M13 bacteriophage displaying amyloid-beta-targeting peptide motifs, methods and uses thereof

Abstract

The present disclosure relates to engineered M13 bacteriophages that display an amyloidogenic peptide motif derived from amyloid beta 42 (Aβ42) on their surface. The disclosure further relates to the use of the disclosed engineered M13 bacteriophages to detect early species of Aβ, i.e., oligomeric and fibrillar Aβ, and prevent their aggregation, thereby helping to slow the progression of Alzheimer's disease and thus contributing to the treatment of this neurodegenerative disease.

Description

[Technical Field]

[0001] The present disclosure relates to engineered M13 bacteriophages that display on their surface an amyloidogenic peptide motif derived from the amyloid beta 42 (Aβ42) peptide. The disclosure further relates to the use of the disclosed engineered M13 bacteriophages to detect early species of amyloid beta (Aβ), i.e., soluble oligomeric and fibrillar Aβ, and prevent and / or inhibit their aggregation into fibrils and plaques, helping to inhibit the progression of Alzheimer's disease (AD), thus contributing to the treatment of this neurodegenerative disease. [Background technology]

[0002] Alzheimer's disease (AD) is a chronic, progressive, degenerative brain disorder and the most common type of dementia. The primary cause of AD is small amyloid-β (Aβ) peptides released by neurons after β- and γ-secretase cleavage of the amyloid precursor protein (APP), a transmembrane protein abundant in the central nervous system (CNS). 1 APP cleavage results in a heterogeneous population of peptides of different lengths, with 40- and 42-amino acid-long isoforms being the two major toxic species. Compared to Aβ40, Aβ42 is more prone to aggregation, and higher abundance of Aβ42 has been shown to correlate with higher neurotoxicity. In pathological conditions, Aβ peptides gradually aggregate into soluble oligomers and protofibrils, which are deposited as insoluble amyloid plaques, one of the hallmarks of AD. 2 It is becoming increasingly clear that Aβ is not immobilized in plaques, but instead exists in soluble, oligomeric forms that are the toxic species of Aβ. 3 Even before amyloid plaques are detected in the brain, Aβ oligomers already induce synaptic loss. 4, which can be strongly correlated with cognitive decline in the early stages of AD. Therefore, it makes more sense to correlate the level of synapse loss and cognitive decline with the presence of Aβ oligomers instead of amyloid plaques in the brain. Multiple lines of evidence suggest that the majority of toxic species are generated from the secondary nucleation of Aβ monomers on the surface of amyloid fibrils, generating Aβ42 oligomers and protofibrils. Therefore, inhibiting this process is a primary target in approaches aimed at limiting Aβ aggregation and toxicity. However, currently available immunohistochemistry tools can only detect the presence of amyloid plaques, while those capable of selectively detecting Aβ oligomers and fibrils in brain samples are lacking.

[0003] The peptides have great potential to target oligomeric and fibrillar Aβ with high affinity and selectivity 5 However, for immunohistochemical use, peptides need to be immunochemically labeled while retaining the structural features that confer target recognition and specificity.

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

[0005] Phages can be genetically and / or chemically modified to display a variety of biomolecules on their surface, making them attractive biotechnological tools for biomedical research. 6 Because they infect bacteria but not eukaryotic cells, phages are safe for humans and easy and cheap to produce on a large scale.

[0006] Researchers agree that treatments that can slow or even halt the progression of AD and preserve brain function are most effective if administered early in the disease continuum, and phages have been widely used in clinical practice since the 1920s to treat bacterial infections. 7 In particular, filamentous phages such as M13 are generally well tolerated by the immune system. 8 , with the ability to cross the BBB 9 In fact, naturally occurring phages are present in relatively high concentrations (up to 10 4 Plaque-forming units (pfu) / ml 10 The ability to be recognized by the immune system and cross the BBB likely depends on the type of phage and the peptide sequence displayed on its surface.

[0007] These facts are disclosed to explain the technical problem addressed by the present disclosure. Summary of the Invention

[0008] The present disclosure relates to an engineered M13 bacteriophage that carries a surface amyloidogenic peptide motif derived from Aβ42.

[0009] Protein aggregation and toxic deposition of amyloid beta (Aβ) are characteristic features of Alzheimer's disease (AD) whose alleviation is important for delaying disease onset.

[0010] One aspect of the present disclosure relates to the genetic engineering of M13 bacteriophage to generate synthetic phages displaying surface peptide motifs known to recognize Aβ42 conformers through homotypic interactions. Using Aβ42 aggregation kinetics, the phages were tested for their effect on Aβ42 aggregation and fibril formation in vitro. Based on immunofluorescence analysis, two of the engineered phages (AB30-39 and AB33-42) were observed to colocalize with Aβ inclusions found in brain tissue from AD patients and mouse models.

[0011] Another aspect of the present disclosure relates to the use of engineered phages as immunohistochemistry tools for detecting Aβ oligomers and fibrils in postmortem brain tissue.

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

[0013] One aspect of the present disclosure relates to an M13 bacteriophage that contains amyloidogenic peptide motifs, AB30-39 (SEQ ID NO: 1-AIIGLMVGGV) and AB33-42 (SEQ ID NO: 2-GLMVGGVVIA), on its surface for use as a pharmaceutical or dye.

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

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

[0016] In one embodiment, the engineered (modified) bacteriophage is used to detect, diagnose, prevent, or treat neurodegenerative diseases.

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

[0018] In one embodiment, the bacteriophage is for use in the detection, diagnosis, prevention or treatment of neurodegenerative diseases or Alzheimer's disease that are positively affected by reduced aggregation of amyloid-beta oligomers and amyloid-beta fibrils.

[0019] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a bacteriophage 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 used for the diagnosis / prevention of neurodegenerative diseases by intravenous (IV) administration.

[0022] In one embodiment, the composition is administered in a daily dose to a person with a neurodegenerative disease that would be positively affected by a reduction in amyloid-beta aggregation, wherein the dose is 10 10 pfu / day (pfu / day) or less. 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 once a week. In either case, the composition can be administered within a given period of time.

