M13 bacteriophage exhibiting peptide motif targeting amyloid beta, methods and uses thereof

Abstract

The present disclosure relates to an engineered M13 bacteriophage displaying on its surface an amyloid producing peptide motif from amyloid beta 42 (A [beta] 42). The present disclosure also relates to the use of the disclosed engineered M13 bacteriophage for detecting and preventing aggregation of early substances of A [beta] (i.e. Oligomeric and fibrillar A [beta]) to promote inhibition of the progression of Alzheimer's disease and thus contribute to the treatment of such neurodegenerative disorders.

Description

Technical Field

[0001] The present disclosure relates to an engineered M13 bacteriophage (bacteriophage, bacterial phage) that displays on its surface an amyloidogenic peptide motif derived from the amyloid-beta 42 (Aβ42) peptide. The present disclosure also relates to the use of the disclosed engineered M13 bacteriophage for detecting early species of amyloid-beta (Aβ), namely soluble oligomeric and fibrillar Aβ, and for preventing and / or inhibiting its aggregation into fibrils and plaques, promoting inhibition of the progression of Alzheimer's disease (AD), and thus contributing to the treatment of this neurodegenerative disorder. Background Art

[0002] Alzheimer's disease (AD) is a chronic, progressive, neurodegenerative brain disease and is the most common type of dementia. The main suspected cause of AD is the small-sized amyloid-beta (Aβ) peptide released after β- and γ-secretase cleavage of the amyloid precursor protein (APP), which is a transmembrane protein abundantly present in the central nervous system (CNS). 1 . APP cleavage generates a heterogeneous set of peptides of different lengths, and the 40- and 42-amino acid-long isoforms are the two main toxic species. Compared to Aβ40, Aβ42 has been shown to be more prone to aggregation, and the presence of higher levels of Aβ42 is associated with higher neurotoxicity. Under pathological conditions, Aβ peptides gradually aggregate into soluble oligomers and protofibrils and deposit as insoluble amyloid plaques 2 , which is one of the hallmarks of AD. It is becoming increasingly clear that the toxic species of Aβ is not the Aβ immobilized in plaques, but rather the still-soluble oligomeric form 3 . Even before amyloid plaques can be detected in the brain, Aβ-oligomers can already trigger the loss of synapses 4 , which is closely related to the cognitive decline during early AD. Therefore, it makes more sense to correlate the levels of synapse loss and cognitive decline with the presence of Aβ-oligomers rather than amyloid plaques in the brain. A large body of evidence suggests that most of the toxic species are generated by secondary nucleation of Aβ monomers on the surface of amyloid fibrils, which gives rise to Aβ42 oligomers and protofibrils. Therefore, inhibition of this step is the main goal in methods aimed at limiting Aβ aggregation and toxicity. However, currently available immunohistochemical tools are only able to detect the presence of amyloid plaques, yet lack those that can selectively detect Aβ-oligomers and fibrils in brain samples.

[0003] Peptides have great potential for targeting oligomeric and fibrillar Aβ with high affinity and selectivity 5 However, for use in immunohistochemistry, the peptides need to be immunochemically labeled while preserving their architecture that confers target recognition and specificity.

[0004] In addition, peptides cannot cross the blood-brain barrier (BBB), which protects the central nervous system from the effects of the systemic circulation. This bottleneck can be overcome by using bacteriophages (simply phages), which are viruses that infect only bacterial cells. It has been demonstrated that the filamentous phage M13 is able to cross the BBB. Thus, a breakthrough in this technology is to engineer M13 to display Aβ-specific peptides.

[0005] Bacteriophages can be genetically and / or chemically modified to display a variety of biomolecules on their surface, which makes them attractive biotechnological tools for biomedical research 6 Since they infect bacteria rather than eukaryotic cells, bacteriophages are safe for humans and are easy and inexpensive to use for large-scale production.

[0006] Researchers agree that a possible treatment to slow or even halt the development of AD and preserve brain function is likely to be most effective when administered early in the disease continuum. Since the 1920s, bacteriophages have been widely used in clinical practice to treat bacterial infections 7 Notably, filamentous phages, such as M13, are generally well tolerated by the immune system 8 and have the ability to cross the BBB 9 In fact, naturally occurring bacteriophages can be found in the cerebrospinal fluid (CSF) of healthy humans at relatively high abundances (up to 10 4 plaque-forming units (pfu) / ml) 10 The ability to be recognized by the immune system and cross the BBB most likely depends on the type of bacteriophage and the peptide sequence displayed on its surface.

[0007] These facts are disclosed to illustrate the technical problems solved by the present disclosure SUMMARY OF THE INVENTION

[0008] The present disclosure relates to engineered M13 bacteriophages having a surface amyloidogenic peptide motif from Aβ42.

[0009] Protein aggregation and toxic deposition of β-amyloid (Aβ) are hallmark features of Alzheimer's disease (AD), and their mitigation is key to delaying the onset of the disease.

[0010] One aspect of the present disclosure relates to the genetic manipulation of bacteriophage M13 to generate synthetic bacteriophages that display surface peptidic motifs known to recognize Aβ42 conformational isomers through homotypic interactions. Using Aβ42 aggregation kinetics, the bacteriophages were tested in vitro based on their effects on Aβ42 aggregation and fibril formation. Based on immunofluorescence analysis, two engineered bacteriophages (AB30-39 and AB33-42) were observed to co-localize with Aβ inclusions found in the brain tissues of AD patients and mouse models.

[0011] Another aspect of the present disclosure relates to the use of engineered bacteriophages as immunohistochemical tools to detect Aβ oligomers and fibrils in postmortem brain tissues.

[0012] In one embodiment, bacteriophage M13 was genetically engineered to display on its surface small amyloidogenic peptidic motifs from Aβ42. These small amyloidogenic peptidic motifs from Aβ42 have previously been reported to recognize Aβ oligomers and fibrils with nanomolar affinity and are capable of neutralizing the toxicity of Aβ oligomers when transplanted into the complementarity-determining regions (CDRs) of antibodies. 11 。

[0013] One aspect of the present disclosure relates to M13 bacteriophages comprising amyloidogenic peptidic motifs AB30-39 (SEQ ID No. 1 - AIIGLMVGGV) and AB33-42 (SEQ ID No. 2 - GLMVGGVVIA) on their surface, which are used as medicaments in medicine or as dyes.

[0014] In one embodiment, the bacteriophage is used for the detection and / or diagnosis of β-amyloid oligomers and β-amyloid fibrils, and for the prevention or inhibition of β-amyloid 42 aggregation.

[0015] In one embodiment, the engineered (or modified) bacteriophage is used for the detection, diagnosis, prevention, or treatment of the aggregation of β-amyloid oligomers and β-amyloid fibrils.

[0016] In one embodiment, the engineered (modified) bacteriophage is used for the detection, diagnosis, prevention, or treatment of neurodegenerative diseases.

