Tests to detect neurodegeneration

Description

1 / 98 Tests to detect neurodegeneration Divided from BR112020018193-5, filed on 04.03.2019. FIELD OF THE INVENTION

[0001] The present invention relates to pharmaceutical compositions and methods for detecting neurodegeneration. In particular, the invention relates to methods for measuring the amount of isolated or multi-phosphorylated tau p217+ protein species in a biological sample and uses thereof, as well as antibodies and kits for use in the methods. BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is a degenerative brain disorder clinically characterized by progressive loss of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental impairment (dementia) in elderly humans and is believed to be the fourth most common medical cause of death in the United States. AD has been observed in ethnic groups worldwide and represents a public health problem now and in the future.

[0003] The brains of individuals with AD exhibit characteristic lesions called senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels), and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments (PHF), are commonly found in various areas of the human brain important for memory and cognitive function in patients with AD.

[0004] Neurofibrillary tangles are primarily composed of aggregates of hyperphosphorylated tau protein. The main physiological function of tau is the polymerization and stabilization of microtubules. Tau binding to microtubules occurs through ionic interactions between positive charges in the tau microtubule-binding region and negative charges in the microtubule network. Petition 870260042583, dated 06 / 05 / 2026, page 10 / 334 2 / 98 (Butner and Kirschner, J Cell Biol. 115(3):717-30, 1991). The tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the primary function of tau. Tau that binds to the axonal microtubule network is in a hypophosphorylated state, while the aggregated tau present in AD is hyperphosphorylated, providing unique epitopes that are distinct from the overall physiologically active tau background (Iqbal et al., Curr Alzheimer Res. 7(8): 656-664, 2010).

[0005] The progression of tauopathy in a brain with AD follows distinct patterns of dissemination. A hypothesis of taupathy transmission and dissemination has been described based on Braak's stages of taupathy progression in the human brain and taupathy dissemination following injections of tau aggregates in preclinical tau models (Frost et al., J Biol Chem. 284:12845-52, 2009; Clavaguera et al., Nat Cell Biol. 11:90913, 2009). Taupathy is believed to spread in a prion-like manner from one brain region to another. This dissemination process would involve an externalization of tau seeds that can be absorbed by neighboring neurons and further induce taupathy.

[0006] Tau protein fragments in neurofibrillary tangles pass into the cerebrospinal fluid (CSF) where they can be collected by lumbar puncture and measured by sensitive assays. The presence of neurological disease can thus be detected using assays that recognize tau-derived protein fragments in the CSF. Such tau assays require the ability to recognize the tau species characteristic of a neurodegenerative condition. Multiphosphorylated tau is the main example of tau protein associated with AD. Therefore, assays that detect multiphosphorylated tau protein in the CSF may be more effective in detecting the presence of AD.

[0007] Phosphorylation is not the only post-translational modification to be considered in tau measurement. Recent studies have shown that in CSF, Petition 870260042583, dated 06 / 05 / 2026, p. 11 / 334 3 / 98 Tau protein exists primarily as fragments rather than as a full-length protein (Meredith et al. PLoS One. 8(10):e76523, 2013). Additionally, the fragmentation pattern of tau can be influenced by disease, such as proteolysis, which is often aberrant in pathological conditions. Consequently, tau-based assays for neurodegeneration need to provide information not only on phosphorylation status (e.g., local phosphorylation) but also on the nature of the tau protein fragments (e.g., tau fragment length, polarity) being measured. However, translating this idea is hampered by the low endogenous levels of phosphorylated tau, especially in samples from healthy individuals.

[0008] In summary, there is a need for sensitive, precise, and accurate methods to detect multiphosphorylated tau in biological fluids. Such methods would be useful for effectively detecting, diagnosing, staging, and tracking the progression of neurodegenerative diseases such as Alzheimer's disease and other tauopathies. The methods would also be useful as pharmacodynamic markers to measure levels of total, free, and therapeutic antibody-bound multiphosphorylated tau. The ability to detect and measure fragments of multiphosphorylated tau is of paramount importance to the field, as the transmissible tau species may be one or more tau fragments. BRIEF SUMMARY OF THE INVENTION

[0009] The invention satisfies the need to detect tau forms in CSFs that are linked to neurodegenerative diseases. The invention makes it possible to detect individually or multiply phosphorylated tau, as well as to detect tau fragments.

[0010] High-sensitivity enzyme-linked immunoassays (ELISAs), according to the embodiment of the invention, have been developed and qualified to measure a tau p217+ comprising a phosphorylated tau epitope (tau p217+ epitope or pT3 epitope) comprising phosphorylated residues T212 and / or T217 having the sequence (212) R(pT)PSLPTPPTR (SEQ ID NO: 25), Petition 870260042583, dated 06 / 05 / 2026, p. 12 / 334 4 / 98 (217) RTPSLP(pT)PPTR (SEQ ID NO: 26) or (212&217) R(pT)PSLP(pT)PPTR (SEQ ID NO: 27).

[0011] Assays according to embodiments of the invention are capable of measuring the p217+ tau species in various fluid matrices, including but not limited to CSF, interstitial fluid (ISF), brain homogenate, serum, plasma, and denatured or enriched versions thereof. Assays according to embodiments of the invention utilize a first monoclonal antibody directed to a pT3 tau epitope as a capture antibody, and a second monoclonal antibody directed to a second tau epitope as a detection antibody. The assays are highly sensitive, precise, accurate, transferable between laboratories, with linear dilution, and applicable to many sample types.In addition to measuring tau p217+ species in crude biological fluid, the assays can be used to measure samples with or without denaturation, or after immunoprecipitation, two complementary techniques for quantifying the amount of free tau p217+ or tau p217+ bound to an endogenous or therapeutically administered antibody. The assays can be used in conjunction with high-performance reversed-phase liquid chromatography (rpHPLC) to measure fractionated CSF, allowing analysis of the tau p217+ fragment profile.

[0012] In a general aspect, the invention relates to a method for measuring the amount of tau p217+ peptides in a sample. The method comprises: (i) placing the sample in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and (ii) placing the captured tau p217+ peptides with a detection antibody directed against an epitope comprising amino acid residues 119 to 126, such as amino acid residues 116 to 127 of the tau protein, or an epitope containing amino acid residues 7 to 20 of the tau protein, thereby measuring an amount of tau p217+ peptides or an amount of long tau p217+ peptides, respectively, wherein the amino acid numbering is relative to the sequence of Petition 870260042583, dated 06 / 05 / 2026, page 13 / 334 5 / 98 amino acids presented in SEQ ID NO: 1.

[0013] In a particular aspect, the invention relates to a method for determining the relative amount of long tau p217+ peptides or short tau p217 peptide fragments in a sample.The method comprises (i) contacting the sample with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, (ii) contacting the captured tau p217+ peptides with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the quantity of tau p217+ peptides, (iii) contacting the captured tau p217+ peptides with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the quantity of long tau p217+ peptides, and (iv) determining a relative quantity of long tau p217+ peptides or short tau p217+ peptides based on the quantity of long tau p217+ peptides and the quantity of short tau p217+ peptides. long, in which the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1.

[0014] In one embodiment of the invention, the quantity of short tau p217+ peptides in a sample is calculated based on the quantity of tau p217+ peptides and the quantity of long tau p217+ peptides in the sample, for example, by subtracting the quantity of long tau p217+ peptides from the quantity of tau p217+ peptides. In another embodiment, a ratio between the quantity of short tau p217+ peptides and the quantity of tau p217+ peptides, a ratio between the quantity of long tau p217+ peptides and the quantity of tau p217+ peptides, or a ratio between the quantity of long tau p217+ peptides and the quantity of short tau p217+ peptides is determined based on the quantity of tau p217+ peptides and the quantity of long tau p217+ peptides in the sample. According to embodiments of the invention, the amount of tau p217+ peptides and / or the Petition 870260042583, dated 06 / 05 / 2026, page 14 / 334 6 / 98 The amount of long p217+ tau peptides in a sample, as well as information based on measured amounts, such as the calculated amount of short p217+ tau peptides and one or more of the ratios described above, can be used for one or more diagnostic purposes.

[0015] Consequently, in a specific aspect, the invention relates to a method for determining a ratio between p217+ tau peptides and total tau peptides in a sample. The method comprises (i) contacting the sample with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and contacting the sample with a phosphorylation-independent capture antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein, to capture total tau peptides in the sample, and (ii) performing at least one of (a) contacting the captured tau p217+ peptides with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure a quantity of tau p217+ peptides,(a) and bring the total capture tau peptides into contact with the first detection antibody in order to measure a quantity of total tau peptides; and (b) and bring the captured p217+ tau peptides into contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure a quantity of long p217+ tau peptides, and bring the total capture tau peptides into contact with the second detection antibody in order to measure a quantity of total tau peptides, and (iii) determine a ratio between the quantity of p217+ tau peptides and the quantity of total tau peptides, or a ratio between the quantity of long p217+ tau peptides and the quantity of total long tau peptides, wherein the amino acid numbering is relative to the sequence of, Petition 870260042583, dated 06 / 05 / 2026, page 15 / 334 7 / 98 amino acids presented in SEQ ID NO: 1. In one embodiment, a quantity of short p217+ tau peptides is calculated by subtracting the quantity of long p217+ tau peptides from the quantity of p217+ tau peptides, a total quantity of short tau peptides is calculated by subtracting the total quantity of long tau peptides from the total quantity of tau peptides, and a ratio between the quantity of short p217+ tau peptides and the total quantity of short tau peptides is determined.

[0016] According to a particular aspect, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, from an individual in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, (ii) performing at least one of (a) placing the captured tau p217+ peptides in contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein in order to measure a quantity of tau p217+ peptides, and (b) placing the captured tau p217+ peptides in contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure a quantity of long tau p217+ peptides,(iii) to determine whether or not the individual suffers from a tauopathy or is at risk of developing a tauopathy based on at least one of the following: the amount of tau p217+ peptides, the amount of long tau p217+ peptides, the amount of short tau p217+ peptides obtained by subtracting the amount of long tau p217+ peptides from the amount of tau p217+ peptides, and ratios thereof, wherein the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1. In one embodiment, the method further comprises administering to the individual a therapeutic agent to treat or prevent the tauopathy.

[0017] According to a particular aspect, a method of the invention comprises (i) contacting a biological sample, preferably a CSF sample, from an individual, with a targeted capture antibody. Petition 870260042583, dated 06 / 05 / 2026, page 16 / 334 8 / 98 against a tau p217+ epitope to capture tau p217+ peptides in the sample, and contact the sample with a phosphorylation-independent capture antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein, to capture the total tau peptides in the sample, (iii) perform at least one of (a) contact the captured tau p217+ peptides with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure a quantity of tau p217+ peptides, and contact the total capture tau peptides with the first detection antibody to,to measure the amount of total tau peptides; (b) and bring the captured p217+ tau peptides into contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure the amount of long p217+ tau peptides, and bring the total captured tau peptides into contact with the second detection antibody in order to measure the amount of total tau peptides, and (iii) determine whether or not the individual suffers from a tauopathy or is at risk of developing a tauopathy based on at least one of (a) a ratio between the amount of p217+ tau peptides and the amount of total tau peptides, (b) a ratio between the amount of long p217+ tau peptides and the amount of total long tau peptides, and (c) a ratio between the amount of short p217+ tau peptides and the amount of total short tau peptides,wherein the quantity of short p217+ tau peptides is obtained by subtracting the quantity of long p217+ tau peptides from the quantity of p217+ tau peptides, and the total quantity of short tau peptides is obtained by subtracting the total quantity of short tau peptides from the total quantity of tau peptides, wherein the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1. In one embodiment, the method further comprises administering to the individual a therapeutic agent to treat or prevent tauopathy. Petition 870260042583, dated 06 / 05 / 2026, page 17 / 334 9 / 98

[0018] According to another particular aspect, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, from an individual undergoing treatment, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and (ii) performing at least one of (a) placing the captured tau p217+ peptides in contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure a quantity of tau p217+ peptides, and (b) placing the captured tau p217+ peptides in contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure a quantity of long tau p217+ peptides, and (iii) determining the effectiveness of the treatment in the individual based on at least one of the quantity of tau p217+ peptides,The quantity of long p217+ tau peptides, a quantity of short p217+ tau peptides obtained by subtracting the quantity of long p217+ tau peptides from the quantity of short p217+ tau peptides, and ratios thereof, wherein the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1. In one embodiment, the method further comprises administering to the individual a therapeutic agent to treat or prevent tauopathy.

[0019] According to another particular aspect, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, from an individual undergoing treatment, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and placing the sample in contact with a phosphorylation-independent capture antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein, to capture the total tau peptides in the sample, and (ii) performing at least one of (a) placing Petition 870260042583, dated 06 / 05 / 2026, page 18 / 334 10 / 98 the captured p217+ tau peptides are brought into contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein, thus measuring a quantity of p217+ tau peptides, and the total captured tau peptides are brought into contact with the first detection antibody, thus measuring a quantity of total tau peptides; and (b) bring the captured p217+ tau peptides into contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure a quantity of long p217+ tau peptides, and bring the total captured tau peptides into contact with the second detection antibody in order to measure a quantity of total tau peptides, and (iii) to determine the effectiveness of the treatment in the individual based on at least one of (a) a ratio between the quantity of p217+ tau peptides and the quantity of total tau peptides,(b) a ratio between the amount of long p217+ tau peptides and the total amount of long p217+ tau peptides, and (c) a ratio between the amount of short p217+ tau peptides and the total amount of short p217+ tau peptides, wherein the amount of short p217+ tau peptides is obtained by subtracting the amount of long p217+ tau peptides from the amount of p217+ tau peptides, and the total amount of short p217+ tau peptides is obtained by subtracting the total amount of short p217+ tau peptides from the total amount of tau peptides, wherein the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1. In one embodiment, the method further comprises administering to the individual a therapeutic agent to treat or prevent tauopathy.

[0020] According to another particular aspect, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, from an individual in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and (ii) performing at least one of (a) placing the captured tau p217+ peptides in contact with a first detection antibody directed against a tau p217+ epitope. Petition 870260042583, dated 06 / 05 / 2026, page 19 / 334 (a) targeting an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of p217+ tau peptides, and (b) targeting the captured p217+ tau peptides with a second detection antibody targeting an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of long p217+ tau peptides, and (iii) determining whether or not the individual is suitable for an anti-tau p217+ antibody based on at least one of the following: the amount of p217+ tau peptides, the amount of long p217+ tau peptides, the amount of short p217+ tau peptides obtained by subtracting the amount of long p217+ tau peptides from the amount of short p217+ tau peptides, and ratios thereof, wherein the amino acid numbering is relative to the presented amino acid sequence. SEQ ID NO: 1.In one embodiment, the method additionally involves administering an anti-tau p217+ antibody to the individual to treat or prevent tauopathy.

[0021] According to another particular aspect, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, from an individual, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and placing the sample in contact with a phosphorylation-independent capture antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein, to capture the total tau peptides in the sample, and (ii) performing at least one of (a) placing the captured tau p217+ peptides in contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to thereby measure an amount of tau p217+ peptides, and placing the total capture tau peptides in contact with the first detection antibody in order to, therefore,(a) measure a quantity of total tau peptides; and (b) place the captured p217+ tau peptides in, Petition 870260042583, dated 06 / 05 / 2026, page 20 / 334 12 / 98 contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of long p217+ tau peptides, and contact the total capture tau peptides with the second detection antibody to measure the amount of total tau peptides, and (iii) determine whether or not the individual is suitable for anti-tau p217+ antibody therapy based on at least one of (a) a ratio of the amount of p217+ tau peptides to the amount of total tau peptides, (b) a ratio of the amount of long p217+ tau peptides to the amount of total long tau peptides, and (c) a ratio of the amount of short p217+ tau peptides to the amount of total short tau peptides, wherein the amount of short p217+ tau peptides is obtained by subtracting the amount of long tau p217+ peptides of the quantity of tau p217+ peptides,The total amount of short tau peptides is obtained by subtracting the total amount of short tau peptides from the total amount of tau peptides, wherein the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1. In one embodiment, the method further comprises administering to the individual an anti-tau p217+ antibody to treat or prevent tauopathy.

