of AD mouse model brain sections with M4 antiserum: SXFAD is mostly Aβ1-42 and plaques, Tg4-42 is only Aβ4-42, and TBA42 is only pyroglutamate Aβ3-42. [Figure 19] FIG. 1 shows the effect of TAP01_04 (cloned as MoG1K) on 18F-FDG uptake in young and aged Tg4-42 mice. [Figure 20] FIG. 1 shows binding ELISA data for binding of (a) TAP01 (MoG1K) antibody and (b) MRCT control IgG1 antibody (cloned as MoG1K) to thioacetal-bridged cyclic peptide variants. [Figure 21] FIG. 1 shows binding ELISA data for binding of (a) TAP01 (MoG1K) antibody and (b) MRCT control IgG1 antibody (cloned as MoG1K) to thioacetal-bridged cyclic peptide variants. [Figure 22]FIG. 1 shows binding ELISA data for the binding of reference antibodies (bapineuzumab, solanezumab, BAN2401, ProBioDrug 6_1_6, ProBioDrug 24_2_3) and TAP01 HuG4K to thioacetal-bridged cyclic peptide variants. [Figure 23] FIG. 1 shows proline mutant peptide binding to TAP01 antibody using Biacore T200. [Figure 24] Figure 1 shows the reduction in cortical plaque burden in immunized 5XFAD mice after passive immunization with TAP01 antibody. Plaque burden analysis of TAP01_4(MoG1K)-immunized 5XFAD mice compared to IgG1-injected 5XFAD mice. (a) Immunostaining with an antibody against total Aβ shows a significant reduction in plaque burden in TAP01_04-immunized mice compared to IgG control, TAP01_01, and TAP01_02-treated mice. (b) Immunostaining with an antibody against pyroglutamate Aβ3-x shows a significant reduction in plaque burden in TAP01_04-immunized mice compared to IgG control. No significant differences were observed for mice immunized with TAP01_01 and TAP01_02. (c) Staining with Thioflavin S. (d) Immunostaining with TAP01(NT4X) shows a significant reduction in plaque burden in TAP01_04-immunized mice compared to IgG control. No significant differences were observed for mice immunized with TAP01_01 and TAP01_02. [Figure 25] FIG. 1 shows binding ELISA data for binding of (a) TAP01 (MoG1K) antibody, (b) MRCT control IgG1 antibody (cloned as MoG1K) to thioacetal-bridged cyclic peptide variants. [Figure 26] (b) Binding ELISA data for bapineuzumab binding to the thioacetal-bridged cyclic peptide variants of (a) compared to the MRCT control IgG1 antibody (cloned as HuG1K). [Figure 27]Figure 1 shows in vivo amyloid plaque imaging of mouse brain cross sections with the tracer florbetaben. (A) Untreated wild-type control mouse brain with no florbetaben retention signal. (B) Untreated 5XFAD with a strong florbetaben retention signal, indicating high amyloid plaque burden in the brain. (C) 5XFAD mice after active immunization show no florbetaben retention signal, indicating significantly reduced amyloid plaque burden in the brain. [Figure 28] Figure 1 shows statistical analysis of florbetaben retention signal as a marker for amyloid plaque burden in mouse brain. Statistical evaluation by ANOVA (p<0.0001, F=21.39) and Bonferroni-corrected comparison between groups. Wild-type (WT) cortex vs. 5XFAD cortex (p<0.001), WT hippocampus vs. 5XFAD hippocampus (p<0.001), WT amygdala vs. 5XFAD amygdala (p<0.001). WT cortex vs. treated 5XFAD cortex (not significant), WT hippocampus vs. treated 5XFAD hippocampus (not significant), WT amygdala vs. treated 5XFAD amygdala (not significant). 5XFAD cortex vs. treated 5XFAD cortex (p<0.01), 5XFAD hippocampus vs. treated 5XFAD hippocampus (p<0.001), 5XFAD amygdala vs. treated 5XFAD amygdala (p<0.001). Treated 5XFAD = 5XFAD immunized with cyclic peptide. [Figure 29]Figure 1 shows immunostaining and quantitative assessment of plaque burden in the cerebral cortex of 5XFAD mice, comparing passive immunization with TAP01_04 (MoG1K) and active immunization with a cyclized peptide. Exemplary staining with total Aβ antibodies (A) is shown for 5XFAD mice after treatment with IgG1, passive immunization with TAP01_04, and active immunization with a cyclized Aβ peptide. Quantification of plaque burden (B) was assessed using antibodies against total Aβ, pyroglutamate Aβ3-X, thioflavin S, and N-truncated Aβ, demonstrating a strong reduction in plaques in 5XFAD mice treated with active immunization. Mice treated with TAP01_04 and active immunization showed similar reductions in plaques stained with all Aβ antibodies and thioflavin S. ANOVA with Bonferroni's multiple comparison test for plaque staining for total Aβ (F=65.20, p<0.0001, R-squared 0.6287), pyroglutamate Aβ3-X (F=23.32, p<0.0001, R-squared 0.3570), thioflavin S (F=17.17, p<0.0001, R-squared 0.3291) and N-excised Aβ (F=89.17, p<0.0001, R-squared 0.6316) are shown (mean + SEM). [Figure 30] Figure 1 shows the effect of active immunization with cyclized Aβ peptide on in vivo cerebral glucose metabolism in 5XFAD mice. (A) Exemplary coronal, transverse, and sagittal views of glucose uptake by 18F-FDG-PET / MRI imaging. (B) Quantitative analysis. Glucose uptake was assessed by 18F-FDG-PET / MRI imaging in actively immunized 5XFAD mice (n=5), two 5XFAD control mice, and two wild-type mice (all female, aged 4.5-5.5 months). Quantitative analysis of signal intensity showed that immunized 5XFAD mice showed significant rescue of cerebral glucose metabolism in most brain regions analyzed, demonstrating therapeutic effects on synaptic and neuronal activity. ANOVA comparison test (F=10.37, p<0.0001, R-squared=0.7352). Significant differences using a t-test are shown (mean + SEM). A, amygdala; Bs, brainstem; C, cortex; Cb, cerebellum; H, hypothalamus; Hc, hippocampus; Hg, Harderian gland; M, midbrain; O, olfactory bulb; S, septum / basal forebrain; St, striatum; T, thalamus. [Figure 31] Figure 1 shows the effect of active immunization with cyclized Aβ peptide compared to passive immunization with TAP01_04 (MoG1K) in Tg4-42 mice. (A) Hippocampus-dependent learning and memory loss in aged Tg4-42 mice is shown by the probe trial of the Morris water maze test. Both passive immunization with TAP01_04 and active immunization with cyclized Aβ peptide rescued memory deficits in Tg4-42 mice. ANOVA with Bonferroni's multiple comparison test (F = 13.27, p < 0.0001, R-squared = 0.646). T-tests are shown with mean + SEM. (B) Aging Tg4-42 mice develop significant neuronal loss in the CA1 layer of the hippocampus. The mean (+SEM) number of neurons on IgG1-treated mice was 128,687 +13,035, while the mean (+SEM) number of neurons in TAP01_04-treated mice was significantly higher at 194,310 +22,572, and the mean (+SEM) number of neurons in actively immunized mice was also significantly higher at 185,858 +39,180. ANOVA with Bonferroni's multiple comparison test shown (F=8.125, p<0.001, R-squared=0.5556). *=p<0.05, **p<0.01, ***=p<0.001. [Figure 32] FIG. 1 shows binding ELISA data for binding of 5XFAD mouse serum to cyclized Aβ peptides. [Figure 33] FIG. 1 shows binding ELISA data for binding of Tg4-42 mouse serum to cyclized Aβ peptides. [Figure 34] FIG. 1 shows binding ELISA data for the binding of control antibodies HuMRCT MoG1K and MoMRCT HUG1K, the reference antibody bapineuzumab and Tap01 MoG1K to cyclic peptide 3,13. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to non-naturally occurring peptides, i.e., synthetic peptides, that mimic conformational epitopes found naturally in the pE3-X amyloid peptide and specifically bind to antibodies that bind to the epitopes of the pE3-X amyloid peptide. These cyclic peptides can be used for active immunization of subjects to generate antibodies specific for amyloid beta, particularly low molecular weight oligomers of amyloid beta. A hairpin structure is found in the N-terminal region of the pE3-X amyloid peptide. This region has been found to be an epitope for antibodies that bind to low molecular weight oligomers of amyloid beta.

[0042] The cyclic peptides of the present invention are based on amino acid residues 1-14 of the amyloid-β protein, where two of the residues found in this sequence have been replaced with cysteine ​​residues, through which the peptide is cyclized. The cyclic peptides of the present invention mimic the hairpin structure found in amyloid-β.

[0043] The cyclic peptide mimics the hairpin structure identified in pE3-X amyloid-β and has been identified as the binding site for antibodies such as the mouse TAP01 antibody (also known as NT4X) and the humanized TAP01_01, TAP01_02, TAP01_03, and TAP01_04 antibodies (also known as NT4X_SA, NT4X_S7A, NT4X_S71A, and NT4X_S71H, respectively, as described in U.S. Patent No. 5,627,291 and U.S. Patent No. 5,627,291). These anti-amyloid-β antibodies have been shown to specifically bind to N-terminally truncated amyloid peptides (AβpE3-x or Aβ4-x). These antibodies do not show significant binding to full-length amyloid peptides or amyloid Aβ1-42.

[0044] The cyclic peptides described herein are variant peptides based on amino acids 1-14 of amyloid-β, DAEFRHDSGYEVHH (SEQ ID NO: 3), in which two amino acids of the naturally occurring sequence have been replaced with cysteine ​​residues, cyclizing the peptide through the cysteine ​​residues. Preferably, one of the cysteine ​​residues replaces the amino acid at position 1, 2, 3, or 5, and the other cysteine ​​replaces the amino acid at position 10, 11, 12, or 13.

[0045] In one embodiment, the cyclic peptide described herein has the formula (I) (SEQ ID NO: 1): X1X2X3FX4HDSGX5X6X7X8H (I) or a variant thereof, During the ceremony, X1 is absent or any amino acid; X2 is alanine or cysteine; X3 is glutamic acid or cysteine, X4 is arginine or cysteine, X5 is tyrosine or cysteine, X6 is glutamic acid or cysteine, X7 is valine or cysteine; X8 is histidine or cysteine, Only one of X1, X2, X3 and X4 is a cysteine, and only one of X5, X6, X7 and X8 is a cysteine, and the peptide is cyclized through the cysteine ​​residues at positions 1, 2, 3 or 5 and 10, 11, 12 or 13. A cyclic peptide may contain only two cysteine ​​residues through which the peptide is cyclized.

[0046] Preferably, there are at least 7 amino acids between any two cysteine ​​residues present in the sequence, more preferably 7 to 11 amino acid residues, and even more preferably 8 to 11 amino acid residues, between any two cysteine ​​residues present in the cyclic peptide.

