WO2026005695A1 - Detection of oligomeric tau and soluble tau aggregates - Google Patents

Abstract

The invention relates to a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to human tau. The monoclonal antibody, or the antigen-binding fragment thereof, can be used in a homogenous immunoassay for the detection and quantification of oligomeric tau and soluble tau aggregates in body fluid samples.

Description

DETECTION OF OLIGOMERIC TAU AND SOLUBLE TAU AGGREGATES

TECHNICAL FIELD

The present invention generally relates to monoclonal antibodies, or antigen-binding fragments thereof, binding specifically to human tau, and to an immunoassay kit and methods involving the use of such monoclonal antibodies, or antigen-binding fragments thereof.

BACKGROUND

One of the principal neuropathological features of Alzheimer’s disease (AD) is the accumulation of aggregated tau in neurofibrillary tangles (NFTs) and in dystrophic neurites surrounding amyloid plaques in the brain. NFTs have been found to correlate more strongly than amyloid plaques with cognitive decline. NFTs are made up of paired helical filaments (PHFs), which are highly ordered long filaments composed of twisted strands of beta-sheet-rich misfolded fibril structures. In turn, fibrils are polymers of smaller aggregates including oligomers and protofibrils. While immunohistochemical characterization of NFTs/PHFs at autopsy is needed for the neuropathological diagnosis of AD, accurate quantification of the pre-NFT/PHF species is critical to evaluating the earlier cascade of pathophysiological events.

Tau PHFs and fibrils from AD brains have distinguishable structural arrangements compared to those from other neurodegenerative tauopathies. Furthermore, fibrillar and prefi brillar tau from AD brains induce neurotoxicity in animal and cell culture models, where they can propagate cellular insult by interacting with endogenous tau and other proteins that can ultimately lead to the dysfunction of different cellular pathways. In line with this, immunotherapy against tau has proven promising, either by promoting further polymerization into less bioactive aggregates or by inducing conformational change that substantially reduces affinity for protein-protein interactions. Thus, pre-NFT tau forms may play fundamental roles in AD pathophysiology. However, their accurate quantification remains challenging due to their heterogeneous and transient nature, and the lack of analytical methods for their measurement.

US 11 ,906,530 discloses methods for determining whether a subject has a tauopathy, such as Pick disease, AD, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) or argyrophilic grain disease (AGD). The methods utilize an amyloid seeding assay.

There is still a need for methods for detection of tau oligomers and soluble tau aggregates.

SUMMARY It is a general objective to provide monoclonal antibodies, or antigen-binding fragments thereof, binding specifically to oligomeric tau and soluble tau aggregates.

It is a particular objective to provide an immunoassay kit capable of detection of oligomeric tau and soluble tau aggregates.

These and other objectives are met by embodiments of the present invention.

The present invention is defined in the independent claims. Further embodiment of the invention are defined in the dependent claims.

An aspect of the invention relates to a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to human tau. The monoclonal antibody, or the antigen-binding fragment thereof, has a VH CDR1 consisting of RYWMN as defined in SEQ ID NO: 1 , a VH CDR2 consisting of QIYPGDGDTNYNSKFKV as defined in SEQ ID NO: 2, a VH CDR3 consisting of SWAY as defined in SEQ ID NO: 3, a VL CDR1 consisting of SASSSVNHTH as defined in SEQ ID NO: 4, a VL CDR2 consisting of DTSKLAS as defined in SEQ ID NO: 5 and a VL CDR3 consisting of FQGSGYPLT as defined in SEQ ID NO: 6.

Another aspect of the invention relates to an immunoassay kit for detection of oligomeric tau and soluble tau aggregates comprising a capture antibody, or an antigen-binding fragment thereof, according to above and a detection antibody, or an antigen-binding fragment thereof, according to above.

A further aspect of the invention relates to a method for determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample. The method comprises contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit according above and determining an amount of oligomeric tau and soluble tau aggregates in the body fluid sample by determining an amount of bound detection antibody, or the antigen-binding fragment thereof.

Yet another aspect of the invention relates to a method for diagnosing Alzheimer’s disease in a subject. The method comprises determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample from the subject according to above and determining the subject as suffering from Alzheimer’s disease or not based on the determined amount of oligomeric tau and soluble tau aggregates.

Another aspect of the invention relates to a method for classifying a neurological disease in a subject. The method comprises determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample from the subject according to above and classifying the neurological disease as Alzheimer’s disease or a neurological disease other than Alzheimer’s disease based on the determined amount of oligomeric tau and soluble tau aggregates.

Further aspects of the invention relate to a monoclonal antibody, or an antigen-binding fragment thereof, according to above for use as a medicament or for use in treatment of Alzheimer’s disease.

The monoclonal antibodies, or the antigen-binding fragments thereof, bind human tau in an epitope present in the MTBR within the tau protein. The monoclonal antibodies, or the antigen-binding fragments thereof, can thereby be used in an immunoassay kit for detection and quantification of oligomeric tau and soluble tau aggregates in body fluid samples. The assay signal from such an immunoassay increased corresponding to total oligomeric tau and soluble tau aggregate content, Braak stating, and insolubility of the sequentially homogenized brain tissue fractions.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments, together with further objects and advantages thereof, may best be understood by referring to the following description taken together with the accompanying drawings, in which:

Fig. 1 : SPR sensorgrams showing binding specificity when different tau peptides (concentration = 3 pM) interacted with the CT19.1 (1A) and CT16.1 (1 B) monoclonal antibodies. The result supports the antibody efficiency of CT19.1 in recognition of epitope 331-361 on the tau sequence. The N-terminal tau fragment 1-224 and C-terminal tau fragment 368-441 are outside the epitope region. (10) The bar chart representing the epitope mapping of the CT19.1 antibody. The reactivity of CT19.1 was assessed with tau peptides 1-11 , covering amino acid residues 326-366. The figure shows significant reactivity with peptides 1 (326-346), 2 (328-348), and 3 (330-350), indicating that the epitope recognized by CT19.1 is within this region. Like blank sample, negligible reactivity was observed with other peptides, confirming the specificity of the antibody. The absorbance readings were analyzed using GraphPad Prism 5 to determine the binding affinity of CT19.1 to the different mapping peptides. Fig. 2: Development and validation of a new ultrasensitive immunoassay for the quantification of aggregated oligomers/soluble tau forms. (2A), Schematic illustration of the full-length tau-441 sequence, indicating where the CT19.1 antibody epitope is located (horizontal line). The cartoon underneath shows the homogenous setup of the SIMOA® assay, which enables the generation of AEB signal when it binds to tau aggregates with multiple epitopes but not single-epitope monomers. (2B), Evaluation of assay specificity and dilution linearity by monitoring AEB signals for preparations of recombinant full-length tau protein in both aggregated and monomeric forms, starting with identical concentrations. AEB signals for different samples monitored over 2 separate days are shown. The assay antibody CT19.1 specifically targets tau aggregates, where a heightened AEB signal correlates with increased quantities of tau aggregates. (2C), Negative-stain transmission electron microscopy of tau aggregates and monomeric tau prepared from aliquots of the same batch of recombinantly produced tau (starting concentration = 50 pM). Scale bar = 500 nm. (2D), The plot shows the mean assay AEB signals for aliquots of a TBS brain fraction from a neuropathologically verified case that was: (left) untreated, that is, nondepleted, or (middle and right) depleted with either the CT19.1 assay antibody used in the assay or the anti- -amyloid control antibody 6E10. AEB, average enzyme per bead; TBS, Tris buffered saline.

Fig. 3: Identification of the binding region of the tau oligomer/soluble aggregate assay. AEB signals of the tau aggregate assay to various concentrations (0-200 pg) of the tau peptides 1-224, 258-368, 302-368, and 368-441. The plots show mean ± standard deviation of n = 7 independent experiments. AEB, average enzyme per bead.

Fig. 4: The tau oligomer/soluble aggregate assay signals in cortical brain samples from the University of Pittsburgh ADRC autopsy-verified cohort. The cohort included cases with autopsy-verified AD and cases with other neurodegenerative diseases, with varying degrees of NFT tau pathology (i.e. , across Braak NFT stages 0-6). Tissues from two different brain regions were used for each case; (4A-4C) show the profiles for inferior temporal cortex and (4D-4F) for medial frontal cortex. For each case and region, equivalent weight of brain material was homogenized and processed into three sequential fractions of decreasing solubility of tau, namely the TBS sonicate (4A, 4D), Na2CO3 (4B, 4E), and urea/detergent (4C, 4F). Patient demographics are shown in Table 1. Braak Stage 0: S23, S6; Braak Stage 1 : S1 , S2, S4, S5; Braak Stage 2: S9, S12; Braak stage 3: S8, S11 ; Braak Stage 4: S7, S10; and Braak Stage 6: S13, S14, S15, S16, S17, S18; Abbreviations: AD, Alzheimer’s disease; AEB, average enzyme per bead; NFT, neurofibrillary tangle; TBS, Tris buffered saline. Fig. 5: Tau oligomer/soluble aggregate profiles in homogenized brain fractions of variable solubility. For each neuropathologically assessed case, tissues from the inferior temporal cortex (5A) and middle frontal cortex (5B) were homogenized and processed into three sequential fractions of decreasing solubility of tau oligomers/aggregates, namely the TBS, Na2CO3, and urea/detergent. The figure shows assay signals across sample fractions and in the same brain region for each participant. Abbreviations: AEB, average enzyme per bead; TBS sonicate, Tris buffered saline.

Fig. 6: Direct comparison of tau oligomer/soluble aggregate levels in equimolar concentrations of homogenized tissue from two different brain regions. The plots show AEB signals (6A-6C) and the corresponding estimated concentrations (6D-6F). The plots show the assay levels in the inferior frontal cortex (IF) and middle frontal cortex (MF) across three sequentially processed tissue fractions; TBS (6A, 6D), Na2CO3 (6B, 6E), and urea/detergent (6C, 6F). Note that the calculated concentration plots (6D, 6E, 6F) have been adjusted for the pre-measurement dilution with the assay diluent while the AEB plots shown in (6A), (6B), and (6C) do not account for this. AEB, average enzyme per bead; TBS, Tris buffered saline.

Fig. 7: Estimated concentrations of the tau oligomer/soluble aggregate assay profile in the neuropathologically defined cohort. Tissues from two different brain regions were used for each case; (7A-7C) show the profiles for inferior temporal cortex and (7D-7F) for medial frontal cortex. For each case and region, equivalent weight of brain material was homogenized and processed into three sequential fractions of decreasing solubility of tau, namely the TBS sonicate (7A, 7D), Na2COs (7B, 7E), and urea/detergent (7C, 7F). Patient demographics are shown in Table 1 . Braak Stage 0: S3, S6; Braak Stage 1 : S1 , S2, S4, S5; Braak Stage 2: S9, S12; Braak stage 3: S8, S11; Braak Stage 4: S7, S10; and Braak Stage 6: S13. S14, S15, S16. S17, S18;

Fig. 8: Evaluation of the TBS brain fraction from a neuropathologically defined case as quality control samples for the tau oligomer/soluble aggregate assay. The plots show AEB values and the corresponding protein content for serial dilutions of the sample. The measurements were repeated on different days, yielding highly consistent results.

Fig. 9: Quantification of relative tau aggregates in CSF. (9A) illustrates the levels of CSF tau aggregates in both AD and non-affected individuals, measured without any pre-incubation or prior treatment with recombinant tau aggregates. (9B) demonstrates the CSF tau aggregates in the same AD and nonaffected individuals following treatment with recombinant tau aggregates before the quantification of SIMOA® signals. (9C) presents the CSF tau aggregates in the same AD and non-affected individuals, wherein the CSF samples underwent treatment with recombinant tau aggregates and were incubated for 36 hours before SIMOA® signal measurement. In each instance, 50 pL of CSF was diluted with 50 pL of Tau 2.0, followed by the for both the AD and non-affected groups comprised 20 individuals. SIMOA® signals AEB were measured using CT19.1 antibody, higher AEB is directly proportional to aggregated tau.