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

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

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

[0026] In one embodiment, the method comprises the following steps: a) incubating brain tissue with the engineered phage of the present disclosure and washing away excess; b) Washed brain tissue is incubated with rabbit anti-fd bacteriophage primary antibody and FITC-labeled goat anti-rabbit IgG secondary antibody, followed by DAPI staining.

[0027] According to one embodiment, the brain tissue is 4 ~10 10 50-100 μl of a solution containing phage (in saline solution of TBS 1x or PBS 1x) at a concentration of pfu / ml is added and incubated overnight at 4°C in a humidified chamber.

[0028] In one embodiment, washed brain tissue is incubated with a 1:1000 diluted rabbit anti-fd bacteriophage primary antibody in a humidified chamber overnight at 4° C. The brain tissue with the rabbit anti-fd bacteriophage antibody is washed and then incubated with a FITC-labeled goat anti-rabbit IgG secondary antibody, then washed and stained with DAPI for fluorescent imaging.

[0029] In one embodiment, the method of the present disclosure further comprises washing the tissue slide with TBST 1× five times for 10 minutes each time.

[0030] In one embodiment, the daily form consists of an intravenous solution (infusion solution) containing a definitive amount of M13 bacteriophage containing the amyloidogenic peptide motif (AB30-39, AB33-42, or both), the entire amount of which is intended to be administered as a single dose or as multiple doses if necessary over a given period of time.

[0031] The present disclosure relates to modified M13 bacteriophages (engineered M13 bacteriophages) comprising an amyloidogenic peptide motif on its surface for use as a pharmaceutical or therapeutic agent, or as a dye, or tag, or label, wherein the peptide motif comprises a sequence at least 90% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical.

[0032] In one embodiment, the bacteriophage comprises a DNA sequence that is at least 90% identical to SEQ ID NO: 5. Preferably, it is 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical.

[0033] In another embodiment, the bacteriophage comprises a DNA sequence that is at least 90% identical to SEQ ID NO: 6. Preferably, it is 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical.

[0034] In one embodiment, the bacteriophage peptide motif comprises a sequence at least 90% identical, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical to SEQ ID NO:1; and a DNA sequence at least 90% identical, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical to SEQ ID NO:5.

[0035] In another embodiment, the bacteriophage peptide motif comprises a sequence at least 90% identical, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical to SEQ ID NO:2; and a DNA sequence at least 90% identical, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or 100% identical to SEQ ID NO:6.

[0036] In one embodiment for better results, the bacteriophage contains 5-8 of the amyloidogenic peptide motifs on its surface. The presence of 5-8 peptides on the surface of each phage has been observed to surprisingly improve the avidity and specificity of the oligomers relative to the monomers rather than the individual Aβ peptides. This improves the diagnosis and prevention of neurodegenerative diseases or Alzheimer's disease, which are positively affected by the reduction of aggregation of amyloid-beta oligomers and amyloid-beta fibrils.

[0037] In one embodiment, the disclosed bacteriophages are used for the detection, diagnosis, prevention and / or treatment of aggregation of amyloid-beta oligomers and / or amyloid-beta fibrils. In a further embodiment, the disclosed bacteriophages are used for the quantification of aggregation of amyloid-beta oligomers and / or amyloid-beta fibrils. In one embodiment, treatment can be achieved by either inhibiting aggregation or disaggregating already formed amyloid-beta aggregates.

[0038] In another embodiment, the disclosed bacteriophage is for use in the detection, diagnosis, prevention, or treatment of neurodegenerative diseases.

[0039] In one embodiment, the disclosed bacteriophages are used to detect, quantify, and / or diagnose the presence of amyloid-beta oligomers and / or amyloid-beta fibrils in a tissue sample.

[0040] In one embodiment, the tissue sample is a brain tissue sample.

[0041] In one embodiment, the bacteriophage is for use in the detection, diagnosis, prevention and / or treatment of neurodegenerative diseases or Alzheimer's disease that are positively affected by reduced aggregation of amyloid-beta oligomers and amyloid-beta fibrils. Surprisingly, the disclosed engineered M13 bacteriophage is capable of detecting and binding to oligomeric and fibrillar Aβ associated with early stages of these diseases, thereby enabling early detection of neurodegenerative diseases or Alzheimer's disease that are positively affected by reduced aggregation of amyloid-beta oligomers and amyloid-beta fibrils.

[0042] An embodiment of the present disclosure relates to an M13 bacteriophage comprising five to eight amyloidogenic peptide motifs on its surface, wherein the peptide motifs comprise a sequence at least 90% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical.

[0043] The present disclosure also relates to pharmaceutical compositions comprising the disclosed bacteriophages.

[0044] In one embodiment, the pharmaceutical composition further comprises a suitable pharmaceutical excipient.

[0045] In one embodiment, the pharmaceutical composition is for use in the prevention and / or treatment of neurodegenerative diseases or Alzheimer's disease that are positively affected by reduced aggregation of amyloid-beta oligomers and amyloid-beta fibrils, wherein the composition is in the form of a solution for intravenous administration.

[0046] In one embodiment, the intravenous dose is 10 10 In another embodiment, the intravenous dose is 10 4 ~10 10 pfu / day.

[0047] Aspects of the present disclosure include kits for detecting, quantifying, and / or diagnosing amyloid-beta oligomers and amyloid-beta fibrils, comprising the disclosed bacteriophages. According to one embodiment, the kits are used for the diagnosis and monitoring of Alzheimer's disease.

[0048] The present disclosure also relates to a method for detecting or diagnosing the presence of amyloid-beta oligomers and / or amyloid-beta fibrils in a tissue sample, comprising incubating the tissue sample with a solution containing the disclosed bacteriophage, and detecting the presence of the bacteriophage in the tissue sample.