[0047] The cyclic peptide is preferably not cyclized through both terminal amino acids of the peptide sequence. Preferably, X1 is present. Preferably, the cyclic peptide is not cyclized through the C-terminal amino acid. In one embodiment, X1 is cysteine, and preferably, the peptide is cyclized through cysteine ​​at position 1 and cysteine ​​at position 13. In a further embodiment, preferably, X1 is proline or aspartic acid, preferably aspartic acid, and the peptide is not cyclized through either of the terminal amino acids of the peptide residues. Cyclization of the peptide through at least one internal cysteine ​​residue, particularly at positions 1, 2, 3, or 5 and 10, 11, 12, or 13, helps provide a stable peptide that can mimic the hairpin structure found in pE3-X amyloid beta.

[0048] In one embodiment, the peptide is cyclized through the cysteine ​​residues at positions 1 (ie, X1 is cysteine ​​and X2 is alanine) and 10, 11, 12 or 13, preferably 13.

[0049] Preferably, the cyclic amino acid comprises a sequence in which X1 is cysteine, X2 is alanine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is valine, and X8 is cysteine, and the peptide is cyclized through the two cysteine ​​residues. For example, the peptide is cyclized through the cysteines at X1 and X8. For example, the cyclic peptide can comprise or consist of the amino acid sequence CAEFRHDSGYEVCH (SEQ ID NO: 14), and the peptide is cyclized through the cysteine ​​residues at positions 1 and 13.

[0050] Alternatively, in one embodiment, preferably, the cyclic amino acid comprises a sequence in which X1 is absent or any amino acid, a) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is cysteine, and X8 is histidine, or b) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is cysteine, and X8 is histidine, or c) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is cysteine, X6 is glutamic acid, X7 is valine, and X8 is histidine, or d) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is valine, and X8 is cysteine, or e) X2 is cysteine, X3 is glutamic acid, X4 is arginine, X5 is tyrosine, X6 is cysteine, X7 is valine, and X8 is histidine, or f) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is tyrosine, X6 is cysteine, X7 is valine, and X8 is histidine, or g) X2 is alanine, X3 is glutamic acid, X4 is cysteine, X5 is tyrosine, X6 is glutamic acid, X7 is cysteine, and X8 is histidine, or h) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is cysteine, X6 is glutamic acid, X7 is arginine, and X8 is histidine, or i) X2 is alanine, X3 is cysteine, X4 is arginine, X5 is tyrosine, X6 is glutamic acid, X7 is valine, and X8 is cysteine; The peptides are cyclized through two cysteine ​​residues. For example, for peptide (a), the sequence is cyclized through the cysteines at X3 and X7; for peptide (b), the sequence is cyclized through the cysteines at X2 and X7; for peptide (c), the sequence is cyclized through the cysteines at X2 and X5; for peptide (d), the sequence is cyclized through the cysteines at X2 and X8; for peptide (e), the sequence is cyclized through the cysteines at X2 and X6; for peptide (f), the sequence is cyclized through the cysteines at X3 and X6; for peptide (g), the sequence is cyclized through the cysteines at X4 and X7; for peptide (h), the sequence is cyclized through the cysteines at X3 and X5; and for peptide (i), the sequence is cyclized through the cysteines at X3 and X8. Preferably, in one embodiment, X1 is present and is selected from proline or aspartic acid. More preferably, X1 is aspartic acid.

[0051] For example, a cyclic peptide may have the amino acid sequence: a) DACFRHDSGYECHH (SEQ ID NO: 4), b) DCEFRHDSGYECHH (SEQ ID NO: 5), c) DCEFRHDSGCEVHH (SEQ ID NO: 10); d) DCEFRHDSGYEVCH (SEQ ID NO: 12); e) DCEFRHDSGYCVHH (SEQ ID NO: 9); f) DACFRHDSGYCVHH (SEQ ID NO: 8); g) DAEFCHDSGYECHH (SEQ ID NO: 7), h) DACFRHDSGCEVHH (SEQ ID NO: 11), or i) DACFRHDSGYEVCH (SEQ ID NO: 13), The peptide may comprise or consist of: The peptide is cyclized through two cysteine ​​residues located at positions 2, 3 or 5, and 10, 11, 12 or 13. Preferably, the cyclic peptide comprises the sequence DACFRHDSGYECHH (SEQ ID NO: 4), in which the peptide is cyclized through the cysteine ​​residues at positions 3 and 12, or DACFRHDSGYEVCH (SEQ ID NO: 13), in which the peptide is cyclized through the cysteine ​​residues at positions 3 and 13.

[0052] The present invention also relates to a cyclic peptide comprising the sequence of formula (I) as described above, wherein the cyclic peptide does not contain cysteine ​​residues at both positions 5 and 12 or at both positions 3 and 10. In particular, the cyclic peptide does not comprise or consist of a peptide having the sequence of SEQ ID NO: 7 or SEQ ID NO: 11.

[0053] Variant cyclic peptides are also provided. A variant cyclic peptide has the same or similar function as its reference peptide, i.e., is a functionally equivalent cyclic peptide that adopts the hairpin structure of amyloid beta. A cyclic peptide as described herein that is a variant of a reference sequence, such as the above-mentioned reference sequence, can have one or more amino acid residues that are modified relative to the reference sequence. For example, up to three amino acid residues may be modified relative to the reference sequence, preferably up to two amino acid residues, or even one amino acid residue. Amino acid residues in the reference sequence can be modified or mutated by insertion, deletion, or substitution, preferably substitution with a different amino acid residue. Preferably, the substitution is a conservative amino acid substitution. A conservative amino acid sequence modification is one that does not affect or alter the properties of the cyclic peptide, for example, maintains the conformation of the cyclic peptide, preferably maintains the immunogenicity of the peptide, preferably maintains the ability to induce an immune response capable of generating anti-amyloid beta antibodies, preferably those that specifically bind to low molecular weight AB oligomers.

[0054] Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Substitutions include substitution with any of the 20 naturally occurring (or "standard") amino acids or variants thereof, such as D-amino acids, or any variant not naturally found in proteins. Non-naturally occurring amino acids have been defined in the art.

[0055] Cyclic peptides as described herein that are variants of a reference sequence may share at least 85% sequence identity with a reference sequence, e.g., SEQ ID NO: 4, 5, 7, 9, 10, 11, 12, 13, or 14, or at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the reference sequence. Cyclic peptides as described herein that are variants of a reference sequence may maintain at least one internal cysteine ​​residue, i.e., contain at least one non-terminal cysteine ​​residue through which the peptide is cyclized. One cysteine ​​residue may be located in the N-terminal region of the peptide and the other in the C-terminal region of the peptide, but both cysteine ​​residues may not be located at the ends of the sequence, i.e., the cyclic peptide contains at least one free N- and C-terminal residue so that the peptide is not cyclized in a head-to-tail manner. In one embodiment, a cysteine ​​residue is not present as the C-terminal residue of the sequence. Cyclic peptides as described herein that are variants of a reference sequence, i.e., SEQ ID NO: 4, 5, 7, 9, 10, 11, 12, 13, or 14, maintain two cysteine ​​residues, one of which is not at the terminus of the sequence, i.e., include at least one non-terminal cysteine ​​residue through which the peptide is cyclized. In one embodiment, neither cysteine ​​residue is located at the terminus of the sequence. Cyclic peptides as described herein that are variants of a reference sequence, i.e., SEQ ID NO: 4, 5, 7, 9, 10, 11, 12, or 13, maintain two internal cysteine ​​residues, i.e., include two non-terminal cysteine ​​residues through which the peptide is cyclized.

[0056] Preferably, cysteine ​​residues are maintained at positions 1, 2, 3 or 5 and 10, 11, 12 or 13, preferably positions 1 and 13, 3 and 12, or 3 and 13. Preferably, the phenylalanine residue at position 4 of the reference sequence is also maintained. For example, the cyclic peptides described herein can comprise an amino acid sequence having at least 85% sequence identity, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the sequence CAEFRHDSGYEVCH (SEQ ID NO: 14), the sequence DACFRHDSGYECHH (SEQ ID NO: 4), or the sequence DACFRHDSGYEVCH (SEQ ID NO: 13), wherein the peptide comprises cysteine ​​residues at positions 1 and 13, 3 and 12, or 3 and 13, and a phenylalanine residue at position 4, and the peptide is cyclized through the cysteine ​​residues at positions 1 and 13, 3 and 12, or 3 and 13. In such variant cyclic peptides, the cysteine ​​residues at positions 1 and 13, 3 and 12, or 3 and 13, and the phenylalanine residue at position 4 are maintained, while conservative amino acid substitutions are introduced into the sequence at other positions. In one embodiment, the cysteine ​​residues at positions 1 and 13 are maintained in the variant sequence. In another embodiment, the cysteine ​​residues at positions 3 and 12 or 13 are maintained in the variant sequence.

[0057] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG Package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences to maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty of 12 and a gap extension penalty of 4. Although the use of GAP may be preferred, other algorithms may also be used, typically using default parameters, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147: 195-197) or the TBLASTN program of Altschul et al. (1990) supra. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may also be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Worcester, Massachusetts, USA). Sequence comparisons are preferably made over the entire length of the relevant sequences described herein.

[0058] As used throughout this application, amino acid positions for cyclic peptides are provided with respect to the sequence of the peptide having the sequence DAEFRHDSGYEVHH (SEQ ID NO: 3). Thus, the phrase "amino acid at position 'x'" of a cyclic peptide, or similar phrases, refers to the amino acid corresponding to the amino acid at position 'x' in the preferred cyclic peptide having SEQ ID NO: 3. Amino acid positions for full-length amyloid β peptides, as well as variants including N-truncated variants, e.g., 1-40, p3-42, and 4-42, are provided with respect to the sequence of the full-length peptide having the sequence of Aβ1-42 (SEQ ID NO: 18). Thus, the phrase "amino acid at position 'x'" of an N-terminally truncated p3-42 peptide, or similar phrases, refers to the amino acid corresponding to the amino acid at position 'x' in the preferred full-length peptide having SEQ ID NO: 18. Note that the numbering system used throughout this application starts from the N-terminal amino acid.

[0059] The cyclic peptides described herein can comprise, consist essentially of, or consist of a variant amino acid sequence of a peptide described herein or a variant thereof. In preferred embodiments, the cyclic peptide comprises 16 or fewer amino acids, preferably 15 or fewer amino acids. More preferably, the peptide comprises 14 or fewer amino acids. In preferred embodiments, the cyclic peptide consists of or consists essentially of an amino acid sequence described herein, i.e., the cyclic peptide consists of or consists essentially of a sequence set forth in Formula (I), Formula (II), or SEQ ID NO: 4, 5, 7, 8, 9, 10, 11, 12, 13, or 14, or a variant thereof.

[0060] Cyclization or "cyclized" or similar expressions means that a peptide is in or made into a cyclic form. The term "cyclic" means that at least some of the constituent residues of the peptide form a ring. The cyclic peptides of the present invention are cyclized through at least one internal amino acid, i.e., are not cyclized in a head-to-tail manner. Preferably, the peptides of the present invention are cyclized through an internal amino acid. Cyclization of the peptide through a cysteine ​​residue constrains the peptide into a structure that mimics the hairpin structure identified in pE3-X amyloid beta.

