Nanobodies for targeting soluble amyloid-beta oligomers

Abstract

Soluble amyloid-b oligomers (SAbOs) accumulate early, prior to amyloid plaque formation. Because SAbOs may provide an earlier and more direct indication of disease progression, this makes them potentially better markers for detecting AD in its preclinical or early stages, when interventions may be more effective. The presently disclosed subject matter describes novel anti-SAbO nanobodies generated from an alpaca immunized with SAbO. The nanobody or fragment thereof that binds SAbO and optionally comprises a cargo molecule. Because of their small size as compared with antibodies, the disclosed nanobodies not only cross the blood brain barrier, but may also be able to cross cell membranes.

Description

Stites Docket No: 11672N / 21359WO NANOBODIES FOR TARGETING SOLUBLE AMYLOID-BETA OLIGOMERS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This PCT application claims priority to U.S. Provisional Application No. 63 / 558,061,filed February 26, 2024, the contents of which are incorporated by reference in its entirety. STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant AG061138 awardedby the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that primarilyaffects memory, thinking, and behavior. AD causes abnormal changes in the brain, including the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, that lead to profound neuronal death. Brain imaging plays a crucial role in diagnosing AD, especially when combined with other diagnostic tools like cognitive tests and medical history. While brain imaging cannot confirm AD on its own, it helps identify structural changes and other abnormalities in the brain that are associated with the disease. Magnetic resonance imaging (MRI) can reveal shrinking brain regions such as the hippocampus (which plays a key role in memory) and brain atrophy. While MRI can show signs of AD, it is often used to rule out other conditions that might be causing similar symptoms, like strokes or brain tumors. Positron emission topography (PET) scans use a small amount of radioactive material to create detailed images of the brain and are more specific for detecting abnormalities associated with AD. In particular, amyloid PET imaging uses a tracer that binds to amyloid plaques. While amyloid plaques are a hallmark of AD, they are also present in other neurodegenerative diseases and conditions, such as cerebral amyloid angiopathy and frontotemporal dementia. Therefore, detecting amyloid in the brain is not specific for AD. In addition, amyloid plaques accumulate over time, but they may not be present in large enough quantities during the very early stages ofStites Docket No: 11672N / 21359WO AD. This means that amyloid PET scans might not detect the disease at its onset, when early intervention could be most beneficial. Furthermore, the amount of amyloid in the brain as detected by PET does not necessarily correspond with the severity of symptoms or cognitive decline, further complicating its clinical usefulness.

[0004] Emerging research has demonstrated that soluble amyloid-^ oligomers (SA^Os)accumulate early, prior to amyloid plaque formation. SA^Os induce memory impairment and disrupt cognitive function independent of amyloid-^ plaques, and even in the absence of plaque formation. Symptoms of AD typically develop slowly and worsen over time. Certain treatments and therapies may help slow the progression of the disease and manage symptoms more effectively when started early. Because SA^Os may provide an earlier and more direct indication of disease progression, this makes them potentially better markers for detecting AD in its preclinical or early stages, when interventions may be more effective.

[0005] The presently disclosed subject matter describes novel anti-SA^O antibodies(nanobodies) were generated from an alpaca immunized with SA^O. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SA^Os and amyloid-^ plaques. The nanobodies cross the blood-brain barrier (BBB) and bind to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SA^O and amyloid-^ plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SA^Os are associated with neurons, while amyloid plaques reside in the extracellular matrix, meaning that the nanobodies were able to cross the cell-membrane. The results of this study demonstrate that the SA^O nanobody can serve as a diagnostic agent with potential theranostic applications in AD. BRIEF DESCRIPTION

[0006] The above-described deficiencies in the art are met by a nanobody or fragmentthereof that binds SA^O, or specifically binds SA^O and Aβ42; and optionally comprises a cargo molecule.

[0007] The above-described deficiencies in the art are met by a nanobody or fragmentthereof including an amino acid sequence and optionally, a cargo molecule, wherein the amino acid sequence includes (a) SEQ ID NO:1; (b) a fragment of SEQ ID NO:1; (c) a variant aminoStites Docket No: 11672N / 21359WO acid sequence of SEQ ID NO:1; or (d) a fragment of a variant amino acid sequence fragment of SEQ ID NO:1.

[0008] A polynucleotide encoding the above-referenced nanobody or fragment thereof isdisclosed.

[0009] An expression vector comprising the above-referenced polynucleotide is disclosed.

[0010] A cell transformed with the above-referenced vector is disclosed.

[0011] A pharmaceutical composition is disclosed including the above-referenced nanobodyor fragment thereof together with a pharmaceutically acceptable carrier.

[0012] A method for treating a subject by administering an anti-SA^O nanobody comprisinga means for binding SA^O.

[0013] A method for detecting soluble amyloid β-oligomer in a subject is disclosed, themethod including administering to the subject an effective amount of the above-referenced nanobody or fragment.

[0014] A method for treating a disease or disorder associated with the presence of solubleamyloid β-oligomer in a subject is disclosed, the method including administering to the subject a therapeutically effective amount of the above-referenced nanobody or fragment.

[0015] A method for monitoring the severity of associated with the presence of solubleamyloid β-oligomer in a subject, the method including administering to the subject an effective amount of the above-referenced nanobody or fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows contemporary and classical hypotheses of the amyloid-^ cascademechanisms.

[0017] FIGS. 2A-2D show evidence of the production of nanobody from SA^O-injectedalpaca. FIG.2A shows coomassie stained SDS-polyacrylamide gel, and FIG.2B shows Western analysis to confirm the production of the low molecular weight SA^Os (16 kDa) and some higher molecular species. Lanes 1 and 2 are from two different SA^O preparations; lane 3 is from free Abeta peptide. FIG.2C shows general procedures for generating antibodies / nanobodies after immunization of alpaca with SA^O antigen. FIG.2D shows dot-blot analysis data, which showed that the plasma from the alpaca immunized with SA^O (CaLee)Stites Docket No: 11672N / 21359WO recognizes SA^O antigen, while no response was observed for other non-immunized alpacas (Twelve, Sweety Pea, Grand Design, and Princess Fawn).

[0018] FIG. 3 shows representative data for the characterization of a dye-labeled clone.(A,B,C) Two-dimensional HPLC assay using a Cytiva HiTrap Sepharose column to characterize E3 nanobody and its labeled product. Unlabeled E3 nanobody. (D,E,F) FAM-labeled E3 nanobody. (G) Western blot analysis to show the purity of E3 (lane 2) and confirm FAM-labeled labeled E3 (lane 3). Molecular weight markers are shown in lane 1.

[0019] FIGS. 4A-4B show screening for the specificity of the nanobody using 5XFADmouse brain slides. FIG.4A shows ex-vivo evaluation of SA^O-binding specificity of the nanobody clones using consecutive brain slides obtained from a WT (6-month-old) and a 5XFAD mouse (11-month-old). FIG.4B shows ranking of nanobody clones after pixel quantification of 5XFAD brain slides. The threshold of each image was adjusted until the background disappeared using ImageJ. This process was repeated 3 times, and the data were presented as a mean ^ SD.

[0020] FIGS. 5A-5C show specificity of E3 nanobody in different regions of the 5XFADbrains. FIG.5A shows representative ex-vivo imaging of different regions of the brains of 5XFAD mice (n=3, 10-month-old) after staining with FAM-labeled E3 nanobody. FIG.5B shows ex-vivo staining of AF488-labeled 6E10 antibody on a 5XFAD brain slide. FIG.5C shows dot-blot analysis of E3 nanobody binding. Lanes 1-3: blots to test the binding specificity of E3 nanobody against different negative control protein and amyloid-^ isoforms. Lane 4: immunoblot using the 6E10 antibody to confirm the presence of SA^O in the supernatant of ultracentrifuge 5XFAD brain sample. All ex-vivo staining of brain slides were incubated for 1 h at room temperature.

[0021] FIG. 6 shows the E3 nanobody can cross the BBB and stain amyloid targetsrepresentative data of hippocampus and cortex per cohort). All treatments were performed by intravenous injection via the tail veins.5XFAD mice (n=3) were treated with E3 nanobody (A,B) resulting in no labeling. WT mice (n=3) were treated with free FAM fluorescent dye (C,D) resulting in no labeling. WT mice (n=3) were treated with FAM-labeled E3 nanobody (E,F) resulting in no labeling.5XFAD mice (n=5) were treated with FAM-labeled E3 nanobody 24 h before brain collection (G,H) resulting in significant numbers of bright green fluorescent profilesStites Docket No: 11672N / 21359WO in cortex and hippocampus.5XFAD mice (n=3) were treated with FAM-labeled E3 Nb 4 h before brain collection (I,J) resulting in fewer bright green fluorescent profiles. The fluorescent pixels were quantified and compared between (A,B) (n=3) versus (G,H) (n=5) at ****p<0.0001 (K). And between (A,B) (n=3) versus (I,J) (n=3) at *p<0.005. Confocal imaging of ex- vivo 5XFAD hippocampus slides: DAPI staining of the nucleus (M). FAM-labeled E3 recognized SA^O (white arrows) and larger amyloid-^ plaques (asterisks) (N). Amyloid-^ plaques stained by Alexa 647-labeled 6E10 antibody (O). Merging of M, N and O (P) indicates that E3-positive staining around the nuclei (white arrows). DETAILED DESCRIPTION

[0022] AD is one of only a few diseases that have been known for over a hundred yearswithout the development of diagnostic or disease-modifying therapies. In an aging population, AD will present one of the greatest challenges to medicine in this century. While the mechanisms underlying neuronal degeneration in AD remain elusive, the cytopathologic hallmarks of AD appear to be the formation of amyloid-^ plaques between neurons and the intracellular accumulation of hyperphosphorylated Tau species. These processes are compounded by the reduction of amyloid-^ clearance from the brain, which ultimately leads to profound neuronal toxicity and tissue atrophy. Originally, the amyloid cascade hypothesis considered soluble amyloid-^ oligomers (SA^Os) as intermediates that would subsequently aggregate into protofibrils, insoluble fibrils and amyloid-^ plaques, which were thought to be the main pathogenic cause of AD (FIG.1). This hypothesis indicates that targeting SA^O could be a beneficial early AD intervention.

[0023] Experimental evidence ranging from cell studies to neuropathological and behavioraldata suggests that elevated SA^O levels in the brain have pathological consequences. As more data have emerged, it is now accepted that SA^Os exist for an extended periods without conversion to fibrillar isoforms, and that SA^Os induce cognitive impairment in AD. Since SA^Os are determinants of the severity of neurodegeneration in AD, they could potentially serve as a biomarker for theranostic applications. SA^O is a synaptotoxin found at elevated levels and associated with synapses in the brains of AD patients, but not in healthy elderly people. While several reports have implicated SA^Os as the cause of synaptic failure and neurodegeneration inStites Docket No: 11672N / 21359WO AD, more detailed studies suggest that SA^Os cause neuronal death due to perturbation of normal membrane functions. SA^Os also induce memory impairment and disrupt cognitive function long before amyloid-^ plaque deposition or even without plaque formation in a mouse model of AD (FIG.1). Together, these findings indicate that SA^Os act upstream of amyloid-^ plaques and Tau to promote neuronal dysfunction. Thus, reagents that target SA^Os are needed, for the prevention of other pathological events, such as activation of Tau- and amyloid-^ plaque- dependent pathways that lead to an increase in severity of the disease.