Fig. 10: Transmission electron microscopy (TEM) characterization of full-length tau aggregates and monomeric tau. TEM images of recombinant full-length tau preparations used for the amplification of CSF tau aggregates in both the discovery and (Translational Biomarkers in Aging and Dementia (TRIAD), McGill University, Canada) validation cohorts. (Left Panel) Fibrillar tau aggregates: Representative TEM image showing filamentous tau assemblies, confirming the formation of fibrillar tau aggregates used as seeding agents in the CSF amplification assay. Scale bar = 500 nm. (Middle Panel) Monomeric tau: TEM image of monomeric tau, displaying diffuse and non-aggregated structures, confirming the absence of fibrillar assembly in monomeric preparations. Scale bar = 500 nm. (Right Panel) Single-unit tau aggregate: TEM image illustrating a distinct protofilament-like tau aggregate, measuring approximately 2 pm in length, further supporting the presence of well-defined tau assemblies. Scale bar = 500 nm. These images validate the structural integrity of tau aggregates and monomers used for the in vitro amplification of CSF tau aggregates, ensuring specificity and reproducibility in the immunoassay detection system.

Fig. 11 : CSF tau aggregates across clinical groups and tau PET Braak stages. (Top Panel): Box plots showing CSF tau aggregate levels across diagnostic groups in the TRIAD validation cohort. CSF tau aggregate concentrations progressively increased along the AD continuum, with significantly higher levels in MCI+ and AD groups compared to CU individuals and Non-ADD controls. Young individuals (OYoung) and biomarker-negative MCI cases (MCI-) showed lower CSF tau aggregate levels, suggesting potential specificity for AD-related tau pathology. (Bottom Panel): Box plots depicting CSF tau aggregate levels stratified by tau PET Braak stages. A stepwise increase in CSF tau aggregates was observed with higher Braak stages, with the most significant elevation occurring from Braak stage 3 onwards, aligning with increased tau pathology detected by tau PET. In both panels, each dot represents an individual participant, and box plots indicate the median, interquartile range, and data distribution. Higher CSF tau aggregate levels correspond to more advanced tau pathology, supporting their potential use as a fluidbased biomarker for AD diagnosis and staging. Fig. 12: Correlation between CSF tau aggregates and established Alzheimer’s disease biomarkers. Scatter plots showing the relationship between CSF tau aggregates and key biomarkers of Alzheimer’s disease (AD) in the TRIAD validation cohort. (12A-12C): CSF tau aggregates strongly correlated with phosphorylated tau (p-tau) and total tau, including p-tau217 (12A, measured using AlzPath), p-tau181 (12B, Lumipulse), and total tau (12C, Lumipulse), suggesting that CSF tau aggregates are linked to tau phosphorylation and total tau burden in AD. (12D): CSF tau aggregates showed a strong association with neurofilament light (NfL), a marker of axonal degeneration, indicating a possible link between tau aggregation and neurodegeneration. (12E-12F): Higher CSF tau aggregate levels were associated with increased tau PET standardized uptake value ratio (SUVR) in global cortical regions (12E) and neocortical regions (12F), demonstrating that CSF tau aggregates reflect in vivo tau deposition. Each point represents an individual participant, and the red line indicates a regression fit with 95% confidence intervals (shaded area). These findings suggest that CSF tau aggregates serve as a fluid-based biomarker of tau pathology, correlating with both biochemical and imaging markers of AD progression.

DETAILED DESCRIPTION

The present invention generally relates to monoclonal antibodies, or antigen-binding fragments thereof, binding specifically to human tau, and to an immunoassay kit and methods involving the use of such monoclonal antibodies, or antigen-binding fragments thereof.

The tau proteins form a group of six highly soluble protein isoforms produced by alternative splicing from the gene MAPT (microtubule-associated protein tau). They have roles primarily in maintaining the stability of microtubules in axons and are abundant in the neurons of the central nervous system (CNS), where the cerebral cortex has the highest abundance. They are less common elsewhere but are also expressed at low levels in CNS astrocytes and oligodendrocytes.

Pathologies and dementias of the nervous system, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are associated with tau proteins that have become hyperphosphorylated insoluble aggregates called neurofibrillary tangles (NFTs). Hyperphosphorylation of the tau protein can result in the self-assembly of tangles of paired helical filaments (PHFs) and straight filaments, which are involved in the pathogenesis of AD, frontotemporal dementia and other tauopathies. These fibrils are polymers of smaller aggregates including oligomers and protofibrils. These soluble pre-NFT/PHF tau forms play fundamental roles in AD pathophysiology by inducing neurotoxicity and by interacting with endogenous tau and other proteins that can ultimately lead to the dysfunction of different cellular pathways. Accordingly, accurate quantification of the soluble pre-NFT/PHF species is critical to evaluating the earlier cascade of pathophysiological events. However, their accurate quantification remains challenging due to their heterogeneous and transient nature, and the lack of analytical methods for their measurement.

The present invention relates to monoclonal antibodies, or antigen-binding fragments thereof, that bind specifically to tau, and in particular to an epitope within the MTBR within the tau protein. The monoclonal antibodies, or the antigen-binding fragments thereof, thereby bind to oligomeric tau and soluble tau aggregates.

The terms “multimers” and “aggregates” as used herein to refer all non-single unit, i.e. , non-monomeric, assemblies of tau protein including oligomers, protofibrils, fibrils, PHFs, and NFTs. Furthermore, the terms “tau oligomers” or “oligomeric tau” and “soluble tau aggregates” are used to refer to the aggregates that are larger than monomers but smaller than fibrils, PHFs, and NFTs. They are the soluble forms of tau protein aggregates that in can be secreted into body fluids, to distinguish them from insoluble PHFs and NFTs.

An aspect of the invention relates to a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to human tau. The monoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain variable region (VH) complementary determining region 1 (CDR1) consisting of RYWMN as defined in SEQ ID NO: 1. The monoclonal antibody, or the antigen-binding fragment thereof, has a VH CDR2 consisting of QIYPGDGDTNYNSKFKV as defined in SEQ ID NO: 2 and a VH CDR3 consisting of SWAY as defined in SEQ ID NO: 3. The monoclonal antibody, or the antigen-binding fragment thereof, has a light chain variable region (VL) CDR1 consisting of SASSSVNHTH as defined in SEQ ID NO: 4, a VL CDR2 consisting of DTSKLAS as defined in SEQ ID NO: 5, and a VL CDR3 consisting of FQGSGYPLT as defined in SEQ ID NO: 6.

The monoclonal antibodies, or the antigen-binding fragments thereof, bind within an aggregation-prone microtuble binding region (MTBR) within the tau protein. This MTBR consists of domains that regulate the aggregation propensity of the tau protein. Specific stretches of amino acids in the MTBR, generally referred to as hexapeptide motifs, can aggregate into oligomers and further into fibrils.

In more detail, experimental data as presented herein shows that the monoclonal antibody, or the antigenbinding fragment thereof, binds specifically to an epitope consisting of amino acids 331 to 361 of human tau as defined in SEQ ID NO: 25. This epitope of amino acids 331 to 361 of human tau is defined in SEQ ID NO: 26. The VH and VL of the monoclonal antibodies, or the antigen-binding fragments thereof, consist of alternative framework regions (FRs) and CDRs in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In an embodiment, VH FR1 comprises, preferably consists of, QVQLQQSGAELVRPGSSVQISCKASGYVFS as defined in SEQ ID NO: 7. In an embodiment, VH FR2 comprises, preferably consists of, WVKQRPGQGLEWIG as defined in SEQ ID NO: 8. In an embodiment, VH FR3 comprises, preferably consists of, KATVTADRSSSTAYMQLTSLTSDDSAVYYCVR as defined in SEQ ID NO: 9. In an embodiment, VH FR4 comprises, preferably consists of, WGQGTTLTVSS as defined in SEQ ID NO: 10.

In an embodiment, the VH comprises the amino acid sequence as defined in SEQ ID NO: 11. In a particular embodiment, the VH consists of the amino acid sequence as defined in SEQ ID NO: 11.

In an embodiment, VL FR1 comprises, preferably consists of, ENILTQSPAIMSASPGEKVTLTC as defined in SEQ ID NO: 12. In an embodiment, VL FR2 comprises, preferably consists of, WYQQKSKTSPKLWIY as defined in SEQ ID NO: 13. In an embodiment, VL FR3 comprises, preferably consists of, GVPGRFSGSGSGKSYSLTISRMEPEDVATYYC as defined in SEQ ID NO: 14. In an embodiment, VL FR4 comprises, preferably consists of, FGGGTKLEIK as defined in SEQ ID NO: 15.

In an embodiment, the VL comprises the amino acid sequence as defined in SEQ ID NO: 16. In a particular embodiment, the VL consists of the amino acid sequence as defined in SEQ ID NO: 16.

In an embodiment, the heavy chain of the monoclonal antibodies, or the antigen-binding fragments thereof, comprises the heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain of the monoclonal antibodies, or the antigen-binding fragments thereof, comprises the light chain variable region (LH) and a light chain constant region (CL).

In an embodiment, the CH comprises the amino acid sequence as defined in SEQ ID NO: 17. In a particular embodiment, the CH consists of the amino acid sequence as defined in SEQ ID NO: 17.

In an embodiment, the CL comprises the amino acid sequence as defined in SEQ ID NO: 18. In a particular embodiment, the CL consists of the amino acid sequence as defined in SEQ ID NO: 18. In an embodiment, the heavy chain comprises the amino acid sequence as defined in SEQ ID NO: 19 or 20. In an embodiment, the heavy chain consists of the amino acid sequence as defined in SEQ ID NO: 19 or 20. SEQ I D NO: 19 defi nes the heavy chain with the VH and CH, whereas SEQ I D NO: 20 additionally comprises an N-terminal signal peptide as defined in SEQ ID NO: 21.

The optional signal peptide destinates the heavy chain, when synthesized in a host cell, toward the secretory pathway. Any such signal peptide that promotes secretion of the heavy chain when produced in a host cell can be used according to the embodiments and the signal peptide as defined in SEQ ID NO: 21 should merely be seen as an illustrative, but non-limiting, example of a signal peptide that could be used according to the embodiments.

In an embodiment, the light chain comprises the amino acid sequence as defined in SEQ ID NO: 22 or 23. In an embodiment, the light chain consists of the amino acid sequence as defined in SEQ ID NO: 22 or 23. SEQ ID NO: 22 defines the light chain with the VL and CL, whereas SEQ ID NO: 23 additionally comprises an N-terminal signal peptide as defined in SEQ ID NO: 24.

Any such signal peptide that promotes secretion of the light chain when produced in a host cell can be used according to the embodiments and the signal peptide as defined in SEQ ID NO: 24 should merely be seen as an illustrative, but non-limiting, example of a signal peptide that could be used according to the embodiments.

The antigen-binding fragment of the monoclonal antibody can be any fragment of a monoclonal antibody capable of binding specifically to oligomeric tau and soluble tau aggregates and can be selected from a group consisting of a single chain antibody, a Fv fragment, a scFv fragment, a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, and a di-scFv fragment.

The monoclonal antibody, or antigen-binding fragment thereof, is preferably an isolated monoclonal antibody, or an antigen-binding fragment thereof, such as isolated from the supernatant of a hybridoma or host cell used for antibody production.

The monoclonal antibody, or the antigen-binding fragment thereof, can be a humanized monoclonal antibody, or a humanized antigen-binding fragment thereof, or a chimeric monoclonal antibody, or a chimeric antigen-binding fragment thereof, capable of binding specifically to oligomeric tau and soluble tau aggregates.

The specificity of a monoclonal antibody, or an antigen-binding fragment thereof, can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with the monoclonal antibody, or the antigen-binding fragment thereof, ( d), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the monoclonal antibody, or the antigen-binding fragment thereof. The lesser the value of d, the stronger the binding strength between the antigenic determinant and the monoclonal antibody, or the antigen-binding fragment thereof. Alternatively, the affinity can also be expressed as the affinity constant ( _a), which is 1/ d. As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

Avidity is the measure of the strength of binding between a monoclonal antibody, or an antigen-binding fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the monoclonal antibody, or the antigen-binding fragment thereof, and the number of pertinent binding sites present on the monoclonal antibody, or the antigenbinding fragment thereof.

Typically, monoclonal antibodies, or antigen-binding fragments thereof, will bind to their antigen with a dissociation constant ( d) of 10⁵ to 10 ¹² moles/liter (M) or less, and preferably 10⁷ to 10 ¹² M or less and more preferably 10⁸ to 10 ¹² M, i.e. with an association constant ( _a) of 10⁵ to 10¹² M ¹ or more, and preferably 10⁷ to 10¹² M ¹ or more and more preferably 10⁸ to 10¹² M ¹.