[0061] Cyclization of the peptide is achieved through the formation of a bridge by incorporating two cysteine ​​residues into the sequence. The cysteine ​​residues replace the corresponding amino acids in the naturally occurring sequence. Preferably, cyclization can be formed by side chain to side chain cyclization. The peptides of the present invention can be cyclized directly or indirectly through the thiol side chains of the cysteine ​​residues. For example, side chain to side chain cyclization can be achieved through the formation of a bridge of the formula -S-(-CH2-)nS- (where n = 0, 1, or 2). Preferably, the bridge has the formula -SS- or -S-CH2-S- (where S is the thiol residue of the linked cysteine ​​residue). More preferably, the bridge has the formula -S-CH2-S-, preferably between the cysteine ​​residues located at positions 1 and 13 of the peptide, between the cysteine ​​residues located at positions 3 and 12 of the peptide, or between the cysteine ​​residues located at positions 3 and 13 of the peptide. Suitable methods for cyclizing peptides through cysteine ​​residues are known in the art, for example, using thiol oxidation, optionally including the introduction of a methylene bridge. See also, for example, Kourra C and Cramer N, Chem. Sci., 2016,7, 7007-7012. Other bridges, such as thioether bridges (-CH2-S-), are also included in the present invention.

[0062] The cyclic peptide exhibits binding specificity to the TAP01 and TAP01_01 antibodies (as described in Patent Documents 2 and 3). The cyclic peptide mimics the N-terminal epitope found on p3-42 amyloid beta, which has a hairpin structure to which these antibodies bind.

[0063] In a preferred embodiment, the cyclic peptide specifically binds to an antibody molecule that specifically recognizes soluble low-molecular-weight AβρE3-X oligomers, i.e., does not bind to an antibody that specifically binds to high-molecular-weight oligomers of the AβρE3 peptide. As used herein, the term "low-molecular-weight oligomer" refers to a soluble oligomer composed of three to six AβρE3-X units, preferably trimeric and tetrameric Aβρ3-X or Aβ4-X oligomers, where X is 38, 40, or 42. Preferably, the low-molecular-weight oligomers of Aβρ3-X or Aβ4-X are at least trimeric oligomers and have a size of less than 15 kDA.

[0064] The antibody may specifically bind to N-terminally truncated amyloid peptides, such as pyroglutamate (pE)-modified amyloid peptides (also referred to as AβpE3-x, AβpGlu3-x, Aβ(Glp3)3-x, and p3-x), such as AβpE3-38, AβpE3-40, AβpE3-14, and AβpE3-42, as well as non-pyroglutamate-modified amyloid peptides, such as Aβ4-38, Aβ4-40, Aβ4-14, and Aβ4-40. The antibodies, such as TAP01 and TAP01_01, may not specifically bind to full-length amyloid peptides or amyloid peptides without N-terminal truncations (Aβ1-x), such as Aβ1-42, Aβ1-38, Aβ1-40, or Aβ1-14.

[0065] In a preferred embodiment, the antibodies to which the cyclic peptides described herein bind can specifically bind to the amyloid peptides AβpE3-42 and Aβ4-42, and may exhibit no or substantially no specific binding to Aβ1-42 monomers and dimers.

[0066] Specific binding or "specific recognition" refers to the situation where an antibody does not exhibit any significant binding to molecules other than the particular epitope on the antigen.

[0067] For example, the term "specific recognition" or similar terms, as used herein, is intended to mean that a binding molecule, i.e., an antibody, specifically binds to and / or detects (i.e., recognizes) soluble low-molecular-weight oligomers of N-terminal truncated amyloid peptides, i.e., Aβρ3-x or Aβ4-x (where X is 42, 40, or 38), such as Aβρ3-42 or Aβ4-42. The antibody does not recognize or bind to Aβ1-40 monomers or dimers, or high-molecular-weight oligomers. Thus, the antibody preferably recognizes a conformational epitope formed in trimeric or tetrameric Aβρ3-42 oligomers. Antibodies that specifically recognize low-molecular-weight oligomers of amyloid β and show no or little binding to full-length amyloid peptides include, but are not limited to, TAP01 and TAP01_01.

[0068] The affinity of an antibody, as described herein, is the degree or strength of antibody binding to an epitope or antigen, including antibody binding to a cyclic peptide as defined herein. The dissociation constant Kd and the affinity constant Ka are quantitative measurements of affinity. Kd is the antibody association rate (k) of an antibody, which indicates how rapidly the antibody binds to its antigen. on ) versus the antibody dissociation rate (k off ) ratio. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate of [antibody][antigen] complex formation is equal to the rate of dissociation into its components [antibody] + [antigen]. Measurements of the reaction rate constants can be used to define the equilibrium or affinity constant Ka (Ka = 1 / Kd). The smaller the Kd value, the greater the affinity of the antibody for its target. Most antibodies have low micromolar (10 -6 ) to nanomoles (10 -7 ~10 -9High affinity antibodies generally have Kd values ​​in the low nanomolar range (10 -9 ), whereas very high affinity antibodies are thought to be in the picomolar (10 -12 ) range.

[0069] In some embodiments, the anti-Aβ antibody (to which the cyclic peptide binds) is at least 2×10 2 M -1 , at least 5 × 10 2 M -1 , at least 10 3 M -1 , at least 5 × 10 3 M -1 , at least 10 4 M -1 , at least 5 × 10 4 M -1 , at least 10 5 M -1 , at least 5 × 10 5 M -1 , at least 10 6 M -1 , at least 5 × 10 6 M -1 , or at least 10 7 M -1 The compound binds (e.g., specifically binds) to amyloid peptides AβpE3-42 and Aβ4-42 with an affinity constant, i.e., Ka, of 0.05 to 0.15.

[0070] In some embodiments, the anti-Aβ antibody (to which the cyclic peptide binds) is 5×10 2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 M -1 Less than 5 x 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M or 5 x 10 -7 M or less.

[0071] Specific binding of an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and generally does not exhibit significant cross-reactivity. An antibody that "does not exhibit significant cross-reactivity" is one that will not appreciably bind to undesired entities (e.g., undesired proteinaceous entities). An antibody specific for a particular epitope will not significantly cross-react with, for example, distant epitopes on the same protein or peptide. Specific binding of an antibody as described herein, i.e., k off , k on , Ka and Kd may be determined according to any art-recognized means for determining such binding.

[0072] The binding of anti-Aβ antibodies can be determined using standard techniques, such as ELISA or surface plasmon resonance. Suitable ELISA techniques are known in the art. For example, the immobilized amyloid peptide can be contacted with an antibody in IgG1 format and washed one or more times with 0.1% non-ionic detergent, such as polysorbate 20 (Tween 20), to remove unbound antibody. The antibody bound to the immobilized peptide can then be detected using any conventional technique, for example, using a secondary antibody linked to a detectable label, such as HRP.

[0073] The present invention further provides a cyclic peptide described herein linked to a carrier, preferably a carrier protein. Preferably, the peptide is linked to the carrier by chemical cross-linking. The cyclic peptide can be conjugated to a carrier protein, including, but not limited to, keyhole limpet hemocyanin (KLH), serum albumin (e.g., bovine serum albumin, BSA) or ovalbumin, immunoglobulin FC domain, tetanus toxoid, diphtheria toxoid, or a combination thereof. The carrier peptide can be linked to the cyclic peptide directly or through a linker. The peptide can be linked to the carrier protein through techniques standard in the art.

[0074] The cyclic peptides described herein can be useful in therapy.For example, cyclic peptide proteins can be administered to individuals for the treatment of neurological diseases.Cyclic peptides will usually be administered in the form of pharmaceutical compositions, which may contain at least one additional component in addition to the cyclic peptide.

[0075] The cyclic peptides and compositions described herein can be administered for therapeutic and / or prophylactic treatment by parenteral, topical, intravenous, oral, gastric, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular methods, as described herein. Intramuscular injection or intravenous infusion is preferred for administration of the cyclic peptides.

[0076] Pharmaceutical compositions can include, in addition to the cyclic peptides described herein, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, and / or other materials known to those of skill in the art. The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for administration to a subject (e.g., a human) and will not cause any undesired or deleterious effects in the subject. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection, or any other suitable route discussed below and known in the art.

[0077] The cyclic peptide may be formulated in a suitable delivery vehicle. For parenteral administration, e.g., by injection, pharmaceutical compositions containing the cyclic peptides described herein may be in the form of a parenterally acceptable aqueous solution or suspension in a physiologically acceptable diluent together with a suitable pharmaceutical carrier. Those skilled in the art are fully capable of preparing suitable solutions using suitable carriers, preservatives, stabilizers, buffers, antioxidants, and / or other additives, which may be used as needed. Suitable carriers, excipients, etc. may be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0078] The term parenteral, as used herein, includes subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, and intrathecal administration of the cyclic peptides or compositions described herein. The cyclic peptides or compositions described herein can also be administered by nasal or gastric methods.

[0079] The cyclic peptides described herein are preferably formulated and administered as a sterile solution, although in some cases it may also be possible to use a lyophilized preparation. The sterile solution is prepared by sterile filtration or other methods known in the art. The solution is then lyophilized or filled into pharmaceutical dosage containers.

[0080] The pharmaceutical composition may be used as a vaccine. The vaccine composition may further comprise an adjuvant. Adjuvants are known in the art to further increase the immune response to the applied antigenic determinant. An adjuvant is defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the cyclic peptides of the present invention. Examples of suitable adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and / or aluminum phosphate, squalane-water emulsions, oil emulsion compositions (or oil-in-water compositions) including, for example, MF59, saponin formulations such as QS21 and immune stimulating complexes (ISCOMS), bacterial or microbial derivatives such as monophosphoryl lipid A (MPL), 3-0-deacylated MPL (3dMPL), CpG motif-containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, for example, E. coli heat-labile enterotoxin LT, cholera toxin CT, and eukaryotic proteins (e.g., antibodies or fragments thereof) that stimulate an immune response upon interaction with recipient cells. In certain embodiments, the compositions of the invention include, as an adjuvant, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, or a combination thereof.

[0081] The present invention provides cyclic peptides that mimic epitopes on AβρE3-x or Aβ4-x, making them suitable for vaccination against amyloid-related diseases. The cyclic peptides as described herein are immunogenic. Immunogenicity means that the cyclic peptide has the ability to induce an immune response. An immunogenic composition comprising the cyclic peptides as described herein can induce an immune response against the cyclic peptide and promote the production of anti-amyloid antibodies, particularly anti-amyloid antibodies that specifically bind to low molecular weight oligomers of AβρE3-x or Aβ4-x.

[0082] Without being bound by theory, it is believed that administration of cyclic peptides as described herein as a vaccine will induce an immune response that leads to the production of anti-Aβ antibodies that specifically bind to low molecular weight AβρE3-x or Aβ4-x oligomers. These anti-Aβ antibodies may neutralize toxic Aβ oligomers generated early in Alzheimer's disease pathology and prevent subsequent plaque formation.