[0024] Two types of approaches have been applied to target pathological amyloid proteins.One is the use of small organic molecules that mimic known amyloid-binding molecules, and the other is the development and use of biologics. Organic molecules that bind to ^-sheets within the amyloid fibrils or plaques inhibit growth by blocking binding sites for additional A^ molecules. The chemical backbone of these organic molecules is restricted to a few structures of the benzothiazoles or stilbenes families. Many PET probes targeting amyloid fibrils / plaques were derived from these templates. For example, [11C]PIB and Flutemetamol are based on the benzothiazole structure, while Florbetapir and Florbetaben are derived from stilbenes. Because SA^Os lack the ordered beta solenoid structure of fibrils, developing small molecules to target SA^Os is more challenging.

[0025] In contrast, the development of biologics could serve as a promising strategy to targetamyloid-^ aggregates. Because SA^Os are made up of a few trimers or tetramers, they lack traditional binding surface for small molecules. Antibodies can recognize conformational epitopes unique to different oligomers. For instance, the mAb158 monoclonal antibody exhibits distinctive selectivity for soluble amyloid-^ protofibrils compared to monomeric amyloid-^. This antibody binds preferentially to soluble protofibrils over mature, insoluble fibrils but has no affinity for amyloid precursor protein (APP). M94 is a polyclonal antibody that was purified from antisera of host animals immunized with synthetic amyloid-^ oligomers. M94 shows high selectivity for pathogenic amyloid-^ oligomers but not for physiological monomers. Furthermore, ex-vivo staining of brain sections using the SA^O-specific antibodies revealed that SA^Os are distributed differently than neuritic plaques. Overall, these studies highlight the prospect of biologics for targeting dementia-associated amyloid oligomers.Stites Docket No: 11672N / 21359WO

[0026] In human AD, pathological amyloid-^ proteins are composed of different assembliesthat included senile plaques including amyloid plaques, protofibrils, fibrils, and SA^Os. The amyloid plaques are formed from amyloid beta peptides. These peptides circulate in the blood and in the cerebrospinal fluid (CSF), typically in complexed form with lipoproteins. The Aβ peptide in circulating form is composed of 39–43 amino acids (mostly 40 (e.g., Aβ40) or 42 (e.g., Aβ42) amino acids) resulting from the cleavage of a common precursor protein, amyloidprecursor protein, often designated APP. It is known that Aβ42 is more prone to aggregate than isAβ40, and when this happens, adverse consequences such as Aβ deposition in amyloid plaques, conversion of Aβ to toxic soluble forms, nerve cell damage, and behavioral impairment such as dementias ensue. There are several amyloid beta (Aβ) antibodies developed and used in both research and clinical settings. These antibodies are primarily designed to target amyloid beta peptides, including Aβ₄₂ and Aβ₄₀, which are implicated in the formation of amyloid plaques in the brains of AD patients. Aβ₄₂ is a 42-amino acid peptide that is known for its tendency toaggregate into toxic oligomers and plaques that disrupt neuronal function and contribute to theneurodegeneration seen in AD. Because of its central role in disease progression, historically, Aβ₄₂ has been a major target for therapeutic strategies aimed at reducing its production, preventing its aggregation, or clearing it from the brain to slow or stop the progression of AD.

[0027] Increasing evidence suggests that SA^Os serve as the toxic species responsible for thepathogenesis and spread of AD. Prior to this work, a number of antibodies or antibody fragments have been used for the specific recognition of SA^Os. These reagents have helped examine the roles of SA^Os in AD brain, leading to the hypothesis that the disruption of particular synapses by SA^Os could be the culprit behind the memory loss in early AD. According to the contemporary model of the amyloid-^ cascade hypothesis (FIG.1), SA^Os are considered stable and independent components, affecting AD pathology without conversion to fibrillar assemblies. Thus, targeting SA^Os is beneficial due to their early role in AD pathogenesis and disease progression. In this work, SA^O-binding nanobodies were prepared. Nanobodies provide numerous advantages as compared with antibodies. Aside from being thermally stable, which makes it amenable for PET labeling chemistry, nanobodies have the added advantage of being able to traverse biological barriers. In fact, it has been reported that SA^Os possess a higher diffusion coefficient compared to large assemblies. Nanobodies (sometimes referred to as VHH)Stites Docket No: 11672N / 21359WO are derived from heavy chain-only IgG isotypes found in animals of the camelid family. Most mammals, which produce typical antibodies with both heavy and light chains, but camelids (i.e.,camels, llamas, and alpacas) produce a distinct type of antibody known as heavy-chain-onlyantibodies (HCAbs). These HCAbs are the basis of nanobodies. Unlike conventional antibodies,which have both heavy and light chains (forming a Y-shaped structure), nanobodies are made up of a single variable domain of the heavy chain, which gives them their small size and unique properties. Nanobodies are approximately 1 / 10ththe size of conventional antibodies (150 kDa). Despite their small size, nanobodies have unique specificities, potent affinities, and also exhibit high thermal stability and solubility. With a mere size of ~15 kDa, 1 / 10ththe size of conventional antibodies (150 kDa), nanobodies are smaller than any antibodies and antibody fragments described to date. For example, Fab (~50 kDa), F(ab’)2 (~110 kDa fragments), single chain (monovalent) Fv (scFv, ~25 kDa) with one antigen-binding site, bivalent scFv dimers (50 kDa), and scFv-fusion proteins (sometimes called minibodies, ~80 kDa), all are larger than nanobodies.

[0028] Nanobodies also exhibit relatively short serum half-lives of approximately 2 hours,making them ideal for imaging applications. Additionally, nanobodies have been shown to cross biological barriers, including the blood-brain barrier (BBB), which makes them attractive for AD applications. Finally, the lack of antibody heavy chain constant regions would limit risk of Amyloid Related Imaging Abnormalities (ARIA). Amyloid Related Imaging Abnormalities (ARIA) refer to a set of changes observed in brain imaging, specifically through MRI scans, that are associated with amyloid-targeting treatments for AD. These abnormalities are linked to the use of monoclonal antibodies or other therapies designed to reduce amyloid plaques in the brain. While amyloid plaques themselves are a hallmark of AD, treatments aimed at removing or reducing these plaques can sometimes lead to unintended side effects, such as ARIA. These abnormalities are important because they can affect the safety of treatment and help doctors monitor potential risks during therapy. ARIA is most often associated with treatments designed to target amyloid plaques, particularly monoclonal antibodies like aducanumab, lecanemab, and donanemab. These treatments aim to clear amyloid plaques from the brain, but in doing so, they can sometimes lead to side effects such as brain edema and hemorrhages.

[0029] Advantageously, the inventors hereof have successfully obtained nanobodies derivedfrom a camelid immunized with human Aβ₄₂. The nanobodies and fragments thereof bind to human SA^O and Aβ₄₂. Using nanobodies for targeting SA^Os can potentially serve as aStites Docket No: 11672N / 21359WO delivery solution for biologics. These nanobodies could be used either as therapeutics or diagnostic pharmaceuticals for AD applications. As used herein, the terms “single domain antibody (VHH)” and “nanobodies” have the same meaning referring to a variable region of a heavy chain of an antibody, and construct a single domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Generally, the antibodies with a natural deficiency of the light chain and the heavy chain constant region 1 (CH1) are first obtained, the variable regions of the heavy chain of the antibody are therefore cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0030] Nanobody production is relatively simple, robust, and economical. Fresh SA^Os wereused for alpaca injections, thus the material had a low propensity to grow larger aggregates. The VHH library was constructed, and a single panning step was performed against the immobilized SA^Os. From the enriched SA^O nanobody-expressing phage, 61 clones were identified by screening for binding to SA^Os via ELISA.

[0031] A robust assay was developed to screen nanobody FAM-labeled nanobody clonesusing consecutive 5XFAD brain slides. The 5XFAD brain refers to a specific transgenic mouse model used in AD research. The “5XFAD” model is genetically engineered to express five familial AD (FAD)-linked mutations in the amyloid precursor protein (APP) and presenilin 1 (PS1) genes, which are associated with the development of early-onset AD in humans. Although signal was obtained for all 61 clones, the degree of staining varied between clones in the brain slices. Among the six best clones, one clone was selected for full characterization (i.e., “E3). Extensive in-vitro and in-vivo characterization demonstrated that the nanobody recognizes SA^Os and amyloid-^ plaques in a preclinical mouse model of AD. Since FAM-labeled E3 can penetrate the BBB and effectively stain amyloid-^ in the brain, the presently disclosed nanobody shows promise for diagnostic applications and potential therapies.

[0032] Disclosed herein is a nanobody or a fragment thereof derived from a camelidimmunized with human Aβ₄₂. The nanobody or a fragment thereof may an amino sequence and optionally, a cargo molecule. Nanobodies are single-domain antibodies that retain the antigen- binding functionality of full antibodies but are much smaller in size. A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of binding anStites Docket No: 11672N / 21359WO antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single- domain antibodies include, for example, camelid VHH antibodies, VNAR antibodies, VH domain antibodies and VL domain antibodies. VNARantibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.

[0033] The amino acid sequence of the nanobodies may include SEQ ID NO:1, a fragment ofSEQ ID NO:1, a SEQ ID NO:1 variant, or a fragment of a SEQ ID NO:1 variant.

[0034] SEQ ID NO:1QVQLQESGGGLVQTGGSLRLSCAASGFTFSSYAMSWYRQAPGKERELVAAISWSGSRIY YSNSVKGRFTMSRDNAKNTVYLQMNSLKPEDSAVYYCLADNQFAPGIPTARAYWGQG TQVTVSS

[0035] The presently-disclosed nanobodies may specifically bind to a soluble amyloid β-oligomer and Aβ42. As used herein, “specifically bind” when referring to an antibody, refers to a binding reaction which determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide and does not bind in a significant amount to other proteins present in the sample or subject. Specific binding can be determined by standard methods. See Harlow & Lane, Antibodies, A Laboratory Manual, 2nded., Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. With reference to an antibody-antigen complex, specific binding of the antigen and antibody may have a KDof less than about 10−7Molar, such as less than about 10−8Molar, 10−9, or even less than about 10−10Molar. KD refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0036] The presently-disclosed nanobodies also encompass fragments. Fragments areportions of nanobodies (e.g., SEQ ID NO:1) that retain the ability to bind to antigens but lack thefull structure of a complete nanobody. Fragments of SEQ ID NO:1 may be truncated at the N-Stites Docket No: 11672N / 21359WO terminus or C- terminus, or may lack internal residues, for example, when compared with a full length SEQ ID NO:1, which has 123 amino acids. Preferably, the fragments include 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 120, 121, or 122 consecutive amino acids of the parentprotein. Truncated nanobodies are engineered versions of nanobodies (single-domain antibodies)in which part of the protein structure is removed or “truncated” to modify its size, stability, or functionality. While nanobodies typically consist of a single VHH domain derived from camelid antibodies, truncation refers to modifications that may remove certain structural regions or alter the nanobody for specific applications.

[0037] The presently-disclosed nanobodies include variant amino acid sequences andfragments thereof. As used herein, a variant amino acid sequence including SEQ ID NO:1 refers to a modified or altered form of a nanobody including SEQ ID NO:1 that has undergone changesin its amino acid sequence. The term “variant amino acid sequences of SEQ ID NO:1” refers toamino acid sequences that are distinguished from a reference amino acid sequence (i.e., SEQ ID NO:1) by the addition, deletion, or substitution of at least one amino acid residue. In some aspects, a variant is distinguished from a reference amino acid sequence by one or more substitutions, which may be conservative or non-conservative. In some aspects, the polypeptide variant comprises conservative substitutions, and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added, deleted, or replaced with different amino acid residues.