[0022] In another particular aspect, the invention relates to a method for monitoring treatment with an anti-tau p217+ antibody in an individual, wherein the method comprises: (i) obtaining a biological sample from the individual, and (ii) separating the biological sample into a first sample containing tau p217+ free of anti-tau p217+ antibody, preferably IgG, and a second sample containing tau p217+ peptides bound to the anti-tau p217+ antibody, (iii) obtaining a third sample containing tau p217+ free of anti-tau p217+ antibody from the second sample, preferably by means of rpHPLC, (iv) placing each of the first and third samples in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in each of the samples, (v) Petition 870260042583, dated 06 / 05 / 2026, page 21 / 334 13 / 98 perform at least one of (a) placing the captured tau p217+ peptides in contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein in order to measure the amount of tau p217+ peptides in each of the samples, and (b) placing the captured tau p217+ peptides in contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure the amount of long tau p217+ peptides in each of the samples, (vi) monitoring the anti-tau p217+ antibody treatment based on at least one of the amount of tau p217+ peptides and the amount of long tau p217+ peptides in each of the samples, wherein the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1.For example, treatment with anti-tau p217+ antibody can be monitored based on a ratio of the amount of long tau p217+ peptides between the first and third samples, a ratio of the amount of tau p217+ peptides between the first and third samples, or a ratio of the amount of short tau p217+ peptides (which can be calculated by subtracting the amount of long tau p217+ peptides from the amount of short tau p217+ peptides) between the first and third samples. In one embodiment, the method further comprises administering an anti-tau p217+ antibody to the individual to treat or prevent tauopathy.

[0023] In another general aspect, the invention relates to a method for monitoring treatment with an anti-tau p217+ antibody in an individual, wherein the method comprises: (i) obtaining a biological sample from the individual, and (ii) obtaining a semi-denatured sample of the biological sample containing total tau p217+, wherein the semi-denatured sample is heated to denature the antibodies in the sample, and obtaining an undenatured sample of the biological sample containing tau p217+ free of anti-tau p217+ antibody, (iii) placing each of the samples Petition 870260042583, dated 06 / 05 / 2026, page 22 / 334 14 / 98 denatured and undenatured samples are contacted with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in each sample; (v) perform at least one of the following: (a) contact the captured tau p217+ peptides with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of tau p217+ peptides in each sample; (b) contact the captured tau p217+ peptides with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of long tau p217+ peptides in each sample; and (vi) monitor the anti-tau p217+ antibody treatment based on at least one of the peptide quantities. tau p217+ and the amount of long tau p217+ peptides in each of the samples,where the amino acid numbering is relative to the amino acid sequence presented in SEQ ID NO: 1. For example, treatment with anti-tau p217+ antibody can be monitored based on a ratio of the amount of long tau p217+ peptides between the semi-denatured and non-denatured sample, a ratio of the amount of tau p217+ peptides between the semi-denatured and non-denatured sample, or a ratio of the amount of short tau p217+ peptides (which can be calculated by subtracting the amount of long tau p217+ peptides from the amount of short tau p217+ peptides) between the semi-denatured and non-denatured sample. In one embodiment, the method further comprises administering an anti-tau p217+ antibody to the individual to treat or prevent tauopathy.

[0024] According to a specific aspect, tauopathy includes, but is not limited to, one or more selected from the group consisting of Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), frontotemporal dementia with parkinsonism linked to Petition 870260042583, dated 06 / 05 / 2026, p. 23 / 334 15 / 98 chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, dementia with tangles only, diffuse neurofibrillary tangles with calcification, dementia with argyrophilic grains, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatza disease, inclusion body myositis, Creutzfelt-Jakob disease, multiple system atrophy, Niemann-Pick type C disease, cerebral amyloid angiopathy caused by prion protein, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia. (boxing disease).

[0025] Preferably, the tauopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), FTDP-17, or progressive supranuclear palsy.

[0026] Preferably, tauopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease).

[0027] According to a specific aspect, the lower limit of quantification of a method of the invention is about 40 fg / ml of p217+ tau peptides and the lower limit of detection of a method of the invention is about 2 fg / ml of p217+ tau peptides.

[0028] In a specific aspect, the sample is a biological sample, such as a blood sample, brain homogenate, or cerebrospinal fluid (CSF) sample, from an individual in need of it. Preferably, the biological sample is a CSF sample from an individual in need of a diagnosis of tauopathy, monitoring of the effectiveness of a tauopathy treatment, or determination of suitability for p217+ anti-tau antibody therapy.

[0029] According to a particular aspect, a capture antibody useful for the methods of the invention is directed against a tau+ epitope. Petition 870260042583, dated 06 / 05 / 2026, page 24 / 334 16 / 98 p217, preferably a tau p217+ epitope containing the amino acid sequence of SEQ ID NOs: 25, 26, or 27. In one embodiment, a capture antibody useful for the methods of the invention comprises the HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively. Preferably, the capture antibody has a variable region of the heavy chain comprising the polypeptide sequence of SEQ ID NO: 28 or 30 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 29 or 31.

[0030] According to a specific aspect, a detection antibody useful for the methods of the invention is directed against an epitope comprising amino acid residues 119 to 126 of the tau protein, preferably an epitope comprising the amino acid sequence of SEQ ID NO: 10, such as the amino acid sequence of SEQ ID NO: 11. In one embodiment, a detection antibody useful for the methods of the invention comprises the HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively. Preferably, the detection antibody is a pT82 antibody comprising a variable heavy chain region having the polypeptide sequence SEQ ID NO: 8 and a variable light chain region having the polypeptide sequence SEQ ID NO: 9.

[0031] According to another specific aspect, a detection antibody useful for the methods of the invention is directed against an epitope containing amino acid residues 7 to 20 of the tau protein, preferably an epitope that has the amino acid sequence SEQ ID NO: 20. In one embodiment, a detection antibody useful for the methods of the invention Petition 870260042583, dated 06 / 05 / 2026, p. 25 / 334 17 / 98 comprises HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having polypeptide sequences with SEQ ID NOs: 12, 13, and 14, respectively; and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having polypeptide sequences with SEQ ID NOs: 15, 16, and 17, respectively. Preferably, the detection antibody is an hT43 antibody comprising a variable region of the heavy chain having the polypeptide sequence with SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence with SEQ ID NO: 19.

[0032] According to another specific aspect, a phosphorylation-independent capture antibody useful for the invention is directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 211 to 221 of the tau protein, or an epitope comprising amino acids 159 to 163 of the tau protein, more preferably an epitope having the amino acid sequence of SEQ ID NO: 21. In one embodiment, a phosphorylation-independent capture antibody useful for the invention is an hT7 antibody.

[0033] According to another particular aspect, the sample used in the methods of the invention is obtained after fractionating a biological sample using reverse-phase high-performance liquid chromatography (rpHPLC).

[0034] In another general aspect, the invention relates to an isolated detection antibody or antigen-binding fragment thereof that binds to a tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein, comprising (a) HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and (b) LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively. According to a specific aspect, the isolated detection antibody or antigen-binding fragment thereof comprises a heavy chain that has the Petition 870260042583, dated 06 / 05 / 2026, p. 26 / 334 18 / 98 polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 19. Preferably, the isolated detection antibody or antigen-binding fragment thereof that binds to a tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein is an hT43 antibody.

[0035] In another general aspect, the invention relates to a kit comprising (a) a capture antibody directed against a p217+ tau epitope and (b) a detection antibody directed against a tau protein epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein. Optionally, the kit further comprises a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein. The kit can be used, for example, to measure the amount of p217+ tau peptides, the amount of long p217+ tau peptides, the amount of short p217+ tau peptides, the ratio between the amount of short p217+ tau peptides and the amount of long p217+ tau peptides, the ratio between the amount of short p217+ tau peptides and the total amount of short tau peptides in a sample, etc.The kit can also be used for various diagnostic or monitoring purposes, for example, to determine whether or not an individual suffers from a tauopathy or is at risk of developing a tauopathy, to monitor the effectiveness of a treatment against a taupathy, such as treatment with an anti-tau p217+ antibody, to determine whether or not the individual is suitable for an anti-tau p217+ antibody, etc.

[0036] According to a specific aspect, a kit of the invention comprises a capture antibody, which has HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36 and 37, respectively. Preferably, the capture antibody has a variable region of the heavy chain comprising the Petition 870260042583, dated 06 / 05 / 2026, page 27 / 334 19 / 98 polypeptide sequence of SEQ ID NO: 28 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 29.

[0037] According to another particular aspect, a kit of the invention comprises a detection antibody, comprising HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively. Preferably, the detection antibody is a pT82 antibody comprising a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 8 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 9.

[0038] According to another particular aspect, a kit of the invention comprises a detection antibody, comprising HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively. Preferably, the detection antibody is an hT43 antibody comprising a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 19.

[0039] Other aspects, features and advantages of the invention will become apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The aforementioned summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. It should be understood that Petition 870260042583, dated 06 / 05 / 2026, page 28 / 334 20 / 98 The invention is not limited to the precise embodiments shown in the drawings.

[0041] Figure 1 shows an example of a standard curve for the pT3xhT43 and pT3xpT82 assays generated using calibrating peptides with mean + / - SD of duplicate measurements shown at each point.

[0042] Figures 2A-2E show the dilution linearity of the pT3xhT43 and pT3xpT82 assays in CSF samples with measurements shown (A, C and E) in pg / ml corrected dilution or (B and D) as % corrected dilution of 1:4 measurement, with dashed lines indicating + / - 20% of 1:4 measurements.

[0043] Figure 3 shows the intra- and inter-test precision of assays (A) pT3xhT43 and (B) pT3xpT82.

[0044] Figures 4A-4B show the accuracy between test sites of the pT3xhT43 and pT3xpT82 assays with the data plotted as signal-to-noise (S / N).

[0045] Figures 5A-5B show the competition of the pT3-based assay signal by soluble antibodies directed to tau p217+ in the (A) pT3xhT43 and (B) pT3xpT82 assays.

[0046] Figure 6 shows the phosphorylation dependence of the pT3xhT43 and pT3xpT82 assays.

[0047] Figure 7 shows a profile of a p217+ tau fragment from CSF of DA, measured using the pT3xhT43 and pT3xpT82 tests, with the data graphically represented as signal-minus-noise.

[0048] Figures 8A-8B show the temperature and stability for freeze-thaw of the tau p217+ signal in a DA CSF sample using the (A) pT3xhT43 and (B) hT7xpT82 assays.

[0049] Figure 9 shows the long-term stability of the tau p217+ signal in CSF samples after storage at -70 °C. No change in the signal was detected.

[0050] Figures 10A-10F show a correlation between tau p217+ and the classic biomarkers of AD Aβ42, tTau (total tau) and pTau181, Petition 870260042583, dated 06 / 05 / 2026, page 29 / 334 21 / 98 as measured by tests of (AC) pT3xhT43 and (DF) pT3xpT82.

[0051] Figures 11A-11B show a correlation between analysis of IHC of brain biopsy and tau p217+, as measured by assays of (A) pT3xhT43 and (B) pT3xpT82.

[0052] Figures 12A-12D show the results of (A) pT3xhT43, (B) pT3xpT82, (C) hT7xpT82 and (D), and the ratio between pT3xpT82 and hT7xpT82 analysis of raw CSF from AD and VS patients.

[0053] Figure 13 shows the predictive power of the pT3xhT43 (343), pT3xpT82 (382) and hT7xpT82 (782) assays in differentiating individuals with DA and VS.

[0054] Figures 14A-14F show the signal from tests of (A, B) pT3xhT43 (343), (C, D) pT3xpT82 (382) and (E, F) hT7xpT82 (782) performed on rpHPLC of CSF fractions from individuals of (A, C, E) DA and (B, D, F) VS.

[0055] Figures 15A-15O show the signal from (AE) pT3xhT43, (FJ) pT3xpT82 and (KO) hT7xpT82 assays performed on rpHPLC fractions of LCR 0 and CDR 0.5 individuals.

[0056] Figures 16A-16B show the results of (A) pT3xpT82 analysis ratio versus hT7xpT82 (short pTau) or pT3xhT43 analysis ratio versus hT7xpT82 (long pTau) in raw CSF and (B) pT3xpT82 vs. hT7xpT82 analysis ratio of rpHPLC fractions of CSF, compared to MMSE score; all from a blinded cohort of CDR 0 and CDR1 individuals.

[0057] Figures 17A-17T show the results of analyses of (A) pT3xhT43, (B) pT3xpT82 and (C) hT7xpT82 in crude CSF, (D) correlation of the two pT3 assays in crude CSF, (E) correlation of pT3xpT82 versus hT7xpT82 in crude CSF, (F) correlation of pT3xhT43 versus Innotest tTau in crude CSF, (G) correlation of pT3xhT43 versus Innotest pTau181 in crude CSF, (H) correlation of pT3xhT43 versus Innotest AB42 in crude CSF, (I) correlation of the ratio of pT3xhT43 versus Innotest AB42 / 40 in crude CSF; (JI) Petition 870260042583, dated 06 / 05 / 2026, page 30 / 334 22 / 98 pT3xhT43 signal, (MP) pT3xpT82 signal or (QT) hT7xpT82 signal in (J, M, Q), all rpHPLC fractions, as well as (K, N, R) the sums of all fractions, (O, S) sums of initial peak fractions (short tau fragments) or (L, P, T) the sums of final peak fractions (longer tau fragments); all from a cohort of VS, MCI and DA individuals.

[0058] Figures 18A-18P show the results of (A) pT3xpT82 (short p217+) versus pT3xhT43 (long p217+), (B) pT3xpT82 versus hT7xpT82 (short tTau), (C) pT3xpT82 vs. NFL and (D) pT3xhT43 or (E) pT3xpT82 versus amyloid status, as well as the correlation of (FI, NP) pT3xhT43 or (JM) pT3xpT82 with (FM) various cognition scores or (NP) change in those scores by 78 weeks; all from the cohort of 235 individuals (90 of whom had 78-week follow-up) from the Janssen ELN115727301 / 302 study of individuals with mild to moderate AD. The individuals were initially enrolled (and classified as AD) based on cognition; however, through biochemical evaluation (AB40 and AB42), it was found that 27 of the individuals were amyloid-negative, and thus likely represent dementia of non-AD causes. These individuals are analyzed as a separate cohort in the figures above and are designated as amyloid-negative individuals = 0 and amyloid-positive individuals = 1.

[0059] Figure 19 shows the signal from a pT3xhT43 assay performed on rpHPLC fractions of CSF samples enhanced with IgG, pT3 mAb, humanized pT3 mAb, or sham control, followed by immunoprecipitation to collect antibody-bound p217+ tau.

[0060] Figures 20A-20B show the antibody dose dependence of the immunocapture / rpHPLC method for quantifying antibody-free (A) and antibody-bound (B) tau p217+, with data plotted as the sum of the signal in rpHPLC fractions 12-16.

[0061] Figures 21A-21C show the differential kinetics of antibody damage vs. tau p217+ (A, C) with or (B) without heat-mediated denaturation. (A) mixture of humanized PT3 mAb / CSF; (B) untreated CSF; (C) PT3 mAb Petition 870260042583, dated 06 / 05 / 2026, p. 31 / 334 23 / 98 humanized.

[0062] Figures 22A-22C show (A) heat-mediated denaturation and (b) immunocapture / rpHPLC methods for quantifying antibody-free vs. antibody-bound p217+ tau; (C) shows a comparison of the methods.

[0063] Figure 23 shows a lack of recognition of the pT3-based assay of tau p217 in cynomolgus monkey CSF.

[0064] Figures 24A-24C show tau p217+ measurements in marmoset CSF, as determined using the (A) pT3xhT43, (B) pT3xpT82 and (C) hT7xpT82 tests.

[0065] Figures 25A-25D show measurements of (A, B) hT7xpT82 (tTau) or (C, D) pT3xpT82 (tau p217+ short) in crude serum from 4 DA and VS individuals. Measurements were performed under a dilution of (A, C) 1:4 or (B, D) 1:16, a lack of linearity and sensitivity of the dilution is observed.

[0066] Figures 26A-26B show measurements of (A) hT7xpT82 (tTau) or (B) pT3xpT82 (tau p217+ short) in sera pre-treated with NaOAc and heat denaturation, from the same 4 DA and VS individuals evaluated in Figures 25A-25D.