[0083] Thus, the present invention provides a method for inducing an immune response against amyloid beta in a subject, comprising administering to the subject a therapeutically effective amount of a cyclic peptide according to the present invention. Also provided are compositions according to the present invention for use in inducing an immune response in a subject, particularly for use as a vaccine. Further provided is the use of a cyclic peptide according to the present invention as described herein for the manufacture of a medicament for use in inducing immune response proteins in a subject. Preferably, the induced immune response is characterized by the production of antibodies capable of specifically binding to low molecular weight oligomers of amyloid beta.

[0084] The present invention also provides a method for treating Alzheimer's disease, particularly Alzheimer's disease, which can be sporadic or familial Alzheimer's disease, as well as other Aβ-related diseases and disorders and other neurological diseases characterized by soluble amyloid. Accordingly, the present invention also relates to a method for treating Alzheimer's disease, comprising administering to a subject a therapeutically effective amount of a cyclic peptide as described herein.

[0085] Treatment includes both prophylactic and therapeutic treatment. The terms "treat," "treating," or "treatment" (or equivalent terms) mean that the severity of an individual's condition is reduced or at least partially improved or ameliorated, and / or some degree of remission, reduction, or reduction is achieved in at least one clinical symptom, and / or there is an inhibition or slowing of the progression of the condition and / or prevention or delay of the onset of a disease or disorder.

[0086] In particular, treating Alzheimer's disease includes preventing or delaying the onset of Alzheimer's disease and / or one or more symptoms associated with Alzheimer's disease in a subject. Treatment includes inhibiting or reducing the accumulation of amyloid-β oligomers in a subject.

[0087] The above-described treatment methods can include administering to an individual an antibody or composition described herein (e.g., a composition comprising a cyclic peptide described herein, a pharmaceutically acceptable excipient, and optionally an additional therapeutic agent) under conditions that result in a beneficial therapeutic response in the individual, e.g., for the prevention or treatment of Alzheimer's disease. Such individuals can have Alzheimer's disease. The treatment methods described herein can be used for both asymptomatic individuals and individuals currently exhibiting symptoms of Alzheimer's disease. The cyclic peptides described herein can be administered prophylactically to individuals who do not have Alzheimer's disease. The cyclic peptides described herein can be administered to individuals who do not have or do not exhibit symptoms of Alzheimer's disease. The cyclic peptides described herein can be administered to individuals who have or are suspected to have Alzheimer's disease. Individuals suitable for treatment include individuals who are at risk for or susceptible to Alzheimer's disease but do not exhibit symptoms, and individuals suspected of having Alzheimer's disease, as well as individuals who currently exhibit symptoms. The cyclic peptides described herein can be administered prophylactically to the general population. In some embodiments, individuals suitable for treatment as described herein can include individuals with early-onset Alzheimer's disease or one or more symptoms thereof, and individuals whose amyloid peptides have been detected in body fluids, such as CSF samples.

[0088] The terms "patient," "individual," or "subject" include humans and other mammalian subjects receiving either prophylactic or therapeutic treatment with one or more cyclic peptides described herein. Mammalian subjects include primates, such as non-human primates. Mammalian subjects also include laboratory animals commonly used in research, including, but not limited to, rabbits and rodents, such as rats and mice.

[0089] The cyclic peptides described herein can be used in methods of preventing or treating Alzheimer's disease, comprising administering to a patient an effective dosage of a cyclic peptide as described herein. As used herein, an "effective amount" or "effective dosage" or "sufficient amount" (or grammatically equivalent terms) of a cyclic peptide described herein refers to an amount of a cyclic peptide or composition described herein that is effective to produce a desired effect, optionally a therapeutic effect (i.e., by administering a therapeutically or prophylactically effective amount). For example, an "effective amount" or "effective dosage" or "sufficient amount" can be such that the severity of an individual's condition, e.g., Alzheimer's disease, is reduced or at least partially improved or ameliorated, and / or a degree of remission, alleviation, or reduction is achieved in at least one clinical symptom, and / or there is an inhibition or delay in the progression of Alzheimer's disease and / or prevention or delay in the onset of Alzheimer's disease.

[0090] In both prophylactic and therapeutic treatment regimens, reagents may be administered in several dosages until a sufficient immune response is achieved. The term "immune response" or "immunological response" includes the development in a recipient subject of a humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response directed against an antigen. Typically, the immune response is monitored, and repeat dosages are administered if the immune response begins to wane.

[0091] Effective doses of the compositions described herein for the treatment of the above-mentioned conditions will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic.

[0092] For active immunization with the cyclic peptides described herein, the dosage ranges from about 0.1 mg / kg to 100 mg / kg of host body weight, more typically 0.1 mg / kg to 50 mg / kg of host body weight. For example, the dosage can be at least 1 mg / kg or at least 10 mg / kg, or within the range of 1 mg / kg to 100 mg / kg. In another example, the dosage is at least 0.5 mg / kg or at least 50 mg / kg, or within the range of 0.5 mg / kg to 50 mg / kg, preferably at least 5 mg / kg. In a preferred example, the dosage can be about 50 mg / kg.

[0093] The treatment described herein can include administering a cyclic peptide to a subject as a single dose, two doses, or multiple doses. The cyclic peptide described herein can be administered multiple times. The interval between single doses can be daily, weekly, monthly, or yearly. The interval can also be irregular, as indicated by measuring the blood level of anti-Aβ antibodies induced in the patient. In some methods, the dosage is adjusted to achieve a desired plasma antibody concentration. The dosage and frequency vary depending on the patient.

[0094] Dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the cyclic peptides described herein are administered to patients not already in a disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose." In this use, the exact amount will again depend on the patient's health and general immune status, but will generally range from 0.1 mg to 25 mg per dose, particularly 0.5 mg to 2.5 mg per dose. Over a longer period of time, lower dosages are administered at less frequent intervals.

[0095] The compositions according to the present invention can be used in the independent treatment and / or prevention of diseases or pathologies caused by amyloid beta protein, i.e., neurodegenerative diseases, such as Alzheimer's disease, or in combination with other prophylactic and / or therapeutic treatments, such as other vaccines, and / or antibodies, and / or other active agents. In certain embodiments, the vaccine may be a combination vaccine further comprising other components, e.g., that induce an immune response against other proteins associated with Alzheimer's disease and / or that induce antibodies directed against other forms of amyloid beta. The administration of the additional active component can be carried out, for example, by separate administration or by administering a combination product of the vaccine of the present invention and the additional active component.

[0096] The cyclic peptides described herein may be provided in the form of a kit. The kit may contain at least one cyclic peptide described herein. The kit may optionally contain a composition described herein in one or more containers, along with one or more other prophylactic or therapeutic agents useful for the prevention, management, or treatment of Alzheimer's disease (AD). If the composition containing the components for administration is not formulated for delivery through the digestive tract, e.g., oral delivery, a device capable of delivering the kit components via some other route, such as a syringe, may be included. The kit may further include compositions containing other therapeutic agents for other diseases or conditions. The kit may further include instructions for preventing, treating, managing, or ameliorating AD, as well as dosing information for side effects and administration methods.

[0097] The present invention also relates to a method for producing a cyclic peptide as described herein. The cyclic peptide as described herein can be prepared by methods known in the art. In one embodiment, the method comprises generating a linear peptide comprising the sequence of a desired peptide and cyclizing the linear peptide through a cysteine ​​residue to obtain a cyclic peptide. The generated linear peptide can be cyclized by methods known in the art, such as thiol oxidation, optionally including the introduction of a methylene bridge. See also Kourra C and Cramer N, Chem. Sci., 2016,7, 7007-7012.

[0098] The present invention also provides a method for producing an antibody that recognizes low molecular weight oligomers of amyloid beta, comprising the steps of: (a) immunizing an animal with a cyclic peptide or variant as described above, a cyclic peptide comprising the sequence of formula (I), preferably the sequence of SEQ ID NO: 14, SEQ ID NO: 4 or SEQ ID NO: 13; (b) obtaining antibodies produced by immunization in step (a); The present invention also relates to a method for detecting amyloid-β peptides, comprising: (a) detecting the antibody obtained in step (b); the method may further comprise step (c), which comprises screening the antibody obtained in step (b). Preferably, the antibody is screened for specific recognition of low molecular weight oligomers of amyloid-β. Preferred antibodies are screened for their ability to specifically recognize N-terminal truncated amyloid peptides, i.e., AβpE3-42 and Aβ4-42, and not significantly bind to Aβ1-42, preferably AβpE3-42 and Aβ4-42.

[0099] Antibodies can be screened for their binding and / or specificity to low-molecular-weight oligomers of amyloid beta, preferably low-molecular-weight oligomers of AβpE3-x and Aβ4-x, using standard methods known in the art, such as ELISA. For example, assays such as those described in U.S. Patent No. 5,999,244 and U.S. Patent No. 5,999,244 can be used. Selection of antibodies that specifically bind to low-molecular-weight oligomers but not to other forms of amyloid beta protein, such as high-molecular-weight oligomers and / or monomeric and dimeric forms of amyloid beta, can be based on positive binding to low-molecular-weight oligomers of amyloid beta and lack of binding to high-molecular-weight oligomers and / or monomeric and dimeric forms of amyloid beta. Selection of antibodies that specifically bind to AβpE3-42 and Aβ4-42 but not Aβ1-42 can be based on positive binding to AβpE3-42 and Aβ4-42 and lack of binding to Aβ1-42.

[0100] Other aspects and embodiments described herein provide for the above aspects and embodiments in which the term "comprising" is replaced with the term "consisting of," as well as the above aspects and embodiments in which the term "comprising" is replaced with the term "consisting essentially of."

[0101] It shall be understood that the present application discloses all combinations of any of the above aspects and embodiments described above with each other unless the context requires otherwise. Likewise, the present application discloses all combinations of preferred and / or optional features, either alone or together with any other aspect, unless the context requires otherwise.

[0102] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to those skilled in the art upon reading this disclosure and, accordingly, are within the scope described herein.

[0103] All documents and sequence database entries referred to herein are incorporated by reference in their entirety for all purposes. [Example]

[0104] Experimental Method 1. Production of cyclized peptides Briefly, a linear peptide containing the desired sequence and containing two cysteine ​​residues is generated using standard techniques in the art. The peptide is cyclized through the cysteine ​​residues present in the peptide.

[0105] The peptides were cyclized by standard methods in the art, for example, by thiol oxidation, optionally incorporating methylene bridges. See also, for example, Kourra C and Cramer N, Chem. Sci., 2016,7, 7007-7012.

[0106] Peptides with the following sequences were generated:

[0107] TIFF0007766040000001.tif133170

[0108] The peptides were cyclized through cysteine ​​residues with either a disulfide bridge having the formula -SS or a thioacetal bridge having the formula -S-CH2-S-.