[0038] Variant amino acid sequences of SEQ ID NO:1 may include the deletion, insertions,and / or substitutions of one or several (e.g., 1-50, 1-30, 1-20, optimally 1-10) amino acids and / or the addition of one or several (generally less than 20, less than 10, or less than 5) amino acids at the C-terminus and / or N-terminus of the nanobody. For example, in the art, the substitution of amino acids with analogical or similar properties usually does not alter the function of the protein. For another example, addition of one or several amino acids at the C-terminus and / or N- terminus usually does not change the function of the protein.

[0039] In some aspects, the variant amino acid sequence including SEQ ID NO:1 is apeptidomimetic. As used herein, “peptidomimetic” means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance someStites Docket No: 11672N / 21359WO property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half-life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Pat. Nos.5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. As used herein, a peptidomimetic is a peptide, wherein at least one naturally-occurring amino acid is replaced with a unnatural amino acid. Some non-limiting examples of unnatural amino acids include β-alanine, L-α-amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L- ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0040] In some aspects, a variant is distinguished from the reference amino acid sequence byone or more substitutions, which may be conservative or non-conservative. As used herein, “conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, the disclosed nanobodies can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for target protein. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Non-conservative substitutions are those that substantially reduce an activity or function of the nanobody, such as the ability to specifically bind to SaβO and Aβ42. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: (1) Alanine (A), Serine(S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0041] The nanobody or fragment thereof may be isolated. As used herein, “isolatednanobody or fragment thereof ” means a nanobody or fragment thereof that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other proteins. Thus, isolated peptides and proteins include proteins purified by standard purification methods. The term also embraces peptides and proteins prepared by recombinant expression in a host cell. TheStites Docket No: 11672N / 21359WO process of isolating a nanobody typically starts by immunizing a camelid (e.g., llama or alpaca) with an antigen of interest. The animal’s immune system produces VHH antibodies against the antigen. After immunization, B cells from the animal’s blood are isolated and the genes encoding the nanobodies are cloned. This can be done by sequencing the VHH genes from the B cells andthen using them to produce phage libraries or yeast libraries that display the nanobodies on theirsurface. One common method for isolating nanobodies is phage display, where phages areengineered to display the nanobodies on their surface. These phages are then screened against atarget antigen to identify the nanobodies that bind specifically to the antigen. Once a nanobodyof interest is identified, it is then produced in bacterial systems or yeast cultures and purifiedusing techniques like affinity chromatography. The isolated nanobodies or fragment thereof can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure using an analytical technique known in the art (e.g., mass spectrometry).

[0042] The nanobodies and fragments thereof may have 100% sequence identity with SEQID NO:1. Sequence identity of amino acid sequence refers to the degree of similarity between two or more amino acid sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs and variants of a nanobody or fragment thereof that specifically binds a target antigen are typically characterized by possession of at least about 75% sequence identity, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. For example, to obtain a nanobody having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. Any suitable method may be used to align sequences for comparison. Non-limiting examples of programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2 (4): 482-489, 1981; Needleman and Wunsch, J. Mol. Biol.48 (3): 443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85 (8): 2444- 2448, 1988; Higgins and Sharp, Gene, 73 (1): 237-244, 1988; Higgins and Sharp, Bioinformatics, 5 (2): 151-3, 1989; Corpet, Nucleic Acids Res.16 (22): 10881-10890, 1988; Huang et al. Bioinformatics, 8 (2): 155-165, 1992; and Pearson, Methods Mol. Biol.24:307-331, 1994., Altschul et al., J. Mol. Biol.215 (3): 403-410, 1990, presents a detailedStites Docket No: 11672N / 21359WO consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215 (3): 403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

[0043] The nanobody or fragment thereof may include a therapeutic agent, a cargo molecule,or a detection moiety. For example, a nanobody or fragment thereof may be conjugated to a a cargo molecule (“conjugated nanobody or fragment thereof,” “nanobody conjugate”). The cargo molecule is the functional payload that the nanobody or fragment thereof delivers to the target. The cargo can vary widely depending on the therapeutic or diagnostic objective. Common cargo molecules include therapeutic agents (e.g., drug), radioisotopes, an enzyme, and fluorescent dyesor bioluminescent markers for imaging.

[0044] Nanobodies can be conjugated to cargo molecules through chemical means (e.g.,covalent bonds) or by recombinant means. Some common conjugation techniques include covalent bonding via chemical linkers, click chemistry, and the like. In some aspects, the nanobodies may be covalently bound directly to the cargo molecule, without an intervening linker. In some aspects, the nanobody and the cargo molecule are connected to one another via an intervening linker. The choice of linker may affect the stability, release mechanism, and efficacy of the nanobody conjugate. The linker should be stable enough to prevent premature release of the cargo while the conjugate is circulating in the body and include both cleavable andnon-cleavable linkers. Some linkers are designed to be cleavable under specific conditions.Cleavable linkers break down under specific conditions such as disulfide bonds (broken byreducing agents) or acid-sensitive linkers (broken by acidic environments). Non-cleavable linkersdo not break down and instead rely on the conjugate’s internalization to get the cargo inside the target cell to release the cargo only once the conjugate has reached the target cell or tissue.

[0045] In some aspects, conjugated nanobody or fragment thereof may be conjugated to atherapeutic agent, such as a drug, in order to deliver drugs and other therapeutic molecules specifically to cells or tissues expressing the target antigen. This targeted approach can reduce off-target effects and increase therapeutic efficacy.Stites Docket No: 11672N / 21359WO

[0046] In some aspects, the conjugated nanobody or fragment thereof may be a nanobody orfragment thereof attached to a cargo molecule, for example, a detectable label for use in imaging techniques, such as SPECT (Single Photon Emission Computed Tomography) imaging, PET, MRI, and fluorescence imaging. As used herein, “detectable label” refers to a detectable molecule (also known as a detectable marker) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, the detectable label can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4thed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0047] SPECT imaging with a labeled nanobody is a non-invasive molecular imagingtechnique used to visualize specific biological processes in vivo, particularly at the cellular andmolecular levels. When nanobodies are conjugated to radioactive isotopes, they can be tracked inthe body using SPECT to identify disease states, such as cancer, inflammation, orneurodegenerative diseases like AD. SPECT imaging detects gamma radiation emitted by the radioactive isotope attached to the nanobody. A gamma camera captures these emissions and constructs images based on the distribution of the radiolabeled nanobody inside the body. SPECT creates 3D images or slices of the subject, which provide valuable information about the location, concentration, and distribution of the radiolabeled nanobody and thus the targetedbiomarker. A radioactive isotope (also called a radiolabel) is attached to the nanobody, allowingit to be detected using SPECT imaging. Common isotopes used for labeling nanobodies include Technetium-99m (99mTc), which is a widely used isotope for SPECT due to its ideal half-life (6hours) and ability to emit gamma radiation suitable for detection, and Iodine-123 (I-123) orIndium-111 (In-111). The radiolabel is attached to the nanobody, often through a chelator (a molecule that binds the metal ion or radioactive isotope) to ensure a stable and safe connection.Stites Docket No: 11672N / 21359WO

[0048] PET uses positron-emitting radioisotopes to track the distribution of a compound inthe body. To enable PET imaging, nanobodies are conjugated to a radioactive isotope, which emits positrons (subatomic particles with a positive charge). Common isotopes used for labeling nanobodies in PET imaging include Fluorine-18 (18F), the most commonly used PET isotope due to its ideal half-life (about 110 minutes) and ease of labeling. It can be incorporated into organic molecules, making it ideal for PET. Other exemplary isotopes include Copper-64(64Cu), Gallium-68 (68Ga), Carbon-11 (11C), Zirconium-89 (89Zr), and Ytterbium-86 (86Y).

[0049] Imaging with a nanobody labeled with a dye is a technique used for visualizingspecific biological processes in real-time, typically through fluorescence imaging. This approach combines the targeting capability of nanobodies with the high sensitivity of fluorescent dyes, enabling researchers and clinicians to monitor the distribution and activity of specific biomarkers in living organisms or tissue samples. To enable visualization using fluorescence imaging, nanobodies are labeled with fluorescent dyes (also called fluorophores). Fluorescent dyes are compounds that absorb light at a specific wavelength and emit light at a longer wavelength. The emitted light is captured by imaging systems to create fluorescent images. Common fluorescentdyes used in nanobody labeling include both dyes from the visible range (e.g., FAM (FluoresceinAmidite), FITC (Fluorescein isothiocyanate), Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, and cyanine dyes (e.g., Cy3 and Cy5)) and those dyes from the near-infrared range (“NIR”). NIR dyes are a class of fluorescent dyes that absorb and emit light in the NIR of theelectromagnetic spectrum, typically between 650 nm and 900 nm. These dyes are highly usefulin molecular imaging, particularly for in vivo applications, because they offer several advantages over dyes that emit in the visible spectrum, such as increased tissue penetration, lowerautofluorescence, and reduced scattering. Non-limiting exemplary NIR dyes include Alexa Fluordyes (e.g., Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790); Cy5.5, ICG(Indocyanine Green), squarylium dyes. Squarylium dyes contain a squarylium ring structure, aheterocyclic system made from a four-membered ring (with alternating double bonds) that includes oxygen or nitrogen atoms. Other dyes that can be used depending on the desired imaging application.

[0050] The dye is typically attached to the nanobody via a covalent bond or through a linkermolecule to ensure stable attachment without interfering with the nanobody’s binding ability. Once the nanobody is labeled with the dye, it is injected into the body or into tissue samples,Stites Docket No: 11672N / 21359WO where the nanobody circulates and binds specifically to its target biomarker or antigen (e.g., SAβO). Once the labeled nanobody has bound to its target, fluorescence imaging can be performed using various imaging platforms such as confocal microscopy, fluorescence microscopy, and in vivo imaging systems.

[0051] Gadolinium-labeled nanobodies are conjugated with gadolinium (Gd), a paramagneticmetal, to be used in Magnetic Resonance Imaging (MRI). Gadolinium is commonly used as a contrast agent in MRI because it enhances the relaxation rate of water protons in tissues, which results in brighter images and improved visualization of structures or abnormalities. The nanobody is conjugated with gadolinium through a chelator molecule, such as DOTA (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-Triazacyclononane-1,4,7- triacetic acid), DTPA (diethylenetriaminepentaacetic acid), BOPTA (bis-(1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid)-N,N'-bis(2-hydroxyethyl)ethylenediamine) benzyl, EDTA (diethylenediaminetetraacetic acid), and DTTA (diethylenetriaminepentaacetic acid).

[0052] The presently-disclosed subject matter includes a pharmaceutical compositioncomprising the nanobody or fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents. In general, these materials can be formulated in non-toxic, inert, and pharmaceutically acceptable aqueous carrier media wherein the pH is generally about 5-8, preferably about 6-8, although the pH can be varied with the nature of the formulation material and the condition to be treated. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration. The pharmaceutical composition of the present invention contains a safe and effective amount (for example, 0.001-99 wt %, or 0.01-90 wt %, or 0.1-80 wt %) of the above-mentioned nanobodies of the present invention (or their conjugates) and pharmaceutically acceptable carriers or excipients. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and the combinations thereof. The drug formulation should be suitable for the mode of administration. The pharmaceutical composition of the present invention may be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvant. Pharmaceutical compositions such asStites Docket No: 11672N / 21359WO injections and solutions are preferably made under aseptic conditions. In addition, the nanobodies and fragments thereof can also be used with other therapeutic agents.