[0067] Figure 27 shows the pT3xpT82 (tau p217+ short) measurements in pT3 immunoprecipitations (IP) from sera of the same 4 DA and VS individuals evaluated in Figures 25A-25D and Figures 26A-26B. DETAILED DESCRIPTION OF THE INVENTION

[0068] Several publications, articles, and patents are cited or described in the background and throughout the descriptive report; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, or the like that has been included in this descriptive report is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these subjects are part of the prior art with respect to any disclosed or claimed inventions. Petition 870260042583, dated 06 / 05 / 2026, p. 32 / 334 24 / 98 Definitions

[0069] Except where otherwise defined, all technical and scientific terms used in the present invention have the same meaning commonly understood by those skilled in the art to which this invention pertains. Otherwise, certain terms used in the present invention have the meanings as defined in the descriptive report. All patents, published patent applications and publications cited herein are incorporated herein by reference as if fully filed herein. It should be noted that, as used in the present invention and appended claims, the singular forms a / an and the / the include the plural reference, unless the context clearly indicates otherwise.

[0070] Except where otherwise specified, any numerical value, such as a concentration or a concentration range described herein, should be understood as being modified in all cases by the term approximately. Thus, a numerical value typically includes ± 10% of the mentioned value. For example, a concentration of 1 mg / ml includes 0.9 mg / ml to 1.1 mg / ml. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used in the present invention, the use of a numerical range expressly includes all possible subranges, all individual numerical values ​​within that range, including whole numbers within those ranges, and fractions of the values, unless the context clearly indicates otherwise.

[0071] As used in the present invention, the term antibody or immunoglobulin is used in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, adapted human, humanized and chimeric monoclonal antibodies and antibody fragments.

[0072] In general, antibodies are proteins or peptide chains that exhibit binding specificity for a specific antigen. The Petition 870260042583, dated 06 / 05 / 2026, page 33 / 334 25 / 98 Antibody structures are well known. Immunoglobulins can be divided into five main classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the constant heavy chain domain. IgA and IgG are further subclassified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Consequently, the antibodies of the invention can be of any of the five main classes or corresponding subclasses. Preferably, the antibodies of the invention are IgG1, IgG2, IgG3, or IgG4. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. Consequently, the antibodies of the invention can contain either a kappa or lambda light chain constant domain.According to specific embodiments, the antibodies of the invention include constant regions of heavy chain and / or light chain antibodies from mice or humans.

[0073] In addition to the constant light and heavy chain domains, antibodies contain variable light and heavy chain regions. An immunoglobulin variable light or heavy chain region consists of a framework region interrupted by antigen-binding sites. Antigen-binding sites are defined using various terms and numbering schemes as follows: (i) Kabat: Complementarity-determining regions or CDRs are based on sequence variability (Wu and Kabat, J Exp Med. 132:211-50, 1970). In general, the antigen-binding site has three CDRs in each variable region (e.g., HCDR1, HCDR2, and HCDR3 in the variable heavy chain (VH) region and LCDR1, LCDR2, and LCDR3 in the variable light chain (VL) region); (ii) Chothia: The terms hypervariable region and HVR refer to regions of an antibody variable domain that are hypervariable in structure, as defined by Chothia and Lesk (Chothia and Lesk, J Mol Biol. Petition 870260042583, dated 06 / 05 / 2026, page 34 / 334 26 / 98 196:901-17, 1987). In general, the antigen-binding site has three hypervariable regions in each HV (H1, H2, H3) and LV (L1, L2, L3). The numbering systems, as well as the annotations of CDRs and HVRs, were reviewed by Abhinandan and Martin (Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008); (iii) IMGT: Another definition of the regions that form the antigen-binding site was proposed by Lefranc (Lefranc et al., Dev Comp Immunol. 27:55-77, 2003) based on a comparison of the V domains of immunoglobulins and T cell receptors. The database of International Immunogenetics (IMGT) (http: / / www.imgt.org) provides a standardized numbering and definition of these regions. The correspondence between CDR, HVR, and IMGT delineations is described in Lefranc et al., 2003, Id.; (iv) The antigen binding site can also be delineated based on Specificity Determining Residue Usage (SDRU) (Almagro, Mol Recognit. 17:132-43, 2004), where SDR refers to amino acid residues of an immunoglobulin that are directly involved in contact with the antigen.

[0074] Framework or framework sequences are the remaining sequences in the variable region of an antibody that are different from those defined to be the antigen-binding site sequences. Since the exact definition of an antigen-binding site can be determined by various delineations, as described above, the exact framework sequence depends on the definition of the antigen-binding site. Framework regions (FRs) are the most highly conserved portions of the variable domains. The variable domains of native light and heavy chains comprise four FRs (FR1, FR2, FR3, and FR4, respectively) that generally adopt a beta-sheet configuration, connected by three hypervariable loops. The hypervariable loops in each chain are held very closely together by the FRs and, with the hypervariable loops of other chains, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies Petition 870260042583, dated 06 / 05 / 2026, page 35 / 334 Study 27 / 98 revealed the relationship between the sequence and the shape of the binding site formed by the complementarity-determining regions (Chothia et al., J. Mol. Biol. 227: 799-817, 1992; Tramontane et al., J. Mol. Biol. 215:175-182, 1990). Despite their high sequence variability, five of the six loops adopt only a small repertoire of main chain conformations, called canonical frameworks. These conformations are, firstly, determined by the length of the loops and, secondly, by the presence of very important residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding, or the ability to assume unusual main chain conformations.

[0075] As used in the present invention, the term antigen-binding fragment refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', an F(ab')2, an Fv fragment (dsFv), an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single-domain antibody (sdab), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single-domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise an antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parental antibody or a fragment of a parental antibody binds.According to specific embodiments, the antigen-binding fragment comprises a variable light chain region, a constant light chain region, and an Fd segment of the constant heavy chain region. According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab'). Petition 870260042583, dated 06 / 05 / 2026, page 36 / 334 28 / 98

[0076] As used in the present invention, the term epitope refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment specifically binds. Epitopes can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by the tertiary fold of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed.1996).

[0077] As used in the present invention, the term tau or tau protein refers to a protein abundant in the central and peripheral nervous system that has multiple isoforms. In the human central nervous system (CNS), six major isoforms with sizes ranging from 352 to 441 amino acids in length exist due to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9, 2009). The isoforms differ from each other by the regulated inclusion of 0 to 2 N-terminal insertion elements and 3 or 4 tandemly arranged microtubule-linking repeats and are called 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R. As used in the present invention, the term control tau refers to the tau isoform of SEQ ID NO: 1 that is devoid of phosphorylation and other post-translational modifications.As used in the present invention, the term tau includes proteins comprising mutations, for example, point mutations, fragments, insertions, deletions, and full-length wild-type tau junctional variants. The term tau also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation. Petition 870260042583, dated 06 / 05 / 2026, p. 37 / 334 29 / 98

[0078] Except where otherwise indicated, as used herein, the amino acid numbering in a tau protein or fragment thereof is with reference to the amino acid sequence presented in SEQ ID NO: 1.

[0079] As used herein, the terms tau p217+ peptides, tau p217+ and tau p217+ protein mean a human tau protein or tau fragment that is phosphorylated at one or both residues 217 (pT217) and 212 (pT212) of the tau protein, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1.

[0080] As used herein, the term p217+ tau epitope refers to a tau epitope containing at least one phosphorylated T217 and phosphorylated T212, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1. Examples of p217+ tau epitopes include, for example, a pT3 epitope. As used herein, the term pT3 epitope refers to an epitope containing amino acids 210 to 220 of the human tau protein, which is phosphorylated to at least one T217 or T212 residue of human tau, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1. Examples of pT3 epitopes include, for example, SEQ ID NOs: 25, 26 and 27.

[0081] As used herein, each of the terms long p217+ tau peptides, long form of p217+ tau peptides or fragment of long p217+ tau peptides has the same meaning, referring to a p217+ tau peptide comprising the p217+ tau epitope and an epitope comprising amino acid residues 7 to 20 of the tau protein. Long p217+ tau peptides, according to embodiments of the invention, may have different lengths. For example, the amino-terminal of a fragment of long p217+ tau peptides may be amino acid residue 1, 2, 4, 5, 6 or 7 of the tau protein.

[0082] As used herein, each of the terms short tau p217+ peptides, short tau p217+, short form of tau p217+ peptides or fragment of short tau p217+ peptides has the same meaning. Petition 870260042583, dated 06 / 05 / 2026, page 38 / 334 30 / 98 refers to a p217+ epitope comprising the dp217+ epitope and an epitope comprising amino acid residues 119 to 126 of the tau protein, but does not contain an epitope comprising amino acid residues 7 to 20 of the tau protein. Short p217+ tau peptides, according to embodiments of the invention, may have different lengths. For example, the amino-terminal of a short p217+ tau peptide may be any of the amino acid residues between the epitope comprising amino acid residues 7 to 20 of the tau protein and the epitope comprising amino acid residues 119 to 126 of the tau protein.

[0083] As used herein, each of the terms long tau peptide, long tau peptide form, or long tau peptide fragment has the same meaning, referring to a tau peptide comprising the tau epitope recognized by a phosphorylation-independent capture antibody and an epitope comprising amino acid residues 7 to 20 of the tau protein. Long tau peptide fragments, according to embodiments of the invention, may have different lengths. For example, the amino-terminal of a long tau peptide fragment may be amino acid residue 1, 2, 4, 5, 6, or 7 of the tau protein.

[0084] As used herein, each of the terms short tau peptide, short tau, short tau peptide form, or short tau peptide fragment has the same meaning, referring to a tau epitope comprising the tau epitope recognized by a phosphorylation-independent capture antibody and an epitope comprising amino acid residues 7 to 20 of the tau protein. Short tau peptide fragments, according to embodiments of the invention, may have different lengths. For example, the amino-terminal of a short tau peptide may be any of the amino acid residues between the epitope comprising amino acid residues 7 to 20 of the tau protein and the epitope comprising amino acid residues 119 to 126 of the tau protein.

[0085] As used herein, the term capture antibody refers to a Petition 870260042583, dated 06 / 05 / 2026, page 39 / 334 31 / 98 an antibody that binds to an antigen of interest and is directly or indirectly linked to a solid support. Examples of solid supports include, but are not limited to, microparticles or capsules, such as magnetic microspheres. Examples of capture antibodies include, but are not limited to, a monoclonal antibody that binds to a p217+ tau epitope. According to embodiments of the invention, the capture antibody may be a monoclonal antibody comprising HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36, and 37. In a particular embodiment, the capture antibody is pT3.As used herein, the term pT3 refers to an antibody that binds to p217+ tau peptides and has a variable heavy chain region with amino acid sequence SEQ ID NO: 28 and a variable light chain region with amino acid sequence SEQ ID NO: 29. In one embodiment, the pT3 monoclonal antibody is expressed by a mouse hybridoma. In another embodiment, the capture antibody is a humanized antibody that has a variable heavy chain region with amino acid sequence SEQ ID NO: 30 and a variable light chain region with amino acid sequence SEQ ID NO: 31.

[0086] According to other embodiments of the invention, the capture antibody may be a monoclonal antibody that binds to an epitope between amino acids 150 and 250 of the tau protein, preferably amino acids 211 to 221 or amino acids 159 to 163 of the human tau protein, in a phosphorylation-independent manner, and the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1. In a specific embodiment, the capture antibody is hT7. As used herein, the term hT7 refers to a publicly available monoclonal antibody that binds to an epitope comprising amino acids 159 to 163 of the human tau protein, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1. An hT7 monoclonal antibody is commercially available, for example, from Petition 870260042583, dated 06 / 05 / 2026, p. 40 / 334 32 / 98 ThermoFisher (e.g., Catalog No.: MN1000).

[0087] As used herein, the term detection antibody refers to an antibody that binds to an antigen of interest and has a detectable marker or is linked to a secondary detection system. Examples of detectable markers include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of detection antibodies include, but are not limited to, a monoclonal antibody that binds to the tau protein, preferably an epitope comprising amino acids 7 to 20 or 116 to 127 of the human tau protein, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1. When a monoclonal antibody that binds to a tau protein at an epitope comprising amino acids 7 to 20 is used as a detection antibody for captured p217+ tau peptides, long tau fragments are detected.When a monoclonal antibody that binds to a tau protein at an epitope comprising amino acids 116 to 127 is used as a detection antibody for captured p217+ tau peptides, both short and long tau fragments are detected.

[0088] In one particular embodiment, the detection antibody is hT43. As used herein, the term hT43 refers to a monoclonal antibody that binds to an epitope comprising amino acids 7 to 20 of the human tau protein, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1, and the antibody has an amino acid sequence from the variable region of the heavy chain of SEQ ID NO: 8 and an amino acid sequence from the variable region of the light chain of SEQ ID NO: 9. In another particular embodiment, the detection antibody is pT82. As used herein, the term pT82 refers to a monoclonal antibody that binds to an epitope comprising amino acids 119 to 126, preferably 116 to 127, of the human tau protein, wherein the numbering of the positions is in accordance with the numbering in SEQ ID NO: 1, and the antibody has an amino acid sequence from the variable region of Petition 870260042583, dated 06 / 05 / 2026, p. 41 / 334 33 / 98 heavy chain of SEQ ID NO: 18 and an amino acid sequence of the variable region of the light chain of SEQ ID NO: 19.

[0089] As used herein, the term pT3-based assay refers to an assay according to an embodiment of the invention, in which the pT3 antibody is used as the capture antibody. As used herein, the term pT3xhT43 refers to an assay according to an embodiment of the invention, in which the pT3 antibody is used as the capture antibody and the hT43 antibody is used as the detection antibody. As used herein, the term pT3xpT82 refers to an assay according to an embodiment of the invention, in which the pT3 antibody is used as the capture antibody and the pT82 antibody is used as the detection antibody.

[0090] As used herein, the term hT7-based assay refers to assays according to embodiments of the invention, in which the hT7 antibody is used as the capture antibody. As used herein, the term hT7xpT82 refers to assays according to embodiments of the invention, in which the hT7 antibody is used as the capture antibody and the pT82 antibody is used as the detection antibody.

[0091] As used in the present invention, the term individual refers to an animal and, preferably, a mammal. According to specific embodiments, the individual is a mammal that includes a non-primate (for example, a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, marmoset or mouse) or a primate (for example, a monkey, chimpanzee or human being). In particular embodiments, the individual is a human being.

[0092] As used herein, a tauopathy encompasses any neurodegenerative disease involving the pathological aggregation of tau in the brain. In addition to familial and sporadic Alzheimer's disease, other exemplary tauopathies include frontotemporal dementia with chromosome 17-linked parkinsonism (FTDP17), progressive supranuclear palsy, corticobasal degeneration, and other similar conditions. Petition 870260042583, dated 06 / 05 / 2026, page 42 / 334 34 / 98 Pick's disease, progressive subcortical gliosis, dementia with tangles only, diffuse neurofibrillary tangles with calcification, dementia with argyrophilic grains, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatza disease, inclusion body myositis, Creutzfelt-Jakob disease, multiple system atrophy, Niemann-Pick type C disease, cerebral amyloid angiopathy caused by prion protein, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy, such as pugilistic dementia (boxing disease) (Morris et al., Neuron, 70:410-26, 2011).

[0093] As used herein, the terms determine, measure, assess, and test are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement and include determining whether a characteristic, trait, or aspect is present or not. Assessment can be relative or absolute. Assessing the presence of includes determining the quantity of something present, as well as determining whether it is present or absent.

[0094] As used herein, the term diagnosis means the detection of a disease or disorder or the determination of the stage or degree of a disease or disorder, such as a tauopathy. Typically, a diagnosis of a disease or disorder is based on the assessment of one or more factors and / or symptoms that are indicative of the disease. A diagnosis may be made based on the presence, absence, or quantity of a factor that is indicative of the presence or absence of the disease or condition, for example, tau p217+. Each factor or symptom that is considered indicative for the diagnosis of a particular disease need not be exclusively related to that particular disease; that is, there may be differential diagnoses that can be deduced from a diagnostic factor or symptom. Similarly, there may be cases where a factor or symptom that is indicative of a specific disease is Petition 870260042583, dated 06 / 05 / 2026, page 43 / 334 35 / 98 present in an individual who does not have the specific disease. The term diagnosis also encompasses the determination of the therapeutic effect of a drug therapy, for example, an anti-tau p217+ antibody therapy, or the prediction of the pattern of response to a drug therapy, for example, an anti-tau p217+ antibody therapy. Diagnostic methods may be used independently, or in combination with other diagnostic and / or staging methods known in medical technique for a given disease or disorder, for example, Alzheimer's disease.