[0017] In one embodiment, bacteriophage M13 is for detecting or diagnosing the presence of β-amyloid oligomers and β-amyloid fibrils in a brain tissue sample.

[0018] In one embodiment, the bacteriophage is for detecting, diagnosing, preventing, or treating Alzheimer's disease or a neurodegenerative disease that is positively affected by a reduction in the aggregation of β-amyloid oligomers and β-amyloid fibrils.

[0019] Another aspect of the present disclosure relates to a pharmaceutical composition comprising the phage of the present disclosure.

[0020] In one embodiment, the composition of the present disclosure further comprises a suitable pharmaceutical excipient.

[0021] In one embodiment, the composition is administered intravenously (IV) for the diagnosis / prevention of neurodegenerative diseases.

[0022] In one embodiment, the composition is administered to a human suffering from a neurodegenerative disease that is positively affected by a reduction in β-amyloid aggregation, wherein the dose is less than 10 10 pfu / day. In another embodiment, the composition is administered every two days. In another embodiment, the composition is administered every three days. In yet another embodiment, the composition is administered at a weekly dose. Administration in all cases can be carried out over a given period of time.

[0023] In one embodiment, the administered dose is in the range of 10 4 -10 10 pfu / day, preferably 10 6 -10 9 pfu / day.

[0024] Another aspect of the present invention relates to a kit for detecting or diagnosing β-amyloid oligomers and / or β-amyloid fibrils, the kit comprising the phage of the present disclosure.

[0025] Another aspect of the present invention relates to a method for in vitro detecting or diagnosing the presence of β-amyloid oligomers and β-amyloid fibrils.

[0026] In one embodiment, the method comprises the following steps:

[0027] a) Incubating a brain tissue with the engineered phage of the present disclosure and washing the excess;

[0028] b) Incubating the washed brain tissue with a rabbit anti-fd phage primary antibody, an FITC-labeled goat anti-rabbit IgG secondary antibody, and a DAPI stain.

[0029] In one embodiment, the brain tissue is incubated overnight at 4 °C in a humidified chamber with a 50 - 100 μl solution (in a 1× TBS or 1× PBS salt solution) containing phage at a concentration of 10 4 to 10 10 pfu / ml.

[0030] In one embodiment, the washed brain tissue was incubated overnight at 4 °C in a humid chamber with a 1:1000 dilution of rabbit anti-fd phage primary antibody. The brain tissue with rabbit anti-fd phage antibody was washed and then incubated with FITC-labeled goat anti-rabbit IgG secondary antibody, and then washed and stained with DAPI for fluorescence imaging.

[0031] In one embodiment, the method of the present disclosure further includes washing the tissue slide 5 times with 1×TBST for 10 minutes each time.

[0032] In one embodiment, the daily form consists of an intravenous solution that contains a defined amount of M13 phage containing an amyloidogenic peptide motif (AB30-39, AB33-42, or both), and the whole form is intended to be administered as a single dose or, if needed, as multiple doses over a given period of time.

[0033] The present disclosure relates to a modified M13 phage (engineered M13 phage) that contains an amyloidogenic peptide motif on its surface that serves as a drug or therapeutic agent, or as a dye, or a label, or a marker, wherein the peptide motif comprises at least a sequence having 90% identity with the following sequences: SEQ ID No1, SEQ ID No 2, and mixtures thereof. Preferably, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

[0034] In one embodiment, the phage contains a DNA sequence having at least 90% identity with SEQ ID No.5. Preferably, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

[0035] In another embodiment, the phage contains a DNA sequence having at least 90% identity with SEQ ID No.6. Preferably, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

[0036] In one embodiment, the peptide motif of the phage comprises a sequence having at least 90% identity with SEQ ID No.1, preferably 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity or 100% identity; and a DNA sequence having at least 90% identity with SEQ ID No.5, preferably 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity or 100% identity.

[0037] In another embodiment, the peptide motif of the phage comprises a sequence having at least 90% identity with SEQ ID No.2, preferably 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity or 100% identity; and a DNA sequence having at least 90% identity with SEQ ID No.6, preferably 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity or 100% identity.

[0038] In one embodiment for better results, the phage comprises 5 to 8 of said amyloidogenic peptide motifs on its surface. It has been observed that 5 to 8 peptides present on each phage surface surprisingly improve the avidity and specificity of the oligomers relative to the monomers compared to the respective Aβ-peptides. This improves the diagnosis and prevention of Alzheimer's disease or neurodegenerative diseases that are positively affected by the reduction of β-amyloid oligomer and β-amyloid fibril aggregation.

[0039] In one embodiment, the phages of the present disclosure are used for detecting, diagnosing, preventing, and / or treating the aggregation of β-amyloid oligomers and / or β-amyloid fibrils. In a further embodiment, the phages of the present disclosure are used for quantifying the aggregation of β-amyloid oligomers and / or β-amyloid fibrils. In one embodiment, the treatment can be carried out by inhibiting aggregation or depolymerizing the aggregates of the formed β-amyloid.

[0040] In another embodiment, the phages of the present disclosure are used for detecting, diagnosing, preventing, or treating neurodegenerative diseases.

[0041] In one embodiment, the phages of the present disclosure are used for detecting, quantifying, and / or diagnosing the presence of β-amyloid oligomers and / or β-amyloid fibrils in a tissue sample.

[0042] In one embodiment, the tissue sample is a brain tissue sample.

[0043] In one embodiment, the phage is used for detecting, diagnosing, preventing, and / or treating Alzheimer's disease or a neurodegenerative disease that is positively affected by a reduction in the aggregation of β-amyloid oligomers and β-amyloid fibrils. Surprisingly, the disclosed engineered M13 phage enables early detection of Alzheimer's disease or a neurodegenerative disease that is positively affected by a reduction in the aggregation of β-amyloid oligomers and β-amyloid fibrils, because it can detect and bind to oligomeric and fibrillar Aβ, which are relevant in the early stages of these diseases.

[0044] One aspect of the present disclosure relates to an M13 phage that comprises, on its surface, 5 to 8 amyloidogenic peptide motifs, wherein the peptide motif comprises a sequence having at least 90% identity to the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof. Preferably, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity.

[0045] The present disclosure also relates to a pharmaceutical composition comprising the phage of the present disclosure.

[0046] In one embodiment, the pharmaceutical composition further comprises a suitable pharmaceutical excipient.

[0047] In one embodiment, the pharmaceutical composition is for preventing and / or treating Alzheimer's disease or a neurodegenerative disease that is positively affected by a reduction in the aggregation of β-amyloid oligomers and β-amyloid fibrils, wherein the composition is in the form of a solution for intravenous administration.

[0048] In one embodiment, the intravenous dose is less than 10 10 pfu / day. In another embodiment, the intravenous dose ranges from 10 4 -10 10 pfu / day.