[0109] 2. 1-14 Disulfide-bridged Cyclic Peptide Binding ELISA 1. Coat a 384-well plate with 30 μL / well of 2.5 μg / ml streptavidin (Thermo Scientific 21122) diluted in PBS (Thermo Fisher 10010-015). 2. Incubate overnight at 4°C 3. Wash the plate (NUNC 384 program) 4. Coat a 384-well plate with 30 μL / well of 2 μg / ml disulfide-bridged cyclic peptide diluted in PBS. 5. Incubate at room temperature for 1 hour. 6. Wash the plate (NUNC 384 program) 7. Block the plate with 80 μL / well of assay buffer 8. Incubate overnight at 4°C 9. Wash the plate (NUNC 384 program) 10. For serum, distribute 70 μL / well of serum sample (diluted 1 / 100 in assay buffer) onto a non-sticky plate and dilute in a 2-fold series (35 μL into 35 μL assay buffer) in assay buffer. For competitor antibodies, control antibodies (diluted to 100.0 μg / ml in assay buffer) are dispensed onto the non-stick plate at 60 μL / well and diluted in a 3-fold series (20 μL into 40 μL assay buffer) in assay buffer. 11. Dispense 60 μL / well of control antibody (diluted to 360.0 μg / ml in assay buffer) onto non-stick plate and dilute in a 3-fold series (20 μL into 40 μL assay buffer) in assay buffer. 12. Transfer 30 μL / well onto the assay plate 13. Incubate at 37°C for 1 hour 14. Wash the plate (NUNC 384 program) 15. Dilute secondary antibody appropriately in assay buffer and add 30 μL / well 16. Incubate at 37°C for 1 hour 17. Wash the plate (NUNC 384 program) 18. Add 20 μL / well of K-BLUE substrate (Neogen 308176) 19. Incubate in the dark at room temperature for 10 minutes. 20. Stop the reaction by adding 10 μL / well of RED STOP solution (Neogen 308176) 21. Read the optical density at 650 nm using a PheraStar Plus (BMG LabTech).

[0110] 3. 1-42 Peptide Binding ELISA 1. Coat a 384-well plate with 30 µL / well of 100 ng / ml PSL amyloid 1-42 peptide (human - Peptide Specialty Laboratories - CEM1904161) diluted in carbonate / bicarbonate buffer 2. Incubate at 37°C for 1 hour 3. Wash the plate (NUNC 384 program) 4. Block the plate with 80 μL / well of assay buffer 5. Incubate overnight at 4°C 6. Wash the plate (NUNC 384 program) 7. Regarding disulfide-bridged immune sera: Dispense 70 μL / well of sample (diluted 1 / 100 in assay buffer) onto a non-stick plate and dilute in a 2-fold series (35 μL into 35 μL assay buffer) in assay buffer. Regarding thioacetal cross-linked immune serum, Serum samples (diluted 1 / 100 in assay buffer) and control antibodies (diluted to 360.0 μg / ml in assay buffer) are dispensed onto a non-stick plate at 60 μL / well and diluted in a 3-fold series (20 μL into 40 μL assay buffer) in assay buffer. 8. For control antibodies, dispense 60 μL / well of control antibody (diluted to 360.0 μg / ml in assay buffer) onto a non-stick plate and dilute in a 2- or 3-fold series (depending on the dilution of the immune serum) in assay buffer (20 μL into 40 μL assay buffer). 9. Transfer 30 μL / well onto the assay plate 10. Incubate at 37°C for 1 hour. 11. Wash the plate (NUNC 384 program) 12. Dilute secondary antibody appropriately in assay buffer and add 30 μL / well 13. Incubate at 37°C for 1 hour 14. Wash the plate (NUNC 384 program) 15. Add 20 μL / well of K-BLUE substrate (Neogen 308176) 16. Incubate in the dark at room temperature for 10 minutes. 17. Stop the reaction by adding 10 μL / well of RED STOP solution (Neogen 308176) 18. Read the optical density at 650 nm using a PheraStar Plus (BMG LabTech).

[0111] 4. pE3-42 Peptide Binding ELISA 1. Coat a 384-well plate with 30 μL / well of 100 ng / ml PSL amyloid pE3-42 peptide (human - Peptide Specialty Laboratories - CEM062210Pyr) diluted in carbonate / bicarbonate buffer. 2. Incubate at 37°C for 1 hour 3. Wash the plate (NUNC 384 program) 4. Block the plate with 80 μL / well of assay buffer 5. Incubate overnight at 4°C 6. Wash the plate (NUNC 384 program) 7. Regarding disulfide-bridged immune sera: Serum samples (diluted 1 / 100 in assay buffer) are dispensed onto a non-stick plate at 70 μL / well and diluted in assay buffer in a 2-fold series (35 μL into 35 μL assay buffer). Regarding thioacetal cross-linked immune serum, Serum samples (diluted 1 / 100 in assay buffer) and control antibodies (diluted to 360.0 μg / ml in assay buffer) are dispensed onto a non-stick plate at 60 μL / well and diluted in a 3-fold series (20 μL into 40 μL assay buffer) in assay buffer. 8. For control antibodies, dispense 60 μL / well of control antibody (diluted to 360.0 μg / ml in assay buffer) onto a non-stick plate and dilute in a 2- or 3-fold series (depending on the dilution of the immune serum) in assay buffer (20 μL into 40 μL assay buffer). 9. Transfer 30 μL / well onto the assay plate 10. Incubate at 37°C for 1 hour. 11. Wash the plate (NUNC 384 program) 12. Dilute secondary antibody appropriately in assay buffer and add 30 μL / well 13. Incubate at 37°C for 1 hour 14. Wash the plate (NUNC 384 program) 15. Add 20 μL / well of K-BLUE substrate (Neogen 308176) 16. Incubate in the dark at room temperature for 10 minutes. 17. Stop the reaction by adding 10 μL / well of RED STOP solution (Neogen 308176) 18. Read the optical density at 650 nm using a PheraStar Plus (BMG LabTech).

[0112] 5. 4-42 Peptide Binding ELISA 1. Coat a 384-well plate with 30 μL / well of 200 ng / ml Anaspec 4-42 peptide (Eurogentec AS-29908-1) diluted in carbonate / bicarbonate buffer. 2. Incubate at 37°C for 1 hour 3. Wash the plate (NUNC 384 program) 4. Block the plate with 80 μL / well of assay buffer 5. Incubate overnight at 4°C 6. Wash the plate (NUNC 384 program) 7. Regarding disulfide-bridged immune sera: Serum samples (diluted 1 / 100 in assay buffer) are dispensed onto a non-stick plate at 70 μL / well and diluted in assay buffer in a 2-fold series (35 μL into 35 μL assay buffer). Regarding thioacetal cross-linked immune serum, Control antibody (diluted to 360.0 μg / ml in assay buffer) is dispensed onto the non-stick plate at 60 μL / well and diluted in a 3-fold series (20 μL into 40 μL assay buffer) in assay buffer. 8. Transfer 30 μL / well onto the assay plate 9. Incubate at 37°C for 1 hour. 10. Wash the plate (NUNC 384 program) 11. Dilute secondary antibody appropriately in assay buffer and add 30 μL / well 12. Incubate at 37°C for 1 hour. 13. Wash the plate (NUNC 384 program) 14. Add 20 μL / well of K-BLUE substrate (Neogen 308176) 15. Incubate in the dark at room temperature for 10 minutes. 16. Stop the reaction by adding 10 μL / well of RED STOP solution (Neogen 308176) 17. Read the optical density at 650 nm using a PheraStar Plus (BMG LabTech).

[0113] 6. 8.5.KLH antigen binding ELISA 1. Coat a 384-well plate with 30 μL / well of 2 μg / ml KLH (Sigma H8283) diluted in PBS (Thermo Fisher 10010-015). 2. Incubate overnight at 4°C 3. Wash the plate (NUNC 384 program) 4. Block the plate with 80 μL / well of assay buffer 5. Incubate at room temperature for 1 hour. 6. Wash the plate (NUNC 384 program) 7. Serum samples (diluted 1 / 1000 in assay buffer) and control antibodies (diluted to 20.0 μg / ml in assay buffer) are dispensed onto a non-stick plate at 60 μL / well and diluted in assay buffer in a 3-fold series (20 μL into 40 μL assay buffer). 8. Transfer 30 μL / well onto the assay plate 9. Incubate at 37°C for 1 hour. 10. Wash the plate (NUNC 384 program) 11. Dilute secondary antibody appropriately in assay buffer and add 30 μL / well 12. Incubate at 37°C for 1 hour. 13. Wash the plate (NUNC 384 program) 14. Add 20 μL / well of K-BLUE substrate (Neogen 308176) 15. Incubate in the dark at room temperature for 5 minutes. 16. Stop the reaction by adding 10 μL / well of RED STOP solution (Neogen 308176) 17. Read the optical density at 650 nm using a PheraStar Plus (BMG LabTech).

[0114] 7. Thioacetal-bridged cyclic peptide binding ELISA 1. Coat a 384-well plate with 30 μL / well of 2.5 μg / ml streptavidin (Thermo Scientific 21122) diluted in PBS (Thermo Fisher 10010-015). 2. Incubate overnight at 4°C 3. Wash the plate (NUNC 384 program) 4. Coat a 384-well plate with 30 μL / well of 2 μg / ml thioacetal-bridged cyclic peptide diluted in PBS 5. Incubate at room temperature for 1 hour. 6. Wash the plate (NUNC 384 program) 7. Block the plate with 80 μL / well of assay buffer 8. Incubate overnight at 4°C 9. Wash the plate (NUNC 384 program) 10. Dispense 60 μL / well of serum samples (diluted 1 / 100 in assay buffer) and control antibodies (diluted to 360.0 μg / ml in assay buffer) onto a non-stick plate and dilute in a 3-fold series (20 μL into 40 μL assay buffer) in assay buffer. 11. Transfer 30 μL / well onto the assay plate 12. Incubate at 37°C for 1 hour. 13. Wash the plate (NUNC 384 program) 14. Dilute secondary antibody appropriately in assay buffer and add 30 μL / well 15. Incubate at 37°C for 1 hour. 16. Wash the plate (NUNC 384 program) 17. Add 20 μL / well of K-BLUE substrate (Neogen 308176) 18. Incubate in the dark at room temperature for 10 minutes. 19. Stop the reaction by adding 10 μL / well of RED STOP solution (Neogen 308176) 20. Read the optical density at 650 nm using a PheraStar Plus (BMG LabTech).

[0115] 8. Protein expression and purification for crystallography studies The Fab fragments of the anti-β-amyloid Fabs TAP01 and TAP01_01 were expressed in Expi293 cells. pE3-14 and the cyclized 3-14 peptide were solubilized at 1 mM in 25 mM Tris-HCl (pH 7.5) and 50 mM NaCl. Fab / peptide complexes were typically mixed at a molar ratio of 1:1.5 in 25 mM Tris-HCl (pH 7.5) and 50 mM NaCl. All Fab / peptide complex samples were concentrated to approximately 14 mg / ml for crystallization.

[0116] 9. Crystallization, Structure Determination, and Refinement All crystals were obtained by vapor diffusion at 19°C by mixing equal volumes of protein plus well solution.