[0095] As used herein, the terms increase and decrease refer to differences in the amount of a specific biomarker in a sample compared to a control or reference level. For example, the amount of a specific peptide may be present in an elevated amount or in a reduced amount in samples from patients with a disease compared to a reference level. In one embodiment, an increase or decrease in a level may be a difference between the biomarker level present in a sample compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more.In one embodiment, an increase or decrease in a level can be a statistically significant difference between the level of the biomarker present in a sample compared to a control. For example, the difference may be statistically significant if the measured biomarker content falls outside approximately 1.0 standard deviation, approximately 1.5 standard deviations, approximately 2.0 standard deviations, or approximately 2.5 standard deviations from the mean of any control or reference group. The reference or control may be, for example, a sample from an individual. Petition 870260042583, dated 06 / 05 / 2026, page 44 / 334 36 / 98 healthy, or from a sample obtained from the same individual at an earlier time, such as a point in time before the administration of a therapeutic agent or an earlier time during a therapeutic regimen.

[0096] As used in the present invention, the term isolate means a biological component (such as a nucleic acid, peptide, or protein) that has been substantially separated, produced apart from, or purified from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal or extrachromosomal DNA and RNA and proteins. Nucleic acids, peptides, and proteins that have been isolated in this way include nucleic acids and proteins purified by standard purification methods. Isolated nucleic acids, peptides, and proteins may be part of a composition and may still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, or protein.The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0097] An isolated antibody that binds to a tau protein or an isolated anti-tau antibody, as used herein, refers to an antibody that specifically binds to the tau protein and is substantially free of other antibodies that have different antigenic specificities (e.g., an isolated anti-tau detection antibody is substantially free of antibodies that specifically bind to antigens other than tau). An isolated anti-tau detection antibody may, however, have cross-reactivity with other related antigens, for example, from other species (such as tau species homologs).

[0098] As used herein, the terms binds specifically and specific binding refer to the ability of an anti-tau antibody of the invention to bind to a predetermined target with a dissociation constant (KD) of about 1x10⁻⁶ M or stronger, for example, about 1x10⁻⁷ M or less, about 1x10⁻⁸ M or less, about 1x10⁻⁹ M or less, about 1x10⁻¹⁰ M Petition 870260042583, dated 06 / 05 / 2026, p. 45 / 334 37 / 98 or less, about 1x10⁻¹¹M or less, about 1x10⁻¹²M or less, or about 1x10⁻¹³M or less. The term KD is obtained from the ratio between Kd and Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values ​​for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD value of an anti-tau antibody can be determined using surface plasmon resonance, such as using a biosensor system, for example, a Biacore® system, a Proteon instrument (BioRad), a KinExA instrument (Sapidyne), ELISA, or competitive binding assays known to those skilled in the art. Typically, an anti-tau antibody binds to a predetermined target (i.e., tau) with a KD that is at least ten times smaller than its KD for a non-specific target, as measured by surface plasmon resonance using, for example, a Proteon instrument (BioRad).Anti-tau antibodies that bind specifically to tau may, however, have cross-reactivity with other related targets, for example, with the same predetermined target from other species (homologous), such as mouse, rat, marmoset, dog, or pig.

[0099] As used in the present invention, the term polynucleotide, referred to as nucleic acid molecule, nucleotides, or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single-stranded and double-stranded regions.Furthermore, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with a modified backbone. Petition 870260042583, dated 06 / 05 / 2026, page 46 / 334 38 / 98 for stability or other reasons. Modified bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, polynucleotide encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. Polynucleotide also encompasses relatively short chains of nucleic acid, often called oligonucleotides.

[0100] As used in the present invention, a vector is a replicon into which another nucleic acid segment can be operationally inserted in order to cause replication or expression of the segment.

[0101] As used in the present invention, the term host cell refers to a cell comprising a nucleic acid molecule of the invention. The host cell can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a host cell is a cell transfected with a nucleic acid molecule of the invention. In another embodiment, a host cell is a progeny or a potential progeny of such a transfected cell. The progeny of a cell may or may not be identical to the original cell, for example, due to mutations or environmental influences that may occur in subsequent generations or integration of the nucleic acid molecule into the host cell genome.

[0102] The term expression, as used herein, refers to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. The term also encompasses the translation of RNA into one or more polypeptides and also includes all naturally occurring post-transcriptional and post-translational modifications. The expressed detection antibody or antigen-binding fragment thereof that binds to tau may be within the cytoplasm of a host cell, in the extracellular medium, as Petition 870260042583, dated 06 / 05 / 2026, page 47 / 334 39 / 98 in the growth medium of a cell culture, or anchored to the cell membrane. Anti-tau antibodies

[0103] In a general aspect, the invention relates to isolated detection antibodies or antigen-binding fragments thereof that bind to tau protein that has been immobilized by a capture antibody. Such anti-tau antibodies may have the binding properties of a phosphorylated epitope on tau or binding to a non-phosphorylated epitope on tau. The detection anti-tau antibodies may be useful as research or diagnostic reagents for detecting tau in biological samples.

[0104] In a particular aspect, the invention relates to an isolated detection antibody or antigen-binding fragment thereof that binds to a tau protein at an epitope comprising amino acid residues 119 to 126, preferably amino acid residues 116 to 127, of the tau protein.

[0105] According to a particular aspect, the isolated detection antibody or antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 116 to 127 of the tau protein comprises (a) HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and (b) LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively.

[0106] According to a specific aspect, the isolated detection antibody or antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 116 to 127 of the tau protein comprises a variable heavy chain region having a sequence with at least 80% of the polypeptide, preferably at least 85% or 90%, more preferably at least 95%, and most preferably 100% identical to SEQ ID NO: 8, and a variable light chain region having the Petition 870260042583, dated 06 / 05 / 2026, p. 48 / 334 40 / 98 polypeptide sequence at least 80%, preferably at least 85% or 90%, more preferably at least 95%, and most preferably 100% identical to SEQ ID NO: 9.

[0107] Preferably, the isolated detection antibody or antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 116 to 127 of the tau protein is a pT82 antibody.

[0108] In one particular aspect, the invention relates to an isolated detection antibody or antigen-binding fragment thereof that binds to a tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein.

[0109] According to a particular aspect, the isolated detection antibody or antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein comprises (a) HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and (b) LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively.

[0110] According to a particular aspect, the isolated detection antibody or antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein comprises a variable region of the heavy chain having a sequence with at least 80% of the polypeptide, preferably at least 85% or 90%, more preferably at least 95% and most preferably 100% identical to SEQ ID NO: 18, and a variable region of the light chain having a polypeptide sequence at least 80%, preferably at least 85% or 90%, more preferably at least 95% and most preferably 100% identical to SEQ ID NO: 19.

[0111] Preferably, the detection antibody alone or fragment of Petition 870260042583, dated 06 / 05 / 2026, p. 49 / 334 41 / 98 antigen binding, which binds to the tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein, is an hT43 antibody.

[0112] The antibodies of the present invention can be produced by a variety of techniques, for example, by the hybridoma method (Kohler and Milstein, Nature. 256:495-7, 1975). Chimeric mAbs containing a variable light and heavy chain region derived from a donor antibody (typically murine) in association with constant light and heavy chain regions derived from an acceptor antibody (typically another mammalian species such as humans) can be prepared by a method disclosed in US4816567. CDR-grafted mAbs that have CDRs derived from a non-human donor immunoglobulin (typically murine) and the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins can be prepared by techniques known to those skilled in the art such as that disclosed in US5225539.Fully human mAbs devoid of any non-human sequences can be prepared from transgenic human immunoglobulin mice using techniques mentioned in (Lonberg et al., Nature. 368:856-9, 1994; Fishwild et al., Nat Biotechnol. 14:84551, 1996; Mendez et al., Nat Genet. 15:146-56, 1997). Human mAbs can also be prepared and optimized from phage display libraries (Knappik et al., J Mol Biol. 296: 57-86, 2000; Krebs et al., J Immunol Methods. 254: 67-84, 2001; Shi et al., J Mol Biol. 397: 385-96, 2010).

[0113] The functional activity of tau-detecting antibodies and antigen-binding fragments thereof can be characterized by methods known in the art. Methods for characterizing antibodies and antigen-binding fragments thereof that bind to tau include, but are not limited to, affinity and specificity assays, including Biacore analysis, ELISA and FACS, immunohistochemical analysis, etc. Petition 870260042583, dated 06 / 05 / 2026, page 50 / 334 42 / 98

[0114] Several well-known methodologies can be employed to determine the binding epitope of the antibodies of the invention. For example, when the structures of both individual components are known, silico protein docking can be performed to identify compatible interaction sites. Deuterium hydrogen exchange (H / D) can be performed with the antigen and antibody complex to map regions on the antigen that are bound by the antibody. Segment and point mutagenesis of the antigen can be used to locate amino acids important for antibody binding. The cocrystalline structure of the antibody-antigen complex is used to identify residues that contribute to the epitope and paratope.

[0115] In another general aspect, the invention relates to an isolated polynucleotide encoding a detection antibody or antigen-binding fragment of the invention. It will be understood by those skilled in the art that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without altering the amino acid sequence of the protein. Consequently, it will be understood by those skilled in the art that nucleic acid sequences encoding detection antibodies or antigen-binding fragments of the invention can be altered without altering the amino acid sequences of the proteins.Exemplary isolated polynucleotides are polynucleotides encoding polypeptides comprising the immunoglobulin heavy chain CDRs HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 2, 3, and 4, respectively, or polypeptides comprising the immunoglobulin light chain CDRs LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 5, 6, and 7, respectively. Other exemplary isolated polynucleotides are polynucleotides encoding polypeptides comprising the immunoglobulin heavy chain CDRs HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 12, 13, and 14, respectively, or polypeptides comprising the CDRs of... Petition 870260042583, dated 06 / 05 / 2026, page 51 / 334 43 / 98 light chain immunoglobulin LCDR1, LCDR2 and LCDR3 shown in SEQ ID Nos: 15, 16 and 17, respectively. Other exemplary isolated polynucleotides are polynucleotides encoding variable antibody regions of the invention. Other polynucleotides that, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the antibodies of the invention, are also within the scope of the invention. The isolated nucleic acids of the present invention can be made using well-known recombinant or synthetic techniques. The DNA encoding monoclonal antibodies is readily isolated and sequenced using methods known in the art. When a hybridoma is produced, such cells can serve as a source of such DNA. Alternatively, display techniques in which the coding sequence and the translation product are linked, such as phage or ribosomal display libraries, can be used.

[0116] In another general aspect, the invention relates to a vector comprising an isolated polynucleotide encoding a detection antibody or antigen-binding fragment of the invention. Any vector known to those skilled in the art, in view of the present disclosure, may be used, such as a plasmid, cosmid, phage vector or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector may include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome-binding element, terminator, enhancer, selection marker and origin of replication. The promoter may be a constitutive, inducible or repressible promoter. Several expression vectors capable of delivering nucleic acids to a cell are known in the art and may be used in the present invention for the production of an antibody or antigen-binding fragment thereof in the cell.Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to the embodiments of the invention. Petition 870260042583, dated 06 / 05 / 2026, page 52 / 334 44 / 98

[0117] In another general aspect, the invention relates to a host cell comprising an isolated polynucleotide encoding a detection antibody or antigen-binding fragment of the invention. Any host cell known to those skilled in the art in view of the present disclosure may be used for the recombinant expression of antibodies or antigen-binding fragments of the invention. Such host cells may be eukaryotic cells, bacterial cells, plant cells or archaeal cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origin.Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines, like murine cell lines SP2 / 0 (American Type Culture Collection (ATCC), Manassas, Va, USA CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580). An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells such as CHO-K1 SV (Lonza Biologics), CHO-K1 (ATCC CRL-61, Invitrogen), or DG44.

[0118] In another general aspect, the invention relates to a method for producing a detection antibody or antigen-binding fragment of the invention, comprising cultivating a cell comprising a polynucleotide encoding the detection antibody or antigen-binding fragment thereof under conditions to produce a detection antibody or antigen-binding fragment of the invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art. Petition 870260042583, dated 06 / 05 / 2026, page 53 / 334 45 / 98 Diagnostic methods

[0119] The invention relates to the measurement of p217+ tau species that are enriched in DA, for example, using a capture antibody, such as a pT3, which selectively immobilizes p217+ tau species, in combination with an anti-tau detection antibody, which is labeled with a reporter element that enables the detection of the captured p217+ tau species. The methods of the invention can be used for various diagnostic purposes, for example, to diagnose DA or other tauopathies in an individual, to monitor the effectiveness of a treatment, to identify an individual suitable for anti-p217+ treatment, etc.

[0120] According to one embodiment of the present invention, tau p217+ peptides in a sample of interest are captured with a capture antibody directed against a tau p217+ epitope, such as an epitope having the amino acid sequence of SEQ ID NOs: 25, 26 or 27. The captured tau p217+ peptides, although all containing the tau p217+ epitope, may have different lengths, which can be detected by detection antibodies that bind to different epitopes.For example, a detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein can only detect captured p217+ tau peptides or fragments thereof that additionally contain amino acid residues 7 to 20 of the tau protein (long p217+ tau peptides), whereas a detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein can detect not only long p217+ tau peptides but also short p217+ tau peptides. The captured p217+ tau peptides can be placed in contact with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein, thus detecting and measuring the quantity of long p217+ tau peptides or of p217+ tau peptides (long and short p217+ tau peptides) in the sample. A quantity of p217+ tau peptides. Petition 870260042583, dated 06 / 05 / 2026, page 54 / 334 The ratio of 46 / 98 short peptides in a sample is calculated by subtracting the amount of long p217+ tau peptides from the amount of p217+ tau peptides.

[0121] According to another embodiment of the invention, in addition to capturing and measuring the amount of p217+ tau peptides in a sample, the total p217+ tau peptides are captured with a phosphorylation-independent capture antibody, such as an antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein. The captured total tau peptides can be brought into contact with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein in order to detect and measure the amount of total long tau peptides or total tau peptides (long and short tau peptide fragments) in the sample. The total amount of short tau peptides in a sample is calculated by subtracting the total amount of long tau peptides from the total amount of tau peptides.

[0122] According to embodiments of the invention, a value relating to p217+ tau peptides in a sample, such as the amount of p217+ tau peptides and the amount of long p217+ tau peptides, optionally the amount of total tau peptides and the amount of long tau fragments, in a sample, as well as information based on the measured amounts, such as the calculated short p217+ tau peptides and total short tau peptides, or a ratio relating to p217+ tau peptides, such as a ratio between the amount of short tau peptide fragments and the amount of long tau peptide fragments, a ratio between the amount of short p217+ tau peptides and the amount of short tau fragments, a ratio between the amount of long p217+ tau peptides and the amount of long tau fragments, etc., can be used for one or more diagnostic purposes.

[0123] The diagnosis is made by comparing a value related to Petition 870260042583, dated 06 / 05 / 2026, p. 55 / 334 47 / 98 tau p217+ peptides in a sample from an individual with corresponding baseline values. Baseline values ​​may represent average levels in a population of healthy individuals. Baseline values ​​may also represent previously determined levels in the same individual. In one embodiment, an individual is determined to be suffering from a tauopathy if a value relating to tau p217+ peptides in the individual's biological sample, such as the amount of long or short tau p217+ peptides, or a ratio relating to tau p217+ peptides, for example, a ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides, is significantly higher than a corresponding baseline value. As used herein, significantly higher refers to a higher value that is statistically significant, not due to chance, and has a p-value of 0.05 or less.Significantly higher can be at least about 1%, 2%, 5%, or 10% greater than that found in healthy volunteers, with a p-value less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc.

[0124] In one embodiment, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, in contact with a capture antibody directed against an epitope comprising phosphorylated tau p217+ to capture tau p217+ peptides in the sample, (ii) placing the captured tau p217+ peptides in contact with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 to measure the amount of long tau p217+ peptides and / or with a detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of long and short tau p217+ peptides in the sample, and (iii) determining whether or not the individual suffers from a tauopathy or is at risk of developing a tauopathy based on the amount of tau p217+ peptides or the ratio between the amount of tau p217+ peptides short p217+ and the amount of long p217+ tau peptides. Petition 870260042583, dated 06 / 05 / 2026, page 56 / 334 48 / 98 Diagnosis can be made by comparing the amount or concentration of p217+ tau peptides in a sample from the individual with corresponding baseline values. Diagnosis can also be performed by comparing the ratio between the amount of short p217+ tau peptides and the amount of long p217+ tau peptides in a sample from the individual with corresponding baseline values.