Generally, any d value greater than 10⁴ M (or any K_a value lower than 10⁴ M ¹) is generally considered to indicate non-specific binding.

Preferably, the monoclonal antibody, or the antigen-binding fragment thereof, will bind oligomeric tau and soluble tau aggregates with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM.

Specific binding of a monoclonal antibody, or an antigen-binding fragment thereof, to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (El A) and sandwich competition assays, and the different variants thereof known per se in the art.

The monoclonal antibodies, or the antigen-binding fragments thereof, can be used in an immunoassay kit for detection of oligomeric tau and soluble tau aggregates. In an embodiment, the immunoassay kit comprises a capture antibody, or an antigen-binding fragment thereof, according to the invention and as described in the foregoing, and a detection antibody, or an antigen-binding fragment thereof, according to the invention and as described in the foregoing.

The immunoassay kit is preferably in the form of a homogenous immunoassay, also referred to as singleantibody immunoassay kit. In such an embodiment, a same monoclonal antibody, or antigen-binding fragment thereof, is used as both capture antibody and detection antibody in the immunoassay kit. Thus, in a preferred embodiment, the capture antibody and the detection antibody of the immunoassay kit comprise the same VH and VL CDRS as defined in SEQ ID NO: 1-6. The capture antibody and the detection antibody may, though, have different VH and/or VL FRS, and/or different CH and CL. In a preferred embodiment, the capture antibody and the detection antibody have the same amino acid sequence.

Oligomeric tau and soluble tau aggregates comprises multiple, i.e., at least two, tau monomers. This means that the oligomeric tau and the soluble tau aggregates comprise multiple epitopes, to which the monoclonal antibody, or the antigen-binding fragment thereof, binds specifically. Thus, the presence of multiple such epitopes in the oligomeric tau and the soluble tau aggregates enables detection of such soluble tau species with an immunoassay kit that uses the same monoclonal antibody, or the antigenbinding fragment thereof, as both capture antibody and detection antibody.

The capture antibody, or the antigen-binding fragment thereof, of the immunoassay kit is employed to capture the oligomeric tau and soluble tau aggregate present in a sample, and preferably to immobilize the antigen, i.e., the oligomeric tau and soluble tau aggregates The detection antibody, or the antigenbinding fragment thereof, can then be used to detect the captured, and preferably immobilized, oligomeric tau and soluble tau aggregate.

Hence, in an embodiment, the immunoassay kit is a sandwich immunoassay kit. This means that the capture and detection antibodies, or the antigen-binding fragments thereof, can simultaneously bind the same oligomeric tau or soluble tau aggregate due to the presence of multiple copies of the epitope as defined in SEQ ID NO: 26 per oligomeric tau and soluble tau aggregate.

In an embodiment, the immunoassay kit comprises a solid support and the capture antibody, or the antigen-binding fragment thereof, is then attached to the solid support.

Illustrative, but non-limiting, examples of such solid supports that could be used according to the embodiments include microtiter plates (MOP), also referred to as microplates, microwell plates or multiwells in the art; beads, such as magnetic or paramagnetic beads, such as SIMOA® beads, or DYNABEADS®; a gel matrix or beads, such as SEPHAROSE®.

In a preferred embodiment, the capture antibody, or the antigen-binding fragment thereof, is attached to beads, preferably magnetic beads, such as paramagnetic beads.

In an embodiment, the immunoassay kit is an enzyme-linked immunosorbent assay (ELISA) kit and preferably a sandwich ELISA.

A sandwich ELISA can be used to detect oligomeric tau and soluble tau aggregates in a sample by preparing a surface of the solid support, to which the capture antibody, or the antigen-binding fragment thereof, is bound or attached. In a preferred embodiment, a known quantity of the capture antibody, or the antigen-binding fragment thereof, is attached to the surface of the solid support. Any nonspecific binding sites on the surface are optionally but preferably blocked. The sample is then applied to the surface so that any oligomeric tau and soluble tau aggregates present therein will be captured by the immobilized capture antibodies, or the antigen-binding fragments thereof. Unbound material is preferably removed by one or multiple washing steps. The detection antibody, or the antigen-binding fragment thereof, is then added and is allowed to bind to any oligomeric tau and soluble tau aggregates captured by the capture antibody, or the antigen-binding fragment thereof.

The amount of bound detection antibody, or the antigen-binding fragment thereof, is then determined in direct or indirection detection methods. For instance, a label or enzyme can be attached directly to the detection antibody, or the antigen-binding fragment thereof, or indirectly via a link, such as a biotinstreptavidin or a biotin-avidin link. It is, alternatively, possible to use a secondary antibody that is labeled or connected to an enzyme and binds specifically to the detection antibody, or the antigen-binding fragment thereof. Hence, in an embodiment the detection antibody, or the antigen-binding fragment thereof, is attached to biotin. Alternatively, the detection antibody, or the antigen-binding fragment thereof, is attached streptavidin or avidin. Thus, the detection antibody, or the antigen-binding fragment thereof, is attached to one of i) biotin and ii) streptavidin or avidin.

The immunoassay kit preferably also comprises an enzyme attached to the other of i) biotin and ii) streptavidin or avidin. The immunoassay kit preferably also comprises a substrate that can be converted by the enzyme into a detectable product.

In an embodiment, the enzyme is horseradish peroxidase (EC 1 .11 .1 .7, also referred to as HRP) and the HRP substrate could then be selected from the group consisting of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate, a 3,3’-diaminobenzidine (DAB) substrate or a 2,2’-azino-bis (3-ethylbenzothiazoline-6- sulphonic acid (ABTS) substrate.

For instance, the immunoassay kit comprises HRP labeled strepatividin or HRP labeled avidin. Alternatively, the immunoassay kit comprises HRP labeled biotin. The immunoassay kit also comprises a HRP substrate. In such a case, the amount of oligomeric tau and soluble tau aggregates in a sample can be determined by spectrophotometric methods that detect the conversion of the substrate by HRP into a colored product that is detectable.

In another embodiment, the enzyme is p-galactosidase (EC 3.2.1.23, also referred to as p-gal or p-D- galactoside galactohydrolase) and the substrate is 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X-gal) that can be converted by p-galactosidase into 5,5'-dibromo-4,4'-dichloro-indigo. In more detail, p- galactosidase cleaves the glycosidic bond in X-gal and form galactose and 5-bromo-4-chloro-3- hydroxyindole, which dimerizes and oxidizes into 5,5'-dibromo-4,4'-dichloro-indigo, which has an intense blue color that is easy to identify and quantify.

For instance, the immunoassay kit comprises p-galactosidase labeled strepatividin or p-galactosidase labeled avidin. Alternatively, the immunoassay kit comprises p-galactosidase labeled biotin. The immunoassay kit also comprises a p-galactosidase substrate. In such a case, the amount of oligomeric tau and soluble tau aggregates in a sample can be determined by spectrophotometric methods that detect the conversion of the substrate by p-galactosidase into a colored product that is detectable. In another embodiment, the immunoassay is a chemiluminescent immunoassay (CLIA).

A CLIA can be used to detect oligomeric tau and soluble tau aggregates in a sample by attaching the capture antibodies, or the antigen-binding fragments thereof, to magnetic beads or particles. The magnetic beads or particles coupled with capture antibodies, or antigen-binding fragments thereof, are added to the sample, or vice versa. Detection antibodies, or antigen-binding fragments thereof, capable of generating a fluorescent product are then added. An immunocomplex is then formed between the a capture antibody, or the antigen-binding fragment thereof, immobilized on a magnetic bead or particle, an oligomeric tau or soluble tau aggregate and the detection antibody, or the antigen-binding fragment thereof.

The immunoassay kit does not necessarily have to be an ELISA or CLIA kit. In a further embodiment, the immunoassay kit uses affinity chromatography where the capture antibody, or the antigen-binding fragment thereof, is bound to the stationary phase, such as to a gel matrix or beads in a column. In such a case, oligomeric tau and soluble tau aggregates present in a sample will be entrapped in the column through binding to the immobilized capture antibodies, or the antigen-binding fragments thereof. Following washing, the bound oligomeric tau and soluble tau aggregates can be eluded and detected using the detection antibody, or the antigen-binding fragment thereof. For instance, the amount of eluded oligomeric tau and soluble tau aggregates can be determined using Western blotting with the detection antibody, or the antigen-binding fragment thereof, using direct or indirect detection methods.

In an embodiment, the sample to be measured for the presence and preferably quantification of oligomeric tau or soluble tau aggregates is preferably contacted with pre-formed tau aggregates and incubated with the pre-formed tau aggregates prior to measuring oligomeric tau and soluble tau aggregates using the immunoassay kit.

Such pre-formed tau aggregates then act as amplifier, i.e. , as a catalytic site that facilitates aggregation of aggregation-prone oligomeric tau forms and small and soluble tau aggregates. Experimental data as presented herein shows that minute amounts of such amplifier, i.e., pre-formed tau aggregates, can be used to get detectable signals showing the presence of oligomeric tau and soluble tau aggregates in body fluid samples from both AD and non-AD individuals. As is shown in Fig. 9, relative amounts of oligomeric tau and soluble tau aggregates in AD and non-AD individuals were substantially the same when measured using the immunoassay without any pretreatment of the body fluid sample (cerebrospinal fluid sample) with pre-formed tau aggregates (Fig. 9A). Further, the body fluid sample with added pre-formed tau aggregates is preferably incubated for a period of time prior to measuring the presence of oligomeric tau and soluble tau aggregates in the body fluid sample. Such an incubation leads to a differentiation in amounts of oligomeric tau and soluble tau aggregates in AD and non-AD individuals as is seen by comparing Figs. 9B and 9C.

Thus, in an embodiment, the immunoassay kit preferably comprises pre-formed tau aggregates.

Such pre-formed tau aggregates are preferably produced using full-length monomeric recombinant tau1 - 441 protein (SEQ ID NO: 25). For instance, the monomeric recombinant tau1 -441 protein can be diluted in or added to a buffer, such as phosphate buffered saline (PBS), optionally comprising a metal ion sequester, such as ethylenediaminetetraacetic acid (EDTA), and then incubated for a period of time, such as at least 24 hours, preferably at least 48 hours, more preferably at least 60 hours, such as at least 72 hours, on a shaker, i.e., under agitation, at room temperature (20-25°C), or preferably above room temperature, such as from 30 up to 45°C, more preferably from 35 up to 40°C, and most preferably about 37°C.

As mentioned in the foregoing, minute amounts of such pre-formed tau aggregates as amplifier can be used to detect and quantify oligomeric tau and soluble tau aggregates in body fluid samples. For instance, a volume of pre-formed tau aggregates at a concentration of no more than 2 nM, preferably no more than 1 .5 nM, more preferably no more than 1 nM, and most preferably no more than 0.75 nM, or even lower, such as no more than 0.5 nM or no more than 0.25 nM, could be used as amplifier. Thus, the amplifier solution preferably comprises pre-formed tau aggregates at a concentration selected within an interval of from 0.05 up to 1 nM, preferably selected within an interval of from 0.1 up to 0.75 nM, more preferably selected within an interval of from 0.2 up to 0.5 nM, and most preferably about 0.25 nM.

In an embodiment, the sample, such as body fluid sample, to be analyzed using the immunoassay kit is preferably treated with and incubated with an amplifier solution comprising pre-formed tau aggregates. In such a case, the sample, such as body fluid sample, could be combined at a volume ratio ((body fluid) sample : amplifier solution) selected within an interval of from 1 :0.1 up to 1 :2, preferably selected within an interval of from 1 :0.2 up to 1 :1 , more preferably selected within an interval of from 1 :0.25 up to 1 : 0.75, such as about 1 :0.5.

Another aspect of the embodiments relates to a method for determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample. The method comprises contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit. The method also comprises determining an amount of oligomeric tau and soluble tau aggregates in the body fluid sample by determining an amount of bound detection antibody, or the antigen-binding fragment thereof.

In an embodiment, contacting the body fluid sample comprises contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigenbinding fragment thereof, of the immunoassay kit comprising the enzyme attached to i) biotin or ii) streptavidin or avidin, and the substrate. In such an embodiment, determining the amount of oligomeric tau and soluble tau aggregates comprises determining the amount of oligomeric tau and soluble tau aggregates in the body fluid sample by determining an amount of the detectable product.