[0049] One aspect of the present disclosure includes a kit for detecting, quantifying, and / or diagnosing β-amyloid oligomers and β-amyloid fibrils, the kit comprising the phage of the present disclosure. In one embodiment, the kit is for diagnosing and monitoring Alzheimer's disease.

[0050] The present disclosure also relates to a method for detecting or diagnosing the presence of β-amyloid oligomers and / or β-amyloid fibrils in a tissue sample, the method comprising the steps of: incubating the tissue sample with a solution comprising a phage of the present disclosure; detecting the presence of the phage in the tissue sample.

[0051] In one embodiment, the method further comprises the step of incubating the tissue sample with a first antibody suitable for binding to the phage. In another embodiment, the first antibody (primary antibody) is a rabbit anti-fd phage antibody.

[0052] In one embodiment, the method further comprises the step of incubating the tissue sample with a second antibody suitable for binding to the first antibody, wherein the second antibody (secondary antibody) is a fluorescent antibody, i.e., an antibody labeled with a fluorescent compound.

[0053] In one embodiment, the second antibody is a fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit immunoglobulin G (IgG) antibody.

[0054] In one embodiment, the tissue sample is a brain tissue sample.

[0055] In one embodiment, the concentration of phage in the solution is in the range of 10 4 pfu / ml - 10 10 pfu / ml.

[0056] One aspect of the present disclosure relates to the use of M13 phage as a dye / tag, or label, wherein the M13 phage comprises an amyloidogenic peptide motif on its surface, and wherein the peptide motif comprises a sequence having at least 90% identity to the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof. Preferably, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The following drawings provide illustrations of preferred embodiments of the present disclosure and should not be construed as limiting the scope of the invention.

[0058] Figure 1 Shows the genetic manipulation process of M13 phage.

[0059] Figure 2 Shows an embodiment of the effect of AB-phage on Aβ42 aggregation.

[0060] Figure 3 Shows an embodiment of the effect of AB-phage on surface-catalyzed secondary nucleation of Aβ42.

[0061] Figure 4 Shows an embodiment of the effect of AB-phage on the protofibril content at the end point of the aggregation curve.

[0062] Figure 5 shows representative fluorescence images of control and APP mouse brain tissue samples in two different age groups: 3 - 4 months and 9 - 12 months ( Figure 5A ), as well as signal quantification ( Figure 5B and C).

[0063] Figure 6 Shows representative fluorescence images and signal quantification of control and AD human brain tissue samples.

[0064] Figure 7 Shows representative fluorescence images of control and APP mouse brain tissue samples with and without antigen retrieval.

[0065] Figure 8 Shows representative fluorescence images of M13 and AB33 - 42 in APP mouse brain tissue samples with different fixation times. Detailed implementation

[0066] The present disclosure relates to engineered M13 phages that display amyloidogenic peptide motifs from amyloid - beta 42 (Aβ42) on their surface. The present disclosure also relates to the use of the disclosed engineered M13 phages for detecting early species of Aβ, namely oligomeric and fibrillar Aβ, and preventing their aggregation, promoting inhibition of the progression of Alzheimer's disease, and thus contributing to the treatment of this neurodegenerative disorder.

[0067] All reagents were of the highest commercially available grade. Thioflavin T (ThT) was obtained from Sigma. Chelex resin (Bio - Rad) was used to remove contaminant trace metals from all solutions. The human Aβ42 expression plasmid (pET - Sac - Abeta(M1 - 42), SEQ ID No.9) was used to prepare recombinant Aβ42 (SEQ ID No.8). According to Walsh et al. 12, recombinant Aβ42 was expressed in Escherichia coli and purified. To obtain the monomeric form, 4 mg of Aβ42 was dissolved in 7 M guanidine hydrochloride and eluted in Superdex S75 (GE Healthcare) with 50 mM HEPES pH 7.4 and used immediately. Amyloid fibrils of Aβ42 were prepared by incubating 5 μM Aβ42 at 37 °C for 24 h under static conditions. Low-bind tubes (Axygen Scientific, Corning) were used in all manipulations of Aβ42.

[0068] In one embodiment, genetic manipulation of M13 phage was performed.

[0069] In one embodiment, preparation of the insert was performed. Primers were designed to clone two 10-amino acid (Aβ-based) amyloidogenic peptide residues into the genome of M13KE phage. The Aβ30-39 (SEQ ID No. 1 - AIIGLMVGGV) and Aβ33-42 (SEQ ID No. 2 - GLMVGGVVIA) peptide motifs from the Aβ42 peptide were fused to the N-terminal gene of gene 3, resulting in the expression of the peptides on the coat protein III of M13 phage ( Figure 1 ).

[0070] Figure 1 The genetic manipulation process of M13 phage is shown. The 10-amino acid Aβ-based peptide motifs Aβ30-39 (AIIGLMVGGV) and Aβ33-42 (GLMVGGVVIA) were cloned into gene 3 of M13 and subsequently displayed on the phage surface protein III.

[0071] Following this procedure, a third engineered phage displaying only 4 amino acids from the Aβ42 peptide - Aβ36-39 (VGGV) was obtained, as only partial correct Aβ sequences were inserted into the M13 phage genome. This phage was named AB36-39.

[0072] In one embodiment, the 10-amino acid Aβ-based peptide motifs Aβ30-39 (AIIGLMVGGV) and Aβ33-42 (GLMVGGVVIA) were cloned into the genome of M13 based on the phagemid cloning system 13 . The basic components of a phagemid include an origin of replication of a plasmid, a selectable marker (usually an antibiotic resistance marker), an intergenic region (IG region, usually containing packaging sequences and an origin of replication for both negative and positive strands), phage coat protein genes, restriction enzyme recognition sites, a promoter, and a DNA fragment encoding a signal peptide 13For phagemid construction, the commercially available plasmid pETDuet-1 (Novagen, Darmstadt, Germany, SEQ ID No. 3) was used. This plasmid contains an ampicillin resistance gene; the T7 promoter (allowing gene transcription that affects the expression level of the fusion gene); the signal peptide pelB (facilitating the translocation of phage proteins across the bacterial membrane and their assembly in phage particles). First, the entire sequence of gene 3 of M13 phage (encoding phage coat protein III) was cloned between the HindIII-NotI restriction sites (multiple cloning site 1) of MCS-1. Then, also on MCS-1, the pelB sequence was cloned between BamHI-EcoRI to obtain an intermediate plasmid with SEQ ID No. 4. The Aβ peptide sequence was cloned between the SalI-SacI restriction sites immediately upstream of gene 3 of M13 on MCS-1 of the pETDuet-1 plasmid to obtain a plasmid with SEQ ID No. 5 (phagemid AB30-39) or SEQ ID No. 6 (phagemid AB33-42). This recombinant plasmid was transformed into competent Escherichia coli (E. coli) cells, and positive clones were confirmed by polymerase chain reaction (PCR) and sequencing.