[0053] The pharmaceutical compositions according to the invention may be administeredorally in the form of a suitable pharmaceutical unit dosage form. The pharmaceutical compositions of the disclosure may be prepared in many forms that include tablets, hard or soft capsules, especially hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels.

[0054] The mode of administration and dosage forms are closely related to the properties ofthe therapeutic agents or compositions which are desirable and efficacious for the given treatment application. Suitable dosage forms include, but are not limited to, oral, intravenous, rectal, sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular, transdermal, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, and lymphatic administration, and other dosage forms for systemic delivery of active ingredients.

[0055] Pharmaceutical compositions of the disclosure may be administered by any methodknown in the art, including, without limitation, transdermal (passive via patch, gel, cream, ointment or iontophoretic); intravenous (bolus, infusion); subcutaneous (infusion, depot); transmucosal (buccal and sublingual, e.g., orodispersible tablets, wafers, film, and effervescent formulations; conjunctival (eye drops); rectal (suppository, enema)); or intradermal (bolus, infusion, depot).

[0056] The solid unit dosage forms can be of the conventional type. The solid form can be acapsule, such as an ordinary gelatin type containing the inhibitor of the present invention, and optionally an anti-bacterial compound of the present invention, and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or corn starch in combination with binders like acacia, corn starch, or gelatin, disintegrating agents such as, corn starch, potato starch, or alginic acid, and a lubricant like stearic acid or magnesium stearate.

[0057] Oral liquid pharmaceutical compositions may be in the form of, for example, aqueousor oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.Stites Docket No: 11672N / 21359WO

[0058] Pharmaceutical compositions of the disclosure may also be formulated for parenteraladministration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the pharmaceutical compositions of the disclosure may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

[0059] Pharmaceutical compositions suitable for rectal administration wherein the carrier is asolid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the pharmaceutical composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0060] For administration by inhalation, the pharmaceutical compositions according to theinvention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the pharmaceutical compositions of the disclosure may take the form of a dry powder composition, for example, a powder mix of the pharmaceutical composition and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

[0061] For intra-nasal administration, the pharmaceutical compositions of the disclosure maybe administered via a liquid spray, such as via a plastic bottle atomizer. Typical of these are the Mistometerg (isoproterenol inhaler-Wintrop) and the Medihaler® (isoproterenol inhaler-Riker).

[0062] It will be further appreciated that the amount of the pharmaceutical compositionsrequired for use in treatment will vary not only with the therapeutic agent selected but also withStites Docket No: 11672N / 21359WO the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0063] The presently-disclosed subject matter includes a polynucleotide that encodes thenanobody or fragment thereof. “A polynucleotide that encodes” means a nucleic acid sequence comprising the coding region of the nanobody or fragment thereof. The coding region may be present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double- stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements. Unless otherwise specified, a “polynucleotide encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may, in some versions, contain an intron(s).

[0064] In some aspects, the polynucleotide encoding the nanobody or fragment thereof is anisolated polynucleotide. The term “isolated polynucleotide” refers to a material that is substantially or essentially free from components that normally accompany it in its native state. The material can be a cell or a macromolecule, such as a protein or nucleic acid. For example, an “isolated polynucleotide,” as used herein, refers to a polynucleotide that has been purified from the sequences that flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and / or purification of a polypeptide from its natural cellular environment and from association with other components of the cell. The presently-disclosed subject matter includes an expression vector comprising the polynucleotide. An “expression vector” includes a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. AnStites Docket No: 11672N / 21359WO expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked. A “recombinant polynucleotide” is a synthetic or modified DNA or RNA molecule that has been artificially created by joining two or more segments of nucleotides that are not found together in nature. This process typically involves genetic engineering techniques where specific genes or sequences of interest are inserted into vectors, plasmids, or other types of DNA constructs to achieve a desired biological function.

[0065] The presently-disclosed subject matter includes a cell transformed with the expressionvector. The transformation process involves introducing foreign DNA (such as a recombinant polynucleotide) into a host cell, allowing the cell to take up, replicate, and express the foreign DNA.

[0066] The presently-disclosed subject matter includes a method for detecting solubleamyloid β-oligomer in a subject, the method comprising administering to the subject an effective amount of the nanobody or fragment. Methods for detecting soluble amyloid β-oligomer in a subject include SPECT, PET, MRI, and fluorescence imaging. An “effective amount” forimaging refers to the quantity of a substance (such as a nanobody, antibody, or other imagingagent) that is needed to achieve optimal imaging results while minimizing any potential sideeffects or toxicity to the subject. The term “effective amount” is context-dependent and varies based on the imaging technique, the agent used, and the biological system being studied. It represents a dose that is sufficient for generating a clear and accurate image without oversaturating or underrepresenting the target. The effective amount of an imaging agent is influenced by its affinity for the target and how well it binds to the target tissue (e.g., a receptor, protein, or disease marker like SAβO). For highly specific and high-affinity agents, lower doses may be effective for imaging, as these agents can bind to their targets with minimalStites Docket No: 11672N / 21359WO concentration. In contrast, for agents with lower specificity or affinity, a higher dose may be needed to ensure sufficient target binding and imaging signal. The effective amount for PET imaging involves radiolabeling a nanobody or antibody with a radioactive isotope. The quantity needed depends on the half-life of the isotope, the target’s expression levels, and the size of thearea being imaged. Typically, doses in the range of 1-10 MBq (Mega Becquerels) are used forimaging in animals, while human doses are carefully calibrated based on radiation safety and imaging goals. Similar to PET, but uses gamma-emitting isotopes like Tc-99m. The effective amount for SPECT imaging is also in the range of 1-10 MBq for animal studies and is adjusted for human imaging. For MRI, gadolinium-based or other contrast agents are typically used. The effective amount is usually in the range of 0.1-0.3 mmol / kg body weight for diagnostic imaging in humans. In animal models, lower doses may be sufficient. For fluorescence-based imaging, a labeled nanobody is used, and the effective amount will depend on the sensitivity of the imaging system. Typical doses range from 0.1 to 1 mg / kg in animals, but it may vary based on the fluorescent dye’s properties and the imaging system’s sensitivity.

[0067] The presently-disclosed subject matter includes a method for treating a disease ordisorder associated with the presence of soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the nanobody or fragment thereof. As used herein, the term “treat”, “treating” or “treatment” of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient. As used herein, the term “a therapeutically effective amount” of a nanobody or a fragment thereof of the present disclosure refers to an amount that will elicit the biological or medical response of a subject, for example, reduction in the amount of target present (e.g., SAβO), or ameliorate symptoms, alleviate conditions, or slow or delay disease progression. As used herein, the term “subject” refers to mammals, primates (e.g., humans, male or female), dogs, rabbits, guinea pigs, pigs, rats and mice. In certain some aspects, the subject is a primate. In yet other some aspects, the subject is a human. A “patient” is a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder,Stites Docket No: 11672N / 21359WO prophylactic or preventative treatment, or diagnostic treatment. In some aspects the patient is a human patient.

[0068] The presently-disclosed subject matter includes a method for monitoring the severityof a disease or disorder associated with the presence of soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject an effective amount of the nanobody or fragment thereof. Diseases or disorders associated with the presence of soluble amyloid β- oligomer include diseases other than AD, including other neurodegenerative diseases. Soluble amyloid-beta oligomers are primarily associated with AD, where they play a critical role in synaptic dysfunction, neuronal damage, and cognitive decline. However, they are also implicated in a range of other neurodegenerative diseases, including cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), Huntingdon’s Disease (HD), Down syndrome, Huntington’s disease, and amyotrophic lateral sclerosis (ALS). These oligomers are believed to be toxic to neurons, contributing to disease progression and cognitive decline.

[0069] The disease or disorder may include cerebral amyloid angiopathy (CAA). In CAA,amyloid-beta accumulates in the walls of blood vessels in the brain, leading to vessel weakening and an increased risk of hemorrhagic stroke. Soluble amyloid-beta oligomers may contribute to the early stages of the disease by disrupting the blood-brain barrier and promoting vessel damage. In addition, CAA can coexist with AD, and the soluble oligomers may have a role in accelerating vascular dysfunction and cerebral hemorrhage in these patients.

[0070] The disease or disorder may include frontotemporal dementia (FTD). In FTD, someforms of frontotemporal dementia, especially those with a familial link, have been associated with amyloid-beta aggregation. In these cases, soluble amyloid-beta oligomers may play a role in neurodegeneration. FTD, which primarily affects the frontal and temporal lobes of the brain, often involves tau pathology, but amyloid-beta oligomers can also be involved, especially in genetic variants of the disease. While tau pathology is the defining feature in many cases of FTD, amyloid-beta oligomers may still contribute to cognitive dysfunction and the onset of neurodegenerative changes.

[0071] The disease or disorder may include Parkinson’s disease (PD) or Lewy BodyDementia (LBD). PD is primarily associated with the accumulation of alpha-synuclein, but there is growing evidence that amyloid-beta oligomers may also play a role in the pathogenesis ofStites Docket No: 11672N / 21359WO neurodegeneration in PD and Lewy Body Dementia (LBD). These oligomers may contribute to synaptic dysfunction and the formation of Lewy bodies in the brain, possibly affecting the cognitive decline seen in these disorders. Amyloid-beta can be present in some regions of the brain affected by PD and LBD, and its oligomers may exacerbate the neurodegenerative process.

[0072] The disease or disorder may include Down syndrome. People with Down syndromehave an extra copy of chromosome 21, which contains the gene for amyloid precursor protein (APP). This leads to an overproduction of amyloid-beta, including soluble oligomers. In individuals with Down syndrome, amyloid-beta oligomers accumulate in the brain, and by the age of 40, many will develop Alzheimer's-like symptoms, including cognitive decline. The presence of amyloid-beta oligomers in Down syndrome is an important factor contributing to the early onset of Alzheimer's-like pathology, even though amyloid plaques do not necessarily form as early as in typical Alzheimer's patients.

[0073] The disease or disorder may include Huntingdon’s Disease (HD). Huntington'sdisease is characterized by the accumulation of mutant huntingtin protein, but there is evidence suggesting that amyloid-beta oligomers may interact with mutant huntingtin and contribute to the neurodegenerative process. While not a core feature of the disease, amyloid-beta oligomers may contribute to the exacerbation of cognitive and motor deficits in some Huntington's disease patients, especially when the disease progresses.

[0074] The disease or disorder may include Amyotrophic Lateral Sclerosis (ALS). There issome evidence suggesting that amyloid-beta oligomers may be involved in ALS, particularly in cases where the disease is accompanied by cognitive dysfunction. Although ALS is primarily associated with motor neuron degeneration, the role of amyloid-beta oligomers in the disease remains an area of ongoing research. They might contribute to the cognitive impairment seen in some ALS patients, especially in those with frontotemporal dementia (ALS-FTD).

[0075] The details of one or more some aspects of the presently-disclosed subject matter areset forth in this document. Modifications to some aspects described in this document, and other some aspects, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary some aspects, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.Stites Docket No: 11672N / 21359WO

[0076] While the terms used herein are believed to be well understood by those of ordinaryskill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0077] Unless defined otherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0078] All patents, patent applications, published applications and publications, GenBanksequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0079] Where reference is made to a URL or other such identifier or address, it understoodthat such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0080] Although any methods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0081] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to“one or more” when used in this application, including the claims, unless the context clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes one or more of such polypeptides, and so forth.