The detection of the detectable product can be done using, for instance, colorimetric, chemiluminescence, fluorescent, or luminescent measurements depending on the particular enzyme and detectable product.

In an embodiment, the method further comprises contacting the body fluid sample with pre-formed tau aggregates and incubating the body fluid sample with the pre-formed tau aggregates prior to contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit.

In an embodiment, the method further comprises producing pre-formed tau aggregates. In a particular embodiment, producing pre-formed tau aggregates comprises diluting or adding monomeric recombinant tau1 -441 protein in a buffer, such as PBS, optionally comprising a metal ion sequester, such as EDTA, and then incubating the monomeric recombinant tau1 -441 protein for a period of time, such as at least 24 hours, preferably at least 48 hours, more preferably at least 60 hours, such as at least 72 hours, on a shaker, i.e., under agitation, at room temperature, or preferably above room temperature, such as from 30 up to 45°C, more preferably from 35 up to 40°C, and most preferably about 37°C, to form an amplifier solution comprising pre-formed tau aggregates. In an embodiment, contacting the body fluid sample with pre-formed tau aggregates comprising contacting the body fluid sample with an amplifier solution comprising the pre-formed tau aggregates at a concentration of no more than 2 nM, preferably no more than 1 .5 nM, more preferably no more than 1 nM, and most preferably no more than 0.75 nM, or even lower, such as no more than 0.5 nM or no more than 0.25 nM. In an embodiment, the amplifier solution preferably comprises pre-formed tau aggregates at a concentration selected within an interval of from 0.05 up to 1 nM, preferably selected within an interval of from 0.1 up to 0.75 nM, more preferably selected within an interval of from 0.2 up to 0.5 nM, and most preferably about 0.25 nM.

In an embodiment, contacting the body fluid sample with pre-formed tau aggregates comprising contacting the body fluid sample with an amplifier solution comprising the pre-formed tau aggregates at a volume ratio of body fluid sample : amplifier solution selected within an interval of from 1 :0.1 up to 1 :2, preferably selected within an interval of from 1 :0.2 up to 1 :1 , more preferably selected within an interval of from 1 : 0.25 up to 1 : 0.75, such as about 1 :0.5.

In an embodiment, incubating the body fluid sample comprises incubating the body fluid sample with the pre-formed tau aggregates for at least 24 hours, preferably at least 30 hours, and more preferably at least 36 hours, prior to contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit.

In an embodiment, the body fluid sample is selected from the group consisting of a cerebrospinal fluid sample, a blood sample, a blood serum sample and a blood plasma sample. In an embodiment, the body fluid sample is a cerebrospinal fluid sample. In another embodiment, the body fluid sample is a blood serum or plasma sample.

A further aspect of the invention relates to a method for diagnosing Alzheimer’s disease in a subject. The method comprises determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample from the subject according to above and determining the subject as suffering from Alzheimer’s disease or not based on the determined amount of oligomeric tau and soluble tau aggregates.

In a particular embodiment, the method also comprises comparing the determined amount of oligomeric tau and soluble tau aggregates with a threshold value and then determining the subject as suffering from Alzheimer’s disease if the determined amount of oligomeric tau and soluble tau aggregates exceeds the threshold value and otherwise the subject as not suffering from Alzheimer’s disease.

The threshold value used for differentiating Alzheimer subjects from subjects not likely to suffer from Alzheimer’s disease depends at least partly on the particular body fluid sample used. Fig. 9C illustrates an example of differentiating AD subjects from non-affected subjects using CSF as body fluid sample.

Yet another aspect of the invention relates to method for classifying a neurological disease in a subject. The method comprises determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample from the subject according to above. The method also comprises classifying the neurological disease as Alzheimer’s disease or a neurological disease other than Alzheimer’s disease based on the determined amount of oligomeric tau and soluble tau aggregates.

Alzheimer’s disease is characterized by the presence of oligomeric tau and soluble tau aggregates whereas other neurological diseases and neurodegenerative tauopathies generally have tau PHFs and fibrils distinguishable from such oligomeric tau and soluble tau aggregates. Thus, a subject suffering from a neurological disease could be classified as likely suffering from AD (high amount of oligomeric tau and soluble tau aggregates) or likely suffering from a neurological disease other than Alzheimer’s disease (low amount of oligomeric tau and soluble tau aggregates) depending on the determined amount of oligomeric tau and soluble tau aggregates in the body fluid sample taken from the subject.

The subject as referred to herein is preferably a human subject.

The monoclonal antibody, or the antigen-binding fragment thereof, of the invention could, when bound specifically to oligomeric tau or a soluble tau aggregate prevents or at least inhibits further aggregation of the oligomeric tau or the soluble tau aggregate into non-soluble tau fibrils and filaments, including PHFs and NFTs. The monoclonal antibody, or the antigen-binding fragment thereof, could therefore be used as a medicament and for treatment of Alzheimer’s disease.

A related aspect is therefore the use of the monoclonal antibody, or the antigen-binding fragment thereof, for the manufacture of a medicament for treatment of Alzheimer’s disease. A further relates aspect defines a method of treating Alzheimer’s disease. The method comprises administering an effective amount of a monoclonal antibody, or an antigen-binding fragment thereof, according to the invention to a subject.

As used herein, effective amount indicates an amount effective, at dosages and for periods of time necessary to achieve a desired result. For example, in the context of treating Alzheimer’s disease an effective amount is an amount that, for example, inhibits or prevents aggregation of oligomeric tau and soluble tau aggregates into non-soluble tau fibrils and filaments, including PHFs and NFTS, compared to the response obtained without administration of the monoclonal antibody, or an antigen-binding fragment thereof. Effective amounts may vary according to factors, such as the disease state, age, sex, weight of the subject. Treating or treatment as used herein and is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results could include, for instance, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized state of disease, i.e., prevent worsening, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission. Treating or treatment may also prolong survival as compared to expected survival if not receiving any treatment.

Treating as used herein also encompass preventing Alzheimer’s disease. Preventing or prophylaxis as used herein and is well understood in the art, means an approach in which a risk of developing a disease or condition is reduced or prevented, including prolonging or delaying disease development. For instance, a subject predisposed to develop Alzheimer’s disease, such as due to genetic or hereditary predisposition, could benefit for administration of the monoclonal antibody, or the antigen-binding fragment thereof, according to the embodiments to prevent, reduce the risk of, delaying and/or slowing development of Alzheimer’s disease.

The embodiments also relate to a pharmaceutical composition comprising a monoclonal antibody, or an antigen-binding fragment thereof, according to the embodiments, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers could be any pharmaceutically acceptable carrier, vehicle and/or excipient, including combinations thereof, that are is or are compatible with the other constituent(s) of the pharmaceutical composition. Non-limiting examples of such pharmaceutically acceptable carriers include injection solutions, such as saline or buffered injection solutions. The monoclonal antibody, or the antigen-binding fragment thereof, and/or the pharmaceutical composition according to the embodiments may be administered to the subject according to various routes including, but not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intracerebral or intracranial administration.

A further aspect of the embodiments includes a nucleic acid molecule encoding a monoclonal antibody, or an antigen-binding fragment thereof, according to the embodiments. Nucleic acid molecule as used herein includes polynucleotide, oligonucleotide, and nucleic acid sequence, and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, non-natural or altered nucleotides, and which may contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

Another aspect of the embodiments relates to an expression vector comprising a promoter and a nucleic acid molecule encoding a monoclonal antibody, or an antigen-binding fragment thereof, according to the embodiments operatively controlled by the promoter.

The expression vector comprises at least one nucleic acid molecule comprising coding sequences that can be expressed, such as transcribed and translated, in a host cell comprising the expression vector. The expression vector is in an embodiment selected among DNA molecules, RNA molecules, plasmids, episomal plasmids and virus vectors.

The nucleic acid molecule encoding the monoclonal antibody, or an antigen-binding fragment thereof, is operatively controlled by the promoter in the expression vector, i.e., is under transcriptional control of the promoter. In an embodiment, the promoter is selected from the group consisting of the human EF1a promoter, the CMV promoter, the CAG promoter, the PGK promoter, the TRE promoter, the U6 promoter and the UAS promoter if the host cell is an eukaryotic cell, such as a human cell. Illustrative, but nonlimiting, examples of a promoter that could be used if the host cell is a bacterial cell include the T5 promoter, the T7 promoter, the rham promoter, the phoA promoter, the Sp6 promoter, the lac promoter, the AraBad promoter, the trp promoter and the Ptac promoter. If the host cell is a yeast cell the promoter could be selected from the group consisting of the CYC1 promoter, the ADH1 promoter, the TEF2 promoter, the pCYC promoter, a PGAL promoter and the GFD promoter as illustrative, but non-limiting, examples. A further aspect of the embodiments relates to a host cell comprising an expression vector according to the embodiments. In such an aspect, the promoter is constitutively active in the host cell or inducible active in the host cell. This means that the nucleic acid molecule encoding that the monoclonal antibody, or an antigen-binding fragment thereof, can then be transcribed in the host cell to produce the monoclonal antibody, or an antigen-binding fragment thereof, in the host cell.

In an embodiment, the host cell is selected from the group consisting of a bacterial cell, a yeast cell, and an eukaryotic cell, such as a human cell.

SEQ ID NO: 27 is an example a nucleotide sequence encoding the heavy chain of the monoclonal antibody with a N-terminal signal peptide, whereas SEQ ID NO: 28 is an example of a nucleotide sequence encoding the light chain of the monoclonal antibody with a N-terminal signal peptide.

EXAMPLES

EXAMPLE 1 - Generation of anti-tau antibodies

19 new monoclonal antibodies (mAbs) against tau were generated by immunizing 8-week-old Balb/c mice with a recombinant tau241 -441 protein (100 pg) in complete Freund’s adjuvant (Sigma). After two to three additional dosages of the recombinant protein (100 pg/mouse) in Freund’s Incomplete adjuvant (Sigma), mice were sacrificed, the spleen removed, and B-cells fused with the myeloma cell line SP2/0 following standard procedures. Approximately 10 days after the fusion, the cell media were screened for tau antibodies using full-length recombinant tau1 — 441 (2N4R) as well as the tau241 — 441 construct by direct enzyme-linked immunosorbent assay (ELISA). Clones that reacted positively with the recombinant tau proteins were further grown, subcloned, and subsequently frozen in liquid nitrogen. Antibody specificity against tau was verified, and the isotype was determined using a commercially available kit (Pierce Rapid Isotyping Kit-Mouse). Finally, antibodies were purified using a Hitrap protein G column (Cytiva) according to the manufacturer’s instructions. Epitope mapping for each mAb was performed by direct ELISA against five recombinant overlapping (10 amino acids) peptides spanning the tau241— 441 sequence (Casio ApS, Denmark), specifically tau241 -291 , tau281— 331 , tau321— 371 , tau361— 411 , and tau 401-441.

The panel of 19 monoclonal antibodies collectively covered the entire primary sequence of the tau protein.

Among these 19 monoclonal antibodies, five subclones recognized the same epitope within the sequence spanning residues 331-361 . Given the binding data, two of the monoclonal antibodies CT16.1 and CT19.1 were selected for further characterization.

To ensure the specificity of epitope recognition, surface plasmon resonance (SPR) experiments were performed using a BIACORE® T1000 biosensor (GE Healthcare) for the monoclonal antibodies CT16.1 and CT19.1. The ligand (CT16.1 or CT19.1 monoclonal antibody) was immobilized on a CM5 surface chip at a flow rate of 5 pL/min using standard amino coupling reagents (Cytiva) to an immobilization level of 4000 RU. The analytes were subsequently injected at a flow rate of 20 pL/minute. All SPR experiments were performed in degassed phosphate-buffered saline (PBS) at 25°C.

The N-terminal tau fragment 1-224 and C-terminal tau fragment 368-441 are outside the epitope region. Therefore, their respective sensorgrams served as baseline signals in Figs. 1A and 1 B. Conversely, the sensorgrams for the 258-368 and 302-368 peptides are expected to generate higher signals. As is shown by comparing Fig. 1A and 1 B, the monoclonal antibody CT19.1 had higher specificity for the epitope fragments and no binding to the N-terminal and C-terminal fragments, whereas monoclonal antibody CT16.1 not only bound the epitope fragments but also interacted with the N-terminal tau fragment 1-224, see Fig. 1 B. Thus, the monoclonal antibody CT19.1 was selected due to its specificity to the epitope fragments and due to no interaction with the N-terminal and C-terminal tau fragments.