[0073] In one embodiment, phage particles were produced. Since phagemids can be converted into phage particles of the same morphology by co-infection with helper phage, the kanamycin-resistant M13KO7 helper phage (N0315S, New England Inc), a derivative of M13 phage containing a kanamycin resistance gene, was used. The infection procedure was carried out according to the protocol of New England Biolabs to fuse the gene 3 of the plasmid with the gene 3 of the helper phage, so that the Aβ sequence was displayed in phage coat protein III. Briefly, cells containing the phagemid were grown in LB medium containing ampicillin at a final concentration of 20 mg / ml and were infected with 50 μL of M13KO7 helper phage (10 8Infect for 90 minutes at 37 °C and 250 rpm with (pfu / ml). Then, add kanamycin to a final concentration of 70 μg / ml and incubate the solution overnight at 37 °C and 250 rpm. To separate the bacterial cells from the phage, centrifuge at 6000 rpm for 10 minutes and transfer the supernatant to a new tube. Precipitate the phage by adding PEG / NaCl solution, then incubate at 4 °C for 2 hours and resuspend in Tris-buffered saline (TBS). Isolate the phage genomic ssDNA using an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, v / v), purify with an equal volume of chloroform, precipitate with 100% ethanol and resuspend in Tris-EDTA (TE).

[0074] In one embodiment, to examine the Aβ sequence, the DNA of the synthetic phage was sequenced using primers (SEQ ID No. 7) that read the region of interest of gene 3. Phage titration was performed after the double agar overlay technique. Briefly, 10 μl of serially diluted phage, 200 μl of host bacterial culture and 3 ml of soft agar were mixed and poured onto an LB plate containing ampicillin. After incubation overnight at 37 °C, the plaque forming units (pfu) were determined.

[0075] Sequence alignment methods for comparison are well known in the art and these include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch to find the global (over the entire sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps 14 . The BLAST algorithm calculates the percentage of sequence identity and performs a statistical analysis of the similarity between two sequences 15 . Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). One of the methods available in the MatGAT software package can also be used to determine the global percentage of similarity and identity: an application that generates a similarity / identity matrix using protein or DNA sequences 16 . Minor manual editing can be performed to optimize the alignment between conserved motifs, which will be obvious to those skilled in the art. Using BLAST with default parameters, sequence identity values are determined over the entire amino acid sequence, which are expressed as a percentage in the subject matter of the present invention.

[0076] In one embodiment, another phage Aβ36-39 (VGGV, SEQ ID NO 10) that displays only a 4-amino acid peptide was also obtained( Figure 1 ).

[0077] In one embodiment, the aggregation kinetics of Aβ42( Figure 2 and3 )。According to the original report, the aggregation kinetics was determined by recording the ThT fluorescence intensity as a function of time in a microplate reader (Fluostar Optima, BMG Labtech) with a 440 nm excitation filter and a 480 nm emission filter. Fluorescence was recorded using the bottom optics in a half-area polyethylene glycol-coated 96-well black polystyrene plate (3881, Corning) with a transparent bottom. Aβ42 monomers were separated by gel filtration (Tricorn Superdex 75 column, GE Healthcare) in 50 mM HEPES at pH 7.4. 10 μM ThT was added to each condition. At the start of the reaction, AB phage and M13 were added at different titers (10 8 、10 9 and 10 10 pfu / ml). The assays were repeated three times at 37 °C without stirring, and fluorescence was read every 400 s. The data were analyzed using Amylofit and processed in Origin. The fibril mass estimated from the ThT intensity at the end point was averaged from the fluorescence recordings during the plateau phase after the transition.

[0078] In one embodiment, immunoblot analysis was performed ( Figure 4 ). According to the manufacturer's instructions, the Aβ42 aggregates obtained at the plateau phase of each aggregation kinetics curve were diluted up to 8-fold and spotted onto a PVDF membrane three times and probed with a 1:1000 dilution of anti-amyloid fibril OC antibody (AB9234, Merck Millipore).

[0079] In one embodiment, an immunofluorescence assay was performed (Figs. 5 to 8). Brains of age-matched APPswe / PS1dE9 transgenic and wild-type mice were dissected in ice-cold phosphate-buffered saline (PBS) at pH 7.45, snap-frozen in liquid nitrogen and stored at -20 °C. Brain tissues were sectioned using a cryostat (CM3050S, Leica) at a chamber temperature of -15 °C and an object temperature of -13 °C. The cutting angle was set at 5 degrees and the section thickness was 10 μm. The tissue was trimmed to reach the hippocampal region. The sections were placed on SuperFrost Plus glass (11950657, Thermo Scientific) and stored at -20 °C. The same procedure was applied to 7-μm postmortem human hippocampal sections (Table 1) from AD patients and corresponding controls. The cryopreserved slides were fixed with PBS containing 4% paraformaldehyde (PFA) for 10 minutes. The slides were incubated with a blocking solution for 1 hour at room temperature, which was a solution containing 2.5% bovine serum albumin (BSA) (Sigma-Aldrich, 10735086001) in Tris-buffered saline with 0.1% Tween-20 (TBST). Next, the slides were incubated with a concentration of 10 8Phage at pfu / ml was incubated overnight at 4 °C in a humidified chamber in TBST. The slides were washed 5 times in TBST for 10 minutes each, and then incubated overnight at 4 °C in a humidified chamber with a 1:1000 dilution of rabbit anti-fd phage antibody (B7786, Sigma). Then, the slides were washed and incubated with a 1:200 dilution of FITC-labeled goat anti-rabbit IgG antibody (F9887, Sigma) for 2 hours at room temperature. For 6E10 antibody staining, antigen retrieval was performed by heat-mediated treatment with sodium citrate buffer 10 mM pH 6.0 (S1894-500G, Sigma-Aldrich), where the boiling state was set for 20 minutes and then cooled to room temperature for 20 minutes. The slides were incubated with a 1:5000 dilution of mouse anti-β-amyloid 6E10 antibody (80300, BioLegend), and then incubated with a 1:700 dilution of donkey anti-mouse IgG (H+L) labeled with Alexa Fluor 594 (A-21203, Thermo Scientific). All antibodies were diluted in TBST containing 1% BSA. The slides were covered with Vectashield mounting medium containing DAPI (VectorLabs). Images were acquired using a fluorescence microscope (Nikon Eclipse E400) and analyzed using ImageJ. Quantification was performed in 40× magnified images, using a set color threshold to identify the fluorescein signal (phage), and all spots within a defined size threshold between 10 - 150 pixels were counted.

[0080] Table 1: Brain tissue samples from human donors. Thal stage based on immunopositive amyloid detection in cortical and subcortical regions: Stage (3) - brainstem / midbrain. Evaluation of AD neuropathological changes (Neuro Change) by ABC score: A - Thal stage of Aβ plaques; B - Braak neurofibrillary tangle score; C - CERAD neuritic plaque score.