[0049] In one embodiment, the method further comprises incubating the tissue sample with a first antibody suitable for binding to the bacteriophage. In a further embodiment, the first antibody (primary antibody) is a rabbit anti-fd phage antibody.

[0050] 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 tagged with a fluorescent compound.

[0051] In one embodiment, the second antibody is a fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit immunoglobulin G (IgG) antibody.

[0052] In one embodiment, the tissue sample is a brain tissue sample.

[0053] In one embodiment, the concentration of bacteriophage in the solution is 10 4 pfu / ml ~10 10 pfu / ml.

[0054] One aspect of the present disclosure relates to the use of M13 bacteriophage as a dye / tag or label, wherein the M13 bacteriophage comprises an amyloidogenic peptide motif on its surface, wherein the peptide motif comprises a sequence at least 90% identical to the sequence of the following list: SEQ ID NO:1, SEQ ID NO:2, and mixtures thereof, preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical.

[0055] The following figures provide preferred embodiments to illustrate the disclosure and should not be considered as limiting the scope of the invention. [Brief explanation of the drawings]

[0056] [Figure 1] Figure 1 illustrates the process of genetic engineering of the M13 phage. [Figure 2] FIG. 2 shows one embodiment of the effect of AB phage on Aβ42 aggregation. [Figure 3] FIG. 3 shows one embodiment of the effect of AB phage on surface-catalyzed secondary nucleation of Aβ42. [Figure 4] FIG. 4 shows one embodiment of the effect of AB phage on fibril content at the end of the aggregation curve. [Figure 5] Figure 5 shows representative fluorescence images (Figure 5A) and signal quantification (Figure 5B and Figure 5C) of brain tissue samples from control and APP mice in two different age groups: 3–4 months and 9–12 months. [Figure 6] Figure 6 shows representative fluorescence images and signal quantification of control and AD human brain tissue samples. [Figure 7] FIG. 7 shows representative fluorescence images of control and APP mouse brain tissue samples with and without antigen retrieval. [Figure 8] Figure 8 shows representative fluorescence images of M13 and AB33-42 in APP mouse brain tissue samples with different fixation times. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present disclosure relates to engineered M13 bacteriophages that display the amyloid-beta 42 (Aβ42) inventive amyloidogenic peptide motif on their surface. The disclosure further relates to the use of the disclosed engineered M13 bacteriophages to detect early species of Aβ, i.e., oligomeric and fibrillar Aβ, and prevent their aggregation, thereby helping to slow the progression of Alzheimer's disease and contributing to the treatment of this neurodegenerative disease.

[0058] All reagents were of the highest quality commercially available. Thioflavin T (ThT) was obtained from Sigma. Contaminating trace metals were removed from all solutions using Chelex resin (Bio-Rad). Recombinant Aβ42 (SEQ ID NO: 8) was prepared using a human Aβ42 expression plasmid (pET-Sac-Abeta(M1-42), SEQ ID NO: 9). Recombinant Aβ42 was expressed in E. coli and purified as described by Walsh et al. 12 Purification was performed according to the method described in the previous section. To obtain the monomeric form, 4 mg of Aβ42 was dissolved in 7 M guanidine hydrochloride, eluted in Superdex S75 (GE Healthcare) with 50 mM HEPES pH 7.4, and used immediately. Aβ42 amyloid fibrils were prepared by incubating 5 μM Aβ42 at 37 °C for 24 h under static conditions. Low-bind tubes (Axygen Scientific, Corning) were used for all Aβ42 manipulations.

[0059] In one embodiment, genetic engineering of the M13 phage was performed.

[0060] In one embodiment, insert preparation was performed. Primers were designed to clone two 10-amino acid amyloidogenic peptide residues (Aβ-based) into the genome of the M13KE phage. The Aβ30-39 (SEQ ID NO: 1-AIIGLMVGGV) and Aβ33-42 (SEQ ID NO: 2-GLMVGGVVIA) peptide motifs derived from the Aβ42 peptide were genetically fused to the N-terminus of gene 3, resulting in peptide expression on coat protein III of the M13 phage (Figure 1).

[0061] The genetic engineering process of the M13 phage is illustrated in Figure 1. 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.

[0062] Following this manipulation, only a portion of the correct Aβ sequence was inserted into the M13 phage genome, resulting in a third engineered phage displaying only four amino acids from the Aβ42 peptide-Aβ36-39 (VGGV), which was designated AB36-39.

[0063] In one embodiment, a phagemid cloning system 13 Based on this, the 10-amino acid Aβ-based peptide motifs Aβ30-39 (AIIGLMVGGV) and Aβ33-42 (GLMVGGVVIA) were cloned into the M13 genome. The basic components of a phagemid include a plasmid replication origin, a selectable marker (usually an antibiotic resistance marker), an intergenic region (IG region, usually containing minus- and plus-strand packing sequences and a replication origin), a gene encoding a phage coat protein, restriction enzyme recognition sites, a promoter, and a DNA region encoding a signal peptide. For phagemid construction, we used the commercially available plasmid pETDuet-1 (Novagen, Darmstadt, Germany; SEQ ID NO: 3). This plasmid contains an ampicillin resistance gene; a T7 promoter (enabling gene transcription, which influences the expression level of fusion genes); and the signal peptide pelB (directing phage proteins through the bacterial membrane and promoting their assembly into phage particles). The entire sequence of gene 3 of the M13 phage (encoding phage coat protein III) was first cloned between the HindIII and NotI restriction sites (multiple cloning site 1) of MCS-1. This pelB sequence was then cloned between the BamHI and EcoRI sites on MCS-1, resulting in an intermediate plasmid with SEQ ID NO:4. The Aβ peptide sequence was cloned immediately before gene 3 of M13 on MCS-1 of the pETDuet-1 plasmid between the SalI and SacI restriction sites, resulting in a plasmid with SEQ ID NO:5 (phagemid AB30-39) or SEQ ID NO:6 (phagemid AB33-42). The recombinant plasmids were transformed into competent E. coli cells, and positive clones were confirmed by polymerase chain reaction (PCR) and sequencing.