EXAMPLE 2 - Epitope mapping using direct ELISA

A total of 11 peptides (Fig. 1 C), collectively covering amino acid residues 326-366 of tau, were serially diluted in coating buffer (50 mM NaHCOs) to the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 ng/mL. Each concentration (100 pL) was added to wells in an ELISA plate and incubated overnight at 4°C. The wells were washed four times with 400 pL of wash buffer (PBS+ 0.05% TWEEN®). Thereafter, the wells were blocked with 200 pL of blocking buffer (PBS-TWEEN®, 1 % BSA) for 1 hour at room temperature. Then, the wells were washed twice with 400 pL of wash buffer. A total 100 pL of biotinylated CT19.1 antibody (1 pg/mL) was added to each well and the plate was incubated at room temperature for 3 hours. The wells were washed four times with 400 pL of wash buffer. A total 100 pL of streptavidin-horseradish peroxidase (Strep-HRP) diluted in PBS-TWEEN® with 1 % BSA was added to each well and incubate for 30 minutes at room temperature. Then, the wells were washed four times with 400 pL of wash buffer. TMB chromogen substrate (100 pL) was added to each well and incubated in the dark at room temperature for 15 minutes. The reaction was terminated by adding 100 pL of sulfuric acid to each well. Finaly, the absorbance was measured using Tecan Sunrise microplate reader (Cat#30087502) at 450 nm with a reference wavelength of 650 nm within 15 minutes of stopping the reaction.

This Example showed differential reactivity of the CT19.1 antibody with various tau peptides, collectively covering amino acid residues 326-366. Peptides 1, 2, and 3 (spanning residues 326-350) demonstrated high reactivity, indicating that the CT19.1 antibody recognized an epitope within these sequences, see Fig. 1 C. Peptides 4-11 showed no reactivity, confirming the specificity of CT19.1 to the identified epitope region, see Fig. 1 C.

EXAMPLE 3 - Monoclonal antibody CT19.1 sequencing

Total RNA was isolated from the hybridoma cells expressing the monoclonal antibody CT19.1 following the technical manual of Zymogen Quick-RNA™ Miniprep Kit. Total RNA was then reverse transcribed into cDNA using isotype-specific anti-sense primers or universal primers following the technical manual of SMARTScribe™ Reverse Transcriptase Kit. The antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No less than five colonies with inserts of correct sizes were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones was provided.

Heavy chain: DNA sequence (396 bp) - SEQ ID NO: 27

Signal sequence-FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4-constant region-stop codon

ATGGAATGGCCTTGTATCTTTCTCTTCCTCCTGTCAGTAACTGAAGGTGTCCACTCTCAGGT TCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGCAGATCTCCTGTA AGGCTTCTGGCTATGTATTCAGTAGGTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAG

GG T C T T GAG T GGAT T GGACAGAT T TATCC TGGAGATGGTGATACCAAC TACAAT TCAAAAT T

CAAGGTTAAAGCCACAGTGACTGCAGACAGATCCTCCAGCACTGCCTACATGCAGCTCACCA GCCTAACGTCTGATGACTCTGCGGTCTATTACTGTGTCAGATCGTGGGCCTACTGGGGCCAA G G GAG GAG T C T GAG AG T C T C C T GAG C C AAAAC AAC AC C C C C AT GAG T C T AT C GAG T G G C C C C TGGGTGTGGAGATACAACTGGTTCCTCCGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCC CTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCA GCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTG GCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAA AACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCAC AAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAA GGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGG AT GAG C C AGAC G T C C AGAT GAG CTGGTTTGT GAAC AAC G T G GAAG TAG AC AC AG C T C AGAC A CAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGCACCCTCCCCATCCAGCA CCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCAC C C AT C GAGAGAAC C AT C T C AAAAAT T AAAG G G C T AG T C AGAG C T C C AC AAG TAT AC AT C T T G CCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTT CAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGG ACACCGCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATATAGCAAGCTCAATATGAAA ACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAA

TTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATGA

Heavy chain: Amino acid sequence (132 aa) - SEQ ID NO: 20

Signal peptide-FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4-constant region

MEWPCI FL FLLSVTEGVHS QVQLQQS GAELVRPGS S VQ I S CKAS GYVFSRYWMNWVKQRPGQ GLE W I GQI YPGDGD TNYNSKFKVKAT VTADRS S S TAYMQL T S L T S DDS AVY YCVRSWAYWGQ G T T L T VS SAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFP ALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPI ST INPCPPCKECH KCPAPNLEGGPSVFI FPPNIKDVLMI SLTPKVTCVWDVSEDDPDVQI SWFVNNVEVHTAQT QTHREDYNST IRWSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERT I SKIKGLVRAPQVYIL PPPAEQLSRKDVSLTCLWGFNPGDI SVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMK TSKWEKTDS FSCNVRHEGLKNYYLKKT I SRSPGK

Light chain: DNA sequence (384 bp) - SEQ ID NO: 28

Signal seguence-FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4-constant region-stop codon

ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAG AGGAGAAAATATTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCA C C T T GAC C T GCAGTGCCAGC TCAAGTGTGAATCACACGCAC T GG T AC CAGCAGAAG T C T AAA ACCTCCCCCAAACTCTGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAGGTCGCTT CAGTGGCAGTGGGTCTGGAAAGTCTTACTCTCTCACGATCAGCCGCATGGAGCCTGAAGATG TTGCCACTTATTACTGTTTTCAGGGGAGTGGATACCCACTCACGTTCGGAGGGGGGACCAAG C TGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCA GTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCA ATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGAT CAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTA T GAAC GAG AT AAC AG CTATACCTGT GAG G C GAG T C AC AAGAC AT C AAC T T GAG C C AT T G T C A AGAGCTTCAACAGGAATGAGTGTTAG

Light chain: Amino acid sequence (128 aa) - SEQ ID NO: 23

Signal peptide-FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4-constant region

MDFQVQI FS FLLI SASVIMSRGENI LTQS PAIMSAS PGEKVTLTCSASSSVNHTHWYQQKSK TSPKLWIYDTSKLASGVPGRFSGSGSGKSYSLT I SRMEPEDVATYYCFQGSGYPLTFGGGTK LE IKRADAAPTVS I FPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC

The isotype of CT19.1 was determined as mouse lgG2b/kappa.

EXAMPLE 4 - Ultrasensitive immunoassay for the selective quantification of tau oligomers and soluble tau aggregates

This Example describes the development and validation of a novel immunoassay that is selective for tau oligomers and related soluble tau aggregates versus the single-unit monomers. Moreover, the immunoassay, which is built on the principle of tau-tau interactions, showed signal increases according to the number of tau molecules forming the soluble tau aggregates. This feature enabled quantification of tau aggregates in solution irrespective of the fibrillization state.

Materials & Methods

Expression and purification of tau variants

Full-length tau1 -441 (Uniprot ID: P10636-8, SEQ ID NO: 25) as well as four shorter tau protein constructs (tau1 — 224, tau258— 368, tau302— 368, and tau368— 441 ) were expressed and purified using a 6xhistidine- Small Ubiquitin Like Modifier (SUMO) fusion system as described previously (Becker et al., 2018) but with modifications as described below.

Cloning

The different tau fragments were polymerase chain reaction (PCR)-amplified using primers representing the 5’ and 3’ sequence of each fragment respectively, with tau full length cDNA (RC213312, Origene) as template. PCR fragments were cloned directly into pET_SUMO plasmid, with a TA cloning site. Constructs containing SUMO-tau fusion protein were sequenced and transformed into Escherichia coli BL21 (DE3) strain for protein expression. SUMO fusion protein expression

E. coli BL21 (DE3) containing the construct was incubated overnight in 20 ml LB medium with Kanamycin at a concentration of 50 g/ml. The overnight culture was used to inoculate 1 L of LB media with Kanamycin (50 pg/ml) at 37°C and when ODeoo reached 0.5-0.7, protein expression was induced with 1.0 mM IPTG and grown overnight (o/n) at 26-28°C. The culture was centrifuged at 7000 rpm for 20 minutes at 4°C and dry weight was calculated. The pellet was stored at -20°C.

Purification

Pellet was resuspended in 1x native buffer (50 mM sodium-phosphate, pH 8.0, 0.5 M NaCI), 8 ml/g dry weight. Lyzosyme was added and the lysate was incubated on ice for 30 minutes, followed by sonication and centrifugation, 12 000 rpm for 20 minutes at 4°C, after which the supernatant was collected.

Protein extract was added to Ni_NTA agarose (Novex) equilibrated with 10 mM imidazole in 1x Native buffer and incubated with rotation at 4°C for 1 h. Ni-NTA agarose was washed with 1x Native buffer + 20 mM imidazole and 6xHis_SUMO_tau fusion protein was eluted with 250 mM imidazole in 1x Native buffer.

The purified fusion protein was dialysed against 50 mM Tris, 150 mM NaCI, pH 8.0 and protein concentration was determined with the BCA method.

SUMO cleavage

2 mg SUMO_tau fusion protein was cleaved at 4°C o/n, using 50 pg SENP-1 (0,7 mg/ml, kindly provided by Dr. Irena Burmann, University of Gothenburg) in 50 mM Tris, 150 mM NaCI, 1 mM DTT. After cleavage, 6xHis-tag and His-tagged SUMO protease was bound to Ni-NTA agarose, flowthrough containing tau protein was collected and any residual bound tau protein was eluted with imidazole gradient, starting at 20 mM. Fractions containing tau protein were collected and dialyzed against PBS.

Preparation of recombinant tau aggregates

Full-length monomeric recombinant tau1— 441 frozen in 1x PBS was used to generate aggregates. Each protein/peptide was diluted in 1x PBS, 2 mM ethylenediaminetetraacetic acid to the desired final concentration and incubated for 72 hours on a shaker (Eppendorf AG, Thermonixer comfort, model no 5355 41412) at 350 rpm at 37°C. Monomer fractions were prepared identically but were stored in cold conditions to prevent aggregation. Aliquots of the protein preparations were stored at -80°C until use. Negative-stain transmission electron microscopy

Aliquots (5 pL) of the recombinant tau aggregates prepared as described above were applied to carbon- coated, glow-discharged cop per grids. Samples were allowed to adhere onto the grids for 1 minute, and the grids were washed in ultrapure distilled water. The grids were then negatively stained for 30 seconds with 0.75% uranyl formate (Electron Microscopy Science) and examined under a Talos L120C 120 kV transmission electron microscopy (TEM) microscope (Thermo Fisher). Transmission electron micrographs were imaged with a BM-CETA camera-4.096 x 4.096, 14 pm pixel CMOS equipped with the TEM image and analysis software (Thermo Fisher).

Development of a homogeneous immunoassay for tau aggregates

To capture tau oligomers and related soluble tau aggregates, the tau CT19:1 antibody (epitope: 331— 361) was coupled to paramagnetic beads (#103207, Quanterix). To make a homogeneous sandwich assay, the same antibody was used for detection, and labeled with biotin (#A3959; Thermo Fisher) following the manufacturer’s recommendation. A three-step SIMOA® assay was established that used 20,000 beads/pL of capture antibody and 2 pg/mL of biotin-conjugated detection antibody. The streptavidin beta-galactosidase concentration was 450 pM. Sample diluent Tau 2.0 (Quanterix, REF# 103847, LOT# 300606) was used to achieve the required concentration of recombinant tau aggregates, monomers, and brain samples. Measurements were performed at the Clinical Neurochemistry Laboratory, University of Gothenburg (Molndal, Sweden) on the SIMOA® HD-X (Quanterix). The average number of enzymes per bead (AEB) signal for each sample was plotted against the concentration of the input biospecimen when known.

Assay specificity was examined by immunodepletion of Tris buffered saline (TBS) extract from an autopsy-verified AD brain using the assay antibody. The procedure has been described previously (Lantero-Rodriguez et al., 2021; Brown et al., 2023). Briefly, 4 pg of the CT19.1 antibody was conjugated to 50 pL M-280 Dynabeads (ThermoFisher Scientific). For the negative control, we conjugated 4 pg of the anti-p-Amyloid antibody 6E10 (BioLegend) to Dynabeads. Samples were brought to 1 mL total volume with the Tau 2.0 assay diluent and incubated overnight at 4°C. Thereafter, the supernatants were recovered and analyzed using the tau oligomer/soluble aggregate assay.