[0081]

[0082] In one embodiment, parametric data for two-group comparisons was evaluated by Student's t-test, and for all analyses, p < 0.05 was considered statistically significant.

[0083] In one embodiment, the presence of Aβ peptide motifs on the surface of M13 phage was determined. Previous studies have shown that corresponding to the 30 to 39 of the c-terminus of β-amyloid (Aβ) ( 30 AIIGLMVGGV 39 ) and 33 to 42 ( 33 GLMVGGVVIA 42) Peptides of amino acids that are reactive to both Aβ-oligomers and protofibrils, but only weakly reactive to monomers 11 . These Aβ peptide motifs were cloned into phagemids for exposure on the coat protein III of M13 filamentous phage ( Figure 1 ). By fusing these phagemids with M13 helper phage, they were packaged as ssDNA into the phage capsid. These synthetic phage particles were named AB36-39, AB30-39, and AB33-42, which display 5 to 8 copies of the peptide motifs Aβ36-39, Aβ30-39, and Aβ33-42, respectively, on their surfaces, and these motifs aggregate at the ends of the filamentous phage ( Figure 1 ).

[0084] In one embodiment, to test whether the engineered phage would inhibit the aggregation of Aβ42, the aggregation kinetics were measured. The aggregation process of Aβ42 leading to the formation of protofibrils can be monitored using thioflavin T (ThT) fluorescence. ThT is an amyloid-sensitive dye, and its intensity can be correlated with the protofibril mass. The reaction started with a highly homogeneous preparation of monomeric Aβ42 17 , enabling the evaluation of the effect of the inhibitor on the reaction rate. The ThT fluorescence kinetics of Aβ42 aggregation showed the characteristic sigmoidal curve of Aβ42 aggregation, which reflects the complex kinetics of the self-assembly of Aβ42 monomers into oligomers and protofibrils ( Figure 2 ).

[0085] In one embodiment, Figure 2 showed the effect of AB-phage on Aβ42 aggregation. At gradually increasing phage titers (10 8 , 10 9 and 10 10 pfu / ml), the aggregation of 5 μM Aβ42 was monitored by ThT emission with and without the addition of M13 phage ( Figure 2 -a), AB36-39 ( Figure 2 -b), AB30-39 ( Figure 2 c) and AB33-42 ( Figure 2 -d). The effect of the engineered phage was compared to the empty M13 phage control. Based on the average endpoint ThT emission, the effect of each AB-phage and M13 on the protofibril mass concentration was determined at the highest phage titer (10 10 pfu / ml) ( Figure 2 -e).

[0086] In one embodiment, a lag phase was observed before the formation of the first batch of primary protofibrils ( Figure 2-a-d). The length of this delay time depends on the concentration of Aβ42 monomers available for fibril formation. Once the first batch of protofibrils is formed, they act as catalysts for generating additional fibrils, as evidenced by the rapid increase in ThT fluorescence. The value at the plateau indicates the total amount of end-stage Aβ42 fibrils formed.

[0087] In one embodiment, the addition of M13 phage (i.e., phage without the displayed peptide) has a sparse (discrete, weak) effect on the lag phase ( Figure 2 -a) and the aggregation half-life (t 1 / 2 ), where t 1 / 2 is defined as the time point when the ThT intensity reaches half of the value between the initial baseline and the final plateau, but they do decrease the level of end-point fibrils with increasing titer ( Figure 2 -e). This may be due to the non-specific interaction between the M13 coat protein and Aβ42 monomers.

[0088] In one embodiment, the engineered phages tested have a significant effect on Aβ42 aggregation. The AB30-39 phage inhibits Aβ42 aggregation and kinetics and results in a lower fibril mass / ThT intensity at the end point ( Figure 2 -c). With increasing titer, as mentioned, AB30-39 gradually delays Aβ42 aggregation by increasing the half-life by ~1 h (Table 2). At the same time, the end-point fibril mass is also significantly reduced ( Figure 2 -e). On the other hand, compared to the M13 phage control ( Figure 2 -b, 2-d), even at the highest tested titer, AB36-39 and AB33-42 have no significant effect on either Aβ fibril mass or the Aβ42 aggregation rate.

[0089] In one embodiment, the fragment AB30-39 interacts more effectively with Aβ monomers and also with a wider set of early Aβ polymorphs, as shown by the stronger inhibition of fibril formation observed.

[0090] In one embodiment, the effect of phage-displayed peptides on the secondary nucleation of monomers on the surface of amyloid fibrils was determined. The aggregation of Aβ includes several microscopic steps, which include primary nucleation of Aβ monomers, secondary nucleation of monomers on the surface of fibrils, and elongation of fibrils due to monomer addition ( Figure 3 -a): primary nucleation (k n ) starting from monomers, elongation (k + ) by monomer attachment to existing aggregates, and secondary nucleation (k 2) 18 。

[0091] In one embodiment, seeding experiments in which preformed Aβ42 fibrils (2%) are added to monomeric Aβ42 enable the assessment of the role of secondary nucleation because the preformed fibrils provide a reactive surface. These assays can identify inhibitors that block the secondary nucleation of Aβ42 monomers on the amyloid fibril surface. In one embodiment, preformed fibrils are added to Aβ42 monomers, resulting in an acceleration of fibril formation independent of primary nucleation. Phage is added to the reaction at a high concentration (10 10 pfu / ml). The results obtained indicate that under the test experimental conditions, seeded Aβ42 aggregation (t 1 / 2 = 1.09 ± 0.01 h) is twice as fast as unseeded Aβ42 aggregation (t 1 / 2 = 2.34 ± 0.1 h)( Figure 3 b-f).

[0092] In one embodiment, the effect of empty M13 phage has a negligible effect on the seeding reaction rate, while when the reaction slows down, AB30-39 appears to be an effective inhibitor of secondary nucleation of monomers on the fibril surface (t 1 / 2 = 2.69 ± 0.22 h). Interestingly, a different situation was observed for AB33-42, which led to a slight increase in the seeded aggregation rate (t 1 / 2 = 0.63 ± 0.04 h) relative to that observed for the M13 control (t 1 / 2 = 1.01 ± 0.05 h)( Figure 3 -f). This indicates that the Aβ33-42 motif itself can seed (aggregate) Aβ42 monomers, which can be explained by the structural features of the Aβ42 amyloid fibril 19 . Indeed, it has been pointed out that a hydrophobic band formed by the extended Val40 and Ala42 residues along the outer surface of the protofilament can enhance secondary nucleation. These are the residues that are present in AB33-42 but not in AB30-39, thus providing a possible explanation for the results obtained.