[0082] Unless otherwise indicated, all numbers expressing quantities of ingredients,properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0083] As used herein, the term “about,” when referring to a value or to an amount of mass,weight, time, volume, concentration or percentage is meant to encompass variations of in some aspects ±20%, in some aspects ±10%, in some aspects ±5%, in some aspects ±1%, in some aspects ±0.5%, and in some aspects ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.Stites Docket No: 11672N / 21359WO

[0084] As used herein, ranges can be expressed as from “about” one particular value, and / orto “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0085] The presently-disclosed subject matter is further illustrated by the following specificbut non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the presently disclosed subject matter.

[0086] Aspect 1. A nanobody or fragment thereof that binds SA^O, or specifically bindsSA^O and Aβ42.

[0087] Aspect 1a. The nanobody or fragment thereof of Aspect 1, wherein the nanobody isisolated.

[0088] Aspect 1b. The nanobody or fragment thereof of any one of the preceding aspects,wherein the nanobody or fragment thereof comprises a cargo molecule.

[0089] Aspect 1c. The nanobody or fragment thereof of any one of the preceding aspects,wherein the nanobody or fragment thereof is conjugated to a cargo molecule to form a conjugate of the nanobody or fragment thereof, wherein the cargo molecule comprises a therapeutic agent,a radioisotope, an enzyme, a fluorescent dye, or a bioluminescent marker, or a combinationthereof.

[0090] Aspect 1d. A polynucleotide encoding the nanobody or fragment thereof of any oneof the preceding aspects.

[0091] Aspect 1e. A expression vector comprising the polynucleotide according to aspect 1d.

[0092] Aspect 1f. A cell transformed with a vector according to aspect 1e.

[0093] Aspect 1g. A pharmaceutical composition comprising the nanobody or fragmentthereof of any one of the preceding aspects, together with a pharmaceutically acceptable carrier.

[0094] Aspect 1h. A pharmaceutical composition for detecting soluble amyloid β-oligomer ina subject, wherein the composition comprises an effective amount of a means for binding soluble amyloid β-oligomer and Aβ42 in the subject, preferably a means for specifically binding soluble amyloid β-oligomer and Aβ42 in the subject.Stites Docket No: 11672N / 21359WO

[0095] Aspect 1i. A method for detecting soluble amyloid β-oligomer in a subject, themethod comprises administering to the subject an effective amount a conjugate of the nanobody or fragment thereof of any one of the preceding aspects.

[0096] Aspect 1j. A method for treating a disease or disorder associated with the presence ofsoluble amyloid β-oligomer in a subject, by administering an anti-SA^O nanobody comprising a means for binding SA^O and Aβ42 , preferably a means for specifically binding SA^O and Aβ42.

[0097] Aspect 1k. A method for treating a disease or disorder associated with the presence ofsoluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the nanobody or fragment thereof of any one of the preceding aspects.

[0098] Aspect 1l. A method for monitoring the severity of a disease or disorder associatedwith the presence of soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject an effective amount of the nanobody or fragment thereof of any one of the preceding aspects.

[0099] Aspect 2. A nanobody or fragment thereof comprising an amino acid sequence andoptionally, a cargo molecule, wherein the amino acid sequence comprises (a) SEQ ID NO:1; or (b) a fragment of SEQ ID NO:1; or (c) a variant amino acid sequence of SEQ ID NO:1; or (d) a fragment of a variant amino acid sequence of SEQ ID NO:1.

[00100] Aspect 2a. The nanobody or fragment thereof of aspect 2, wherein the nanobody isisolated.

[00101] Aspect 2b. The nanobody or fragment thereof of aspect 2, wherein the amino acidsequence comprises SEQ ID NO:1.

[00102] Aspect 2c. The nanobody or fragment thereof of aspect 2, wherein the amino acidsequence comprises a fragment of SEQ ID NO:1.

[00103] Aspect 2d. The nanobody or fragment thereof of aspect 2, wherein the amino acidsequence comprises a variant amino acid sequence comprising at least 85% sequence identity with SEQ ID NO:1.

[00104] Aspect 2e. The nanobody or fragment thereof of aspect 2, wherein the amino acidsequence comprises a variant amino acid sequence comprising at least 95% sequence identity with SEQ ID NO:1.Stites Docket No: 11672N / 21359WO

[00105] Aspect 2f. The nanobody or fragment thereof of aspect 2, wherein the amino acidsequence comprises a fragment of a variant amino acid sequence, wherein the fragment of a variant amino acid sequence comprises at least 85% sequence identity with SEQ ID NO:1.

[00106] Aspect 2g. The nanobody or fragment thereof of aspect 9, wherein the amino acidsequence comprises a fragment of a variant amino acid sequence, wherein the fragment of a variant amino acid sequence comprises at least 95% sequence identity with SEQ ID NO:1.

[00107] Aspect 2h. The nanobody or fragment thereof of aspect 1, wherein the nanobody orfragment thereof comprises a cargo molecule.

[00108] Aspect 2i. The nanobody or fragment thereof of aspect 10, wherein the nanobody orfragment thereof is conjugated to a cargo molecule to form a conjugate of the nanobody or fragment thereof, wherein the cargo molecule comprises a therapeutic agent, a radioisotope, anenzyme, a fluorescent dye, or a bioluminescent marker, or a combination thereof.

[0109] Aspect 2j. A polynucleotide encoding the nanobody or fragment thereof of aspect 2.

[0110] Aspect 2k. A expression vector comprising the polynucleotide according to aspect2j.

[00111] Aspect 2l. A cell transformed with a vector according to aspect 13.

[0112] Aspect 2m. A pharmaceutical composition comprising the nanobody or fragmentthereof of aspect 1, together with a pharmaceutically acceptable carrier.

[00113] Aspect 2n. A pharmaceutical composition for detecting soluble amyloid β-oligomerin a subject, wherein the composition comprises an effective amount of a means for binding soluble amyloid β-oligomer in the subject.

[00114] Aspect 2o. A method for detecting soluble amyloid β-oligomer in a subject, themethod comprises administering to the subject an effective amount a conjugate of the nanobody or fragment thereof of aspect 1.

[00115] Aspect 2p. A method for treating a disease or disorder associated with the presenceof soluble amyloid β-oligomer in a subject, by administering an anti-SA^O nanobody comprising a means for binding SA^O.

[00116] Aspect 2q. A method for treating a disease or disorder associated with the presenceof soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the nanobody or fragment thereof of aspect 1.Stites Docket No: 11672N / 21359WO

[00117] Aspect 2r. A method for monitoring the severity of a disease or disorder associatedwith the presence of soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject an effective amount of the nanobody or fragment thereof of aspect 1. EXAMPLES

[00118] Generation of SA^O as an antigen. Human SA^Os as an antigen for alpacaimmunization were prepared and isolated as follows from Aβ1–42. Aβ1–42 is a 42-amino acid peptide that is a cleavage product of the amyloid precursor protein (APP). Based on prior work (see Ryan, D. A., et al. J Neurosci Methods 190, 171-179 (2010), the inclusion of 0.05% SDS in the preparation helped to induce stable oligomers. Synthetic Aβ1–42 peptide was purchased from alternate vendors, Anaspec (Fremont, CA) and California Peptide Research (Napa, CA) for comparison to AβOs generated from Aβ1–42 purchased from American Peptide.

[00119] After confirmation of the generation of SA^Os by Coomassie Blue gel staining, andthe material was stored at -20°C before use. The assembled amyloid-^ preparation contained three major components that included monomers, low molecular weight oligomers (~16 kDa), and high molecular weight oligomers (see FIG.2A-2B). The monomer concentration is inversely proportional to assembly time. At the beginning of the seeding process, the monomer dominates the solution and migrates at 4-4.5 kDa. However, over time, this band gets lighter, indicating the formation of higher-order oligomers at the expense of their monomer counterparts. The low molecular weight oligomers are primarily composed of tetramers migrating at approximately 16 kDa. A small amount of higher molecular weight SA^O species in the 37-150 kDa range was also observed.

[00120] Development and characterization of SA^O nanobodies. The overall procedure forgenerating anti-SA^O nanobodies is highlighted in FIG.2C. An alpaca was immunized, CaLee, with SA^O antigen 7 times over the course of 84 days. Blood collections were performed 1 week after the 5thand 7thinjections, and the plasma was tested for the presence of antibodies recognizing SA^O. The plasma obtained from CaLee, but not the control plasma from other alpacas, recognized the SA^O by dot-blot analysis (FIG.2D). Nanobodies were isolatedfollowing an established protocol (Pardon, E. et al. A general protocol for the generation ofnanobodies for structural biology. Nat Protoc 9, 674-693 (2014)). A phage display library usingStites Docket No: 11672N / 21359WO cDNA derived from the PBMC mRNA was generated and affinity panning of the phage library was performed. Positive clones were identified by ELISA and clones identified multiple times were prioritized. Six clones were tested in 8-point dilution series and positive clones were prioritized. From this operation, the 6 clones were identified that bind SA^O (D11, E3, F9, B10, D4 and H5).

[00121] An ex-vivo assay was developed using mouse brain tissue sections mounted ontoglass slides to screen for nanobody binding specificity. To facilitate the screening process, the nanobody clones were all labeled with fluorescein-NHS (FAM) dye. After removal of excess dye using a desalting spin column (Zepa columns, 7K, MWCO, 2 mL), confirmation of the labeling was done via 2D-HPLC (FIGS.3A-3F). As shown in the representative data of the E3 nanobody, the chromatogram profile of FAM-labeled E3 nanobody is nearly identical to that of the unlabeled counterpart, albeit with a longer retention time. Aside from having similar absorbance characteristic of a protein at 220-280 nm, the FAM-labeled product has an additional absorbance of FAM dye at 498 nm (FIG.3E, F). Western analysis also confirmed the FAM-labeled E3 product (FIG.3G). The FAM-labeled nanobody clones were used to stain 5XFAD and WT brain slides. To compare the binding specificity of the clones, FAM-labeled nanobodies were incubated with consecutive brain sections for 1h at room temperature and the slides were imaged using a fluorescent microscope (FIG.4A). The screening data were exported to ImageJ to quantify the fluorescent signal and rank the nanobodies. Based on these data, the nanobodies were ranked from best to worst: E3>H5>G11=F9>D4>B10 (FIG.4B). This ex-vivo staining process was repeated using two different 5XFAD brains, and the data confirmed E3 was the best among the clones.

[00122] FAM-labeled E3 nanobody recognizes SA^Os and amyloid-^ plaques in an ex-vivostaining of brain sections. FAM-labeled E3 nanobody was used to investigate the spatial distribution of SA^O in ex-vivo brain sections. The data in FIG.5A demonstrate that the FAM- labeled E3 nanobody (1.8 ^g / mL) stained several regions of the brains obtained from 5XFAD mice (n=3, 10-month-old). The stained amyloid-^ species were heterogeneous and had distinct size, shape, and morphology features. More dense populations were found within the hypothalamus and thalamus, followed by cortex (Table 1). Aside from the smaller size species (~2 ^m), the large-sized amyloid-^ species (~14 ^m) detected with FAM-labeled E3 wereStites Docket No: 11672N / 21359WO similar to those detected by Alexa Fluor-488-labeled 6E10 antibody (FIG.5B). The 6E10 staining data are consistent with a past report that described amyloid-^ plaques in 5XFAD miceto be approximately 20 ^m (Oakley, H. et al. J Neurosci 26, 10129-10140 (2006)).Table 1.physical characteristics of amyloid species in the brain stained with FAM-labeled E3 nanobody were analyzed. Overall, the density of amyloid-^ species present in the thalamus and hypothalamus was higher than in the hippocampus or cortex (Table 1). It was also notable that the size of these SA^Os was nearly 7-fold smaller than that of amyloid-^ plaques.