[0117] TAP01-pE3-14 crystals were grown in 20% PEG3350 and 0.2 M ammonium citrate. TAP01_01-pE3-14 crystals were grown in 10% PEG 20K, 20% PEG550MME, 0.1 M MOPS / HEPES, pH 7.5, and 0.03 M each of sodium nitrate, disodium hydrogen phosphate, and ammonium sulfate.

[0118] TAP01-cyclized 3-14 cocrystals were grown in 20% PEG 6K, 0.1 M HEPES, pH 7.0, and 0.01 M zinc chloride. For cryoprotection, crystals were typically transferred to a mother liquor solution containing 22% ethylene glycol.

[0119] Data sets were collected at the European Synchrotron Radiation Facility (beamline ID30B (TAP01 + pE3-14)) or the Diamond Light Source (beamline I04 (TAP01_01 + pE3-14 and TAP01 + cyclized 3-14)). Cocrystals of TAP01 and TAP01_01 with pE3-14 peptide were refined to 1.4 Å and 2.5 Å resolution, respectively, while TAP01 with cyclized 3-14 peptide diffracted to 2.1 Å. Data were processed using XDS (Kabsch, W. (2010a / b) Acta Cryst D66, 125-132) and AIMLESS of the CCP4 Suite (Winn, M., et al. (2011) Acta Cryst D67, 235-242).

[0120] All crystal structures were solved by molecular replacement using Phaser (McCoy et al., (2005) Acta Cryst D 61, 458-64). The TAP01 structure was solved using homology models generated using SWISSMODEL (Waterhouse, A., et al. (2018) Nucleic Acids res. 46 W296-W303) with the deposited antibody structures 4F33 (Ma, J., et al. (2012) JBC, 287: 33123-33131) and 1I7Z (Larsen NA, et al. (2001) JMB, 311: 9-15), which were used to model the heavy and light chains, respectively. The refined coordinates of the TAP01 structure served as a search model for the subsequent TAP01_01 structure. Atomic models were constructed using Coot (Emsley, P. & Cowtan, K. Coot, (2004) Acta Cryst D60, 2126-32) and refined with Refmac (Murshudov, et al., (1997) Acta Cryst D53, 240-255). All structures were solved by molecular replacement and are reported with excellent stereochemistry and final Rworked / Rfree values ​​of less than 20 / 25% (Table 1).

[0121] TIFF0007766040000002.tif175170

[0122] Results and Discussion 1. Identification of novel epitopes and generation of "constrained" cyclic peptides Novel epitopes of the amyloid peptide for the TAP01 antibody, TAP01 and TAP01_01 (also known as NT4X and NT4X_SA), have been identified. X-ray crystallography studies were performed using the murine TAP01 antibody and the humanized TAP01_01 antibody in the presence or absence of the pE3-14 peptide (Table 1).

[0123] The structures of TAP01 Fab alone (Figure 1) and in the presence of pE3-14 peptide (Figure 2) were determined. These studies showed that the TAP01 antibody binds to a hairpin structure in the amyloid peptide (Figure 3). This binding site for the antibody has not been previously identified.

[0124] The results also show that the apo structure is identical to the antibody-peptide structure, thereby demonstrating that no conformational changes occur upon amyloid peptide binding. Furthermore, the structure of the TAP01 antibody and epitope is maintained during the humanization process of the TAP01 antibody (Figure 4).

[0125] The 1-14 amyloid peptide (Table 2) was generated, containing cysteine ​​residues at positions 3 and 12, forming a "constrained" version of the cyclic peptide.

[0126] Two different structures for constraining the cyclic peptide were generated: a disulfide-bridged peptide and a thioacetal-bridged peptide. The peptide sequences and structures are shown in Table 2 below. The thioacetal-bridged peptide provides a more chemically stable analog of the disulfide-bridged cyclic peptide. Analysis of the cyclic peptide with the sequence DACFRHDSGYECHH showed that it mimicked the hairpin structure identified in the structural studies.

[0127] TIFF0007766040000003.tif60170

[0128] X-ray crystallography studies were able to generate this "cyclic" conformation and confirmed that the TAP01 antibody binds to cyclic peptides in a manner similar to the pE3-42 peptide.

[0129] Both cyclic peptide structures revealed similar binding modes and conformations to the native structures (Figure 4). It was also shown that the cyclic peptides adopt the same hairpin conformation as the epitope of the native pE3-14 peptide (Figures 5 and 6).

[0130] Although several benchmark antibodies were able to bind to the pE3-42 amyloid peptide, the results show that TAP01 was the only antibody capable of binding to this novel hairpin epitope (Figure 7). The binding of the benchmark antibodies (bapineuzumab, solanezumab, BAN2401, ProBioDrug 6_1_6, and ProBioDrug 24_2_3) to the identified epitope was examined by ELISA using a 1-14 thioacetal-bridged cyclic peptide constrained to the epitope conformation. ProBioDrug 6_1_6 (accession number DSM ACC 2924) and ProBioDrug 24_2_3 (accession number DSM ACC 2926) are described in WO 2010 / 009987. None of the benchmark antibodies tested was able to bind to this "cyclic" peptide conformation.

[0131] 2. Immunization of mice and rabbits with cyclic peptides 2.1. Immunization with disulfide-bridged peptides and binding to amyloid peptides Immunization studies were conducted in rabbits and mice using the 1-14 amyloid peptide sequence, which contains cysteine ​​residues at positions 3 and 12 and has a disulfide bridge, to investigate the potential of a vaccine approach for the treatment of AD. Animals (5 mice, 2 rabbits) were immunized with the disulfide-bridged cyclic peptide, and sera were collected at pre-immunization (day 1), intermediate (day 35), and final (day 63) time points, as shown in Table 3.

[0132] TIFF0007766040000004.tif49170

[0133] Sera were screened for binding to biotinylated cyclic, 1-42, pE3-42, and 4-42 amyloid peptides, and the results are shown in Figures 8-12.

[0134] The results showed that mouse 5 produced an optimal immune response, producing a titer of 1 / 3200 against the disulfide-bridged cyclic peptide (Figure 8). Higher levels of background binding (pre-immunization) were produced by rabbits against the disulfide-bridged cyclic peptide (Figure 8). The resulting sera were examined for binding to the "cyclic" peptide, as well as the 1-42, 4-42, and pE3-42 amyloid peptides. Minimal binding to the 4-42 and pE3-42 amyloid peptides was observed by ELISA (Figures 9-11).

[0135] 2.2. Immunization with thioacetal-bridged peptides and binding to amyloid peptides Immunization studies were conducted in rabbits and mice using the 1-14 amyloid peptide sequence, which contains cysteine ​​residues at positions 3 and 12 and has a thioacetal bridge, to investigate its potential as a vaccine approach for the treatment of AD. Animals were immunized with the thioacetal-bridged cyclic peptide, and sera were collected at pre-immunization (day 1), intermediate (day 35), and terminal (day 63) time points, as shown in Table 3.

[0136] After immunization with the thioacetal-bridged cyclic peptide (Table 3), an immune response was generated in both rabbits and mice, with higher titers obtained in mice (Figure 13).

[0137] The resulting sera were examined for binding to the "cyclic" peptide and to the 1-42, 4-42, and pE3-42 amyloid peptides (Figures 13-16). Results showed that mice 2, 3, and 4 generated optimal immune responses, with titers of 1 / 72900 (mouse 2) and 1 / 24300 (mouse 3 and 4), respectively, against the thioacetal-bridged cyclic peptide (Figure 13). Consistent with the results obtained after immunization with the disulfide-bridged cyclic peptide, higher levels of background binding were observed in rabbits.

[0138] Testing of both forms of the "constrained" cyclic peptide showed that the thioacetal-bridged peptide was more stable and produced a higher titer response in mice, and therefore was used in downstream experiments.

[0139] 3. Screening of sera on human AD brain, and 5X FAD and Tg4-42 brain sections Sera from mouse immunizations (M2 and M4 sera) were used to stain human AD brain sections, as well as brain sections from 5× FAD and Tg4-42 mouse models (FIGS. 17 and 18).

[0140] 4. Biomarker identification and the effect of TAP01 antibody on glucose metabolism Imaging of 18F-FDG uptake in young and aged Tg4-42 mice showed a decrease in cerebral glucose metabolism in aged Tg4-42 mice (Figure 19). The results indicate that this decrease in cerebral glucose metabolism can be rescued with the TAP01 humanized antibody.

[0141] 5. Generation and evaluation of binding of TAP01_04 antibody to 1-14 cyclic peptide (thioacetal bridge) variants The 1-14 thioacetal-bridged cyclic peptide evaluated in the above experiments has cysteine ​​residues at positions 3 and 12 for the thioacetal bridge, which constrains the peptide. To evaluate the role of the placement of cysteine ​​residues at positions 3 and 12 on TAP01 antibody binding, additional peptides with cysteine ​​residues at different positions within the peptide sequence were generated (Table 4).

[0142] TIFF0007766040000005.tif107170

[0143] The binding of the TAP01 antibody to these thioacetal-bridged cyclic peptide variants has been assessed by ELISA (Figures 20-22 and Table 5). Binding to a reference antibody was also assessed.

[0144] TIFF0007766040000006.tif54170

[0145] The results showed that the affinity of the TAP01 (MoG1K) antibody was higher for the cyclic peptides 2,10, 2,12, and 2,13 compared to the cyclic peptide 3,12 (Figures 22 and 23), with calculated EC50 values ​​of 1.33 nM, 0.24 nM, and 1.56 nM, respectively, compared to 13.33 nM for 3,12. However, the reference antibody bapineuzumab could also bind to the 2,10, 2,12, and 2,13 cyclic peptide variants (Figure 22). Furthermore, BAN2401 and solanezumab could also bind to the 2,10 peptide variant with low affinity (Figure 22). This suggests that the novel hairpin epitope recognized by the TAP01 antibody is primarily in the 3,12 conformation.

[0146] To assess the role of different positional combinations of cysteine ​​residue placement, particularly when a cysteine ​​is provided at position 1, on TAP01 antibody binding, additional peptides were generated with cysteine ​​residues at different positions within the peptide sequence (Table 6).

[0147] TIFF0007766040000007.tif66170

[0148] The binding of the TAP01 antibody to these thioacetal-bridged cyclic peptide variants, as well as to cyclic peptide 3,12, cyclic peptide 2,10, cyclic peptide 2,12, and cyclic peptide 2,13, has been assessed by ELISA (Figure 25, Table 7). Binding to a reference antibody was also assessed (Figure 26).

[0149] TIFF0007766040000008.tif48170

[0150] The results showed that the affinity of the TAP01 (MoG1K) antibody was highest for cyclic peptide 1,13 compared to cyclic peptide 1,10, cyclic peptide 1,11, and cyclic peptide 1,12 (Figure 25), with calculated EC50 values ​​of 3.5 compared to 12, 111, and 9.5, respectively.