[0125] In another embodiment, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, or with a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein to capture the total tau peptides in the sample, (ii) placing the captured tau p217+ peptides, or the captured total tau peptides, in contact with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein to thereby measure the amount of long and short tau p217+ peptides, or the amount of total short tau peptides, in the sample,(iii) to determine whether or not the individual suffers from a tauopathy or is at risk of developing a tauopathy based on the ratio of the amount of short p217+ tau peptides to the total amount of short tau peptides in the biological sample. The diagnosis can be made by comparing the ratio of short p217+ tau peptides to the total amount of short tau peptides comprising the same tau protein region as recognized by the pT3 antibody, i.e., amino acids 211 to 221 of tau, in a sample from the individual to the corresponding baseline values.

[0126] In another embodiment, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, in Petition 870260042583, dated 06 / 05 / 2026, page 57 / 334 49 / 98 contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, (ii) place the captured tau p217+ peptides with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 to measure the amount of long tau p217+ peptides, and / or with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein to measure the amount of long and short tau p217+ peptides in the sample, and (iii) determine the effectiveness of the treatment in the individual based on the amount of tau p217+ peptides or the ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides.

[0127] In yet another embodiment, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, or with a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein to capture the total tau peptides in the sample, (ii) placing the captured tau p217+ peptides, or the captured total tau peptides, in contact with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein to thereby measure the amount of long and short tau p217+ peptides, or the amount of total short tau peptides,in the sample and (iii) determine the effectiveness of the treatment in the individual based on the ratio of the amount of short p217+ tau peptides to the total amount of short tau peptides in the biological sample.

[0128] In yet another modality, the effectiveness of the treatment in the individual is determined by monitoring the amount of tau p217+ peptides, the ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides, or the ratio between the amount of Petition 870260042583, dated 06 / 05 / 2026, page 58 / 334 50 / 98 short p217+ tau peptides and the amount of total short tau peptides, before, during, or after treatment. A decrease in values ​​from baseline signals a positive response to treatment. Values ​​may also temporarily increase in biological fluids as pathological tau is cleared from the brain.

[0129] According to a specific aspect, tauopathy includes, but is not limited to, one or more selected from the group consisting of Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), frontotemporal dementia with chromosome 17-linked parkinsonism (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, dementia with tangles only, diffuse neurofibrillary tangles with calcification, dementia with argyrophilic grains, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatza disease, inclusion body myositis, Creutzfelt-Jakob disease, multiple system atrophy, Niemann-Pick type C disease, cerebral amyloid angiopathy caused by prion protein, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles,Post-encephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (boxing disease).

[0130] Preferably, the tauopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), FTDP-17, or progressive supranuclear palsy.

[0131] Most preferably, tauopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease).

[0132] According to one embodiment, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, (ii) Petition 870260042583, dated 06 / 05 / 2026, page 59 / 334 51 / 98 place the captured tau p217+ peptides in contact with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 in order to measure the amount of long tau p217+ peptides, and / or with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein in order to measure the amount of long and short tau p217+ peptides in the sample and (iii) determine whether or not the individual is suitable for anti-tau p217+ antibody therapy based on the amount of tau p217+ peptides or the ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides.

[0133] According to a particular aspect, an individual is determined to be suitable for anti-tau p217+ antibody therapy if the amount of tau p217+ peptides in the biological sample, or the ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides in the biological sample is significantly higher than a corresponding baseline value.

[0134] According to another particular aspect, a method of the invention comprises (i) placing a biological sample, preferably a CSF sample, in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, or with a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein to capture the total tau peptides in the sample, (ii) placing the captured tau p217+ peptides, or the captured total tau peptides, with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein to thereby measure the amount of long and short tau p217+ peptides, or the amount of total short tau peptides, in the sample,and (iii) determine whether or not the individual is suitable for anti-tau p217+ antibody therapy based on the ratio of the amount of short tau p217+ peptides to the amount of, Petition 870260042583, dated 06 / 05 / 2026, page 60 / 334 52 / 98 total short tau peptides in the biological sample.

[0135] According to one embodiment, it is determined whether an individual is suitable for anti-tau p217+ antibody therapy if the ratio of the amount of short tau p217+ peptides to the amount of total short tau peptides is significantly higher than a corresponding baseline value.

[0136] The invention also relates to the measurement of tau p217+ that is in complex with antibody in a biological sample, as well as free tau p217+ in the sample that is not bound to the antibody. In one embodiment, the total antibody is captured using affinity techniques, followed by denaturation conditions including chaotrophs, heat inactivation, or other protein disruption techniques. The tau p217+ is separated from the antibody using rpHPLC, and is measured using the methods of the invention, which allow the quantification of antibody-bound tau p217+.

[0137] According to a general aspect, the invention relates to a method for monitoring treatment with an anti-tau p217+ antibody in an individual, the method comprising: (i) obtaining a biological sample from the individual, (ii) separating the biological sample into an IgG-enriched sample containing antibody-bound tau p217+ and an IgG-depleted sample containing antibody-free tau p217+, (iii) purifying the tau p217+ away from the IgGs by rpHPLC to obtain an antibody-free tau p217+ sample, (iv) placing each IgG-enriched sample and the antibody-free tau p217+ sample in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in each of the samples, (v) placing the captured tau p217+ peptides in each of the samples in contact with an antibody directed against an epitope comprising amino acid residues 7 to 20 are used to measure the amount of long p217+ tau peptides.or with a detection antibody directed against an epitope comprising amino acid residues, Petition 870260042583, dated 06 / 05 / 2026, p. 61 / 334 53 / 98 116 to 127 of the tau protein, thus measuring the amount of long and short tau p217+ peptides in each of the samples, (vi) calculating the ratio between the amount of antibody-bound tau p217+ and the amount of antibody-free tau p217+, and (vii) monitoring treatment with anti-tau p217+ antibody in the individual based on the calculated ratio.

[0138] According to another general aspect, the invention relates to a method for monitoring treatment with an anti-tau p217+ antibody in an individual, wherein the method comprises: (i) obtaining a biological sample from the individual, (ii) obtaining a semi-denatured sample of the biological sample containing total tau p217+ and obtaining an undenatured sample of the biological sample containing antibody-free tau p217+, wherein the semi-denatured sample is heated to denature the antibodies in the sample, (iii) placing each of the denatured and undenatured samples in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in each of the samples, (iv) placing the captured tau p217+ peptides in each of the samples in contact with an antibody directed against an epitope comprising amino acid residues 7 to 20 in order to measure the amount of peptides. tau p217+ long,or with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein, thus measuring the amount of long and short tau p217+ peptides in each of the samples, (v) calculate the amount of antibody-bound tau p217+ in the sample by subtracting the amount of antibody-free tau p217+ from the total amount of tau p217+, (vi) calculate the ratio between bound tau p217+ and antibody-free tau p217+, and (vii) monitor treatment with anti-tau p217+ antibody in the individual based on the calculated ratio.

[0139] According to a particular aspect, the effectiveness of the treatment in the individual is determined by monitoring the amount of antibody-bound and antibody-free tau p217+ peptides before, during, or after treatment. A decrease in antibody-free tau p217+ values Petition 870260042583, dated 06 / 05 / 2026, page 62 / 334 A value of 54 / 98 relative to baseline, or an increase in antibody-bound tau p217+ values ​​relative to baseline and therefore an increase in the ratio of antibody-bound tau p217+ to antibody-free tau p217+ relative to baseline, signals a positive response to treatment. Antibody-free tau p217+ values ​​may also temporarily increase in biological fluids as pathological tau is cleared from the brain.

[0140] According to specific aspects, the capture antibody of the invention's methods is conjugated to a microsphere, such as a magnetic microsphere. According to other specific aspects, the detection antibody is biotinylated.

[0141] According to specific aspects, the amount of tau p217+ peptides measured in the methods of the invention can be determined using any suitable techniques known in the art, including ELISA and single-molecule array platform. According to specific aspects, the methods of the invention utilize a high-sensitivity array platform, such as Quanterix Simoa or MSD S-plex, to measure the amount of tau p217+ peptides in a sample. According to a specific aspect, the lower limit of quantification of the methods of the invention is about 40 fg / ml and the lower limit of detection of the method is about 2 fg / ml.

[0142] According to a particular aspect, the samples used in the methods of the invention are a biological sample, such as a blood sample, brain homogenate, or cerebrospinal fluid (CSF). Preferably, the sample is a CSF sample. According to a specific aspect, the sample is a crude CSF sample. According to another specific aspect, the sample is obtained after fractionation of a biological sample, such as CSF, using reverse-phase high-performance liquid chromatography (rpHPLC), which separates full-length tau protein and differentially sized tau fragments. Petition 870260042583, dated 06 / 05 / 2026, page 63 / 334 55 / 98

[0143] According to a particular aspect, the capture antibody of the invention's methods comprises the HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively. Preferably, the capture antibody is a pT3 antibody comprising the variable region of the heavy chain comprising the polypeptide sequence of SEQ ID NO: 28 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 29.

[0144] According to a specific aspect, the detection antibody of the invention's methods comprises HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively; and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7, respectively. Preferably, the detection antibody is a pT82 antibody comprising a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 8 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 9.

[0145] According to another specific aspect, the detection antibody of the invention's methods comprises HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 12, 13, and 14, respectively; and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 15, 16, and 17, respectively. Preferably, the detection antibody is an hT43 antibody comprising a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 19. Petition 870260042583, dated 06 / 05 / 2026, p. 64 / 334 56 / 98 Kits

[0146] In another general aspect, the invention relates to a kit comprising (a) a capture antibody directed against a tau p217+ epitope, optionally a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein, and (b) at least one detection antibody directed against a tau protein epitope comprising amino acids 7 to 20 or 116 to 127 of the tau protein. The kit is used to measure the amount of tau p217+ peptides, which is used as the ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides, and / or the ratio between the amount of short tau p217+ peptides and the total amount of short tau peptides in a sample.

[0147] The detection antibody may contain any detectable marker (e.g., fluorescent molecule, biotin, etc.) that is detectable directly or through a detectable secondary reaction (e.g., reaction with streptavidin). Alternatively, a second reagent containing the detectable marker may be used, where the second reagent has binding specificity for the primary antibody. In a diagnostic kit suitable for measuring tau p217+ in a biological sample, the antibodies in the kit may be supplied pre-bound to a solid phase, such as to the wells of a microtiter plate or to microspheres.

[0148] According to a specific aspect, the capture antibody of a kit of the invention comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36 and 37, respectively. Preferably, the capture antibody is a pT3 antibody comprising the variable region of the heavy chain comprising the polypeptide sequence of SEQ ID NO: 28 and a variable region of the light chain having the polypeptide sequence. Petition 870260042583, dated 06 / 05 / 2026, page 65 / 334 57 / 98 of SEQ ID NO: 29.

[0149] According to a particular aspect, the detection antibody of a kit of the invention comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively. Preferably, the detection antibody is a pT82 antibody comprising a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 8 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 9.

[0150] According to another particular aspect, the detection antibody of a kit of the invention comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively. Preferably, the detection antibody is an hT43 antibody comprising a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 19.

[0151] According to another specific aspect, a kit of the invention is used to measure the amount of tau p217+ peptides, the ratio between the amount of short tau p217+ peptides and / or the ratio between the amount of short tau p217+ peptides and the amount of short tau p217+ peptides in a sample using a method of the invention.

[0152] The contents of all references cited (including literature references, granted patents, published patent applications, and all copending patent applications) throughout this application are expressly incorporated herein by reference. Modalities Petition 870260042583, dated 06 / 05 / 2026, p. 66 / 334 58 / 98

[0153] The disclosure also provides the following non-limiting options.

[0154] Embodiment 1 is a method for measuring the amount of p217+ tau peptides in a sample, comprising: (i) bringing the sample into contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and (ii) bringing the captured tau p217+ peptides into contact with the detection antibody directed against an epitope comprising amino acid residues 119 to 126, such as amino acid residues 116 to 127 of the tau protein, or an epitope containing amino acid residues 7 to 20 of the tau protein, in order to measure an amount of tau p217+ peptides or an amount of long tau p217+ peptides, respectively.

[0155] Embodiment 2 is a method for determining a relative amount of long tau p217+ peptides or short tau p217+ peptide fragments in a sample, comprising (i) contacting the sample with a capture antibody directed against a tau p217+ epitope to capture the tau p217+ peptides in the sample,(ii) bring the captured p217+ tau peptides into contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein in order to measure the quantity of p217+ tau peptides, (iii) bring the captured p217+ tau peptides into contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure the quantity of long p217+ tau peptides, and (iv) determine a relative quantity of long p217+ tau peptides or short p217+ tau peptides based on the quantity of p217+ tau peptides and the quantity of long p217+ tau peptides. Petition 870260042583, dated 06 / 05 / 2026, page 67 / 334 59 / 98

[0156] Modality 3 is a method in accordance with modality 1 a 2, in which the capture antibody is conjugated with a microsphere, where the detection antibody is biotinylated.

[0157] Modality 4 is the method of any of the modalities 1 to 3, in which the amount of tau p217+ peptides in the sample is measured using a high-sensitivity platform.

[0158] Embodiment 5 is a method in accordance with any of embodiments 1 to 4, wherein the lower limit of quantification of the method is about 40 fg / ml of tau p217+ peptides and the lower limit of detection of the method is about 2 fg / ml of tau p217+ peptides.

[0159] Embodiment 6 is the method of any of the embodiments from 1 to 5, in which the sample is a biological sample, preferably a CSF sample, from an individual, and the method further comprises determining whether or not the individual suffers from a tauopathy or is at risk of developing a tauopathy based on the amount of p217+ tau peptides, the ratio of the amount of short p217+ tau peptides to the amount of long p217+ tau peptides, or the ratio of the amount of short p217+ tau peptides to the total amount of short tau peptides in the biological sample.

[0160] Modality 7 is the method of modality 6, in which the individual is determined to have a tauopathy or to be at risk of developing a tauopathy if the amount of tau p217+ peptides in the biological sample, the ratio of the amount of short tau p217+ peptides to the amount of long tau p217+ peptides, or the ratio of the amount of short tau p217+ peptides to the total amount of short tau peptides is significantly higher than corresponding reference values, such as the average corresponding value of healthy volunteers.

[0161] Modality 8 is the method of any of the modalities from a to 5, in which the sample is a biological sample, preferably a CSF sample, from an individual undergoing treatment for a tauopathy, and the Petition 870260042583, dated 06 / 05 / 2026, page 68 / 334 The 60 / 98 method further comprises determining the effectiveness of the treatment in the individual based on the amount of p217+ tau peptides, the ratio between the amount of short p217+ tau peptides and the amount of long p217+ tau peptides, or the ratio between the amount of short p217+ tau peptides and the total amount of short p217+ tau peptides in the biological sample.

[0162] Modality 9 is the method of modality 8, in which treatment is determined to be effective if the amount of p217+ tau peptides in the biological sample decreases over the course of treatment.

[0163] Modality 10 is a method of any of the modalities to 9, in which the tauopathy is selected from the group consisting of Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), frontotemporal dementia with chromosome 17-linked parkinsonism (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, dementia with tangles only, diffuse neurofibrillary tangles with calcification, dementia with argyrophilic grains, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatza disease, inclusion body myositis, Creutzfelt-Jakob disease, multiple system atrophy, Niemann-Pick type C disease, cerebral amyloid angiopathy caused by prion protein, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles,Post-encephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (boxing disease).

[0164] Modality 11 is the method of modality 10, in which tauopathy is Alzheimer's disease.

[0165] Modality 12 is the method of any of the modalities from 1 to 5, in which the sample is a biological sample, preferably a CSF sample, from a human individual, and the method further comprises determining whether or not the individual is suitable for a Petition 870260042583, dated 06 / 05 / 2026, page 69 / 334 61 / 98 anti-tau p217+ antibody therapy based on the amount of tau p217+ peptides, the ratio between the amount of short tau p217+ peptides and the amount of long tau p217+ peptides, or the ratio between the amount of short tau p217+ peptides and the total amount of short tau peptides in the biological sample.