[00124] Next, a dot-blot assay was developed to test the specific recognition of E3 nanobodyversus 6E10 antibody (widely used monoclonal antibody that specifically binds to amyloid-beta) for different Abeta species, including free Abeta peptide, extracted SA^O from 5XFAD brains, or the in-vitro produced counterparts (FIG.5C). For this study, the Abeta peptide was prepared fresh using human amyloid-beta (1-42) peptide, while extracted SA^Os were obtained from the supernatant of homogenized 5XFAD mouse brains after ultracentrifugation. The in-vitro SA^O was generated as described in FIG.2. All three samples of the amyloid isoforms were quantified by a BCA assay, enabling all isomers to be “spotted” at equal concentrations. The dot-blot data (cropped and magnified for presentation) suggests that the E3 nanobody recognizes in-vitro SA^Os weakly. In contrast, the E3 nanobody recognizes extracted SA^Os from 5XFAD brains strongly. The detected signal of this interaction is concentration dependent (figure not shown) The data also showed that the 6E10 antibody is less specific compared to the E3 nanobody as the antibody recognizes all three Abeta species.

[00125] FAM-labeled E3 nanobody can cross the BBB and detect SA^Os and amyloid-^plaques in the brain after intravenous injection. To determine if the E3 nanobody could be used to image SA^O in-vivo, we injected the FAM-labeled E3 nanobody (1.8 mg / mL, 150 ^L) intoStites Docket No: 11672N / 21359WO 5XFAD mice (n=5) and WT (n=3) mice. To ensure the fluorescent signal detected was not due to free dye diffusion to the brain or autofluorescence generated from the tissue or nanobody, two additional control cohorts of animals were included in the study. These controls included WT and 5XFAD mice that were intravenously injected with either free FAM dye (1 mg / mL, 150 ^L) or unlabeled E3 nanobody (1.8 mg / mL, 150 ^L) (n=3, each), respectively.

[00126] Many different areas of the brain were imaged, but the cortex and hippocampalregions were chosen for each cohort since they are relevant to AD. As shown in FIG.6, no fluorescent signals were detected in 5XFAD mice injected with E3 nanobody alone (FIG.6A,B) or in WT mice injected with either free FAM dye (FIG.6C, D) or FAM-labeled E3 nanobody (FIG.6E,F). In contrast, FAM-labeled E3 nanobody detected copious amyloid-^ deposits in the brain of 5XFAD mice at 24h (FIG.6G, H) and 4h (FIG.6 I, J) post-injection. Pixel quantification of the signals demonstrated that the 24h-post-injection group was significantly higher than that of control mice (FIG.6K) (p<0.0001). Although by a smaller amount than the 24h-cohort, the accumulation of signal in the 4h-post-injection cohort (n=3) was significantly higher than control counterparts (p<0.05) (FIG.6L).

[00127] With the superior resolution provided by confocal microscopy, we also showed thatSA^O and amyloid-^ plaques recognized by the E3 nanobody not only differ in size, shape, and morphology, but they have distinct spatial distributions in the brain. As shown in FIG.6M-P, SA^Os are associated with the neuronal nuclei (white arrows). This observation agrees with a previous report showing the association of oligomers with neuronal nuclei. In contrast, the E3 nanobody-positive amyloid-^ plaques (asterisks) reside in the cellular matrix. These results indicate that E3 nanobody, similar to other nanobodies, crosses the BBB, most likely due to its small size and relatively high isoelectric point (calculated to be 9.2 using Expasy).

[00128] Quantitative analysis. The in-vivo data obtained using FAM-E3 nanobody revealedthe spatial distribution of SA^Os in 5XFAD brains. In contrast, no registered fluorescence signal was detected in WT brains. While SA^Os and amyloid-^ plaques were distributed across many regions of the brain of 5XFAD mice, they appeared to be more concentrated in some brain regions than others. Thus, we were interested in quantifying these differences using our algorithms developed in MATLAB. The data are summarized in Table 1 and show that the presence and the density of amyloid-^ species in the hypothalamus (170.90 / mm2) wasStites Docket No: 11672N / 21359WO surprisingly high, followed by the thalamus (122.44 / mm2), compared to other regions. On the other hand, the hippocampus had the least number of amyloid-^ species with a recorded density of 38.87 / mm2). On average, the diameter of SA^O-associated FAM-E3 was about 2 ^m, with average measured eccentricities of approximately 0.5.

[00129] The accumulation of amyloid-^ aggregates in the brain is a hallmark of AD. Inhuman AD, pathological amyloid-^ proteins are composed of different assemblies that included senile plaques, protofibrils, fibrils, and SA^Os. Increasing evidence suggests that SA^Os serve as toxic species responsible for the pathogenesis and spread of AD. Prior to this work, a number of antibodies or antibody fragments have been used for the specific recognition of SA^Os. These reagents have helped examine the roles of SA^Os in AD brain, leading to the hypothesis that the disruption of particular synapses by SA^Os could be the culprit behind the memory loss in early AD. According to the contemporary model of the amyloid-^ cascade hypothesis (FIG.1), SA^Os are considered stable and independent components, affecting AD pathology without conversion to fibrillar assemblies. Thus, targeting SA^Os is beneficial due to their early role in AD pathogenesis and disease progression. In this work, we report the development, production, and characterization of a novel SA^O-specific nanobody. Aside from being thermally stable, which makes it amenable for PET labeling chemistry, a nanobody has the added advantage of being able to traverse biological barriers. In fact, it has been reported that SA^Os possess a higher diffusion coefficient compared to large assemblies. With a mere size of ~15 kDa, 1 / 10ththe size of conventional antibodies (150 kDa), nanobodies are smaller than any antibodies and antibody fragments described to date. For example, Fab (~50 kDa), F(ab’)2(~110 kDa fragments), single chain (monovalent) Fv (scFv, ~25 kDa) with one antigen-binding site, bivalent scFv dimmers (50 kDa), and scFv-fusion proteins (sometimes called minibodies, ~80 kDa), all are larger than nanobodies. It is anticipated that using nanobodies for targeting SA^Os could serve as a valuable tool for responding to the delivery solution for biologics. These molecules could be used either as therapeutics or diagnostic pharmaceuticals for AD applications.

[00130] Nanobody production is relatively simple, robust, and economical. The quality of theSA^O antigen used for immunization was confirmed by Coomassie staining and Western blot analysis (FIG.2A,B). Fresh SA^Os were prepared for alpaca injections, thus the material had a low propensity to grow larger aggregates. The VHH library was constructed, and a singleStites Docket No: 11672N / 21359WO panning step was performed against the immobilized SA^Os. From the enriched SA^O nanobody-expressing phage, 61 clones were identified by screening for binding to SA^Os via ELISA.

[00131] A robust assay was developed to screen nanobody FAM-labeled nanobody clonesusing consecutive 5XFAD brain slides. Data in FIG.4 showed that no signals were detected in the WT brain sections. In stark contrast, all the clones stained oligomers in the hippocampus and cortex of a 5XFAD mouse, although the degree of staining varied between clones in the brain slices. Among the six best clones, D11, F9, H5 and E3 showed considerable signal with at least 100 pixel counts per cortex, as quantified using ImageJ. From these results, E3 nanobody was selected for full characterization. The ex-vivo staining was repeated with FAM-labeled E3 nanobody using brain slides from different 5XFAD mice (n=3). The data showed that the FAM- E3 nanobody stained amyloid-^ in the 5XFAD brains with a mixed morphology (FIG.5A). It is apparent that two populations of amyloid-^ isoforms dominate many parts of the brain, with the highest abundance found in the hypothalamus, followed by the thalamus, cortex, and then hippocampal regions (Table 1). The size, shape, and morphology of the smaller population are similar to what has been seen with anti-SA^O ACU193 and NU4 antibodies. Meanwhile, the larger population observed in the 6E10 antibody-treated 5XFAD brain slides resembles amyloid- ^ plaques (FIG.5 B). To explain the mixed results of our in-vivo amyloid-^ staining, we tested the specificities of E3 nanobody against amyloid-^ isomers that included monomers, SA^Os, in- vitro generated SA^O, and SA^O, which were isolated from 5XFAD brain homogenates using ultracentrifugation. The results of this work confirmed the ex-vivo staining data showing that E3 nanobody is more specific than 6E10 antibody for SA^O in that it can recognize SA^Os extracted from the brain of 5XFAD mice but not the free Abeta peptide monomer (FIG.5C). The data suggest that the nanobody recognizes the SA^Os not only based on the specific epitope but depending also on the secondary structure. A complete understanding of the molecular mechanism by which E3 nanobody preferentially recognizes oligomeric forms of Abeta will require detailed structural analysis. However, an enticing possibility is suggested by AlphaFold models of E3. The complementary determining regions of E3 may make a large, relatively flat surface, capable of binding across multiple rungs of the beta-solenoid structure of amyloidStites Docket No: 11672N / 21359WO oligomers (figure not shown). Such a binding mechanism would lead to strong preference for oligomers over monomers.

[00132] Importantly, the E3 nanobody could localize in the brain after intravenous injection.Using MALDI-TOF, E3 nanobody was detected in the brain extracts of 5XFAD mice 24h post- injection (figure not shown). The in-vivo application of FAM-labeled nanobody (via intravenous injection) indicated that it could cross the BBB and stain amyloid-^ in the brain of 5XFAD mice (FIG.6). The FAM-labeled E3 nanobody is distributed significantly in the brain of 5XFAD mice, 4 h post-intravenous injection (FIG.6I, J). These data point to an advantage of nanobodies as compared to larger antibodies. It has been reported that an uptake time of 4h post-intravenous injection of anti-SA^O antibody labeled with64Cu PET radioisotope in 5XFAD mice resulted in poor signal distribution and was not robust for signal detection. In this work, increased signal in the 24h-post-injection cohort was observed (FIG.6G, H). Based on the pharmacokinetics of the probe, it appears that the probe enters the brain of injected mice, binds SA^O targets and its accumulation, leading to increased signal. The confocal imaging data showed the distinct locations between SA^Os (white arrows) and amyloid-^ plaques (asterisks) (FIG.6M-P). The former is associated with neurons, while the latter resides in the extracellular matrix. This regiospecific distribution may explain the distinct role of how they contribute to AD pathology.

[00133] In conclusion, we report a robust, large-scale, and reproducible generation of a novelSA^O nanobody. Extensive in-vitro and in-vivo characterization demonstrated that the nanobody recognizes SA^Os and amyloid-^ plaques in a preclinical mouse model of AD. Since FAM-labeled E3 can penetrate the BBB and effectively stain amyloid-^ in the brain, work is currently being undertaken to utilize this nanobody for diagnostic applications and potential therapies. Because SA^Os are involved in the early stage of AD pathology, we anticipate that this nanobody can be utilized to develop a novel and powerful platform for precision in-vivo molecular fingerprinting of SA^Os for AD early detection.