Human brain samples, neuropathological assessment, and serial fractionation of brain tissue

Frozen autopsy brain samples of the inferior temporal cortex (IT) and the middle frontal cortex (MF) were obtained from the University of Pittsburgh Alzheimer’s Disease Research Center brain bank. Written informed consent for research and brain autopsy was obtained for all subjects. The diagnosis of AD was made using standard diagnostic criteria (McKhann et al., 1984). The neuropathology evaluation included immunohistochemical analyses of amyloid beta (A|3), phosphorylated tau, a-synuclein, and transactive response DNA-binding protein (TDP-43), as well as routine hematoxylin and eosin and Bielschowsky silver staining. Cases with stroke, Parkinson’s disease, or hippocampal sclerosis were excluded from the study. Neuropathologic diagnosis was based on the National Institute on Aging (NIA)-Reagan Institute (Rl) criteria (NIA-RI, Consensus recommendations 1997), recommendations of the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD, Mirra et al., 1991), and Braak staging of neurofibrillary tangles (Braak and Braak 1991). Demographic and neuropathological details of the nine cases included in the study are listed in Table 1. All cases were de-identified and randomly assigned a unique lab code that was used throughout the study. Investigators were blinded to case demographics and diagnosis throughout the experiment.

Table 1 - Demographic and neuropathological characteristics of the neuropathology cohort from the University of Pittsburgh Alzheimer’s Disease Research Center

Abbreviations: AD, Alzheimer’s disease; APOE, apolipoprotein E; CAA, cerebral amyloid angiopathy; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; F, female; FTLD-TDP, frontotemporal lobar degeneration with TPD-43-immunoreactive pathology; g, grams; M, male; TDP, transactive response DNA-binding protein;

Brain tissue was homogenized using a glass dounce homogenizer at a concentration of 300 mg/mL in TBS (50 mM Tris pH 8.0, 100 mM NaCI) with protease inhibitors (AEBSF, 40 x dilution; Sigma Protease Inhibitor Cocktail P8340, 100 x dilution; Sigma Phosphatase Inhibitor Cocktail 2 P5726, 100 x dilution; Sigma Phosphatase Inhibitor Cocktail P0044, 100 x dilution). The sample was sonicated on ice for 10 seconds, centrifuged at 135,000 x g for 30 minutes in a bench-top, refrigerated (4°C) microcentrifuge, and the supernatants were collected as the TBS-Soluble Fraction. The pellet was resuspended in the same volume as centrifuged in the previous step, using ice cold 100 mM Na2CO3 (pH 11.0) with protease inhibitors, sonicated for 10 seconds on ice, and left to incubate on ice for 20 minutes. The sample was centrifuged at 135,000 x g for 30 minutes at 4°C, and the supernatant was saved as the Na2CO3-Soluble Fraction. The pellet was resuspended in the same volume as centrifuged in the previous step, using Urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM bicine, pH 8.5), sonicated on ice for 10 seconds, and incubated on ice for 20 minutes. This sample was centrifuged at 135,000 x g for 30 minutes at 4°C, and the supernatants were saved as the Urea-Soluble Fraction. All soluble fractions were aliquoted at time of collection and stored at -80°C until assayed by Simoa. Chemicals and reagents were from Sigma.

Measurement of tau oligomers and related soluble aggregates in the autopsy cohort The brain homogenate fractions were first diluted with the assay diluent at a dilution fold informed by an initial testing; 50-fold for all fractions except the urea/detergent for which a 1 :400 dilution was used. The samples were measured using the tau aggregate assay setup described above.

Measurement of tau oligomers and related soluble aggregates in CSF

CSF samples obtained from both AD and non-AD individuals (50 pL each) were subjected to treatment with 0.25 nM of pre-formed recombinant tau aggregates. The treated CSF samples were then incubated for a duration of 36 hours while placed on a shaker operating at 450 rpm and maintained at a temperature of 37°C. The amplified tau aggregates were detected using an immunoassay performed on the SIMOA® HD-X platform (Quanterix, Billerica, MA, USA), employing the CT19.1 antibody.

Statistical analysis

AEB (or relative AEB; sample signal over blank value) signals were plotted as the mean or mean ± standard deviation (SD) of the number of replicates as indicated in the figure legends. Biosensor responses were processed using the Bl Aeval uation Software, and sensorgrams were prepared using the statistical software GraphPad Prism version 9. Other figures were prepared with R version 4.2.2.

Results

Development and validation of a homogenous assay for tau oligomers and related soluble aggregates The tau aggregate assay followed the homogeneous format, using the same antibody for both capture and detection. Because a protein monomer unit has a single epitope, the reaction needed when the same antibody is used for both capture and detection in a homogeneous assay cannot happen. Contrarily, oligomers/soluble tau aggregates consist of multiple tau protein monomers; hence, the capture and detection antibodies can each have access to an epitope on an oligomer/soluble tau aggregate, leading to the formation of a stable sandwich complex and a signal in the assay (Fig. 2A).

We used the CT19:1 antibody clone (epitope: 331-361, within the aggregation-prone tau microtubule binding region [MTBR]) because it demonstrated superior specificity to recombinant tau peptides covering the MTBR versus the flanking N- and C-terminal regions (Fig. 1A). As a proof of concept, we compared assay specificity to tau aggregates versus monomers prepared from identical recombinant full-length tau1 -441 (2N4R) samples of the same starting concentration. While the AEB signals for aggregated tau consistently increased from 0 to 458.5 ng/mL, the AEB values for the corresponding monomer aliquots did not change correspondingly to sample concentration (Fig. 2B). These results were replicated over 2 separate days with highly consistent results (Fig. 2B). To verify the presence of tau oligomers and related soluble aggregates in solution, recombinant tau aggregate versus monomer preparations were evaluated for the presence of aggregated tau structures using negative-stain TEM. Fibrils were observed in the recombinant soluble aggregate tau multimer preparation, whereas no structures with fibril-like morphology were observed in the monomeric tau preparations (Fig. 2C).

To examine assay specificity, aliquots of TBS brain homogenates from an autopsy-verified AD patient (Braak VI) were measured with the assay with or without prior immunodepletion with the CT19.1 antibody or with a control antibody (6E10, BioLegend) that targets A0. The choice of 6E10 was informed by it being of the same isotype (immunoglobulin G) as the CT19.1 used in the present assay. Depletion with CT19.1 removed « 80% of the original signal while 6E10 did not lead to any detectable signal changes (Fig. 2D).

Together, these findings show that the assay specifically recognizes tau aggregates, and that the relative signal directly corresponds to the structure of tau oligomers and related soluble tau aggregates, i.e., the number of monomers present in the oligomer or soluble tau aggreagate.

Mapping the binding site of the oligomer/soluble tau aggregate assay on full-length tau

The findings in Fig. 2 show that the assay recognizes tau oligomers and related soluble aggregates formed from full-length tau. However, previous publications have suggested that specific shorter sequences from full-length tau, specifically the MTBR domain (« amino acids 244-368) and shorter fragments from this sequence, might be responsible for the aggregation/toxicity reaction. We therefore sought to deduce potential sequences accounting for the antibody binding to tau oligomers and other soluble aggregates. We used four recombinant peptide constructs that together cover the full-length tau sequence. The assay did not recognize the N- and C-terminal sequences tau 1-224 and tau 368-441 ; the signal did not change with increasing concentration up to 200 pg (Fig. 3A). Contrarily, the AEB signals increased proportionally to protein concentration for the MTBR-containing constructs 258-368 and 302— 368 (Fig. 3A), which also matches results from the epitope mapping in which CT19:1 had the epitope within tau331— 361 .

SPR was used to further verify these findings. The CT19.1 antibody was immobilized onto a CM5 chip surface and the binding response of each protein was then recorded in cycles of binding and regeneration. The 258-368 and 302-368 peptides gave the highest signals while the N- and C-terminal sequences had no appreciable increases from the baseline (Fig. 1A). These results indicate that the assay recognizes specific sequences of soluble aggregated tau forms within MTBR tau 331-361 .

Validation of the assay on autopsy-verified brain tissue samples

We used cortical brain samples from a well-characterized cohort from the University of Pittsburgh Alzheimer’s Disease Research Center including cases with autopsy-verified AD and cases with other neurodegenerative diseases, with varying degrees of NFT tau pathology (i.e., across Braak NFT stages 0— VI). The demographic and neuropathological characteristics are provided in Table 1. We examined tissues from two brain regions: inferior temporal cortex and middle frontal cortex. For each case and region, equivalent weight of brain material was homogenized and processed into three sequential fractions of decreasing solubility of tau, namely the TBS, Na2CO3, and urea/detergent fractions. The tau oligomer/soluble aggregate assay signal was higher in patients with more severe NFT pathology as indexed by the Braak staging; those with Braak Vl-rated NFT pathology had the strongest signal. These findings were consistently observed for all brain fractions and in both brain regions (Fig. 4). Comparing the assay in the different brain fractions for the same patient, we found that the signal was higher in the brain fraction extracting more insoluble tau, being generally strongest in the urea/detergent fraction followed by the Na2CO3 fraction and lowest in the TBS fraction especially in the MF tissue samples (Fig. 4). Comparing the assay signal in the two brain regions, the readings were higher in the IT than the MF samples (Fig. 5), which agrees with reported regional differences in the severity of NFT pathology. Together, these results indicate that the assay recognizes brain-derived tau oligomers and other soluble tau aggregates and that the signal is equivalent to the amount of tau that can be extracted from these complexes (i.e., low signal from soluble, diffusible tau oligomers and high signal from protofibrillar and fibrillar tau forms in insoluble aggregates).

According to Figs. 4, 5 and 6, the tau assay signal was low (and sometimes undetectable) in most of the low- and moderate-Braak NFT stage cases. We therefore considered the primary neuropathological diagnosis given at autopsy (Table 1). Notably, the Braak VI cases had the strongest assay signals and were all diagnosed with AD at autopsy. Additionally, two cases in Braak II and Braak IV (samples S7, S9 and S10, S12 in the IT and MF specimens, respectively) who were diagnosed with AD at autopsy had high tau oligomer/soluble aggregate signals that were most prominent in the urea/detergent fractions. The lowest signals were recorded for the remaining cases who were diagnosed with Lewy body dementia, Huntington’s disease, and frontotemporal lobal degeneration with TDP-43 pathology (Figs. 4-6).

Establishment of assay calibrators and quality control samples We next sought to establish assay calibrators and quality control samples to enable signal conversion to relative concentration and to monitor the day-to-day reproducibility of the method. We used the curve obtained by diluting recombinantly produced tau aggregates (which also showed lack of reactivity toward monomers; Fig. 2B) as the assay standard curve. Using pre-formed recombinant tau aggregates with a known initial concentration of monomeric tau prepared according to a defined protocol ensures that the material can be reproduced when needed.

Fitting the AEB values obtained from the autopsy brain tissue experiments onto the standard curve gave ng/mL concentration results that were equivalent to the AEB plots (Figs. 6 and 7).

To assess the day-to-day and run-to-run variability, monitor instrument performance, and ensure reproducibility, the assay employed a brain sample with known AEB signal as an internal quality control (iQC) sample, which was measured at the start and end of each analytical run. This iQC brain sample from the parietal cortex of an AD patient was initially diluted 50 times with the sample diluent to obtain the material that was further diluted to generate the results shown in Fig. 8. Results over separate days showed highly consistent results, which allows three separate concentrations of low, medium, and high values to be selected for future use. Because only a small starting volume of the brain material is required, we estimate that the available iQC material will be enough to analyze > 2000 analytical runs of the assay.