[0064] In one embodiment, phage particles were produced. Because phagemids can be converted into phage particles of the same morphology by co-infection with helper phage, we used kanamycin-resistant M13KO7 helper phage (N315S, New England BioLabs® Inc.), a derivative of M13 phage containing a kanamycin resistance gene. The infection protocol, which fuses gene 3 of the plasmid with gene 3 of the helper phage and enables display of the Aβ sequence in the phage coat protein III, was performed according to the protocol from New England BioLabs. Briefly, cells containing the phagemid were grown in LB medium containing a final concentration of 20 mg / ml ampicillin and co-infected with 50 μl of M13KO7 helper phage (10 μg / ml) at 37°C and 250 rpm for 90 minutes. 8 The cells were infected with 1000 pfu / ml of phage. Kanamycin was then added to a final concentration of 70 μg / ml, and the solution was incubated overnight at 37°C and 250 rpm. To separate the bacterial cells from the phages, the cells were centrifuged at 6000 rpm for 10 minutes, and the supernatant was transferred to a new tube. The phages were then incubated for 2 hours at 4°C with PEG / NaCl solution and resuspended in Tris-buffered saline (TBS). The phage genomic ssDNA was isolated using an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, v / v), purified with an equal volume of chloroform, precipitated with 100% ethanol, and resuspended in Tris-EDTA (TE).

[0065] In one embodiment, to check the Aβ sequence, the DNA of the synthetic phage was sequenced using a primer (SEQ ID NO: 7) that reads the region of interest in gene 3. Phage titration was performed using the double agar overlay method. 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 overnight incubation at 37°C, plaque-forming units (pfus) were measured.

[0066] Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the Needleman and Wunsch algorithm to find a global alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. 14 The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between two sequences. 15 Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). Global percentages of similarity and identity can also be determined using one of the methods available in the MatGAT software package, an application that uses protein or DNA sequences to generate similarity / identity matrices. 16 As will be apparent to those skilled in the art, minor manual editing can be performed to optimize alignments between conserved motifs. Sequence identity values ​​shown in the present subject matter as percentages were determined over the entire amino acid sequence using BLAST with default parameters.

[0067] In one embodiment, another phage displaying only a four amino acid peptide, Aβ36-39 (VGGV, SEQ ID NO: 10) was also obtained (FIG. 1).

[0068] In one embodiment, the aggregation kinetics of Aβ42 were determined (Figures 2 and 3). The aggregation kinetics was determined by recording ThT fluorescence intensity as a function of time on a plate reader (Fluostar Optima, BMG Labtech) equipped with an excitation filter of 440 nm and an emission filter of 480 nm, according to the original report. Fluorescence was recorded using bottom optics in a half-area 96-well polyethylene glycol-coated black polystyrene plate (3881, Corning) with a clear bottom. Aβ42 monomers were isolated by gel filtration (Tricorn Superdex75 column, GE Healthcare) in 50 mM HEPES, pH 7.4. 10 μM ThT was added to each condition. Various titers (10 8 , 10 9 , 10 10 AB phage and M13 were added to the start of the reaction at 100 pfu / ml. Assays were performed in triplicate at 37°C without stirring, and fluorescence readings were taken every 400 seconds. Data were analyzed using Amylofit and processed in Origin. Fibril mass, estimated from ThT intensity at the endpoint, was averaged from fluorescence recordings during the post-transition plateau phase.

[0069] In one embodiment, immunoblot analysis was performed (Figure 4). Aβ42 aggregates obtained during the plateau phase of each aggregation rate curve were diluted up to 8-fold, dotted in triplicate onto PVDF membranes, and probed with a 1:1000 dilution of anti-amyloid fibril OC antibody (AB9234, Merck Millipore) according to the manufacturer's instructions.

[0070] In one experiment, immunofluorescence assays were performed (Figures 5-8). Brains from age-matched APPswe / PS1dE9 transgenic and wild-type mice were isolated in ice-cold phosphate-buffered saline (PBS) pH 7.45, deep-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°, and the section size was set at 10 μm. Tissues were trimmed to reach the hippocampal region. Sections were mounted on SuperFrost Plus glass (11950657, Thermo Scientific) and stored at -20°C. The same procedure was applied to 7 μm sections of post-mortem human hippocampi from AD patients and respective control groups (Table 1). Cryopreserved slides were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes. Slides were then incubated with blocking solution in TBS + 0.1% Tween-20 (TBST) containing 2.5% bovine serum albumin (BSA) (Sigma-Aldrich, 10735086001) for 1 hour at room temperature. Slides were then incubated for 1 hour at room temperature with 10% paraformaldehyde in TBST. 8The slides were incubated overnight at 4°C in a humidified chamber with phage at a concentration of 1 pfu / ml. The slides were washed five times for 10 min in TBST and then incubated overnight at 4°C in a humidified chamber with a 1:1000 diluted rabbit anti-fd phage antibody (B7786, Sigma). The slides were then washed and incubated for 2 h at room temperature with a 1:200 diluted FITC-labeled goat anti-rabbit IgG antibody (F9887, Sigma). For 6E10 antibody staining, antigen retrieval was performed by heat-mediated treatment in 10 mM pH 6.0 sodium citrate buffer (S1894-500G, Sigma-Aldrich), where boiling was set for 20 min, followed by cooling to room temperature for 20 min. Slides were incubated with a 1:5000 dilution of mouse anti-β-amyloid 6E10 antibody (80300, BioLegend) followed by 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 with 1% BSA. Slides were covered with Vectashield mounting medium with DAPI (VectorLabs). Images were acquired using a fluorescence microscope (Nikon Eclipse E400) and analyzed using ImageJ. Quantification was performed on 40x amplified images using a color threshold set to identify fluorescein signals (phage), and all puncta within a defined size threshold between 10 and 150 pixels were counted.