[00134] Methods and Materials. All commercially-available reagents and solvents were usedas received without further purification. FAM-NHS was purchased from Lumiprobe Corp (Hunt Valley, Maryland, USA), sealed with parafilm and stored at -20ºC. The purity and stability of the dye were monitored every 6 months using liquid chromatography mass spectrometry (LC-MS). Human amyloid-^ peptide (1-42) was obtained from Anaspec (Fremont, CA, Cat. AS-20276).Stites Docket No: 11672N / 21359WO Ultrapure water (18.2 M^) was obtained from a Millipore Direct Q-5 water purification system. A Hitachi HPLC system (Lachrom Elite) equipped with a diode array detector was used for purification, and employed a Cytiva HiTrap Desalting column with a mobile phase of 25 mM sodium phosphate, 150 mM sodium chloride, pH7.4. Three fluorescence microscopes were used in our studies, including a manual Zeiss Axio Observer, an automated Zeiss Axio Observer, and a Zeiss LSM 710 confocal laser scanning microscope. MALDI-TOF analysis was performed on a Bruker Rapiflex with sinapinic acid matrix.

[00135] Animals. All animal experiments performed complied with institutional guidelinesand were conducted according to the protocol approved by the Vanderbilt Institutional Animal Care and Use Committee. The 5XFAD mice were maintained at Vanderbilt University under standard conditions, in a 12h-light / dark cycle, and with free access to food and water. The 5XFAD mice overexpress both mutant human APP and PS1 genes and exhibit high APP levels correlating with high burden and accelerated accumulation of amyloid-^. A colony of 5XFAD transgenic mice obtained from Jackson Laboratories was maintained by crossing 5XFAD mice with a wild-type (WT) C57BL / 6J strain. The mice were genotyped by a standard polymerase chain reaction (PCR) using DNA isolated from tail tips and the following primers provided by Jackson Laboratory (Bar Harbor, Maine): Mutant reverse, 5’-CGG GCC TCT TCG CTA TTA C -3’; Common, 5’-ACC CCC ATG TCA GAG TTC CT -3’; Wild Type reverse, 5’-TAT ACA ACC TTG GGG GAT GG-3’. Amplified PCR products were then analyzed by size fractionation using agarose (1.5%) gel electrophoresis; band sizes for wild type = 216bp, 5XFAD heterozygous = 129bp and 216bp, and 5XFAD homozygous = 129 bp. The 5XFAD mice were maintained as heterozygotes and all mice used in these experiments were approximately 6- to 11- month-old.

[00136] Alpaca immunizations and bleeds were performed at Turkey Creek Biotechnology,LLC (Waverly, TN) in accordance with their Institutional Animal Care and Use Committee protocol 18-02.

[00137] Generation of SA^O as an antigen. This work was performed using a reportedprocedure with some modifications (Ryan, D. A., et al. J Neurosci Methods 190, 171-179 (2010). Human amyloid-^ peptide (1-42) (5.0 mg, Anaspec, Fremont, CA, Cat. AS-20276) was dissolved in DMSO to a final concentration of 5 mM. An equivalent volume of DMSO served asStites Docket No: 11672N / 21359WO a negative control. The materials were sonicated at room temperature for 10 minutes in Eppendorf tubes. A small portion of the solution (20 ^L) was set aside and stored at -20°C for future monomer analysis. The remaining solution was diluted further to 100 ^M with cold PBS (1x) containing 0.05% SDS followed by a brief vortex (30 seconds). At the same time, the DMSO negative control was diluted with an equivalent of volume of cold PBS (1x) containing 0.05% SDS. The resulting stock solution of 100 ^M peptide solution and control DMSO were then incubated overnight at 4°C. Approximately 24h later, the stock solution was further diluted to 25 ^M with PBS (1x), and an equivalent volume of PBS (1x) was again added to the DMSO negative control. The samples were incubated for two weeks at 4°C to enable amyloid-^ oligomerization.

[00138] Following incubation, the amyloid-^ concentration was found to be ~150 ^g / mL asdetermined by the BCA protein assay (Thermo Fisher Scientific, Waltham, MA, Cat.23225). The amyloid-^ was then concentrated using Amicon Ultra-4 Centrifugal Filter Units with a cutoff of 3 kDa NMWL (Millipore Sigma, Burlington, MA, Cat. UFC800324) to reach a final concentration of 500 ^g / mL. The purity of the SA^O was confirmed by SDS-PAGE using 12% precast polyacrylamide gels (BioRad, Hercules, CA, Cat.4561044) followed by Coomassie staining (FIG.2A). The data showed that with this method, we could generate a mixture of low and high molecular weight oligomers. Freshly prepared samples were made prior to each animal immunization, and the quality of each preparation was confirmed by SDS-PAGE and Coomassie staining of the gels.

[00139] Coomassie staining. Soluble amyloid-β oligomer preparations were solubilized inLaemmli Sample buffer (1X final concentration), boiled for 5 minutes at 96^C, and analyzed by SDS-PAGE. The prepared samples (20 ^L) were loaded into a 12-well 4-20% gradient gel (BioRad Laboratories, Hercules CA, Cat No.4561095) along with BioRad Precision Plus Protein Kaleidoscope Prestained Protein Standards (BioRad Laboratories, Hercules CA, Cat No. 1610375) and electrophoresed at 170V for 50 minutes. The gel was washed three times for 5 minutes each with deionized water, stained with BioSafe Coomassie G-250 (BioRad Laboratories, Hercules CA, Cat No.1610786) with gentle shaking for 1h, destained overnight, and imaged on a LiCOR Odyssey M (LiCOR Biosciences, Lincoln NE). Images were collected and extracted using Empiria software (LiCOR Biosciences, Lincoln NE).Stites Docket No: 11672N / 21359WO

[00140] Western analysis. Samples were prepared for immunoblotting by adding LaemmliSample buffer (1X final concentration) and 20 µL of each sample was loaded onto a 12-well 4- 20% gradient gel (BioRad Laboratories, Hercules CA, Cat No.4561095) along with BioRad Precision Plus Protein Kaleidoscope Prestained Protein Standards (BioRad Laboratories, Hercules CA, Cat No.1610375). Following electrophoresis at 170V for 50 minutes, the gel proteins were transferred onto a 0.2 ^m supported nitrocellulose membrane (BioRad Laboratories, Hercules CA, Cat No.1620097) using the TransBlot Turbo System (BioRad Laboratories, Hercules CA, Cat. No.1704150) and TransBlot Turbo Buffer (BioRad Laboratories, Hercules CA, Cat No.10026938) at 2.5A, 25V, for 10 minutes. The blots were rinsed in deionized water for 5 minutes, then placed between two pieces of Whatman filter paper (Cytiva, Malborough MA, Cat No.10427818) to dry for 15 minutes at 37^C. Dried blots were rehydrated in TTBS for 5 minutes and blocked for 1h in LiCOR Odyssey Intercept Buffer TBS (LiCOR Biosciences, Lincoln NE, Cat No.27-60001) at room temperature. Blots were heat- sealed in ULine Poly Tubing bags containing 5 mL of 1:1000 (v / v) 6E10 antibody (BioLegend, San Diego CA, Cat No.803004) in ULine Poly Tubing and incubated overnight at 4^C. Blots were then washed 4 times with large volumes of TTBS, and incubated with 1:5000 (v / v) LiCOR Donkey anti-Mouse IRDye800CW (LiCOR Biosciences, Lincoln NE, 926-32212) for 1h at room temperature. Blots were washed 4 times with TTBS and imaged on a LiCOR Odyssey M LiCOR Odyssey M (LiCOR Biosciences, Lincoln NE) at 800nm and 700nm channels, and images were collected and processed using LiCOR Empiria Software (LiCOR Biosciences, Lincoln NE).

[00141] Alpaca immunization. The SA^O preparation (300 ^g) was emulsified in Gerbuadjuvant for the initial injections (day 0), followed by boosters at days 14, 28, 42, 56, 70 and 84 with 150 ^g SA^O in Gerbu adjuvant. Blood was drawn for analysis on day 63 (first bleed) and day 91 (second bleed). The plasma was monitored by dot-blot analysis to test for the presence of antibodies recognizing SA^O. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation from 17.5 mL of alpaca blood using SepMate centrifugal devices, following the manufacturer’s protocol (Stemcell Technologies, Vancouver, CA).

[00142] Phage library construction. A cDNA library was made by reverse transcription witholigo dT primers and Superscript IV (ThermoScientific). A nested PCR strategy was used toamplify VHH coding regions of fragments, as described (Pardon et al.; and Oakley, H. et al. JStites Docket No: 11672N / 21359WO Neurosci 26, 10129-10140 (2006)). The resulting PCR fragments were ligated into pBBR3, a modified pADL22 vector (Antibody Design Labs). All clones contain in-frame, C-terminal, HA and hexahistadine tags. The library was electroporated into high-efficiency TG1 cells (Lucent), and phage were produced using CM13 helper phage. Phage were harvested from the supernatant by PEG precipitation and suspended in PBS.

[00143] Panning for SA^O nanobodies and isolation of individual clones. A single round ofpanning against SA^O immobilized on a Nunc MaxiSorp (Nalge Nunc Intl., Rochester, NY) plates was performed. Three wells of a 96-well MaxiSorp plate were coated overnight with 10 ^g of SA^O in PBS (100 ^L); three PBS-coated wells served as controls. After removing unbound antigen, the wells were blocked with 2% nonfat milk in PBS for 2h, washed, and incubated with 2x1012phage particles in blocking buffer for 1h. After extensive alternating washes with PBS and PBS + 0.5% Tween 20, phage were eluted with 100 µL of 100 mM glycine pH2.2 and then immediately neutralized with 100 ^L of 1 M Tris pH8. Recovered phage wereused to infect TG1 E. coli, and single clones were picked and inoculated in deep-well 96 wellblocks containing 1 mL Terrific Broth and 100 ^g / mL ampicillin. Plates were grown at 37^C for 5h, followed by 28^C overnight. Then, 1 mM IPTG was added, and the growth was continued for 3h. Bacteria were pelleted and lysed by two freeze-thaw cycles with a total of 400 µL of PBS. After centrifugation, the periplasmic extracts were analyzed by ELISA for the presence of nanobody-recognizing SA^O.

[00144] Detection of positive SA^O nanobody clones. A MaxiSorp plate was coatedovernight at 4^C with 1 µg / well of SA^O in PBS (100 ^L), washed, blocked with LI-COR Intercept buffer (PBS), and then incubated with 50 µL of periplasmic extracts lysate from above and 50 µL of PBS for 2h at room temperature or overnight at 4^C. Wells were washed with TTBS and incubated with a HRP-conjugated anti-HA tag (Roche, Basel, CH) antibody for 1h at room temperature. After washing, the plate was developed with TMB ultra (Sigma, St. Louis, MO) to identify positive clones.

[00145] In an alternative procedure, a 96-well Costar plate (black with clear bottom) wascoated overnight at 4^C with 0.5 ^g / well of SA^O (100 ^L), washed with TTBS, blocked with LI-COR Intercept buffer (TBS), and then incubated with 50 ^L of periplasmic extracts from above and 50 ^L of PBS for 2h at room temperature or overnight at 4^C. The wells were washedStites Docket No: 11672N / 21359WO with TTBS and incubated with fluorophore 800-conjugated anti-HA antibody for 1h at room temperature. After washing with TTBS, the plate was imaged using the LI-COR Odyssey Imager to identify positive clones.