[0093] In one embodiment, the effect of engineered phage on the fibril content at the end point of the aggregation curve (10 10 pfu / ml) was qualitatively evaluated in immunoblots using anti-amyloid fibril OC antibody and phage at a high concentration (10 10 pfu / ml)( Figure 4)。In the detection, it was observed that in the presence of engineered phages AB30 - 39 and AB33 - 42, rather than phage AB36 - 30, the amount of fibrillar Aβ was reduced( Figure 4 )。

[0094] In one embodiment, both AB30 - 39 and AB33 - 42 phages are effective in affecting the formation of Aβ42 fibrils from monomeric Aβ42, but AB36 - 39 cannot, so it is not effective for the intended purpose.

[0095] In one embodiment, the ability of AB - phages to recognize Aβ aggregates in hippocampal slices of APP / PS1 transgenic mice was determined. It was examined whether engineered phages could detect native Aβ aggregates in the brain tissue of mice with overproduction of Aβ42. Mice expressing human APP and mutant presenilin 1 were used as a model of early - onset AD 20 。These APP / PS1 transgenic mice began to show spine loss and altered network activity in the hippocampus as early as 3 - 4 months old, accompanied by hippocampus - dependent memory impairment 20 。However, amyloid plaques could only be detected in their hippocampus when these mice were ≥6 months old, indicating that Aβ - oligomers can well affect neuronal function before plaque formation. Brains of 3 - 4 - month - old and 9 - 10 - month - old APP / PS1 transgenic mice and wild - type mice were isolated, and immunohistochemical analysis was performed on brain sections (Figure 5). Immunostaining with an anti - Aβ antibody (6E10) revealed plaques in the CA1 region of 9 - 10 - month - old APP / PS1 - mice, but not in 3 - 4 - month - old mice( Figure 5A -d4)。

[0096] Figure 5A Representative fluorescence images and signal quantification of brain tissue samples of control mice and APP mice in two different age groups: 3 - 4 months and 9 - 12 months are shown. The samples were incubated with 10 8 pfu / ml of M13( Figure 5A -a), AB30 - 39( Figure 5A -b) and AB33 - 42 phages( Figure 5A -c). The anti - fd phage was diluted 1:1000, and 6E10 (1:1000 dilution) antibody staining was performed to identify Aβ plaques( Figure 5A -d). Phages are shown in green, Aβ substances in red, and cell nuclei in blue. Samples of WT and APP mice in two age groups( Figure 5B e and f) and the cell body region and the radial layer( Figure 5BSignal quantification analysis was performed on each individual phage in (g, h, and 5C - i, j). The results plotted show the mean ± SE of n = 4 samples. Statistical comparisons were made using the Student's t - test, with ** when p < 0.001 and * when p < 0.05.

[0097] Brain sections were exposed to phages and subsequently stained with anti - M13 mAb. M13 phages without the Aβ peptide motif did not show any staining in WT or APP / PS1 mice ( Figure 5A -a). In contrast, both AB30 - 39 and AB33 - 42 phages showed punctate staining in the CA1 of brain samples from APP / PS1 - mice ( Figure 5A -b2, b4, c2, c4). The size of these puncta was significantly smaller than amyloid plaques, and the density of these puncta increased significantly with age ( Figure 5B -f). In 3 - 4 - month - old mice, these puncta were mainly present in the cell body region, with a lower level in the radial layer, while in 9 - 12 - month - old mice, the puncta were evenly distributed in the CA1 cell body and dendritic regions ( Figure 5B -g, h). Compared with the AB30 - 39 phage, the AB33 - 42 phage showed consistently higher numbers of puncta in the CA1 region of APP / PS1 - mice ( Figure 5B -e, f). This observation is consistent with the previous analysis of the binding affinities of Aβ30 - 39 and Aβ33 - 42 peptides for Aβ fibrils and oligomers: Aβ30 - 39 was only able to recognize fibrils and oligomers above 36 ng, while the Aβ33 - 42 peptide sequence recognized fibrils above 2.4 ng and oligomers above 5.8 ng 11 . Compared with APP / PS1 - mice, staining with AB30 - 39 and AB33 - 42 was essentially absent in brain tissue from WT mice ( Figure 5A -b1, A - b3, A - c1, A - c3 or 4B - e, f). Low levels of AB30 - 39 and AB33 - 42 phage staining were detected in the older WT animals ( Figure 5A -b3, c3), which corresponded to the presence of oligomeric Aβ in older WT mice 26 . These data suggest that phages displaying Aβ peptides can serve as an immunohistochemical tool to detect small Aβ aggregates in mouse brain sections.

[0098] In APP / PS1 mice, AB30-39 and AB33-42 phages stained only small (<1 μm) Aβ aggregate material and could not detect amyloid plaques. Direct analysis of the staining of AB30-39 phage or AB33-42 phage compared to 6E10 was not possible because antigen retrieval involves heat denaturation of secondary and tertiary protein structures and is necessary for the detection of plaques by the primary antibody 6E10, and antigen retrieval prevented the binding of AB30-39 phage and AB33-42 phage to the brain tissue of APP / PS1 mice( Figure 7 ). When brain samples were fixed with paraformaldehyde for an extended period, AB33-42 staining was also shown to be unsuccessful( Figure 8 ). These observations suggest that Aβ oligomers and (protofibrillar) fibrils in tissues need to be in their native conformation to be recognized by AB30-39 or AB33-42 phages.

[0099] In one embodiment, the ability of AB-phages to recognize Aβ aggregates in the hippocampus of AD patients was determined. The ability of AB30-39 phage and AB33-42 phage to detect Aβ aggregates in human brain samples was evaluated. Cryopreserved brain samples of the hippocampus from three AD patients and three age-matched healthy controls (Table 1) were stained with AB30-39, AB33-42, and control M13 phages( Figure 6 ).

[0100] Figure 6 Representative fluorescence images and signal quantification of control and AD human brain tissue samples are shown. Samples were incubated with 10 8 pfu / ml of M13( Figure 6 -a), AB30-39( Figure 6 -b), and AB33-42 phages( Figure 6 -c). Anti-fd phage was diluted 1:1000, and 6E10 (1:1000 dilution) antibody staining was performed for the identification of Aβ plaques( Figure 6 -d). Phages are shown in green, Aβ material in red, and nuclei in blue. Signal quantification( Figure 6 -e) and green spot quantification( Figure 6 -f) were performed for each individual phage in control and AD human samples. The results plotted show the mean ± SE of n = 3 samples. Statistical comparisons were made using the Student's t-test, with ** for p < 0.001 and * for p < 0.05.

[0101] Both phage AB30-39 and phage AB33-42, but not the control phages, showed substantial staining in AD samples, which was significantly higher than their staining in hippocampal samples from control individuals (p < 0.05 and p < 0.001, respectively). Similarly, in mouse samples, phage AB33-42 provided enhanced staining in AD samples compared to phage AB30-39. Phage AB30-39 and phage AB33-42 also detected slightly larger aggregates (∼10 μm), in addition to the small aggregates also observed in APP / PS1 - mice, but no plaque-sized aggregates were detected, as identified by 6E10 staining in these human samples ( Figure 6 -d2). Although the presence of Aβ plaques is not uncommon in cognitively healthy individuals of advanced age 21 , 6E10 staining showed only minimal plaques in brain samples from three age-matched control individuals, which corresponded to the lower levels of AB30-39 and AB33-42 staining. These data suggest that phage AB30-39 and phage AB33-42 can be used to detect small Aβ aggregates in postmortem human brain tissue.