[0071] Table 1: Brain tissue samples from human donors. ThalPhase, based on the detection of immunopositive amyloid in cortical and subcortical regions: Phase (3) - Brainstem / midbrain. Neuropathological changes in AD were assessed by the ABC score, A-Aβ plaque thal phase, B-Braak neurofibrillary tangles score, and C-CERAD neuritic plaques score.

[0072] [Table 1]

[0073] In embodiments, parametric data were evaluated by Student's t-test for two-group comparisons, and p<0.05 was considered statistically significant for all analyses.

[0074] In one embodiment, the presence of an Aβ peptide motif on the surface of M13 phage was determined. Previous studies have shown that the c-terminal amyloid-beta (Aβ) amino acids 30-39 ( 30 AIIGLMVGGV 39 ) and 33-42( 33 GLMVGGVVIA 42 ) was shown to be reactive to both Aβ oligomers and fibrils, but only weakly reactive to monomers. 11 These Aβ peptide motifs were cloned into phagemids and exposed on the coat protein III of M13 filamentous phage (Fig. 1). By merging these phagemids with M13 helper phage, they were packaged as ssDNA into the phage capsid. These synthetic phage particles, designated AB36-39, AB30-39, and AB33-42, displayed five to eight copies of the peptide motifs Aβ36-39, Aβ30-39, and Aβ33-42, respectively, on their surface, clustered at the ends of the filamentous phage (Fig. 1).

[0075] In one embodiment, aggregation kinetics were determined to determine whether the engineered phage inhibits aggregation on Aβ42. The aggregation process of Aβ42 leading to fibrils can be monitored using thioflavin T (ThT) fluorescence, an amyloid-sensitive dye that can be correlated with fibril mass. The reaction was started from a highly homogenous preparation of monomeric Aβ42. 17The effect of inhibitors on the reaction rate could be estimated. ThT fluorescence kinetics of Aβ42 aggregation showed a characteristic sigmoidal curve, reflecting the complex kinetics of Aβ42 self-assembly into oligomers and fibrils (Figure 2).

[0076] In an embodiment, Figure 2 shows the effect of AB phage on Aβ42 aggregation. The aggregation of 5 μM Aβ42 was measured by ThT luminescence 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). Phage titer (10 8 , 10 9 and 10 10 pfu / ml) were monitored. The effects of the engineered phages and the empty M13 phage control were compared. The effect on fibril mass concentration was measured at the highest phage titer (10 pfu / ml) of each AB phage and M13. 10 pfu / ml) as determined from mean endpoint ThT luminescence (Figure 2-e).

[0077] In one embodiment, a lag phase is observed before the first protofibrils are formed (Figure 2a-d). The length of this lag time depends on the concentration of Aβ42 monomers available for fibril formation. Once the first protofibrils are formed, they act as catalysts for the generation of further fibrils, reflected by a rapid increase in ThT fluorescence. The plateau value indicates the total amount of end-stage Aβ42 fibrils formed.

[0078] In one embodiment, addition of M13 phage (i.e., phage without a displayed peptide) reduces the aggregation half-life (t 1 / 2) had distinct effects on the lag phase (Figure 2-a), defined as the time point at which ThT intensity reaches halfway between the initial baseline and final plateau values, but they reduced the level of endpoint fibrils as titers increased (Figure 2-e), likely due to nonspecific interactions between M13 coat protein and Aβ42 monomers.

[0079] In one embodiment, the engineered phages tested had a clear effect on Aβ42 aggregation. AB30-39 phage inhibited Aβ42 aggregation and kinetics, resulting in a decrease in endpoint fibril mass / ThT intensity (Figure 2-c). With increasing antibody titers, AB30-39 progressively delayed Aβ42 aggregation, extending the half-life by approximately 1 hour (Table 2). Concurrently, endpoint fibril mass was significantly reduced (Figure 2-e). In contrast, AB36-39 and AB33-42 showed no significant effect on Aβ fibril mass or Aβ42 aggregation rate compared to the M13 bacteriophage control, even at the highest titers tested (Figures 2-b and 2-d).

[0080] In one embodiment, the AB30-39 segment interacts more effectively with Aβ monomers and also with a broader collection of early Aβ polymorphs, as indicated by the stronger inhibition of fibril formation observed.

[0081] In one embodiment, the effect of phage-displayed peptides on the secondary nucleation of monomers on the surface of amyloid fibrils was determined. Aβ aggregation is characterized by the primary nucleation of Aβ monomers, the secondary nucleation of monomers on the fibril surface, and the extension of fibrils by the addition of monomers (Figure 3-a): primary nucleation (k) initiated from monomers. n ), and elongation due to the dependence of monomers on pre-existing aggregates (k + ), which involves several microscopic steps, including secondary nucleation (k2) by nucleation of monomers on the fibril surface. 18 .

[0082] In one embodiment, seeding experiments in which preformed Aβ42 fibrils (2%) are added to monomeric Aβ42 allow the contribution of secondary nucleation to be assessed, as the preformed fibrils provide a reactive surface. These assays allow the identification of inhibitors that block secondary nucleation of Aβ42 monomers on the surface of amyloid fibrils. In one embodiment, preformed fibrils were added to Aβ42 monomers to accelerate fibril formation independently of primary nucleation. Phages were added at high concentrations (10 10 pfu / ml) were added to the reaction. The results obtained showed that under the experimental conditions tested, seed aggregation of Aβ42 (t 1 / 2 = 1.09 ± 0.01 h) was observed for unseeded aggregation (t 1 / 2 = 2.34 ± 0.1 h) (Fig. 3b–f).