[00146] Clone identification. A total of four 96-well plates of clones were picked from twodifferent nanobody libraries. All clones showing signal above background were sequenced. Six clones were both identified multiple times and found to be positive in multiple assays. These six clones were expressed and purified from 3 L bacterial cultures. A portion of each purified nanobody was biotinylated with 20-fold molar excess of NHS-biotin (Pierce) and 8 point, 5-fold, binding titrations were done for each clone using streptavidin-HRP or anti-HA-HRP (Thermo Fisher).

[00147] Nanobody purification. For large scale purifications of E3, the plasmid was isolatedand used to transform T7 shuffle bacteria. An overnight culture was used to inoculate fresh media which was grown at 37^C until a OD600 of ~1.2 was reached. At this point the temperature was lowered to 18^C, 1mM IPTG was added, and protein expression was continued overnight. Cells were harvested by centrifugation and lysed with an Emulsiflex. Nanobodies were purified using Talon affinity resin (Takara Bio, Ann Arbor, MI). Aliquots of E3 were chemically biotinylated using NHS-Biotin (Thermo Fisher, Waltham, MA).

[00148] Labeling E3 nanobody with FAM-NHS dye. The E3 nanobody (1.8 mg / mL, 57nmol) in PBS (1x, 50 ^L) was treated with Na2CO3(1 M, 6 ^L, pH8) prior to the addition of FAM-NHS dye (20 eq., 1140 nmol, 0.54 mg) in argon-purged anhydrous DMSO (12 ^L). The reaction vial was rotated overnight at 4ºC. Excess of activated dye was neutralized with Tris-HCl buffer (10 ^L, pH7), and the labeled product was separated from the excess dye using a 7k MWCO Zepa spin column and reconstituted in PBS (1x, pH7). The resulting FAM-E3 conjugate was characterized by 2D-HPLC using a Cytiva HiTrap desalting column (5 mL, cat.29048684) under the isocratic conditions of 25 mM sodium phosphate, 150 mM sodium chloride, pH7.4, and a flow rate of 0.5 mL / min.

[00149] Immunohistochemistry staining. Brains embedded in OCT were cut into coronalsections (10 µm) using a Tissue-Tek (Sakura Finetek USA, Torrance, CA) cryostat and mounted onto charged glass slides. Prior to staining, slides were washed with PBS (10 minutes) and then treated with blocking buffer (5% normal goat serum, 0.2% Triton X-100, 0.5% bovine albuminStites Docket No: 11672N / 21359WO in PBS) for 1h at room temperature. The treated sections were then incubated overnight at 4°C or1h at room temperature with the E3-labeled nanobody diluted 1:100 in PBS. The sections werethen washed with PBS twice for 10 minutes and once for 30 minutes, and cover-slipped with Vectashield plus antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, Cat. H2000-2) before observation using fluorescence microscopy.

[00150] For slides that required the use of secondary antibodies, the process was performedthe same way as above except that after incubation with nanobody, the sections were subsequently washed in PBS 3 times for 10 minutes each and then incubated for 30 minutes at room temperature with a secondary antibody diluted in PBS containing 3% NGS (normal goat serum), 0.2% Triton X-100 and 0.5% bovine albumin. The sections were then washed with PBS twice for 10 minutes and once again for 30 minutes before being cover-slipped with Vectashield plus and examined by fluorescence microscopy.

[00151] Isolation of amyloid-^ fibrils / plaques from 5XFAD mouse brains. This procedurewas modified based on the Abcam “Extraction of amyloid-^ plaques from mouse brain” protocol (https: / / www.abcam.com / protocols / extraction-of-amyloid-beta-from-mouse-brain). The brain of a 5XFAD mouse (n=2, 6-month-old) was homogenized in PBS (5 mL), followed by the addition of formic acid (10 mL). The mixture was sonicated for 1 minute on ice and centrifuged at 135,000 g for 1h at 4ºC. The collected supernatant was neutralized with a neutralizing buffer composed of 1M Tris base, 0.5M Na2HPO4, and 0.05% NaN3. The solution was aliquoted, and flash frozen on dry ice before storage at -80ºC. Aliquots were thawed prior to loading onto nitrocellulose membranes for dot-blot analysis.

[00152] Dot-blot assays. For testing the plasma, one microliter of 0.1, 0.01, 0.001, and0.0001 mg / mL stock solutions of SA^O in SDS sample buffer or PBS were spotted on Amersham Protran Supported 0.2 mm Nitrocellulose (GE Healthcare Life Sciences). The membranes were allowed to dry, wetted in Tris-buffered saline (TBS), blocked in Intercept Blocking Buffer (LI-COR), and then incubated overnight with alpaca plasma diluted 1:200 in Intercept Blocking Buffer. After washing with TBS / 0.1% Tween, the membranes were incubated with an 800 fluorophore-labeled goat anti-alpaca secondary antibody, washed with TBS / 0.1% Tween, and then imaged on the Odyssey Infrared Imaging system (LI-COR).

[00153] For testing the specificity of the E3 nanobody, samples of bovine serum albumin(BSA), amyloid-b monomer, in-vitro-generated SA^O, and mouse brain SA^O extract wereStites Docket No: 11672N / 21359WO spotted onto a nitrocellulose membrane and allowed to dry at room temperature. The membrane was then blocked for 30 minutes using Intercept (TBS) blocking buffer (LI-COR Bioscience, Lincoln, Nebraska) before three 5-minute washes with TTBS. Next, the blot was incubated for30 minutes in a solution containing E3 nanobody (10 mg / mL) in TTBS before three 5-minutewashes with TTBS. The membrane was then incubated with goat anti-VHH antibody(12.5 mg / mL in TTBS; Jackson ImmunoResearch, West Grove, PA) for 30 minutes and washedwith TTBS three times for 5 minutes each again. Finally, the blot was incubated with donkeyanti-goat AF488 (2 mg / mL in TTBS; Abcam, Cambridge, UK) and washed three more times for5 minutes each with TTBS before imaging. All incubations and washes were performed at roomtemperature with gentle shaking. A separate control blot to test for non-specific binding wasprepared likewise as above, except that no E3 nanobody was used as a primary antibody againstthe samples. Imaging was performed on a ChemiDoc MP Imaging System (Bio-Rad, Hercules,CA).

[00154] Quantitative analysis program written on MATLAB. Semi-automated quantificationof amyloid-β burden was performed for each slide using a custom image analysis script written in MATLAB (Mathworks, Natick, MA). First, an image was loaded into the program. Second, adaptive threshold was used to decrease the impact of background noise. Third, a tissue mask was created based on that adaptive threshold and then cleaned up using a morphological opening. Finally, regions-of-interest (ROIs) were manually drawn, enabling us to avoid histological artifacts. From the ROI plaque masks, plaque morphologic parameters can be calculated using the function “regionprops”. Plaque density was reported in plaques per mm2and was defined as the number of plaques divided by the ROI area.

[00155] Confocal imaging. The brain sections obtained from 5XFAD mice (n=3) wereincubated with a mixture of FAM-labeled E3 (1.8 mg / mL) or mouse 6E10 (BioLegend, San Diego, CA, USA) for 1h at room temperature with gentle rocking. Then, the slides were washed with PBS three times before being incubated with goat anti-mouse IgG cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, Waltham, MA, USA). The slides were then counter- stained with DAPI incorporated in Vectashield (Vector Laboratories, Newark, CA, USA). The slides were sealed with a coverslip before microscopy. The images were obtained with 20x and 63x objectives using a Zeiss LSM 710 confocal laser scanning microscope. Then, the images were reconstructed with Zeiss Zen Blue software.Stites Docket No: 11672N / 21359WO

[00156] Protein structure analysis. AlphaFold (Jumper, J. et al., Nature 596, 583-589 (2021))as implemented through the AlphaFold Colab was used to calculate 20 models of E3 using 4 different starting seeds. The predicted value of the local distance difference test (pLDTT), a per residue confidence value output by AlphaFold, was used for the coloring scheme in panel C in (data not shown) as described in Oeffner, R. D. et al., Acta Crystallogr D Struct Biol 78, 1303- 1314 (2022).

[00157] Statistical analysis. We used t-tests to compare the differences between two groups.The data were expressed as means ± SE of the difference. The statistical significance of the differences between sample means was determined via unpaired t-tests, the results of which were considered significant when P < 0.05 as reported with GraphPad Prism software.

Claims

Stites Docket No: 11672N / 21359WO CLAIMS What is claimed is:

1. A nanobody or fragment thereof that binds SA^O and optionally comprises a cargo molecule.

2. A nanobody or fragment thereof comprising an amino acid sequence and optionally, a cargo molecule, wherein the amino acid sequence comprises (a) SEQ ID NO:1; or (b) a fragment of SEQ ID NO:1; or (c) a variant amino acid sequence of SEQ ID NO:1; or (d) a fragment of a variant amino acid sequence of SEQ ID NO:

1.

3. The nanobody or fragment thereof of claim 2, wherein the nanobody is isolated.

4. The nanobody or fragment thereof of claim 2, wherein the amino acid sequence comprises SEQ ID NO:

1.

5. The nanobody or fragment thereof of claim 2, wherein the amino acid sequence comprises a fragment of SEQ ID NO:

1.

6. The nanobody or fragment thereof of claim 2, wherein the amino acid sequence comprises a variant amino acid sequence comprising at least 85% sequence identity with SEQ ID NO:

1.

7. The nanobody or fragment thereof of claim 2, wherein the amino acid sequence comprises a variant amino acid sequence comprising at least 95% sequence identity with SEQ ID NO:1.Stites Docket No: 11672N / 21359WO 8. The nanobody or fragment thereof of claim 2, wherein the amino acid sequence comprises a fragment of a variant amino acid sequence, wherein the fragment of a variant amino acid sequence comprises at least 85% sequence identity with SEQ ID NO:

1.

9. The nanobody or fragment thereof of claim 9, wherein the amino acid sequence comprises a fragment of a variant amino acid sequence, wherein the fragment of a variant amino acid sequence comprises at least 95% sequence identity with SEQ ID NO:

1.

10. The nanobody or fragment thereof of claim 1, wherein the nanobody or fragment thereof comprises a cargo molecule.

11. The nanobody or fragment thereof of claim 10, wherein the nanobody or fragment thereof is conjugated to a cargo molecule to form a conjugate of the nanobody or fragment thereof, wherein the cargo molecule comprises a therapeutic agent, a radioisotope, an enzyme, afluorescent dye, or a bioluminescent marker, or a combination thereof.

12. A polynucleotide encoding the nanobody or fragment thereof of claim 2.

13. A expression vector comprising the polynucleotide according to claim 12.

14. A cell transformed with a vector according to claim 13.

15. A pharmaceutical composition comprising the nanobody or fragment thereof of claim 1, together with a pharmaceutically acceptable carrier.

16. A pharmaceutical composition for detecting soluble amyloid β-oligomer in a subject, wherein the composition comprises an effective amount of a means for binding soluble amyloid β-oligomer in the subject.

17. A method for detecting soluble amyloid β-oligomer in a subject, the method comprises administering to the subject an effective amount a conjugate of the nanobody or fragment thereof of claim 1.Stites Docket No: 11672N / 21359WO 18. A method for treating a disease or disorder associated with the presence of soluble amyloid β-oligomer in a subject, by administering an anti-SA^O nanobody comprising a means for binding SA^O.

19. A method for treating a disease or disorder associated with the presence of soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the nanobody or fragment thereof of claim 1.

20. A method for monitoring the severity of a disease or disorder associated with the presence of soluble amyloid β-oligomer in a subject, wherein the method comprises administering to the subject an effective amount of the nanobody or fragment thereof of claim 1.

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