[0166] Modality 13 is the method of modality 12, in which the individual is determined to be suitable for anti-tau p217+ antibody therapy if the amount of tau p217+ peptides in the biological sample, the ratio of the amount of short tau p217+ peptides to the amount of long tau p217+ peptides, or the ratio of the amount of short tau p217+ peptides to the total amount of short tau peptides is significantly higher than corresponding reference values, such as the average corresponding value of healthy volunteers.

[0167] Modality 14 is a method for monitoring treatment with an anti-tau p217+ antibody in an individual, the method comprising: i. Obtain a biological sample from the individual, ii. Separate the biological sample into an IgG-enriched sample containing antibody-bound tau p217+, and an IgG-depleted sample containing antibody-free tau p217+, iii. Place each of the IgG-enriched and IgG-depleted samples in contact with a capture antibody directed against an epitope comprising phosphorylated T212 and / or phosphorylated T217 of the tau protein to capture the tau p217+ peptides in each of the samples, iv. Place the captured tau p217+ peptides in contact with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein to measure the amount of antibody-bound tau p217+ and the amount of tau Petition 870260042583, dated 06 / 05 / 2026, page 70 / 334 62 / 98 p217+ antibody-free in biological sample, v. calculate the ratio between antibody-bound tau p217+ and antibody-free tau p217+, and vi. monitor treatment with anti-tau p217+ antibody in the individual based on the calculated ratio.

[0168] Modality 15 is a method for monitoring treatment with an anti-tau p217+ antibody in an individual, the method comprising: i. obtain a biological sample from the individual, ii. obtain a semi-denatured sample of the biological sample containing total tau p217+, and obtain an undenatured sample of the biological sample containing antibody-free tau p217+, wherein the semi-denatured sample is heated to denature the antibodies in the sample, iii. place each of the semi-denatured and undenatured samples in contact with a capture antibody directed against an epitope comprising phosphorylated T212 and / or phosphorylated T217 of the tau protein to capture tau p217+ peptides in each of the samples, iv. The captured tau p217+ peptides are placed in contact with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein, in order to measure the amount of total tau p217+ and the amount of antibody-free tau p217+ in the biological sample. v. Calculate the amount of antibody-bound tau p217+ in the sample by subtracting the amount of antibody-free tau p217+ from the total amount of tau p217+, vi. Calculate the ratio between antibody-bound tau p217+ and antibody-free tau p217+, and vii. Monitor the individual's treatment with anti-tau p217+ antibody based on the calculated ratio. Petition 870260042583, dated 06 / 05 / 2026, page 71 / 334 63 / 98

[0169] Embodiment 16 is a method of any of the embodiments 1 to 15, wherein the capture antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36 and 37, respectively; preferably, the capture antibody has a variable region of the heavy chain comprising the polypeptide sequence of SEQ ID NO: 28 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 29.

[0170] Embodiment 17 is a method of any of the embodiments 1 to 16, wherein the detection antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively; preferably, the detection antibody comprises a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 8 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 9.

[0171] Embodiment 18 is a method of any of the embodiments in 16, wherein the detection antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively; preferably, the detection antibody comprises a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 19.

[0172] Modality 19 is the method of any of the modalities Petition 870260042583, dated 06 / 05 / 2026, p. 72 / 334 64 / 98 to 18, where the sample is a blood sample, brain homogenate, or cerebrospinal fluid (CSF).

[0173] Embodiment 20 is the method of any of the embodiments to 19, in which the sample is obtained after fractionating a biological sample using reversed-phase high-performance liquid chromatography (rpHPLC).

[0174] Modality 21 is a single detection antibody or antigen-binding fragment thereof that binds to a tau protein at an epitope comprising amino acid residues 116 to 127 of the tau protein, comprising: a. HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences SEQ ID NOs: 2, 3 and 4, respectively; and b. LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences SEQ ID NOs: 5, 6 and 7, respectively.

[0175] Embodiment 22 is the isolated detection antibody or antigen-binding fragment of embodiment 21, comprising a variable heavy chain region having a polypeptide sequence of SEQ ID NO: 8 and a variable light chain region having a polypeptide sequence of SEQ ID NO: 9, preferably.

[0176] Modality 23 is a single detection antibody or antigen-binding fragment thereof that binds to a tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein, comprising: a. HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences with SEQ ID NOs: 12, 13 and 14, respectively; and b. LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 15, 16 and 17, Petition 870260042583, dated 06 / 05 / 2026, p. 73 / 334 65 / 98 respectively.

[0177] Embodiment 24 is the isolated detection antibody or antigen-binding fragment of embodiment 23, comprising a variable heavy chain region having a polypeptide sequence of SEQ ID NO: 18 and a variable light chain region having a polypeptide sequence of SEQ ID NO: 19, preferably.

[0178] Embodiment 25 is an isolated nucleic acid encoding the detection antibody or antigen-binding fragment of either embodiment 21 to 24.

[0179] Modality 26 is a vector comprising the nucleic acid isolated from modality 25.

[0180] Modality 27 is a host cell comprising the nucleic acid of modality 25.

[0181] Embodiment 28 is a method for producing the detection antibody or antigen-binding fragment of any of claims 21 to 24, comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment under conditions to produce the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the cell or cell culture.

[0182] Modality 29 is a kit that includes: a. a capture antibody directed against an isolated or multiphosphorylated tau protein epitope comprising phosphorylated T212 and / or phosphorylated T217 of the tau protein, and b. a detection antibody directed against a tau protein epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein; where the kit is used to measure the amount of p217+ tau peptides in a sample.

[0183] Modality 30 is a modality 29 kit, in which the antibody Petition 870260042583, dated 06 / 05 / 2026, p. 74 / 334 The 66 / 98 capture antibody comprises HCDR1, HCDR2, and HCDR3 of the immunoglobulin heavy chain having polypeptide sequences of SEQ ID Nos: 32, 33, and 34, respectively, and LCDR1, LCDR2, and LCDR3 of the immunoglobulin light chain having polypeptide sequences of SEQ ID Nos: 35, 36, and 37, respectively; preferably, the capture antibody has a variable region of the heavy chain comprising the polypeptide sequence of SEQ ID No: 28 and a variable region of the light chain having the polypeptide sequence of SEQ ID No: 29.

[0184] Modality 31 is the kit for modality 29 or 30, where the detection antibody is the detection antibody isolated from any of the modalities 20 to 23. Examples

[0185] The following examples of the invention are provided to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and that the scope of the invention is determined by the appended claims. Example 1. High-sensitivity assay for tau p217+ detection

[0186] The specific reagents for the assay were as follows: Kit Simoa Homebrew (Quanterix, cat. no. 101351), Helper Beads (Quanterix, cat. no. 101732), mouse monoclonal antibody pT3 (mAb), mAb hT43, mAb pT82, and mAb hT7. pT3 is the parent antibody developed at Janssen that recognizes tau p217+, and its humanized version is referred to in this document as humanized mAb pT3.

[0187] The samples were diluted in 50 mM Tris, 50 mM NaCl, 5 mM EDTA, 2% bovine serum albumin, 0.1% Tween 20, 0.05% ProClin 300, pH 7.8.

[0188] Three custom peptides made by New England Peptide were used to calibrate the assay (calibrating peptides).

[0189] The pT3xhT43 peptide contains hT43, PT51 and pT3 epitopes connected by PEG4 ligands and has a molecular weight of 6893 g / mol. Petition 870260042583, dated 06 / 05 / 2026, page 75 / 334 67 / 98 The amino acid sequence of a pT3xhT43 peptide is PRQEFEVMEDHAGTYGLGDR(dPEG4)GKTKIATPRGAAPPGQKG(dPEG4 )GSRSR(pT)PSLP(pT)PPTREPKKV-amide (SEQ ID NO: 22).

[0190] The pT3xpT82 peptide contains pT82 and pT3 epitopes connected by a PEG4 linker and has a molecular weight of 4551 g / mol. The amino acid sequence of the pT3xpT82 peptide is AcSLEDEAAGHVTQARMVSK(dPEG4)GSRSR(pT)PSLP(pT)PPTREPKKVamida (SEQ ID NO: 23).

[0191] The hT7xpT82 peptide contains pT82 and hT7 epitopes connected by a PEG4 linker and has a molecular weight of 3619 g / mol. The amino acid sequence of the hT7xpT82 peptide is AcSLEDEAAGHVTQARMVSK(dPEG4)PRGAAPPGQKGQANA-amide (SEQ ID NO: 24). Reagent preparation

[0192] The capture microspheres were coated with 0.3 mg / ml of Capture microspheres were prepared following the protocol provided in the Quanterix manual. The coated capture microspheres were diluted in microsphere diluent buffer to 200,000 microspheres / ml, and 200,000 microspheres / ml of Helper Beads were added so that the total concentration of microspheres was 400,000 microspheres / ml.

[0193] Detection antibodies were biotinylated 60x following the protocol provided in the Quanterix manual and were diluted in Homebrew Detector / Sample Diluent to 1.8 ug / ml.

[0194] Calibrating peptides were reconstituted at 5 mg / ml in 0.1% phosphoric acid / water, separated into 20 µl aliquots, and frozen. When ready for use, the calibrating peptide aliquots were thawed and diluted 1:1000 (e.g., 1.5 µl in 1498.5 µl), and the dilutions were further diluted 1:1000 so that the final peptide concentration was 5000 pg / ml. A 3x jump standard curve was made, starting at 30 pg / ml. Petition 870260042583, dated 06 / 05 / 2026, page 76 / 334 68 / 98

[0195] CSF samples were diluted at least 1:4 in Diluent of Sample. Samples from healthy volunteers (HV) were diluted 1:5 or 1:10, and samples from DA were diluted at least 1:20. Simoa Essay

[0196] A customized Simoa assay was created comprising a two-step protocol, comprising 35 minutes with capture, sample, and detection antibodies, and washing, followed by 5 minutes with streptavidin-β-galactosidase (SBG). Each reaction comprised 25 µl of microsphere solution, 100 µl of sample or calibrator, 20 µl of detection solution, and 100 µl of SBG. Antibodies were assigned names, and up to five capture antibodies and five detection antibodies could be loaded at once. Reactions were performed in Simoa cuvettes by the instrument, washed a final time, and loaded onto measuring discs with β-galactosidase (RGP) substrate before measurements were taken by the instrument. Example 2. Separation of native tau fragments in rpHPLC

[0197] The reagents were as follows: Trifluoroacetic acid (grade HPLC), water (HPLC grade), acetonitrile (HPLC grade), phosphoric acid (analytical grade), HPLC and binary gradient system, immunoassay buffer (100 mM TrisHCl, 100 mM NaCl, 0.05% Tween & BSA, pH 7.8).

[0198] The protocol was as follows: 500 µl of frozen CSF were thawed on ice for 30 minutes. The thawed CSF was added to 1.5 ml of 100 mM sodium phosphate at pH 2.5 containing 100 mM sodium chloride and mixed. 1.8 ml of the resulting mixture was applied to a C18 column or similar reversed-phase chromatography column equilibrated with 0.1% trifluoroacetic acid in water. The HPLC column was then developed in an increasing acetonitrile gradient. Fractions were collected by elution. The fractions were adjusted to 10 mM guanidine HCl and then subjected to drying in a vacuum concentrator. The dried fractions were resuspended in immunoassay buffer and Petition 870260042583, dated 06 / 05 / 2026, page 77 / 334 69 / 98 submitted to measurement of the tau peptide in the fraction based on a pair of anti-tau capture and detection antibodies of the invention. Example 3. Quantification of tau p217+ free or bound by antibodies.

[0199] With additional upstream sample manipulation, highly sensitive pT3-based assays can be used to measure tau p217+ binding by antibodies that were produced within a patient or are administered exogenously, for example, humanized pT3 mAb. This technique can be used as a pharmacodynamic assay to study therapeutic anti-tau p217+ antibodies such as humanized pT3 Ab. For example, the following methods can be used to measure antibody-free tau p217+ versus antibody-bound tau p217+. Assay 1: Quantification of free vs. bound tau p217+ in biological fluids using immunocapture / depletion followed by rpHPLC.

[0200] Biological fluid (e.g., CSF) was incubated with protein A / G coated magnetic microspheres (15 µl of microsphere fluid paste per 0.5 mL of CSF) for 2 hours with shaking at room temperature to capture immunoglobulins in the sample. The microspheres were precipitated by magneto and the supernatant was transferred to a second tube (sample = IgG-depleted supernatant). The microspheres were washed 4x with 1 mL of phosphate-buffered saline (PBS). 0.5 mL of 6 M GuHCl was then added to the tubes containing (a) washed microspheres and (b) IgG-depleted supernatant, and the tubes were incubated for 20 minutes with shaking at room temperature. The microspheres were then precipitated by magneto, and the resulting supernatant was transferred to a third tube (sample = IgG supernatant).Finally, 0.1 M phosphoric acid (pH 2) was added to both solutions (1.0 mL of phosphoric acid was added to the depleted supernatant of denatured IgG and 1.5 mL of phosphoric acid was added to the IgG supernatant, to bring the samples to a final volume of 2.0 mL) before separation by rpHPLC. Petition 870260042583, dated 06 / 05 / 2026, page 78 / 334 70 / 98 performed as in Example 2. The resulting rpHPLC fractions were reconstituted as described in Example 2 and measured using Simoa's tau p217+ assays from Example 1. The signal from the IgG-depleted supernatant represents free tau p217+ (i.e., that which is not bound by antibodies), while the signal from the concentrated IgG supernatant represents bound tau p217+ (i.e., that which is bound by antibodies, such as humanized pT3 mAb). rpHPLC separation and Simoa's p217+ measurement of the same parental biological fluid (e.g., CSF) that was not subjected to the depletion / immunocapture process were analyzed simultaneously to assess the total tau p217+ signal, either as a control or as a normalizer for both free and bound measurements. Assay 2: Quantification of free vs. bound tau p217+ in biological fluids using heat denaturation of antibodies.

[0201] An aliquot of biological fluid of interest (e.g., CSF) was heated to 95 °C for 4 minutes, followed by cooling on moist ice for 4 minutes (sample = semi-denatured fluid). In parallel, a second aliquot of the same fluid was cooled on moist ice for 8 minutes (sample = undenatured fluid). Both samples were then measured using the Simoa p217+ tau assays of Example 1. The semi-denatured fluid signal represents total p217+ tau, while the undenatured fluid represents free p217+ tau. Subtracting the second from the first results in the measurement of bound tau. The precise heating time and temperature were determined to irreversibly modify any antibodies in the fluids, so that they could not continue to interfere with the Simoa p217+ tau assays, while the p217+ tau signal itself was spared from any impact.This assay is not a direct measure of whether antibodies are bound to tau p217+; instead, it demonstrates the presence of the assay's competing antibodies. However, the assay produced similar results to the more laborious Assay 1. Example 4. Biological samples Petition 870260042583, dated 06 / 05 / 2026, p. 79 / 334 71 / 98 Samples used for assay development and technical qualification

[0202] The assays in Examples 1 to 3 were developed using pooled CSF from human subjects with elevated tau levels. Some experiments were also performed with pooled CSF from human subjects with low tau levels to ensure assay sensitivity, which would be necessary for testing healthy volunteers in Phase 1 trials. CSF from cynomolgus monkeys (Macaca fascicularis) and common marmosets (Callirix jacchus), obtained from Neu Enripharm GmbH (animal testing CRO), were also measured using the assays in Examples 1 to 3. Additionally, rapidly frozen brain samples from cognitively normal human subjects and common marmosets were homogenized and measured using the assays in Examples 1 and 3. Some experiments were performed with individual sera from clinically defined VS and DA subjects. Samples used for preliminary clinical qualification

[0203] Cohort 1 (inter-assay correlation cohort): CSF samples of ventricular fluid (VF) and lumbar fluid (LF) were obtained from individuals with normal pressure hydrocephalus (NPH) (n=11) (University of Kuopio, Professor Ville Lenoinen). These samples were separated by CSF Aβ42, total tau (tTau), and pTau181 measurements, as determined by Innotest assays performed at Sahlgrenska University (Professor Kaj Blennow), and by immunohistochemistry (IHC) measurements of tau and amyloid from brain biopsy. Tau p217+ measurements by rpHPLC and Simoa were performed at Janssen Neuroscience Biomarkers, La Jolla.