[0083] In one embodiment, the effect of empty M13 bacteriophage is that it has little effect on the rate of the seeded reaction, whereas AB30-39 appears to be an efficient inhibitor of secondary nucleation of monomers on the surface of fibrils as the reaction slows (t 1 / 2 = 2.69 ± 0.22 hours). Interestingly, a clear picture was observed in AB33-42, whereas the M13 control (t 1 / 2 = 1.01 ± 0.05 h) compared to that observed for seed aggregation rates (t 1 / 2 A slight increase in the time (time = 0.63 ± 0.04 h) occurred (Fig. 3f). This means that the Aβ33-42 motif itself can seed Aβ42 monomers, which can be explained by the structural features of Aβ42 amyloid fibrils. 19 Indeed, it has been suggested that a hydrophobic strip formed by residues Val40 and Ala42 running along the outer surface of the protofilament can enhance binucleation. These are the exact residues present in AB33-42 but absent in AB30-39, providing a possible explanation for the results obtained.

[0084] In one embodiment, the engineered (recombinant) phage is expressed at the endpoint of the aggregation curve (10 10 The effect of high concentrations (10 pfu / ml) on the fibril amount was investigated. 10 The amount of fibrillar Aβ was qualitatively assessed by immunoblotting using phages (pfu / ml) and anti-amyloid fibril OC antibodies (Fig. 4). A reduction in the amount of fibrillar Aβ was observed in the assay in the presence of both engineered phages AB30-39 and AB33-42, but not in the presence of AB36-30 phage (Fig. 4).

[0085] In one embodiment, both AB30-39 and AB33-42 phages effectively affect the formation of Aβ42 fibrils detached from monomeric Aβ42, while AB36-39 is not effective for the intended purpose.

[0086] In one experiment, we determined the ability of the AB phage to recognize Aβ aggregates in hippocampal slices from APP / PS1 transgenic mice. We investigated whether the engineered phage could detect native Aβ aggregates in brain tissue from Aβ42-overproducing mice. Mice expressing human APP and mutant presenilin 1 were used as a model of early-onset AD. 20 These APP / PS1 transgenic mice begin to show spine loss and altered network activity in the hippocampus, accompanied by hippocampal-dependent memory impairment, as early as 3–4 months of age. However, amyloid plaques could only be detected in the hippocampus of these mice at 6 months of age or older, suggesting that Aβ oligomers affect neuronal function well before plaque formation. Brains from 3–4-month-old and 9–10-month-old APP / PS1 transgenic and wild-type mice were isolated and immunohistochemistry was performed on brain slices (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).

[0087] Figure 5A shows representative fluorescence images of control and APP mouse brain tissue samples in two different age groups: 3-4 months and 9-12 months, as well as signal quantification. 8 The cells were incubated with pfu / ml of M13 (Fig. 5A-a), AB30-39 (Fig. 5A-b), and AB33-42 (Fig. 5A-c) phages. Anti-fd phages were diluted 1:1000, and 6E10 (1:1000 dilution) antibody staining was performed to identify Aβ plaques (Fig. 5A-d). Phages are shown in green, Aβ species in red, and cell nuclei in blue. Signal quantification was performed for each individual phage in WT and APP mouse samples from both age groups (Fig. 5B-e and -f), as well as in the cell body region and stratum radiatum (Fig. 5B-g, h and 5C-i, j). Results are shown as the mean ± SE of n = 4 samples. Statistical comparisons were performed using Student's t-test, with ** for p < 0.001 and * for p < 0.05.

[0088] Brain sections were exposed to phages and subsequently stained with anti-M13 mAb. M13 phage, which lacks the Aβ-peptide motif, showed no staining in either WT or APP / PS1 mice (Fig. 5A-a). In contrast, both AB30-39 and AB33-42 phages showed punctate staining in the CA1 region of brain samples from APP / PS1 mice (Fig. 5A-b2, b4, c2, c4). These puncta were significantly smaller in size than amyloid plaques, and the density of these puncta significantly increased with age (Fig. 5B-f). In 3- to 4-month-old mice, these puncta were primarily present in the cell body region, with lower levels in the stratum radiatum, whereas in 9- to 12-month-old mice, the puncta were evenly distributed throughout the CA1 cell body and dendritic regions (Fig. 5B-g, h). The AB33-42 phage consistently showed higher concentrations in the CA1 region of APP / PS1 mice compared with AB30-39 (Fig. 5B-e, f). This finding is consistent with previous analyses of the binding affinity of the Aβ30-39 and Aβ33-42 peptides to Aβ fibrils and oligomers: Aβ30-39 can only recognize fibrils and oligomers at ≥36 ng, whereas the Aβ33-42 peptide sequence recognizes fibrils at ≥2.4 ng and oligomers at ≥5.8 ng. 11 Compared to APP / PS1 mice, AB30-39 and AB33-42 staining was almost absent in brain tissue from WT mice (Fig. 5A-b1, A-b3, A-c1, A-c3 or Fig. 5B-e, f). In WT animals of the older age group, low levels of staining with AB30-39 and AB33-42 phages were observed (Fig. 5A-b3, c3), which corresponds to the presence of oligomeric Aβ in WT animals of the older age group. 26 These data indicate that phage displaying Aβ peptides can be used as an immunohistochemical tool to detect small Aβ aggregates in mouse brain slices.

[0089] AB30-39 and AB33-42 phages stained only small (<1 μm) Aβ aggregates in APP / PS1 mice and failed to detect amyloid plaques. Direct analysis of AB30-39 or AB33-42 phage versus 6E10 contamination was not possible because the primary antibody 6E10 was required to detect plaques, and antigen retrieval, which involves heat denaturation of secondary and tertiary protein structures, prevented binding of AB30-39 and AB33-42 phages to APP / PS1 mouse brain tissue (Figure 7). AB33-42 contamination was unsuccessful, even when brain samples were fixed for extended periods with paraformaldehyde (Figure 8). These observations suggest that Aβ oligomers and (proto)fibrils in tissue must be in their native (natural) form to be recognized by AB30-39 or AB33-42 phages.

[0090] In one embodiment, we measured the ability of AB phages to recognize Aβ aggregates in the hippocampus of AD patients. We evaluated whether AB30-39 and AB33-42 phages could detect Aβ aggregates in human brain samples. Cryopreserved hippocampal brain samples 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).