[0151] No binding of cyclic peptide 3,12 to the reference antibody bapineuzumab was observed (Figure 26). Furthermore, no binding of cyclic peptide 3,13 to the reference antibody bapineuzumab was observed (Figure 34). Binding to the reference antibody bapineuzumab was observed for cyclic peptide 2,10, cyclic peptide 2,12, cyclic peptide 2,13, cyclic peptide 1,10, cyclic peptide 1,11, cyclic peptide 1,12, and cyclic peptide 1,13 (Figure 26A). However, this data further suggests that the novel hairpin epitope recognized by the TAP01 antibody is primarily in the 3,12 conformation and that the cyclic peptide in the 3,13 conformation mimics this hairpin epitope.

[0152] 6. Generation of 1-14 mutant peptide variants and evaluation of TAP01 antibody binding to these variants To determine the mechanism of action of the amyloid peptide on the TAP01 antibody, five peptides (Table 8) were generated in which proline residues replaced the actual amino acids found in the peptides, and the binding of these peptides to the TAP01 antibody was examined (Figure 23).

[0153] No binding was observed with the DPEFRHDSGYEVHH and DAPFRHDSGYEVHH peptides, suggesting that residues 2 (A) and 3 (E) are important for binding. Peptides PAEFRHDSGYEVHH, PPPFRHDSGYEVHH, and PPEFRHDSGYEVHH bound in a dose-dependent manner, suggesting that residue 1 (D), the combination of residues 1, 2, and 3 (DAE), and the combination of residues 1 and 2 (DA) are not essential for binding.

[0154] TIFF0007766040000009.tif59170

[0155] 7. Immunization of mice with TAP01_01, TAP01_02, and TAP01_4 and the effect on plaque burden in 5XFAD mice 5XFAD mice were treated i.p. with 10 mg / kg of antibodies (TAP01_01, TAP01_02, and TAP01_4) between 6 and 18 weeks of age. Passive immunization with the TAP01_4 (cloned as MoG1K) (also known as NT4X_S71H) antibody reduced plaque burden for distinct Aβ species compared with the isotype control IgG1 antibody. TAP01_4 (MoG1K) significantly reduced plaques staining for total Aβ, pyroglutamate Aβ3-x, thioflavin, and TAP01.

[0156] In total Aβ-positive plaques, no effect was detected with TAP01_01(MoG1K), and a weak effect was detected with TAP01_02(MoG1K) compared to the IgG control. TAP01_02(MoG1K) significantly reduced plaques stained for pyroglutamate Aβ3-x. TAP01_01(MoG1K) and TAP01_02(MoG1K) treatment groups were shown to significantly reduce fibrillar Aβ deposits, as indicated by thioflavin staining (Figure 24).

[0157] 8. Active immunization of mice with constrained cyclic peptides Six-week-old 5XFAD mice were injected with antigen [thioacetal-bridged amyloid β peptide 1-14-KLH conjugate, DAC] emulsified in complete Freund's adjuvant (CFA). * FRHDSGYEC * HH[Cys]-amide ( * After 12 weeks of immunization with the antigen (S-CH-S bridge, cyclized at positions 3 and 12), mice were immunized with a booster dose of the protein emulsified in incomplete Freund's adjuvant (IFA). Mice were allowed to acclimate for at least 7 days in our facility before immunization. Mice were injected subcutaneously at two sites on their backs with the antigen emulsified in CFA, 0.05 mL to 0.1 mL injected at each site (total volume 0.1 mL to 0.2 mL per mouse).

[0158] Booster injections of antigen emulsified in IFA were administered 14, 28, 42 days, and 10 weeks after immunization with the antigen / CFA emulsion. The booster was administered as a single subcutaneous injection of 0.1 mL of IFA emulsion at a single site on the back. After the mice were sacrificed (at 18 weeks of age), serum samples were isolated from the mice and tested for antibody concentrations.

[0159] 18F-FDG-PET / MRI imaging 18F-FDG-PET / MRI was performed on 5xFAD mice and age-matched C57Bl / 6J wild-type mice. Mice were fasted overnight, and blood glucose levels were measured before tracer injection. 11.46 MBq to 20.53 MBq (mean 16.81 MBq) of 18F-FDG was intravenously injected into the tail vein in a maximum volume of 200 μl, followed by a 45-minute uptake period. Mice remained awake throughout the uptake process. PET scans were performed for 20 minutes using a small animal 1 Tesla nanoScan PET / MRI (Mediso, Hungary). Mice were anesthetized with isoflurane supplemented with oxygen and maintained on a heated bed (37°C) during the scan. Respiration rate was measured throughout the imaging process. Material maps (matrix 144 × 144 × 163, voxel size 0.5 × 0.5 × 0.6 mm) were prepared. 3 MRI-based attenuation correction was performed with the following parameters: matrix 136 × 131 × 315, voxel size 0.23 × 0.3 × 0.3 mm (TR: 15 ms, TE 2.032 ms, and flip angle 25°). 3 PET images were reconstructed using

[0160] 18F-florbetaben-PET / MRI of amyloid plaque burden 7.5 MBq to 24 MBq (mean 14 MBq) of 18F-florbetaben was administered intravenously into the tail vein in a maximum volume of 200 μl. After a 40-minute uptake period, mice were anesthetized and scanned as described above. PET acquisition time was 30 minutes. Material maps (matrix 144 × 144 × 163, voxel size 0.5 × 0.5 × 0.6 mm) were used. 3 MRI-based attenuation correction was performed with the following parameters: matrix 136 × 131 × 315, voxel size 0.23 × 0.3 × 0.3 mm (TR: 15 ms, TE 2.032 ms, and flip angle 25°). 3 PET images were reconstructed using (Bouter et al, (2019), Frontiers in Aging Neuroscience vol. 10:425).

[0161] Image analysis All images were analyzed using PMOD v3.9 (PMOD Technologies, Switzerland) as previously described (Bouter et al.). Briefly, a predefined MRI-based mouse brain atlas template was used to define different volumes of interest (VOIs), including the whole brain volume and the amygdala, brainstem, cerebellum, cortex, hippocampus, hypothalamus, midbrain, olfactory bulb, septum / basal forebrain, striatum, and thalamus. PET VOI statistics (kBq / cc) were generated for all brain regions, and standardized uptake values ​​(SUV) were calculated for semiquantitative analysis [SUV = mean tissue activity concentration (kBq / cc) × body weight (g) / injected dose (kBq)]. SUV for 18F-FDG-PET scans was corrected for measured blood glucose levels [SUVGlc = SUV × blood glucose level (mg / dL)]. The SUV of the 18F-florbetaben scan was further normalized by the SUV within the cerebellar VOI, and the resulting ratio (SUVr) was used for further analysis.

[0162] result Amyloid plaque imaging using the amyloid plaque tracer florbetaben was performed in immunized 5XFAD mice (n = 5), two 5XFAD control mice, and two wild-type mice (all female, aged 4.5 to 5.5 months). The results are shown in Figures 27 and 28. Immunized 5XFAD mice showed no florbetaben retention in the cortex, hippocampus, or amygdala, clearly demonstrating a dramatic reduction in amyloid plaque signal. The cyclic peptide used for immunization is specific for N-truncated amyloid-β oligomers, and the antibodies induced by this cyclic peptide do not react with full-length amyloid-β 1-42. The cyclic peptide used for immunization (a mimic of the hairpin structure of the truncated peptide against which the antibody was raised) resulted in clearance of amyloid plaques in 5XFAD brains, which were mostly composed of full-length amyloid-β 1-42 and only a small proportion of N-truncated amyloid-β. Therefore, this indicates that the cyclic peptide generates antibodies that bind to excised amyloid beta and dissolve the plaques. The hairpin structures are seeding factors for Alzheimer's plaques, and these can be removed by active immunization with the cyclic peptide.

[0163] summary Novel peptides bound by the TAP01 and TAP01_01 antibodies have been identified. These antibodies bind only to low molecular weight oligomers and not to plaques, compared to several control antibodies that also bind to plaques. While several antibodies can bind to different regions of the amyloid peptide sequence, only TAP01 can bind to the cyclic / hairpin conformation of the amyloid peptide. Thus, the generated cyclic peptides that mimic the hairpin epitope of Aβp3-42 can be used in active immunization to induce the production of antibodies specific to low molecular weight oligomers in subjects.

[0164] 9. Active immunization of cyclized Aβ peptides in 5XFAD mice: amyloid burden by immunohistochemistry in brain sections and in vivo glucose metabolism by 18F-FDG-PET / MRI imaging 5XFAD mice were treated with antigen [thioacetal-bridged amyloid β peptide 1-14-KLH conjugate, DAC] as previously described. * FRHDSGYEC * HH[Cys]-amide ( * S-CH-S bridge, cyclized through positions 3 and 12)].

[0165] In vivo imaging As described above, in vivo glucose metabolism in Alzheimer's mice (5XFAD) and age-matched C57Bl / 6J wild-type mice was analyzed by 18F-FDG-PET / MRI imaging.

[0166] Immunohistochemical staining of paraffin sections After CO2 anesthesia, mice were sacrificed by cervical dislocation. Brain samples were carefully excised and post-fixed in 4% phosphate-buffered formalin at 4°C. Human and mouse tissue samples were processed as previously described. 4 Briefly, 4 μm paraffin sections were deparaffinized in xylene and then rehydrated in a series of ethanol. To block endogenous peroxidase, sections were treated with H2O2. For antigen retrieval, sections were boiled in 0.01 M citrate buffer and then incubated in 88% formic acid for 3 min. After blocking nonspecific binding sites with skim milk and fetal bovine serum in PBS, primary antibodies were added. The following antibodies were used: polyclonal antibody 24311 against total Aβ5, monoclonal antibody 1-57 against pyroglutamate Aβ3-X (Synaptic Systems, Göttingen, Germany, 1 mg / ml, 1:500), and TAP01_4 (1:200, 2 mg / ml). Corresponding biotinylated secondary anti-human and anti-mouse antibodies (1:200) were purchased from DAKO (Glostrup, Denmark). Staining was visualized using the ABC method with a Vectastain kit (Vector Laboratories, Burlingame, USA) and diaminobenzidine (DAB) as a chromogen. Counterstaining was performed with hematoxylin.

[0167] Quantification of Aβ burden Plaque burden was quantified as previously described (G. Antonios et al., Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X. Scientific Reports 5, 17338 (2015)). Five to six paraffin-embedded sections, spaced at least 80 μm apart, were simultaneously stained with DAB as a chromogen. For thioflavin S fluorescent staining, tissue sections were deparaffinized, rehydrated, washed twice with deionized water, and then treated with 1% (w / v) thioflavin S in aqueous solution and counterstained with 1% (w / v) 4'6-diamidine-2-phenylindole in water. Relative Aβ burden was assessed using an Olympus BX-51 microscope equipped with an Olympus DP-50 camera and ImageJ software (NIH, USA). Representative photographs at 100x magnification were systematically captured. Using ImageJ, photographs were binarized to 8-bit black and white pictures and a fixed intensity threshold was applied to define DAB staining.

[0168] Percent area covered by DAB staining, as well as mm 2 Measurements were made regarding the number of particles per 1000 particles and the average size of the particles.