In this Example, we report the development and preliminary validation of an immunoassay that specifically recognizes and quantifies oligomers and related soluble tau aggregates containing the tau331— 361 sequence from the MTBR domain in recombinant protein and brain tissue homogenates. The assay demonstrated selectivity to tau oligomers and related soluble tau aggregates (including those obtained from solubilizing pathological aggregates obtained from sequential brain fractionation) versus monomers, and the signal intensity was proportional to the relative concentration of tau aggregates present. Furthermore, the assay did bind to sequences of tau within the aggregation-prone MTBR but not to the adjoining N- and C-terminal regions that by themselves lack sequences thought to be required for aggregation. In both autopsy-verified brain tissue and recombinant preparations of tau, the assay signal decreased proportionally to the extent of dilution of the sample, including very low protein concentrations. Together, these results indicate that the assay will be useful to quantify the aggregated forms of tau in both human and in vitro biospecimens. The assay detected tau oligomers and related soluble aggregates in brain tissue homogenates from AD cases but not from cases with other chronic neurodegenerative diseases, which had low levels of pathological tau aggregates in the brain regions examined. While multiple tau-based immunoassays for the measurement of phosphorylated and nonphosphorylated forms of tau30— 37 as well as other assays that use the same homogenous assay approach employed herein exist, assays specifically for oligomeric and soluble aggregated forms of tau are lacking. Moreover, the specificity of several so-called tau oligomer or fibril antibodies and assays have been called into question. To address these challenges, we employed the homogeneous/single antibody immunoassay approach to detect oligomeric and related soluble aggregate forms of tau protein as they consist of multiple epitopes compared to the single epitope in monomeric forms. The assay signal toward recombinant tau monomer preparations did not change when the concentration was increased, indicating that it does not recognize monomers. Contrarily, the signals showed stepwise increases toward oligomers/soluble tau aggregates across the same concentration range as the monomers, further verified by TEM.

Epitope mapping experiments showed that in agreement with the CT19.1 antibody epitope within tau331 — 361, and in agreement that the assay signal was highest for the 302-368 peptide, which corresponds to the core region of insoluble tau fibrils. The signal remained high against the 258-368 peptide, which covers most of the MTBR, meaning that multimers made of longer fragments will be recognized as long as the sequence includes the antibody epitope. On the other hand, the assay did not recognize tau 1- 224 and 368-441 that fall outside the MTBR. These results are supported by previous findings that demonstrated that the MTBR consists of domains that regulate the protein’s aggregation propensity. In fact, specific stretches of amino acids in the MTBR, referred to as the hexapeptide motifs, are sufficient to aggregate into oligomers and fibrils in vitro. Moreover, recombinant tau peptides covering the MTBR aggregate at a much faster rate than full-length tau, suggesting that the sequences that are located island C-terminal to the MTBR do not promote aggregation.

SPR has been used as a biosensor to evaluate the kinetics of biological processes, including proteinprotein interactions. Interaction occurs on the sensor surface, typically a gold surface that is coated with a monolayer of carboxymethylated dextran covalently attached to the surface. The binding of an analyte to an immobilized molecule on the sensor chip increases mass, which generates SPR response signals expressed as resonance units (RU). The two tau MTBR peptides gave high SPR responses while the other peptides gave no signal. Together with the TEM and epitope mapping results, the SPR findings indicate that the assay antibody CT19.1 is selective for the tau aggregation-prone region.

The stronger assay signals in brain tissue samples with more abundant pathological tau aggregates according to Braak NFT staging indicate that the assay measures aggregates of various sizes that increase with disease severity in the same brain region. These findings suggest that the assay will be valuable not only for the quantification of mature tau fibrils but also for pre-fi brillar structures, such as oligomers, protofibrils, and early-stage fibrils. Moreover, the much higher assay signal in autopsy-verified cases with AD diagnosis versus barely detectable signals in those with other aggregated proteins indicates specificity to AD versus non-AD degenerative disorders and, thus, will be suitable for differential diagnosis.

Detection of the oligomer/soluble tau aggregates in cerebrospinal fluid (CSF)

The abundance of oligomer/soluble tau aggregates in bodily fluids, such as CSF and blood is typically significantly lower, which presents a challenge for their detection, necessitating the use either an amplification method or a highly sensitive assay or a combination of both. The recombinant tau and brain studies form the foundation upon which we employed the assay in CSF with a modification. This modification combines tau amplification targeting the microtubule-binding region (MTBR) domain prior to detection on SIMOA® platform.

The immunoassay for CSF tau aggregates levels in both AD and non-affected individuals without any pre-incubation or prior treatment with recombinant tau aggregates generates baseline SIMOA® signal as buffer control (Fig. 9A). This result confirms inability to detect low abundance tau aggregates using the SIMOA® platform that is considered more than 1000 times more sensitive than conventional enzyme- linked immunosorbent assay (ELISA).

The CSF tau aggregates in the same AD and non-affected individuals were measured after treatment with recombinant tau aggregates (Fig. 9B). Like untreated groups, the result shows no increase in SIMOAR® signals, indicating no presence of amplified tau aggregates following treatment.

The CSF tau aggregates in the same AD and non-affected individuals were measured after treatment with recombinant tau aggregates and a 36-hour incubation period (Fig. 9C). This extended incubation period facilities soluble tau to elongate which results in higher SIMOA® signals, demonstrating further amplification of tau aggregates.

The strengths of this Example include the development of a novel immunoassay specific to tau oligomers and related soluble aggregates and its analytical and neuropathological validation in both recombinant tau preparations and brain-derived homogenates from AD and other neurodegenerative disorders. Moreover, deducing the primary sequence of tau that the assay recognizes allowed us to link its signal to the presence of specific stretches of amino acids in the MTBR. The evaluation in a pilot set of brain samples provided a proof-of-concept demonstration that the assay recognizes brain-derived oligomers and related soluble tau aggregates including pathological tau aggregates that increase with disease severity. Future studies will aim to evaluate the assay performance in a larger cohort of brain tissue samples from AD, non-AD tauopathy, and other neurodegenerative diseases.

Together, we have developed an oligomer/soluble aggregate-specific tau immunoassay on the ultrasensitive SIMOA® platform and validated its performance using recombinant tau aggregate preparations versus monomer preparations and homogenized human brain fractions from cases with AD and non-AD neurodegenerative diseases patients. The assay signal intensity corresponded to the total protein content and Braak NFT staging in human soluble brain tissue extracts, and the signal diluted linearly down to very low protein concentrations demonstrating the high sensitivity of the method. The new assay will be useful for the quantification and differentiation of tau oligomers and related soluble tau aggregates including pathological tau in human brain biospecimens.

EXAMPLE 5

This Example is a cross-sectional biomarker validation study designed to evaluate CSF tau aggregates as a potential diagnostic biomarker for AD. We assessed CSF tau aggregation levels across the Alzheimer’s disease continuum, compared them with established AD biomarkers, and investigated their diagnostic and pathological relevance.

Materials & Methods

Study design and population

The discovery cohort included patients with Alzheimer’s disease, with a typical Alzheimer’s disease core CSF biomarker profile (CSF tau, amyloid 01-42, amyloid 01-40, p-tau, and total-tau), and age-matched controls, who were patients examined at the memory or neurology clinics in the catchment area of the Sahlgrenska University Hospital (Gothenberg, Sweden) for minor neurological or psychiatric symptoms, and who had both basic and core CSF biomarker levels within normal ranges.

Participants were selected from the TRIAD cohort, (visits October 2017— August 2021), a well- characterized research cohort consisting of young adults (20-30 years), cognitively unimpaired (CU) individuals (>60 years), mild cognitive impairment (MCI) participants, and clinically diagnosed AD cases, along with individuals with non-AD neurodegenerative disorders. Participants underwent standardized clinical, neuropsychological, imaging, and biomarker assessments, including CSF (amyloid 01-42, p- tau181 , and total-tau) and tau PET imaging. Amyloid 0 positivity in the TRIAD cohort was independently determined using amyloid 0 PET uptake, on the basis of visual rating and a consensus of two neurologists blinded to the diagnosis for TRIAD (Pascoal et al., 2019).

A novel SIMOA@-based immunoassay with an integrated tau aggregation amplification step was developed to detect low-abundance CSF tau aggregates. The primary objectives of this Example were to:

• Determine the levels of CSF tau aggregates across clinical and pathological groups;

• Compare CSF tau aggregates with established AD biomarkers, including CSF p-tau181 , p- tau217, total tau, neurofilament light (NfL), and tau PET standardized uptake value ratio (SUVR); and

• Assess the diagnostic performance of CSF tau aggregates in differentiating AD from CU individuals and from non-AD neurodegenerative disorders.

The study was conducted following ethical guidelines, and all participants provided written informed consent. The CSF samples obtained under stringent ethical guidelines and approvals from the University of Gothenburg (#EPN 140811). The CSF samples from the TRIAD cohort has received ethical approval from the Montreal Neurological Institute Ethics Board.

Generation of new anti -tau antibodies

We generated monoclonal antibodies (mAbs) against tau using a standard hybridoma approach, as described in Example 1. Briefly, Balb/c mice were immunized with recombinant tau241 -441, followed by booster doses before spleen cell fusion with SP2/0 myeloma cells. Hybridoma supernatants were screened using ELISA against full-length tau (2N4R) and tau241— 441 , and positive clones were expanded and subcloned. Antibody specificity and isotype were confirmed, and purification was performed using protein G affinity chromatography. Epitope mapping was conducted via direct ELISA against overlapping recombinant tau peptides spanning tau241— 441 as described in Example 2.

Expression and purification of tau variants

Full-length tau 441 (Uniprot ID: P10636-8) and four truncated tau constructs (tau1— 224, tau258— 368, tau302— 368, and tau368— 441 ) were expressed and purified using a 6* histidine-SUMO fusion system, following a previously established protocol (Islam et al., 2024). Preparation of recombinant tau aggregates

Full-length monomeric recombinant tau1— 441 , stored in 1 x PBS, was used for tau aggregation. Proteins and peptides were diluted in 1 x PBS with 2 mM EDTA, adjusted to the desired concentration, and incubated at 37°C for 72 hours on a shaker (Eppendorf AG, Thermomixer Comfort, model 5355 41412) at 350 rpm to induce aggregation. Monomeric tau fractions were prepared under identical conditions but kept at low temperatures to prevent aggregation. Aliquots were stored at -80°C until use.

Negative-stain transmission electron microscopy

Aliquots (5 p L) of recombinant tau aggregates were applied to carbon-coated, glow-discharged copper grids and allowed to adhere for 1 minute. The grids were then washed with ultrapure distilled water and negatively stained with 0.75% uranyl formate (Electron Microscopy Sciences) for 30 seconds. Imaging was performed using a Talos L120C 120 kV transmission electron microscope (TEM) (Thermo Fisher), equipped with a BM-CETA 4,096 x 4,096, 14 pm pixel CMOS camera and TEM imaging and analysis software (Thermo Fisher).

Characterization of full-length tau aggregates used for CSF amplification

To facilitate the detection of low-abundance tau aggregates in CSF, we employed a seeded amplification approach using pre-formed recombinant full-length tau aggregates. Transmission electron microscopy (TEM) imaging confirmed the structural properties of these aggregates (Fig. 10). Fibrillar tau aggregates (Left panel in Fig. 10): TEM revealed extensive filamentous tau assemblies, characteristic of pathological tau fibrils. These aggregates served as seeding agents in the CSF amplification assay. Monomeric tau (Middle panel in Fig. 10): In contrast, tau monomers appeared as diffuse, non-aggregated structures, confirming the absence of fibrillar assembly in monomeric preparations. Single-unit tau aggregate (Right panel in Fig. 10): A distinct tau protofilament measuring approximately 2 pm in length was observed, supporting the presence of well-defined tau assemblies. These pre-formed tau aggregates were utilized to amplify endogenous CSF tau aggregates in both the discovery and TRIAD validation cohorts, ensuring enhanced detection using the ultrasensitive immunoassay.

Immunoassay for CSF tau aggregates

CSF samples from Alzheimer’s disease (AD) and non-AD individuals (50 pL per sample) were treated with pre-formed tau aggregates to facilitate seeding. The treated samples were then incubated for 30 hours at 37°C on a shaker set at 450 rpm to promote tau elongation and aggregation. Following the incubation, amplified tau aggregates were detected using a homogeneous immunoassay on the SIMOA® HD-X platform (Quanterix, Billerica, MA, USA). The assay employed the CT19.1 antibody (mouse monoclonal, epitope: 331-361) for both capture and detection, ensuring high specificity for aggregated tau species. Immunoassay signals were measured as average enzyme beads (AEB) values, with higher signals indicating increased tau aggregation.