[0091] Figure 6 shows representative fluorescence images and signal quantification of control and AD human brain tissue samples. 8 The cells were incubated with pfu / ml of M13 (Fig. 6-a), AB30-39 (Fig. 6-b), and AB33-42 (Fig. 6-c) phages. Anti-fd phages were diluted 1:1000, and 6E10 (1:1000 dilution) antibody staining was performed for Aβ plaque identification (Fig. 6-d). Phages are shown in green, Aβ species in red, and cell nuclei in blue. Signal quantification (Fig. 6-e) and green dot quantification (Fig. 6-f) were performed for individual phages in control and AD human samples. Plotted results represent the mean ± SE of n = 3 samples. Statistical comparisons were performed using Student's t-test, with ** for p < 0.001 and * for p < 0.05.

[0092] Both AB30-39 and AB33-42 phages, but not control phages, showed substantial staining in AD samples that was significantly higher than their staining in individual hippocampal samples from controls (p<0.05 and p<0.001, respectively). Similarly, it was observed in mouse samples that AB33-42 phages conferred enhanced staining in AD samples compared to AB30-39 phages. In addition to the small aggregates also observed in APP / PS1 mice, both AB30-39 and AB33-42 phages also detected slightly larger aggregates (~10 μm), but not plaque-sized ones, as identified by 6E10 staining in these human samples (Fig. 6d2). While the presence of Aβ plaques in cognitively healthy individuals is not uncommon in older adults, 21 6E10 staining revealed only a small amount of plaques in brain samples from three age-matched controls, which corresponds to the lower levels of AB30-39 and AB33-42 staining. These data indicate that AB30-39 and AB33-42 phages can be used to detect small Aβ aggregates in human postmortem brain tissue.

[0093] In one embodiment, the ability of Aβ-derived peptides displayed on M13 phage to interact with Aβ aggregates was measured: low (μM) affinity for Aβ monomers and Aβ oligomers and fibrils. 11 We used 10-aa-long peptides corresponding to the carboxy-terminal region of Aβ, which have previously been shown to have high (nM) affinity for Aβ. Because each phage displays 5–8 peptides on its surface, the AB30-39 and AB33-42 phages bind to Aβ aggregates with high avidity and may exhibit higher specificity for oligomers than for individual Aβ peptides relative to monomers. The AB-specific phages were tested for their ability to prevent and / or inhibit Aβ aggregation. The M13 phage itself already has a slight ability to destabilize Aβ fibrils, as previously reported. 22, AB30‐39 demonstrates its potency primarily via inhibition of secondary nucleation of Aβ42 monomers on the surface of amyloid fibrils.

[0094] In one embodiment, the ability of both the AB30-39 and AB33-42 phages to recognize native Aβ aggregates was also measured. In brain tissue from APP / PS1 transgenic mice, both phages detected Aβ aggregates that were substantially smaller than amyloid plaques. These phages can detect Aβ oligomers and fibrils by immunofluorescence in brain slices, and because they are inexpensive and easy to manufacture, they offer a good alternative to commercially available antibodies. The AB33-42 phage can detect larger amounts of Aβ aggregates than AB30-39 in both mouse and human AD brain samples, which is likely due to the higher affinity of the Aβ33-42 peptide for Aβ oligomers. These phages were observed to detect only Aβ oligomers, provided that secondary protein structures in brain tissue remained largely intact, as prolonged exposure to paraformaldehyde or antigen retrieval procedures substantially obscured this phage staining of Aβ aggregates.

[0095] In one embodiment, AB33-42 phage was used as an immunohistochemical tool to examine Aβ aggregates in postmortem brain tissue. It was observed that Aβ aggregates were present in the CA1 region of the hippocampus in 3-month-old APP / PS1 mice, which is likely responsible for the synaptic memory deficits that these mice may experience at this age. Furthermore, surprisingly, it was observed that at this early age, Aβ oligomers were mainly found in the stratum pyramidalis compared to the stratum radiatum.

[0096] Example As an example, for use as a diagnostic tool in immunoassays, 4 ~10 10The engineered phage at a concentration of pfu / ml is stored at 4°C in a buffer solution of TBS 1x or PBS 1x. 4 ~10 8 Incubate with tissue samples at a concentration of pfu / ml (if necessary, dilute phage in TBST 1x). The amount of phage added to the tissue varies between 50 and 100 μl. The reaction is carried out overnight at 4°C in a humidified chamber.

[0097] As an example, for a diagnostic method using engineered phages, tissue slides were washed five times in TBST for 10 minutes each time and then incubated overnight at 4°C in a humidified chamber with a 1:1000 diluted rabbit anti-fd phage antibody. They were then washed and incubated with a 1:200 diluted FITC-labeled goat anti-rabbit IgG antibody at room temperature for 2 hours. The tissue slides were covered with Vectashield mounting medium with DAPI and images were acquired under a fluorescent microscope.

[0098] Prevention in Action As an example, engineered phage AB30-39 was used as a prophylactic tool. 4 ~10 10 It is administered in saline solution (PBS 1x) at a concentration of 1 pfu / ml. It should not exceed 20-100 mL / day. Multiple doses may be required over a period of time. Cognitive and behavioral tests are performed to assess the ability of the phage to prevent spinal cord loss and memory impairment.

[0099] Treatment example For example, for use as a therapeutic tool, engineered phage AB30-39 was incubated at 10 4 ~10 10 It is administered at a concentration of pfu / ml. It should not exceed 20-100 mL / day. Multiple doses may be required over a period of time. Cognitive and behavioral tests are performed to assess the ability of the phage to prevent spinal cord loss and memory impairment.

[0100] In one embodiment, the AB30-39 phage was observed to have the ability to inhibit Aβ42 aggregation in vitro.

[0101] Whenever used in this document, the term "comprises" is intended to indicate the presence of stated features, integers, steps, components, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0102] The present disclosure is not limited to the described embodiments, and those skilled in the art foresee many possibilities for modification thereof. The above-described embodiments can be combined.