[0169] result We evaluated the impact of active immunization on plaque burden in brain sections (FIG. 29) and on in vivo glucose metabolism using PET / MRI imaging (FIG. 30).

[0170] Active immunization of the AD mouse model 5XFAD with the 3-12 linked Aβ1-14 cyclic peptide resulted in a reduction in amyloid plaque burden in brain tissue.

[0171] Immunostaining of plaque burden in the cortex of 5XFAD mice treated with active immunization with TAP01_04 antibody or cyclized Aβ peptide (Figure 29) closely matched the florbetaben retention signal seen for the cortex, hippocampus, and amygdala shown in Figure 28. 5XFAD mouse cortical sections were stained with antibodies against total Aβ, pyroglutamate Aβ3-X, thioflavin S, and Aβ4-X. Actively immunized 5XFAD mice showed a significant reduction in plaque burden accompanied by antibody and thioflavin S staining (Figure 29).

[0172] Glucose uptake was assessed in WT and 5XFAD mice by 18F-FDG imaging. Rescue of glucose uptake signals was observed in the cortex, hippocampus, thalamus, forebrain, and midbrain of actively immunized 5XFAD mice (Figure 30B).

[0173] 10. Therapeutic effects of cyclized Aβ peptide on active immunization and hippocampal function in Tg4-42 mice Six-week-old Tg4-42 mice were injected with antigen [thioacetal-crosslinked Aβ peptide 1-14-KLH conjugate, DAC] emulsified in complete Freund's adjuvant (CFA). * FRHDSGYEC * HH[Cys]-amide ( * After 12 weeks of immunization with the antigen / CFA emulsion (S-CH-S crosslinked), mice were immunized with a booster dose of protein emulsified in incomplete Freund's adjuvant (IFA). Booster injections of antigen emulsified in IFA were administered 14, 28, and 42 days after immunization with the antigen / CFA emulsion, and monthly thereafter (three times at 4, 5, and 6 months). Mice were acclimated for at least 7 days in our facility before immunization. Mice were injected subcutaneously at two sites on their backs with antigen emulsified in CFA or IFA, with 0.05 mL to 0.1 mL injected at each site (total volume 0.1 mL to 0.2 mL per mouse). Boosters were administered as a single subcutaneous injection of 0.1 mL of IFA emulsion at one site on the back. For titer determination, mice were sacrificed and serum samples were isolated from the mice.

[0174] Spatial reference memory using the Morris water maze Spatial reference memory in mice was assessed using the Morris water maze (R. Morris, Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47-60 (1984)), as previously described (Y. Bouter et al., N-truncated amyloid beta (Abeta) 4-42 forms stable aggregates and induces acute and long-lasting behavioral deficits. Acta Neuropathol 126, 189-205 (2013)).

[0175] Quantifying neuron number using unbiased stereology Stereological analysis was performed as previously described (G. Antonios et al., Alzheimer's therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X. Scientific Reports 5, 17338 (2015)). The hippocampal CA1 cell layer (bregma -1.22 mm to -3.52 mm) was delineated on cresyl violet-stained sections and analyzed using a stereological workstation (Olympus BX51 with a motorized specimen stage for automated sampling) and a StereoInvestigator 7 (MicroBrightField, Williston, TN, USA) under a 100× oil immersion lens (NA = 1.35).

[0176] result Active immunization of the AD mouse model Tg4-42 with the 3-12 linked Aβ1-14 cyclic peptide revealed a substantial rescue of learning and memory deficits accompanied by significantly reduced neuronal loss.

[0177] The therapeutic effects of active immunization were evaluated in 6.5-month-old Tg4-42 mice on hippocampal-dependent learning and memory by the Morris water maze test (Figure 31A) and by counting the total number of CA1 neurons in the hippocampus (Figure 31B). This was compared with the effects of passive immunization with TAP01_04 and an IgG1 control antibody. Active immunization and passive immunization with TAP01_04 antibody significantly improved spatial reference memory deficits and CA1 neuron counts in aged Tg4-42 mice that had received active or TAP01_04 immunization.

[0178] 11. Active immunization with cyclized Aβ peptides as a potential vaccine approach for the treatment of AD Animals (5XFAD and Tg4-42 mice) were immunized with the thioacetal-bridged cyclic peptide 1-14, and sera were screened for binding to the biotinylated cyclized peptide as previously described above. All mice generated a strong immune response (Figures 32 and 33).

[0179] summary The therapeutic potential of active immunization for the treatment and prevention of Alzheimer's disease using cyclic peptides that mimic the hairpin epitope of Aβp3-42 has been demonstrated by the above results.

Claims

1. Formula (I): X １ X ２ X ３ FX ４ HDSGX ５ X ６ X ７ X ８ H (I) A cyclic peptide comprising an amino acid sequence having the structure: During the ceremony, X １ is absent or any amino acid, X ２ is alanine or cysteine, X ３ is glutamic acid or cysteine, X ４ is arginine, X ５ is tyrosine or cysteine, X ６ is glutamic acid, X ７ is valine or cysteine, X ８ is histidine or cysteine, X １ , X ２ and only one of X3 is cysteine, ５ , X ７ and X ８ is a cysteine, and the peptide is cyclized through the cysteine ​​residues at positions 1, 2, or 3 and the cysteine ​​residues at positions 10, 12, or 13.

2. X １ is absent or any amino acid, a) X １ is cysteine, and X ２ is alanine, and X ３ is glutamic acid, and X ４ is arginine, and X ５ is tyrosine, and X ６ is glutamic acid, and X ７ is histidine, and X ８ is cysteine ​​or b) X ２ is alanine, and X ３ is cysteine, and X ４ is arginine, and X ５ is tyrosine, and X ６ is glutamic acid, and X ７ is cysteine, and X ８ is histidine, or c) X ２ is cysteine, and X ３ is glutamic acid, and X ４ is arginine, and X ５ is tyrosine, and X ６ is glutamic acid, and X ７ is cysteine, and X ８ is histidine, or d) X ２ is cysteine, and X ３ is glutamic acid, and X ４ is arginine, and X ５ is cysteine, and X ６ is glutamic acid, and X ７ is valine, and X ８ is histidine, or e) X ２ is cysteine, and X ３ is glutamic acid, and X ４ is arginine, and X ５ is tyrosine, and X ６ is glutamic acid, and X ７ is valine, and X ８ is cysteine ​​or f) X ２ is alanine, and X ３ is cysteine, and X ４ is arginine, and X ５ is cysteine, and X ６ is glutamic acid, and X ７ is histidine, and X ８ is histidine, or g) X ２ is alanine, and X ３ is cysteine, and X ４ is arginine, and X ５ is tyrosine, and X ６ is glutamic acid, and X ７ is histidine, and X ８ is cysteine, The cyclic peptide of claim 1 , wherein the peptide is cyclized via the two cysteine ​​residues.

3. a) DACFRHDSGYECHH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 12; b) DACFRHDSGYEVCH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 13; c) CAECFRHDSGYEVCH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 1 and 13; d) DCEFRHDSGYECHH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 2 and 12; e) DCEFRHDSGCEVHH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 2 and 10; f) DCEFRHDSGYEVCH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 2 and 13; and, g) DACFRHDSGCEVHH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 10; The cyclic peptide of claim 1 or 2, comprising an amino acid sequence selected from the group consisting of:

4. X １ The cyclic peptide of claim 1 or 2, wherein is proline or aspartic acid.

5. 5. The cyclic peptide of claim 1, wherein the peptide is cyclized through a bridge connecting the two cysteine ​​residues.

6. The peptide has, between the two cysteine ​​residues, a group of the formula -S-S- or -S-CH ２ The cyclic peptide according to any one of claims 1 to 5, which is cyclized through a bridge having -S-.

7. a) the amino acid sequence DACFRHDSGYECHH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 12; b) the amino acid sequence DACFRHDSGYEVCH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 3 and 13; or c) the amino acid sequence CAECFRHDSGYEVCH, wherein the peptide is cyclized through the cysteine ​​residues located at positions 1 and 13; The cyclic peptide according to any one of claims 1 to 6, comprising:

8. a) the peptide has, between the two cysteine ​​residues at positions 3 and 12, a cysteine ​​residue of the formula -S-CH ２ The amino acid sequence DACFRHDSGYECHH, which is a cyclic peptide formed by a bridge having -S-; or b) the peptide has, between the two cysteine ​​residues at positions 3 and 13, a cysteine ​​residue of the formula -S-CH ２ The amino acid sequence DACFRHDSGYEVCH, which is a cyclic peptide formed by a bridge having -S-; or c) the peptide has, between the two cysteine ​​residues at positions 1 and 13, a cysteine ​​residue of the formula -S-CH ２ the amino acid sequence CAEFRHDSGYEVCH, which is a cyclic peptide formed by a bridge having -S-; The cyclic peptide according to any one of claims 1 to 6, comprising:

9. a) the peptide has, between the two cysteine ​​residues at positions 3 and 12, a cysteine ​​residue of the formula -S-CH ２ The amino acid sequence DACFRHDSGYECHH, which is a cyclic peptide formed by a bridge having -S-; or b) the peptide has, between the two cysteine ​​residues at positions 3 and 13, a cysteine ​​residue of the formula -S-CH ２ The amino acid sequence DACFRHDSGYEVCH, which is a cyclic peptide formed by a bridge having -S-; or c) the peptide has, between the two cysteine ​​residues at positions 1 and 13, a cysteine ​​residue of the formula -S-CH ２ the amino acid sequence CAEFRHDSGYEVCH, which is a cyclic peptide formed by a bridge having -S-; The cyclic peptide according to any one of claims 1 to 8, comprising:

10. the peptide comprises the amino acid sequence DACFRHDSGYECHH, and between the cysteine ​​residues at positions 3 and 12, the peptide has a structure of the formula -S-CH ２ The cyclic peptide according to any one of claims 1 to 9, which is a cyclic peptide formed by a bridge having -S-.

11. A pharmaceutical composition comprising the cyclic peptide of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11 further comprising an adjuvant.

13. A pharmaceutical composition according to claim 11 or 12 for treating a neurodegenerative disease in an individual in need thereof.

14. The pharmaceutical composition of claim 13, wherein the neurodegenerative disease is Alzheimer's disease.

15. The pharmaceutical composition according to claim 11 or 12 for inducing an immune response in a subject.

16. 16. The pharmaceutical composition of claim 15, wherein the immune response produces antibodies against amyloid beta in the form of low molecular weight amyloid beta oligomers.

17. A method for producing a cyclic peptide according to any one of claims 1 to 10, comprising: (a) synthesizing a linear peptide comprising the sequence of said peptide as defined in any one of claims 1 to 10; (b) cyclizing the linear peptide through a cysteine ​​residue to obtain a cyclic peptide according to any one of claims 1 to 10; A method comprising:

18. 1. A method for producing an antibody that specifically recognizes low molecular weight oligomers of amyloid beta, comprising: (a) immunizing an animal with the cyclic peptide of any one of claims 1 to 10; (b) obtaining the antibody produced by the immunization in step (a); and Including, The method, wherein the animal is a non-human animal.

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