[0204] Cohort 2 (clear VS versus clear DA cohort): Samples of Fluid-rich fluid (CSF) from individuals with biochemically defined Alzheimer's Disease (AD) versus healthy volunteers (HV) (n=20 per group) were obtained from Sahlgrenska University (Professor Kaj Blennow). CSF Aβ42, tTau, and pTau181 measurements using Innotest assays were performed at Sahlgrenska University. Samples were selected from a large pool of individuals with biochemically defined Alzheimer's Disease (AD) versus healthy volunteers (HV) (n=20 per group). Petition 870260042583, dated 06 / 05 / 2026, page 80 / 334 72 / 98 sample panel based on segregation at predetermined DA versus VS cutoff measurements (DA = CSF Aβ42 < 400 pg / ml AND CSF tTau > 600 pg / ml, VS = CSF Aβ42 > 400 pg / ml AND CSF tTau < 600 pg / ml). Tau p217+ measurements by rpHPLC and Simoa were performed at Janssen Neuroscience Biomarkers, La Jolla.

[0205] Cohort 3 (cohort of VS versus ARAD versus early-stage DA): Fluid-rich cerebrospinal fluid (CSF) samples from clinically defined normal individuals (Clinical Dementia Classification 0; CDR 0) versus those with mild memory complaints (CDR 0.5) (n=20 per group) were obtained from the Janssen ALZ1005 / 1002 study. CSF Iβ42, tTau, and pTau181 measurements using Innotest assays were performed at Sahlgrenska University. Based on CDR and LCR Iβ42 scores, individuals were classified into (a) VS = CDR 0 and Iβ42 > 600 pg / ml, (b) at risk of AD (ARAD) = CDR 0 and Iβ42 > 600 pg / ml, (c) potentially non-AD dementia = CDR 0.5 and Ae42 > 600 pg / ml, and (d) early-stage AD = CDR 0.5 and Ae42 > 600 pg / ml. Tau p217+ measurements by rpHPLC and Simoa were performed at Janssen Neuroscience Biomarkers, La Jolla.

[0206] Cohort 4 (CDR 0 vs CDR1 cohort): CSF FL samples from clinically defined normal individuals (Clinical Dementia Classification 0; CDR 0) vs. mild memory complaint (CDR 1) (n=5 per group) were obtained at the University of Washington. CDR and MMSE, as well as CSF Ae42, tTau, and pTau181 measurements by Innotest assays were obtained at the University of Washington. Prior to dispatch, samples were coded so that Janssen was blinded to the identity or characterization of the samples. tTau and p217+ measurements by rpHPLC and Simoa were performed at Janssen Neuroscience Biomarkers, La Jolla, and sent to the University of Washington for analysis.

[0207] Cohort 5 (VS versus MCI versus DA cohort): Clinically and biochemically defined VS FL CSF samples (Innotest AB42 > 600 pg / ml) (n=7) were obtained from Precision Medicine, San Diego. Clinical FL CSF samples Petition 870260042583, dated 06 / 05 / 2026, page 81 / 334 73 / 98 and biochemically defined (Innotest AB42 < 600 pg / ml) MCI (n=28) and DA (n=12) were obtained from the University of Antwerp. Tau p217+ measurements by rpHPLC and Simoa were performed at Janssen Neuroscience Biomarkers, La Jolla.

[0208] Cohort 6 (disease severity and progression cohort): CSF samples from clinically defined AD individuals (Clinical Assessment of Dementia 1+) (n=235) were obtained from the Janssen ELN115727301 / 302 study. These samples were baseline (pre-dose) samples from all individuals in the trial. In addition, CSF samples from 78 weeks of follow-up from placebo individuals (n=90) were included to assess biomarkers of disease progression. Cognitive assessment (ADAS-COG, MMSE, NTB, CDR.SOB), ApoE genotype, gender, and age were obtained from the trial. The Innotest AB42, Innotest AB40, Simoa NFL, pT3xpT82, pT3xhT43, and hT7xpT82 trials were conducted at Janssen Neuroscience Biomarkers, La Jolla. Individuals were confirmed as amyloid positive or negative based on the AB42 / 40 ratio cutoff of 0.09 (i.e., individuals with a ratio < 0.09 = amyloid positive = AD, while those > 0.09 = amyloid negative = dementia of non-AD cause).27 out of 235 individuals were determined to be amyloid negative; both groups were analyzed separately. Samples used for evaluation of target engagement after treatment with anti-p217+ agents.

[0209] FCR FL of individuals VS (n=40) treated with placebo or JNJ63733657 (single IV injection) assays were obtained from the Janssen JNJ63733657EDI1001 assay. pT3xpT82 assays were performed at Janssen Neuroscience Biomarkers, La Jolla. pT3xhT43 assays were performed at Quanterix Corporation, Lexington, MA. Example 5. Screening of capture and detection antibody pairs using the Simoa platform.

[0210] Previous reports from Janssen Neuroscience Discovery and Petition 870260042583, dated 06 / 05 / 2026, page 82 / 334 74 / 98 literature (e.g., Meredith et al. PLoS One. 8(10):e76523, 2013; Barthelemy et al., J Alzheimers Dis. 51(4):1033-43, 2016; Russell et al., J Alzheimers Dis. 55(1):303-313, 2017; Hanger et al. J Biol Chem. 282(32): 23645-54, 2007) indicated that tau fragments containing amino acids 200 to 220, and especially some combination of phosphorylation at amino acids 212, 214, 217, are enriched in DA. Developing an assay to measure this particular type of tau (tau p217+) could thus yield an enhanced biomarker for the diagnosis and / or staging of Alzheimer's disease (AD), as well as a potential predictive and / or pharmacodynamic assay for targeting new drugs to this portion of tau. However, tau can be present at low levels (< 200 pg / ml) in healthy volunteers, and tau p217+ is a minor component of total tau, so tau p217+ assays require ideal antibody pairings and high sensitivity.

[0211] To achieve this goal, a set of anti-tau mAbs discovered at Janssen, as well as some high-affinity commercial anti-tau mAbs, were evaluated for their ability to produce a signal in a sandwich ELISA (sELISA) format when paired with pT3. The antibody pairs were screened on the Simoa HD-1 Analyzer platform (Quanterix Corporation) to provide the necessary sensitivity, using a serial dilution of a CSF group from DA individuals. Assay performance was based on signal / noise = average enzymes per microsphere (AEB) of the sample diluted in the sample diluent / AEB of the assay diluent alone. Ideal detection antibodies to pair with pT3 were hT43, pT82, the Quanterix tau 2.0 detector reagent, and BT2 in that order of sensitivity (Table 1). hT43 and the Quanterix tau 2.0 detector reagent recognize the N-terminal region of tau, while pT82 and BT2 recognize sequences closer to the intermediate region of tau.The N-terminal (hT43) and intermediate region (pT82) mAbs were selected for further optimization. Screening was performed in parallel at Janssen Neuroscience Biomarkers and Quanterix Corporation, producing similar results. Petition 870260042583, dated 06 / 05 / 2026, p. 83 / 334 75 / 98 Table 1. Specificity screening in DA CSF of tau detection antibodies paired with pT3. Antibody Epitope Detection (tau aa) S / N @ dilution 1:2 S / N @ dilution 1:4 S / N @ dilution 1:8 S / N @ dilution 1:16 S / N @ dilution 1:32 S / N @ dilution 1:64 hT43 7-20 955 587 313 183 102 55 Quanterix tau 2.0 test detector 16-24 525 262 147 75 35 19 pT82 116- 127 733 400 211 105 48 23 PT51 151- 158 165 52 17 6 ​​​​3 1 PT98 159- 163 NT NT 17 7 3 2 pT89 166- 182 87 24 9 4 2 1 BT2 193- 198 732 245 78 28 10 4 HT52 393- 398 NT NT 1 1 1 1 HT60 423- 440 NT NT 1 1 1 1 The antibody epitope in the signal-to-noise (S / N) ratios of tau in pooled CSF measurement from AD individuals is shown; NT = not tested. Example 6. Optimization of pT3xhT43 and pT3xpT82 assays

[0212] A series of optimization experiments were performed based on the overall Quanterix experience with test optimization on the Simoa platform. 10% mouse serum or 500 pg / ml of IgG of Petition 870260042583, dated 06 / 05 / 2026, p. 84 / 334 76 / 98 mice were added to the detector diluents, but there was no improvement in assay sensitivity. Titrations of the detector mAb concentration (0.15, 0.3, 0.6, 1.2, and 1.8 μg / ml), SGe concentration (100, 200, or 300 pM), and concentrations of capture mAb microspheres (300K / well, 150K + 200K auxiliary microspheres) were evaluated. The protocol incubation times (65 minutes versus 35 minutes) and sample volume (100 versus 150 pL) were also evaluated. The ideal reagent concentrations for both assays were 150K of capture microspheres + 200K of auxiliary microspheres, 1.8 pg / ml of detector, and 200 pM of SBG, respectively. Sample volume and incubation time had minimal impact on the assay, so lower conditions of 100 µl sample and 35 minutes of incubation were chosen. Example 7. Technical qualification of pT3xhT43 and pT3xpT82 linear range tests with calibrating material.

[0213] The calibrating peptides described in Example 1 were produced. The calibrating peptides contained the main epitopes of pT3 and hT43, or pT3 and pT82 separated by PEG4 ligands, and they were used to generate the standard curves. A representative standard curve is shown in Figure 1. The calibrating peptides were titrated from 30 pg / ml to 0.041 pg / ml in 1:3 steps in assay buffer and measured with the pT3xhT43 and pT3xpT82 assays. A 4-parameter curve fitting data reduction method (4PL, 1 / y2 weighted) was used to generate the calibration curve. The lower limit of detection (LLOD) was defined as the calibrated calculated level producing an AEB equal to the mean of the zero calibrator + 2.5 standard deviations (SD), including a 10% coefficient of variation (CV). Based on these criteria, the representative data yielded an LLOD of ~0.002 pg / ml.The linear range of the assay, the lower limit of quantification (LLOQ), and the upper limit of quantification (ULOQ) were defined as the lowest and highest points on the standard curve achieving CV < 20% and 80-120% expected recovery. With these... Petition 870260042583, dated 06 / 05 / 2026, page 85 / 334 77 / 98 criteria, the linear range for both pT3xhT43 and pT3xpT82 assays was 0.041 for 30 pg / ml (Figure 1, Table 2). Table 2. Representative calibration curve for the optimized pT3xhT43 test, with LLOD calculations. pg / ml of AEB calibrant Mean SD CV (%) S / B 0 0.0185 0.0019 10 0.034 0.1369 0.0260 19 7 0.103 0.1952 0.0039 2 11 0.309 0.5221 0.0626 12 28 0.926 1.4545 0.0145 1 78 2.78 5.4789 0.1096 2 295 8.33 11.4588 1.0313 9 617 25 24.0576 0.2406 1 1297 LLOD=0.0185 + (2.5 x 0.0019)= 0.0231 AEB, which is calculated at a theoretical concentration of 0.002 pg / ml. Linearity of dilution with CSF

[0214] To assess dilution linearity and determine the ideal dilution for test CSF samples, a panel of 4 CSF samples from AD individuals (high tau, low AB42) was titrated from dilutions of 1:2 to 1:4096 in assay buffer and measured in p217+ assays. Samples diluted beyond 1:512 typically measured below LLOQ. Measurements from 1:4 to 1:512 were linear in dilution, so this was the range defined for measuring CSF samples. For confirmation in cognitively normal individuals, a pool of CSF from individuals with low tau and high AB42 was similarly measured. Dilution linearity was again observed for dilutions from 1:4 to 1:256, and beyond this range measurements fell below LLOQ (Figure 2). Precision

[0215] To assess the accuracy of the measurements, the standard curve for Petition 870260042583, dated 06 / 05 / 2026, page 86 / 334 78 / 98 pT3xhT43 was prepared and measured on 3 separate days (Figure 3 and Table 3). The calibrating peptide was diluted from 30 to 0.041 pg / ml in serial 1:3 jumps and measured in duplicate in the pT3xhT43 assay. The procedure was repeated over 3 successive days at the same site and by the same technician. Analysis of the 4 midpoints of the curve (where CSF samples are measured) indicated that the precision within an operation (intratest CV %) was always < 10% and calculated from 2.46–5.18% CV, and the precision between tests averaged 6.46% CV. These are well within the accepted limits of 20% CV for a research-only (RUO) assay and are partly attributed to the automated nature of all ELISA steps in the Simoa HD-1 Analyzer. Table 3. Intra- and inter-test precision of the pT3xhT43 assay. Operation 1 Operation 2 Operation 3 Sum of operations fg / ml of calibrant AEB Average SD CV % AEB Average SD CV % AEB Average SD CV % AEB Average SD CV % 49 0.0228 0.00 05 2.0 3 0.0248 0.00 24 9.6 4 0.0319 0.00 05 1.4 1 0.0265 0.00 48 18.0 4 195 0.0762 0.00 19 2.5 5 0.0777 0.00 11 1.3 9 0.0785 0.00 28 3.5 2 0.0775 0.00 12 1.54 781 0.2130 0.00 84 3.9 5 0.2161 0.02 00 9.2 6 0.0210 0.01 29 6.1 4 0.2130 0.00 30 1.43 3125 0.8012 0.01 05 1.3 1 0.8193 0.00 36 0.4 4 0.7460 0.02 89 3.8 8 0.7888 0.03 82 4.84 Average 2.4 6 5.1 8 3.7 4 6.46 Petition 870260042583, dated 06 / 05 / 2026, p. 87 / 334 79 / 98 Transferability between laboratories

[0216] To evaluate the accuracy of the p217+ tau assays between test sites, the same DA CSF cluster was measured during titration using the same reagent batch from Janssen Neuroscience Biomarkers and Quanterix Corporation. Figure 4 shows that the measurements are very similar for the pT3xhT43 and pT3xpT82 assays at both test sites. Accuracy

[0217] To assess the accuracy of the assays, two different pools of VS CSF were boosted with known concentrations of the calibrating peptides (0, 2, or 20 pg / ml), diluted to the recommended 1:4 dilution, and then measured in the pT3xhT43 and pT3xpT82 assays. This is a measure of the potential interference presented by the sample matrix components. Endogenous signal levels were subtracted from the measurements with 2 and 20 pg / ml boosts, and then the observed concentration of the calibrating material was compared to the expected concentration to calculate the percentage recovery. The measured concentrations were compared to the expected concentrations to calculate the boost concentrations, yielding an average recovery of 114% (Table 4). This is well within the accepted limits of 80 to 120% recovery for a RUO assay, indicating that there was no significant interference in the CSF when tested at a dilution >1:4. Table 4. Reinforcement recovery from the pT3xhT43 test. CSF Sample Calibrant Boost (pg / ml) AEB pg / ml calculated pg / ml CV% Average pg / ml Corrected dilution pg / ml % recovery 1 0 1.4863 0.945 3 0.924 3.69 NA 1.4209 0.902 2 2.2451 1.44 2 1.42 5.69 100 2.1776 1.40 20 8.9800 6.33 2 6.40 25.6 110 Petition 870260042583, dated 06 / 05 / 2026, page 88 / 334 80 / 98 9.1679 6.48 2 0 1.9960 1.28 6 1.23 4.91 NA 1.8397 1.18 2 2.8076 1.82 2 1.85 7.39 124 2.8886 1.88 20 10.163 6 7.28 1 7.34 29.4 122 10.302 6 7.40 AVERAGE RECOVERY 114% Competition for signaling by antibodies directed against p217+ in the CSF

[0218] To confirm the accuracy of the pT3xhT43 and pT3xpT82 assay signals in CSF, and to assess their potential utility as a pharmacodynamic assay in clinical studies of antibodies targeting tau p217+, a pooled CSF sample from DA was boosted with titers of humanized pT3 mAb or humanized pT3 mAb and measured in the pT3xhT43 and pT3xpT82 assays after 2 hours of incubation at room temperature (Figure 5). Administration of soluble and humanized pT3 antibodies reduced the signal in the pT3-based assays in a dose-dependent manner. Boosting with msIgG (negative control) at comparable concentrations had no impact on either measurement. The inferior competitive ability of humanized pT3 mAb vs. pT3 may be attributed to the higher affinity of pT3 for tau p217+. Phosphorylation dependence

[0219] To confirm that the signal in CSF obtained with the pT3xhT43 and pT3xpT82 assays was, in fact, based on a phosphorylated epitope, DA CSF was treated with alkaline phosphatase to dephosphorylate all residues. Samples were then analyzed in pT3 assays and in two hT7-based assays, hT7xpT82 or hT7xBT2. hT7 is known to be phosphorylation-independent, so it was used as a negative control.