[0102] In one embodiment, the ability of Aβ-derived peptides displayed on M13 phage to interact with Aβ aggregates was determined. A 10-amino acid length peptide corresponding to the carboxyl-terminal region of Aβ was used, which was previously shown to have low (μM) affinity for Aβ monomers, while having high (nM) affinity for Aβ oligomers and protofibrils 11 . Since each phage presents 5 to 8 peptides on its surface, phage AB30-39 and phage AB33-42 bind to Aβ aggregates with higher avidity compared to individual Aβ peptides, and may also have higher specificity for oligomers relative to monomers. The ability of AB-specific phages to prevent and / or inhibit Aβ aggregation was tested, and as previously reported, although M13 phage itself already has the ability to slightly destabilize Aβ protofibrils 22 , phage AB30-39 showed better ability in this regard, mainly by inhibiting the secondary nucleation of Aβ42 monomers on the surface of amyloid protofibrils.

[0103] In one embodiment, the ability of phages AB30-39 and AB33-42 to recognize native Aβ aggregates was also determined. In the brain tissues of APP / PS1-transgenic mice, both phages detected Aβ aggregates that were significantly smaller than amyloid plaques. These phages were able to detect Aβ oligomers and protofibrils by immunofluorescence in brain sections, and since they are actually inexpensive and easy to produce, these phages can provide a good alternative compared to commercially available antibodies. In mouse and human AD brain samples, phage AB33-42 was able to detect a larger amount of Aβ aggregates compared to AB30-39, which may be due to the higher affinity of the Aβ33-42 peptide for Aβ oligomers. It was observed that these phages could detect Aβ oligomers only when the secondary protein structure in brain tissues was basically intact, because prolonged exposure to paraformaldehyde or antigen retrieval procedures would significantly obscure the staining of Aβ aggregates by this phage.

[0104] In one embodiment, phage AB33-42 was used as an immunohistochemical tool to study Aβ aggregates in postmortem brain tissues. It was observed that Aβ aggregates were present in the CA1 region of the hippocampus of 3-month-old APP / PS1 mice, which may be the cause of the synaptic memory deficit that these mice can experience at this age. Further unexpectedly, it was observed that at this early age, Aβ oligomers were mainly present in the pyramidal layer compared to the radial layer.

[0105] Examples

[0106] As an example of a diagnostic tool used in immunoassays, engineered phages at a concentration of 10 4 to 10 10 pfu / ml were stored in a buffer solution of TBS1× or PBS1× at 4°C. The phages were incubated with tissue samples at a concentration of 10 4 -10 8 pfu / ml (diluting the phages in TBST 1× if necessary). The amount of phages added to the tissues varied between 50 - 100 μL. The reaction was carried out at 4°C in a humid chamber overnight.

[0107] As an example of a diagnostic method using engineered phages, tissue slides were washed 5 times in TBST for 10 minutes each, and then incubated overnight at 4°C in a humid chamber with a 1:1000 dilution of rabbit anti-fd phage antibody. Then, it was washed and incubated with a 1:200 dilution of FITC-labeled goat anti-rabbit IgG antibody for 2 hours at room temperature. The tissue slides were covered with Vectashield mounting medium containing DAPI, and images were acquired using a fluorescence microscope.

[0108] Preventive Example

[0109] As an example of using as a preventive tool, engineered phage AB30-39 is administered in a salt solution (PBS 1×) at a concentration between 10 4 and 10 10 pfu / ml. The administration does not exceed 20 - 100 ml / day. Over a given period of time, multiple doses may be necessary. Cognitive and behavioral tests are conducted to evaluate the ability of the phage to prevent spinal loss and memory deficits.

[0110] Therapeutic Example

[0111] As an example of using as a therapeutic tool, engineered phage AB30-39 is administered in a salt solution (PBS 1×) at a concentration between 10 4 and 10 10 pfu / ml. The administration does not exceed 20 - 100 ml / day. Over a given period of time, multiple doses may be necessary. Cognitive and behavioral tests are conducted to evaluate the ability of the phage to prevent spinal loss and memory deficits.

[0112] In one embodiment, it was observed that phage AB30-39 has the ability to prevent Aβ42 aggregation in vitro.

[0113] Whenever the term "comprising" is used in this document, it is intended to mean the presence of the recited features, integers, steps, components, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0114] The disclosure of the present invention should not in any way be regarded as limited to the described embodiments, and those skilled in the art will foresee various possibilities of modification thereof. The above embodiments are combinable.

[0115] The appended dependent claims further state specific embodiments of the present disclosure.

[0116] Sequence Listing:

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] References

[0127] 1. Kamenetz F, et al. APP processing and synaptic function. Neuron 37, 925 - 937 (2003).

[0128] 2. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353 - 356 (2002).

[0129] 3. Huang YR, Liu RT. The Toxicity and Polymorphism of beta - Amyloid Oligomers. International Journal of Molecular Sciences 21, (2020).

[0130] 4. Mucke L, et al. High - level neuronal expression of abeta 1 - 42 in wild - type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. The Journal of neuroscience: the official journal of the Society for Neuroscience 20, 4050 - 4058 (2000).

[0131] 5. Jokar S, et al. Amyloid beta-Targeted Inhibitory Peptides for Alzheimer's Disease: Current State and Future Perspectives. In: Alzheimer's Disease: Drug Discovery (ed Huang X) (2020).

[0132] 6. Farr R, Choi DS, Lee SW. Phage-based nanomaterials for biomedical applications. Acta Biomaterialia 10, 1741 - 1750 (2014).

[0133] 7. Schmidt C. Phage therapy′s latest makeover. Nature Biotechnology 37, 581 - 586 (2019).

[0134] 8. Popescu M, Van Belleghem JD, Khosravi A, Bollyky PL. Bacteriophages and the Immune System. Annual Review of Virology 8, 415 - 435 (2021).

[0135] 9. Barr JJ. A bacteriophages journey through the human body. Immunological Reviews 279, 106 - 122 (2017).

[0136] 10. Ghose C, et al. The Virome of Cerebrospinal Fluid: Viruses Where We Once Thought There Were None. Frontiers in Microbiology 10, 2061 (2019).

[0137] 11. Perchiacca JM, Ladiwala AR, Bhattacharya M, Tessier PM. Structure-based design of conformation- and sequence-specific antibodies against amyloid beta. Proceedings of the National Academy of Sciences of the United States of America 109, 84 - 89 (2012).