Results

Detection of CSF tau aggregates in the discovery cohort

To assess the presence of tau aggregates in CSF, we applied the amplification-based SIMOA® assay to measure relative tau aggregate levels across groups (Fig. 9). CSF samples from AD and non-affected individuals showed no detectable SIMOA® signals, similar to buffer control levels, indicating that endogenous tau aggregates were below the detection limit without amplification (Fig. 9A). After treatment with pre-formed tau aggregates, CSF samples from AD and non-affected individuals still showed minimal SIMOA® signal increase, indicating that seeding alone was insufficient for robust tau aggregation detection (Fig. 9B). Following pre-formed tau treatment and 36-hour incubation, CSF tau aggregates were significantly elevated in AD compared to non-affected individuals (Fig. 9C). The increased SIMOA® signal in AD demonstrated that tau aggregation was amplified over time, showing that CSF from AD patients contained tau species that were prone to further aggregation. These results confirmed that direct detection of CSF tau aggregates was limited without amplification, and that AD-derived CSF exhibited enhanced tau aggregation upon seeding and incubation. This supports the role of CSF tau aggregates as a biomarker for AD pathology.

Assay validation

CSF tau aggregates across the Alzheimer’s disease continuum: In the TRIAD validation cohort, CSF tau aggregate levels progressively increased along the Alzheimer’s disease (AD) continuum, with the highest levels observed in individuals with mild cognitive impairment (MCI+) and AD (Fig. 11 , top panel). Compared to cognitively unimpaired (CU) individuals, CSF tau aggregate levels were significantly elevated in MCI+ (p < 0.01) and AD (p < 0.001). Individuals classified as MCI- (biomarker-negative) and non-AD neurodegenerative disease (Non-ADD) cases showed significantly lower CSF tau aggregate levels, indicating potential specificity for AD-related tau pathology.

Association between CSF tau aggregates and tau PET Braak staging: CSF tau aggregate levels demonstrated a stepwise increase with advancing tau PET Braak stages, further supporting their relevance to in vivo tau pathology (Fig. 11 , bottom panel). Individuals with higher Braak stage tau PET burden exhibited significantly elevated CSF tau aggregates, with the most pronounced differences observed between Braak stages 0-2 (low tau pathology) and Braak stages 4-6 (high tau pathology) (p < 0.001).

Clinical and diagnostic implications: The observed progressive increase in CSF tau aggregates from CU to MCI+ and AD, alongside its strong correlation with tau PET Braak staging, showed that CSF tau aggregates reflected pathological tau accumulation in AD. These findings indicate that CSF tau aggregates serve as a biomarker for staging tau pathology, providing a fluid-based alternative to tau PET for assessing disease progression and pathological burden in AD.

Assay correlation

Correlation between CSF tau aggregates and established Alzheimer’s disease biomarkers: To assess the biological relevance of CSF tau aggregates, we examined their correlation with established cerebrospinal fluid (CSF) and imaging biomarkers of tau pathology and neurodegeneration in the TRIAD validation cohort. Strong positive correlations were observed between CSF tau aggregate levels and phosphorylated tau species, including p-tau217 (Fig. 12A) and p-tau181 (Fig. 12B), both measured using AlzPath and Lumipulse assays, respectively. Similarly, CSF tau aggregates correlated with total tau (Fig. 12C), further reinforcing their association with tau burden in AD. Additionally, CSF tau aggregates were significantly associated with markers of neurodegeneration, including neurofilament light chain (NfL, Fig. 12D), showing a potential link between tau aggregation and neuronal injury.

Association between CSF tau Aggregates and tau PET imaging: CSF tau aggregate levels also exhibited strong correlations with tau PET standardized uptake value ratio (SUVR) across different brain regions. Notably, higher CSF tau aggregate levels were associated with increased tau PET burden in both global cortical regions (Fig. 12E) and neocortical regions (Fig. 12F).

These findings demonstrate that CSF tau aggregates strongly correlated with in vivo tau pathology as measured by CSF p-tau, total tau, and tau PET imaging, supporting their potential as a fluid-based biomarker reflecting tau pathology in AD.

Table 2 - sequence information

The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. REFERENCES

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Brown et al., Tau in cerebrospinal fluid induces neuronal hyperexcitability and alters hippocampal theta oscillations. Acta Neuropathologica Commun. 2023; 11 : 67

Consensus recommendations for the postmortem diagnosis of Alzheimer's disease. The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease. Neurobiol Aging. 1997; 18: S1-S2

Islam et al., Novel ultrasensitive immunoassay for the selective quantification of tau oligomers and related soluble aggregates. Alzheimers Dement. 2024; 20: 2894-2905

Lantero-Rodriguez et al., P-tau235: a novel biomarker for staging preclinical Alzheimer’s disease. EMBO Mol Med. 2021 ; 13: e15098

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Claims

1 . A monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to human tau, wherein the monoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain variable region, VH, complementarity determining region 1 , CDR1 , consisting of RYWMN as defined in SEQ ID NO: 1 ; a VH CDR2 consisting of QIYPGDGDTNYNSKFKV as defined in SEQ ID NO: 2; a VH CDR3 consisting of SWAY as defined in SEQ ID NO: 3; a light chain variable region, VL, CDR1 consisting of SASSSVNHTH as defined in SEQ ID NO: 4; a VL CDR2 consisting of DTSKLAS as defined in SEQ ID NO: 5; and a VL CDR3 consisting of FQGSGYPLT as defined in SEQ ID NO: 6.

2. The monoclonal antibody, or the antigen-binding fragment thereof, according to claim 1 , wherein the monoclonal antibody, or the antigen-binding fragment thereof, binds specifically to an epitope consisting of amino acids 331 to 361 of human tau as defined in SEQ ID NO: 25.

3. The monoclonal antibody, or the antigen-binding fragment thereof, according to claim 1 or 2, wherein the VH comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 11.

4. The monoclonal antibody, or the antigen-binding fragment thereof, according to any one of claims 1 to 3, wherein the VL comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 16.

5. The monoclonal antibody, or the antigen-binding fragment thereof, according to any one of claims 1 to 4, wherein the heavy chain comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 19 or 20.

6. The monoclonal antibody, or the antigen-binding fragment thereof, according to any one of claims 1 to 5, wherein the light chain comprises, preferably consists of, the amino acid sequence as defined in SEQ ID NO: 22 or 23.

7. An immunoassay kit for detection of oligomeric tau and soluble tau aggregates comprising: a capture antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 6; and a detection antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to

6.

8. The immunoassay kit according to claim 7, wherein the capture antibody, or the antigen-binding fragment thereof, is attached to a solid support, preferably beads, and more preferably magnetic beads.

9. The immunoassay kit according to claim 7 or 8, wherein the detection antibody, or the antigenbinding fragment thereof, is attached to one of i) biotin and ii) streptavidin or avidin.

10. The immunoassay kit according to claim 9, further comprising an enzyme, preferably p-galactosidase, attached to the other of i) biotin and ii) streptavidin or avidin; and a substrate, preferably 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside, that can be converted into a detectable product, 5,5'-dibromo-4,4'-dichloro-indigo, by the enzyme, preferably p-galactosidase.

11 . The immunoassay kit according to any one of claims 7 to 10, further comprising pre-formed tau aggregates.

12. The immunoassay kit according to claim 11 , wherein the pre-formed tau aggregates are preformed tau aggregates of recombinant tau1 -444 protein as defined in SEQ ID NO: 25.

13. The immunoassay kit according to claim 12 wherein the pre-formed tau aggregates are obtainable by adding monomeric recombinant tau1 -441 protein as defined in SEQ ID NO: 25 in a buffer, preferably phosphate buffered saline (PBS), optionally comprising a metal ion sequester, preferably ethylenediaminetetraacetic acid (EDTA), and then incubating for at least 24 hours, preferably at least 48 hours, more preferably at least 60 hours, and most preferably at least 72 hours, under agitation at room temperature (20-25°C) or at a temperature from 30 up to 45°C, preferably from 35 up to 40°C, and more preferably about 37°C.

14. The immunoassay kit according to any one of claims 11 to 13, wherein the pre-formed tau aggregates comprises the pre-formed tau aggregates at a concentration of no more than 2 nM, preferably no more than 1.5 nM, more preferably no more than 1 nM, and most preferably no more than 0.75 nM, such as no more than 0.5 nM or no more than 0.25 nM.

15. The immunoassay kit according to any one of claims 11 to 14, wherein the pre-formed tau aggregates comprises the pre-formed tau aggregates at a concentration selected within an interval of from 0.05 up to 1 nM, preferably selected within an interval of from 0.1 up to 0.75 nM, more preferably selected within an interval of from 0.2 up to 0.5 nM, and most preferably about 0.25 nM.

16. A method for determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample, comprising: contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit according to any one of claims 7 to 15; and determining an amount of oligomeric tau and soluble tau aggregates in the body fluid sample by determining an amount of bound detection antibody, or the antigen-binding fragment thereof.

17. The method according to claim 16, wherein contacting the body fluid sample comprises contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit according to claim 10; and determining the amount of oligomeric tau and soluble tau aggregates comprises determining the amount of oligomeric tau and soluble tau aggregates in the body fluid sample by determining an amount of the detectable product.

18. The method according to claim 17 or 18, further comprising: contacting the body fluid sample with pre-formed tau aggregates; and incubating the body fluid sample with the pre-formed tau aggregates prior to contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit according to any one of claims 7 to 11.

19. The method according to claim 18, wherein incubating the body fluid sample comprises incubating the body fluid sample with the pre-formed tau aggregates for at least 24 hours, preferably at least 30 hours, and more preferably at least 36 hours, prior to contacting the body fluid sample with the capture antibody, or the antigen-binding fragment thereof, and the detection antibody, or the antigen-binding fragment thereof, of the immunoassay kit according to any one of claims 7 to 15.

20. The method according to claim 19, further comprising producing the pre-formed tau aggregates.

21 . The method according to claim 20, wherein producing the pre-formed tau aggregates comprises: diluting or adding monomeric recombinant tau1 -441 protein as defined in SEQ ID NO: 25 in a buffer, preferably phosphate buffered saline (PBS), optionally comprising a metal ion sequester, preferably ethylenediaminetetraacetic acid (EDTA); and incubating the monomeric recombinant tau1 -441 protein for a period of time or at least 24 hours, preferably at least 48 hours, more preferably at least 60 hours, and most preferably at least 72 hours, under agitation at room temperature or at a temperature from 30 up to 45°C, preferably from 35 up to 40°C, and more preferably about 37°C, to form an amplifier solution comprising the pre-formed tau aggregates.

22. The method according to any one of claims 19 to 21 , wherein contacting the body fluid sample with the pre-formed tau aggregates comprising contacting the body fluid sample with an amplifier solution comprising the pre-formed tau aggregates at a concentration of no more than 2 nM, preferably no more than 1.5 nM, more preferably no more than 1 nM, and most preferably no more than 0.75 nM, such as no more than 0.5 nM or no more than 0.25 nM.

23. The method according to any one of claims 19 to 22, wherein contacting the body fluid sample with the pre-formed tau aggregates comprising contacting the body fluid sample with an amplifier solution comprising the pre-formed tau aggregates at a concentration selected within an interval of from 0.05 up to 1 nM, preferably selected within an interval of from 0.1 up to 0.75 nM, more preferably selected within an interval of from 0.2 up to 0.5 nM, and most preferably about 0.25 nM.

24. The method according to any one of claims 19 to 23, wherein contacting the body fluid sample with the pre-formed tau aggregates comprising contacting the body fluid sample with an amplifier solution comprising the pre-formed tau aggregates at a volume ratio of body fluid sample : amplifier solution selected within an interval of from 1 :0.1 up to 1 :2, preferably selected within an interval of from 1 :0.2 up to 1 :1 , more preferably selected within an interval of from 1 :0.25 up to 1 : 0.75, such as about 1 :0.5.

25. The method according to any one of claims 16 to 24, wherein the body fluid sample is selected from the group consisting of a cerebrospinal fluid sample, a blood sample, a blood serum sample and a blood plasma sample.

26. A method for diagnosing Alzheimer’s disease in a subject, comprising: determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample from the subject according to any one of claims 16 to 25; and determining the subject as suffering from Alzheimer’s disease or not based on the determined amount of oligomeric tau and soluble tau aggregates.

27. A method for classifying a neurological disease in a subject, comprising: determining an amount of oligomeric tau and soluble tau aggregates in a body fluid sample from the subject according to any one of claims 16 to 25; and classifying the neurological disease as Alzheimer’s disease or a neurological disease other than Alzheimer’s disease based on the determined amount of oligomeric tau and soluble tau aggregates.

28. A monoclonal antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 6 for use as a medicament.

29. A monoclonal antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 6 for use in treatment of Alzheimer’s disease.