[0103] The following dependent claims further describe particular embodiments of the present disclosure.

[0104] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

Table 2-11

Table 2-12

[0105] References 1. Kamenetz F, et al. APP processing and synaptic function. Neuron 37, 925-937 (2003). 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). 3. Huang YR, Liu RT. The Toxicity and Polymorphism of beta-Amyloid Oligomers. International Journal of Molecular Sciences 21, (2020). 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). 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). 6. Farr R, Choi DS, Lee SW. Phage-based nanomaterials for biomedical applications. Acta Biomaterialia 10, 1741-1750 (2014). 7. Schmidt C. Phage therapy's latest makeover. Nature Biotechnology 37, 581-586 (2019). 8. Popescu M, Van Belleghem JD, Khosravi A, Bollyky PL. Bacteriophages and the Immune System. Annual Review of Virology 8, 415-435 (2021). 9. Barr JJ. A bacteriophages journey through the human body. Immunological Reviews 279, 106-122 (2017). 10. Ghose C, et al. The Virome of Cerebrospinal Fluid: Viruses Where We Once Thought There Were None. Frontiers in Microbiology 10, 2061 (2019). 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). 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). 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). 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). 15. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology 215, 403-410 (1990). 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). 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). 18. Tornquist M, et al. Secondary nucleation in amyloid formation. Chemical Communications 54, 8667-8684 (2018). 19. Gremer L, et al. Fibril structure of amyloid-beta(1-42) by cryo-electron microscopy. Science 358, 116-119 (2017). 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). 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. 1. An engineered M13 bacteriophage containing an amyloidogenic peptide motif on its surface for use as a pharmaceutical or dye, A bacteriophage wherein the peptide motif comprises a sequence at least 90% identical to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, and mixtures thereof.

2. 2. The bacteriophage of claim 1, wherein the peptide motif comprises a sequence at least 95% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof.

3. 3. The bacteriophage of claim 1 or 2, wherein the peptide motif comprises a sequence that is at least 100% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof.

4. 4. The bacteriophage of claim 1, wherein the peptide motif comprises a sequence that is at least 90% identical to SEQ ID NO:1 and a DNA sequence that is at least 90% identical to SEQ ID NO:

5.

5. 4. The bacteriophage of claim 1, wherein the peptide motif comprises a sequence at least 90% identical to SEQ ID NO:2 and a DNA sequence at least 90% identical to SEQ ID NO:

6.

6. The bacteriophage according to any one of claims 1 to 5, which comprises 5 to 8 of said amyloidogenic peptide motifs on its surface.

7. A bacteriophage according to any one of claims 1 to 6 for use in the detection, diagnosis, prevention and / or treatment of aggregation of amyloid-beta oligomers and / or amyloid-beta fibrils.

8. A bacteriophage according to any one of claims 1 to 7 for use in the detection, diagnosis, prevention and / or treatment of a neurodegenerative disease.

9. A bacteriophage according to any one of claims 1 to 8 for use in detecting, quantifying or diagnosing the presence of amyloid-beta oligomers and / or amyloid-beta fibrils in a tissue sample.

10. The bacteriophage of claim 9, wherein the tissue sample is a brain tissue sample.

11. A bacteriophage according to any one of claims 1 to 10 for use in the detection, diagnosis, prevention, monitoring and / or treatment of neurodegenerative diseases or Alzheimer's disease which are positively affected by reduced aggregation of amyloid-beta oligomers and amyloid-beta fibrils.

12. An engineered M13 bacteriophage containing 5 to 8 amyloidogenic peptide motifs on its surface, A bacteriophage wherein the peptide motif comprises a sequence at least 90% identical to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, and mixtures thereof.

13. 13. The bacteriophage of claim 12, wherein the peptide motif comprises a sequence at least 95% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof.

14. 14. The bacteriophage of claim 12 or 13, wherein the peptide motif comprises a sequence at least 100% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof.

15. A pharmaceutical composition comprising the bacteriophage according to any one of claims 1 to 14.

16. 16. The composition of claim 15, further comprising a suitable pharmaceutical excipient.

17. 17. A composition according to claim 15 or 16 for use in the prevention and / or treatment of neurodegenerative diseases or Alzheimer's disease which are positively influenced by a reduction in the aggregation of amyloid-beta oligomers and amyloid-beta fibrils, The composition is in the form of a solution for intravenous administration.

18. The amount of intravenous administration is 10 10 The composition of claim 17, wherein the pfu / day is less than or equal to 1000 pfu / day.

19. The amount of intravenous administration is 10 4 ~10 10 The composition according to any one of claims 17 to 18, wherein the pfu / day

20. Use of a bacteriophage according to any one of claims 1 to 14 or a composition according to any one of claims 15 to 19 for the manufacture of a medicament for the prevention and / or treatment of neurodegenerative diseases or Alzheimer's disease which are positively affected by a reduction in the aggregation of amyloid-beta oligomers and amyloid-beta fibrils.

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

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

23. 1. A method for detecting or diagnosing the presence of amyloid-beta oligomers and / or amyloid-beta fibrils in a tissue sample, comprising: Incubating a tissue sample with a solution containing a bacteriophage according to any one of claims 1 to 14; detecting the presence of bacteriophage in said tissue sample; A method comprising:

24. 24. The method of claim 23, further comprising the step of incubating the tissue sample with a first antibody suitable for binding to the bacteriophage.

25. 25. The method of claim 24, wherein the first antibody is a rabbit anti-fd phage antibody.

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

27. 27. The method of claim 26, wherein the second antibody is a fluorescein isothiocyanate-labeled goat anti-rabbit IgG antibody.

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

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

30. 1. Use of M13 bacteriophage as a dye, M13 bacteriophage contains an amyloidogenic peptide motif on its surface, The use wherein the peptide motif is at least 90% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and mixtures thereof.

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