[0138] 12. Walsh DM, et al. A facile method for expression and purification of the Alzheimer′s disease-associated amyloid beta-peptide. The FEBS Journal 276, 1266 - 1281 (2009).

[0139] 13. Qi H, Lu H, Qiu HJ, Petrenko V, Liu A. Phagemid vectors for phage display: properties, characteristics and construction. Journal of Molecular Biology 417, 129 - 143 (2012).

[0140] 14. Needleman SB and Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 48, 443 - 453 (1970).

[0141] 15. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology 215, 403 - 410 (1990).

[0142] 16. Campanella JJ, Bitinka L, Smalley J. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences. BMC Bioinformatics 4:29 (2003).

[0143] 17. Cohen SI, et al. Proliferation of amyloid-beta42 aggregates occurs through a secondary nucleation mechanism. Proceedings of the National Academy of Sciences of the United States of America 110, 9758 - 9763 (2013).

[0144] 18. Tornquist M, et al. Secondary nucleation in amyloid formation. Chemical Communications 54, 8667 - 8684 (2018).

[0145] 19. Gremer L, et ol. Fibril structure of amyloid-beta(1 - 42) by cryo-electron microscopy. Science 358, 116 - 119 (2017).

[0146] 20. Trinchese F, Liu S, Battaglia F, Walter S, Mathews PM, Arancio O. Progressive age-related development of Alzheimer-like pathology in APP / PS1 mice. Annals of Neurology 55, 801-814 (2004). 21. Nelson PT, et al. Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. Journal of neuropathology and experimental neurology 71, 362-381 (2012).

[0147] 22. Levenson JM, et al. NPT088 reduces both amyloid-beta and tau pathologies in transgenic mice. Alzheimer’s & Dementia (N Y) 2, 141-155 (2016).

Claims

1. An engineered M13 phage, wherein the engineered M13 phage comprises on its surface an amyloidogenic peptide motif that serves as a drug or a dye, wherein, the peptide motif comprises a sequence having at least 90% identity with the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof.

2. The phage according to the preceding claim, wherein, the peptide motif comprises a sequence having at least 95% identity with the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof.

3. The phage according to any one of the preceding claims, wherein, the peptide motif comprises a sequence having 100% identity with the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof.

4. The phage according to any one of the preceding claims, wherein, the peptide motif comprises a sequence having at least 90% identity with SEQ ID No.1 and a DNA sequence having at least 90% identity with SEQ ID No.

5.

5. The phage according to any one of claims 1-3 of the preceding claims, wherein, the peptide motif comprises a sequence having at least 90% identity with SEQ ID No.2 and a DNA sequence having at least 90% identity with SEQ ID No.

6.

6. The phage according to any one of the preceding claims, wherein, the phage comprises five to eight of the amyloidogenic peptide motifs on its surface.

7. The phage according to any one of the preceding claims, for detecting, diagnosing, preventing, and / or treating the aggregation of β-amyloid oligomers and / or β-amyloid fibrils.

8. The phage according to any one of the preceding claims, for detecting, diagnosing, preventing, and / or treating neurodegenerative diseases.

9. The phage according to any one of the preceding claims, for detecting, quantifying, or diagnosing the presence of β-amyloid oligomers and / or β-amyloid fibrils in a tissue sample.

10. The phage according to the preceding claim, wherein, the tissue sample is a brain tissue sample.

11. The phage according to any one of the preceding claims, for detecting, diagnosing, preventing, monitoring, and / or treating Alzheimer's disease or a neurodegenerative disease that is positively affected by a reduction in the aggregation of β-amyloid oligomers and β-amyloid fibrils.

12. An engineered M13 phage, wherein the engineered M13 phage comprises five to eight amyloidogenic peptide motifs on its surface, wherein, the peptide motif comprises a sequence having at least 90% identity with the following sequences: SEQ ID No1, SEQ ID No 2, and mixtures thereof.

13. The phage according to the preceding claim, wherein, the peptide motif comprises a sequence having at least 95% identity with the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof.

14. The phage according to any one of the preceding claims 12 - 13, wherein, the peptide motif comprises a sequence having 100% identity with at least the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof.

15. A pharmaceutical composition comprising the phage according to any one of the preceding claims.

16. The composition according to the preceding claim, further comprising a suitable pharmaceutical excipient.

17. The composition according to any one of the preceding claims 15 - 16, for the prevention and / or treatment of Alzheimer's disease or a neurodegenerative disease, which is positively affected by a reduction in the aggregation of β - amyloid oligomers and β - amyloid fibrils, wherein, the composition is in the form of a solution for intravenous administration.

18. The composition according to the preceding claim 17, wherein, Intravenous dose less than 10 10 pfu / day.

19. The composition according to any one of the preceding claims 15 - 18, wherein, The intravenous dose is 10 4 -10 10 pfu / day.

20. Use of the phage according to any one of claims 1 - 14 or the composition according to any one of the preceding claims 15 - 19 for the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease or a neurodegenerative disease, which is positively affected by a reduction in the aggregation of β - amyloid oligomers and β - amyloid fibrils.

21. A method for treating or preventing Alzheimer's disease or a neurodegenerative disease in a subject, which is positively affected by a reduction in the aggregation of β - amyloid oligomers and β - amyloid fibrils, the method comprising administering to the subject the phage according to any one of claims 1 - 14 or the composition according to any one of the preceding claims 15 - 19.

22. A kit for detecting, quantifying, and / or diagnosing β - amyloid oligomers and / or β - amyloid fibrils in a tissue sample, the kit comprising the phage according to any one of the preceding claims 1 - 14.

23. A method for detecting or diagnosing the presence of β - amyloid oligomers and / or β - amyloid fibrils in a tissue sample, the method comprising the steps of: incubating the tissue sample with a solution comprising the phage according to any one of the preceding claims 1 - 14; detecting the presence of the phage in the tissue sample.

24. The method according to the preceding claim, further comprising the step of incubating the tissue sample with a first antibody suitable for binding to the phage.

25. The method according to the preceding claim, wherein, the first antibody is a rabbit anti - fd phage antibody.

26. The method according to any one of the preceding claims 23 - 25, further comprising the step of incubating the tissue sample with a second antibody suitable for binding to the first antibody, wherein, the second antibody is a fluorescent antibody.

27. The method according to the preceding claim, wherein, the second antibody is a fluorescein isothiocyanate - labeled goat anti - rabbit IgG antibody.

28. The method according to any one of the preceding claims 23-27, wherein, the tissue sample is a brain tissue sample.

29. The method according to any one of the preceding claims 23-28, wherein, The concentration of phage in the solution is in the range of 10 4 pfu / ml - 10 10 pfu / ml.

30. Use of M13 bacteriophage as a dye, wherein, the M13 bacteriophage comprises an amyloidogenic peptide motif on its surface, wherein the peptide motif comprises a sequence having at least 90% identity with the following sequences: SEQ ID No 1, SEQ ID No 2, and mixtures thereof.

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