[0220] Pooled CSF from AD patients was treated with Petition 870260042583, dated 06 / 05 / 2026, page 89 / 334 81 / 98 increasing amounts of alkaline phosphatase (AP) at 37 °C for 4 hours in a buffer containing zinc and magnesium chloride. The effect on the epitope directed against pT3 was measured using the pT3xhT43 and pT3xpT82 assays. The pT3xhT43 and pT3xpT82 signal was reduced by alkaline phosphatase treatment in a dose-dependent manner. However, the non-phosphorylation-dependent assays, hT7xpT82 or hT7xBT2, did not show a decrease in signal. They actually showed an increase, as expected since pT7 binding is reduced by phosphorylation (Figure 6). Tau fragment profile p217+

[0221] To explore the nature of the p217+ tau signal derived from crude CSF measurement, a CSF sample was fractionated by rpHPLC using a method similar to that described in Meredith et al. PLoS One. 8(10):e76523, 2013. Fractions were collected and measured using the pT3xhT43 and pT3xpT82 assays (Figure 7). In this chromatography format, smaller tau fractions eluted rapidly (smaller number of fractions), while larger fractions eluted later (larger number of fractions). Full-length tau eluted in fraction 19. The tau fragment profile indicated that very little full-length tau was detected by either assay, consistent with previous reports (Meredith et al. PLoS One. 8(10):e76523, 2013, Barthelemy et al., J Alzheimers Dis. 51(4):1033-43, 2016). The pT3xpT82 assay detected two large peaks (tau-like) that were smaller than the full-length tau (fractions 12 and 14), while the pT3xhT43 assay detected only one of these large peaks (fraction 14). This indicated that p217+ tau in CSF exists in at least two fragments, a larger fragment encoding at least the hT43 to pT3 region (tau amino acids 7-220) and a smaller fragment encoding at least the pT82 to pT3 region (tau amino acids 116-220) but not extending all the way to the hT43 epitope. That is, there is probably a proteolytic cleavage site between amino acids 20 and 116 that is cleaved only in a subset of Petition 870260042583, dated 06 / 05 / 2026, page 90 / 334 82 / 98 tau molecules at a given time. The profile is not specific for p217+, since measurements with other tau assays that recognize a similar region of tau but are not specific for phosphorylation yield similar findings (data not shown). Analyte stability

[0222] The stability of the endogenous tau p217+ epitope was evaluated at various temperatures. DA CSF pooling was aliquoted and each aliquot was subjected to storage at 4°C, 22°C, or 37°C for 1, 2, or 4 hours. In addition, a subset of aliquots was thawed (80°C to 22°C) 2 or 3 times. All samples were then diluted 1:20 and analyzed using pT3xhT43 and hT7xpT82 assays (Figure 8). No significant signal change was observed under any of the tested conditions, indicating that all 4 epitopes recognized by these assays are sufficiently stable to allow standard storage / testing procedures. Finally, CSF was prospectively collected from 4 donors, then aliquoted and frozen at -70°C. Samples were taken every 3 months for measurement with the pT3xpT82 assay. No significant change in signal was observed at the 3, 6, or 9-month time points (Figure 9). Example 8. Clinical quantification of pT3xhT43 and pT3xpT82 assays

[0223] To evaluate the usefulness of pT3xhT43 and pT3xpT82 assays in the diagnosis and positioning of AD, three cohorts of CSF samples were obtained for measurement of p217+ tau. The measurements were analyzed by correlation with cognitive scores and other classic AD biomarkers. Cohort 1: Inter-trial correlation cohort

[0224] CSF, FV and FL samples, and brain (ventricle) biopsy were obtained from 10 individuals with the neurodegenerative disorder normal pressure hydrocephalus (NPH), a condition characterized by the overproduction of interstitial fluid in the brain and by presenting a Petition 870260042583, dated 06 / 05 / 2026, page 91 / 334 83 / 98 high incidence of AD. Tau p217+ measurements were performed in raw CSF and analyzed for correlation with traditional AD biomarkers.

[0225] Levels of Aβ42 (Figures 10A, 10D), tTau (Figures 10B, 10E), pTau181 (Figures 10C, 10F) in FV were determined by Innotest ELISA (classical measurement). The same samples were measured with pT3xhT43 assays (Figures 10A, 10B, 10C) and pT3xpT82 (Figures 10D, 10E, 10F), and correlations were evaluated. Both pT3xhT43 and pT3xpT82 assays showed a negative correlation with Αβ42 in CSF (r2=0.609, p=0.0077 and r2=0.590, p=0.0095, respectively), and a positive correlation with tTau in CSF (r2=0.525, p=0.0177 and r2=0.435, p=0.0381, respectively), but they did not correlate significantly with pTau181 in CSF (Figure 10).

[0226] Brain biopsies from the same 10 individuals with NPH were analyzed by IHC and classified by the pathologist as amyloid positive / negative and tau positive / negative. When positive for both, the sample was designated biopsy + and was a classic diagnosis for AD. When negative for both, the sample was designated biopsy - and was a classic diagnosis for non-AD. Samples designated biopsy + (Amil) were positive for amyloid but negative for tau. CSF obtained from ventricular shunt (VF=black dots) or lumbar shunt (FL=red dots) was measured with the pT3xhT43 and pT3xpT82 assays, and correlations were evaluated (Figure 11). Both pT3xhT43 and pT3xpT82 assays were able to separate the negative brain biopsy (amyloid / tau-) from the positive samples (amyloid+ / tau+) (p=0.04 and 0.02, respectively). Samples positive for amyloid but not for tau often measured between positive and negative biopsy samples.Amyloid plaques in the brain are believed to precede tau tangles; therefore, amyloid+ / tau- samples may represent early Alzheimer's disease or another disease. Cohort 2: VS vs. DA Cohort Petition 870260042583, dated 06 / 05 / 2026, page 92 / 334 84 / 98

[0227] CSF (CSF) samples from biochemically defined AD versus VS individuals (n=20 per group) were obtained from Sahlgrenska University. Αβ42 and tTau levels were determined by Innotest ELISA (classical measurement) to subdivide the groups (AD=CSF Aβ42<400 pg / ml and CSF tTau>600 pg / ml, VS=CSF Aβ42>400 pg / ml and CSF tTau<600 pg / ml). Measurements were performed using pT3xhT43, pT3xpT82, and hT7xpT82 assays on raw CSF and a subset of CSF fractionated by rpHPLC. The results were analyzed by correlation with traditional AD biomarkers (Figure 12). The data in panels A and B of Figure 12 demonstrated that the pT3 epitope is an indicator of patients at high risk of rapid progression to incipient AD. The pT3 epitope was highly elevated in patients who demonstrated high total tau levels and low Aβ42 levels. Conversely, the pT3 epitope was present at low levels in individuals with low total tau and high Aβ42.Figure 11C confirmed that the elevated pT3 epitope containing tau was driven at least in part by elevated total tau levels, as demonstrated by the hT7xpT82 total tau assay, but not entirely (Figure 12D). This indicates that the amount of tau, and the extent to which it is phosphorylated in the p217+ epitope, is elevated in DA.

[0228] The data in Figure 11 were used to create ROC curves regarding the ability of the pT3xhT43, pT3xpT82, and hT7xpT82 assays to differentiate DA from VS samples. All three assays showed excellent specificity and sensitivity. Meanwhile, the two pT3-based assays (pT3xhT43, pT3xpT82, which detect p217+ tau) had better diagnostic power than the hT7-based assay (Figure 13).

[0229] A subset of the same CSF samples measured in Figure 12 (n=11 per group) was fractionated by rpHPLC and then measured with pT3xhT43, pT3xpT82, hT7xpT82 assays (the last one was measured from the same tau fragments in a phosphorylation-independent manner). Petition 870260042583, dated 06 / 05 / 2026, page 93 / 334 85 / 98 (Figure 14). The profile of the observed tau fragments was similar to that observed in Example 7 and Figure 7. That is, two main species were seen with both pT82-based assays, pT3xpT82 and hT7xpT82, while only one peak was seen with the pT3xhT43 assay. Both main species were present in higher concentrations in the DA group than in the VS group. Additionally, the pT3 (p217+ tau)-based assays showed a greater difference between the groups than the hT7 (total tau)-based assay, as detected in the raw CSF analysis. The larger p217+ tau species (fractions 13-14) provided the greatest difference between DA versus VS (Figure 14).

[0230] The sum of all major tau fragments in Figure 14 (fractions 11-14) was calculated, then compared between the DA and VS subgroups. The percentage increase in the tau-containing pT3 epitope (pT3xhT43 or pT3xpT82) in DA was more than double that seen with the non-pT3 tau-containing epitope (hT7xpT82) (Table 5). Table 5. Total tau signal in pT3 vs. non-pT3 assays Average AEB assay in DA Standard Deviation in DA Average AEB in VS Standard Deviation in VS % increase in DA pT3 / pT82 0.74 0.32 0.12 0.05 620 pT3 / hT43 1.65 1.88 0.22 0.14 750 hT7 / pT82 2.00 1.66 0.84 0.66 250

[0231] Signal analysis of each fraction in Figure 14 independently vs. as a sum, as done in Table 5, was performed to reveal which fraction generated the strongest DA vs. VS signal. The most informative fragment cluster was detected using the pT3 antibody in fragment clusters 13 and 14 (Table 6). Table 6. Tau signal in different tau fragment clusters in pT3 vs. pT3 assays. Petition 870260042583, dated 06 / 05 / 2026, page 94 / 334 86 / 98 The pT3 assay identifies AD patients better than a non-pT3 assay: Differences by Fragment Clustering Assay Fragment Clustering Average AEB in DA Average AEB in VS % of DA over VS pT3xhT43 11 0.22 0.105 210 12 0.333 0.113 295 13 1.78 0.235 757 14 4.25 0.407 1044 pT3xpT82 11 0.447 0.079 566 12 0.631 0.098 644 13 0.681 0.121 563 14 1.2 0.193 622 hT7xpT82 11 0.51 0.29 176 12 2.8 1.51 185 13 0.73 0.26 281 14 3.94 1.31 301 Cohort 3: Cohort of VS versus ARAD versus early-stage DA

[0232] CSF (CSF) samples from clinically normal individuals (CDR 0) vs. mild memory complaint (CDR 0.5) (n=20 per group) were obtained from the Janssen ALZ1005 / 2002 study. Αβ42, tTau, and pTau181 levels were determined by Innotest ELISA. Based on CDR and CSF Αβ42 scores, individuals were classified into (a) VS=CDR>0 and Aβ42 ≥ 600 pg / ml, (b) ARAD=CDR 0 and Aβ42<600 pg / ml, (c) potentially non-AD dementia=CDR 0.5 and Aβ42>600, and (d) early-stage AD = CDR 0.5 and Aβ42<600 pg / ml.

[0233] CSF samples were also fractionated by rpHPLC and measured with pT3-based assays (pT3xhT43, Figures 15A-15E and pT3xpT82, Figures 15F-15J) and total tau-based assays (hT7xpT82, Figures 15K-15O). All pT3-based and hT7-based assays showed high signal at CDR 0 versus 0.5 (Figures 15A, 15F and 15K) and in samples with Petition 870260042583, dated 06 / 05 / 2026, page 95 / 334 87 / 98 Aβ42 <600 pg / ml versus >600 pg / ml (Figures 15B, 15G, and 15L). Discrimination by CDR level x Aβ42 is shown in Figures 15C, 15D, 15H, 15I, 15M, and 15N, and the added signal in all fractions is illustrated in Figures 15E, 15J, and 15O. Signal levels were highest in the Aβ42 <600 pg / ml + CDR 0.5 subgroup, consistent with the elevated tau p217+ signal in early-stage AD versus VS or AARAD. Subgroup separation was superior in pT3-based assays vs. hT7-based assays, indicating that pT3 epitope hyperphosphorylation is particularly enriched (above simple total tau elevation) in the disease. Cohort 4 (CDR 0 vs CDR1 cohort)

[0234] CSF FL samples from clinically defined normal individuals (Clinical Dementia Classification 0; CDR 0) vs. mild memory complaint (CDR 1) (n=5 per group) were obtained at the University of Washington. CDR and MMSE, as well as CSF Aβ42, tTau, and pTau181 measurements by Innotest assays were obtained at the University of Washington. Prior to dispatch, samples were coded so that Janssen was blinded to the identity or characterization of the samples. Simoa's tTau and p217+ tau measurements were performed at Janssen Neuroscience Biomarkers, La Jolla, and sent to the University of Washington for analysis.

[0235] CSF samples were measured in their raw state or after fractionation by rpHPLC, using assays based on both pT3 (pT3xhT43 and pT3xpT82) and tTau (hT7xpT82). Data were expressed as a ratio between the two pT3 assays (Table 7) to assess the relative impact of the short tau species, or as (Figures 16A-16B) a ratio between the pT3 and tTau assays to assess the relative impact of this phosphorylation event. In both cases, the result accurately diagnosed CDR status for 9 of 10 individuals. The individual outside the limits 1 was determined by Innostest to also have abnormally low Tau, and thus may represent non-tauopathy dementia. Intriguingly, a correlation between the Tau ratio Petition 870260042583, dated 06 / 05 / 2026, page 96 / 334 88 / 98 p217+ / tTau and MMSE were observed, suggesting that the signal detected by pT3 assays may correlate with cognition. Table 7. Ratio of analysis of pT3xpT82 (p217+ short) versus pT3xhT43 (p217+ long) and gross CSF Innotest Αβ, tTau, pTau P217 + tau DI CD R MM SE Gén ero idade_e m_LP Innotes t Aβ Inno Tau InnopTau P217 + short / P217 + long 55 + 816.42 7 104.47 1.25 2571 1 1 28 M 79 + 450.00 5 65.486 1.78 6249 6 1 24 M 85 + 1126.9 19 153.024 143 F 72 - 261.84 6 44.805 0.81 6472 2 0 30 F 77 - 247.99 55.951 0.99 6499 6 0 30 M 80 - 427.87 1 96.301 0.8 6583 F 6083 -6 180.45 2 39.263 0.99 6592 2 0 28 M 58 - 539.75 96.29 low 6803 1 1 27 M 68 + 1080.0 48 120.47 1.22 * CDR 11 is positive Αβ but has low Tau and pTau in Innotest and Simoa Petition 870260042583, of 06 / 05 / 2026, p. 97 / 334 89 / 98 Cohort 5 (VS versus MCI versus DA cohort)

[0236] Clinically and biochemically defined FL CSF samples (Innotest>AB42 600 pg / ml) (n=7) were obtained from Precision Medicine (San Diego, CA). Clinically and biochemically defined FL CSF samples (Innotest AB42 < 600 pg / ml) MCI (n=28) and DA (n=12) were obtained from the University of Antwerp. Tau p217+ measurements by rpHPLC and Simoa were performed at Janssen Neuroscience Biomarkers, La Jolla.

[0237] CSF samples were measured in their raw state or after fractionation by rpHPLC, using assays based on both pT3 (pT3xhT43 and pT3xpT82) and tTau (hT7xpT82). All pT3-based and hT7-based assays showed a progressively increasing signal in the VS vs MCI vs DA groups (Figures 17A and C) and correlated well with each other (as seen in cohort 1, Figure 9) (Figures 17D and 17E). The pT3 assays also correlated to some extent with Innotest tTau and pTau181 (Figures 17F and 17G), but not with Innotest AB42 or the AB42 / 40 ratio (Figures 17H and 17II). Similar results for diagnostic positioning were observed in measurements of raw CSF (Figures 17A-17C) or fractionated material by rpHPLC (Figures 17J-17T).As seen in cohort 3, the separation of VS versus MCI versus DA was more pronounced (greater statistical significance) using pT3-based assays than the tTau assay, highlighting the pathological relevance of this pT3 assay measurement. Cohort 6 (Disease severity and progression cohort)

[0238] CSF (CSF) samples from clinically defined AD individuals (Clinical Assessment of Dementia 1+) (n=235) were obtained from the Janssen ELN115727301 / 302 study. These samples were baseline (pre-dose) samples from all individuals in the trial. In addition, CSF samples from 78 weeks of follow-up from placebo individuals (n=90) were Petition 870260042583, dated 06 / 05 / 2026, page 98 / 334 90 / 98 individuals were included to assess biomarkers of disease progression. Cognitive assessment (ADAS-COG, MMSE, NTB, and CDR.SOB), ApoE genotype, sex, and age were obtained from the assay. The Innotest AB42, Innotest AB40, Simoa neurofilament light (NFL), pT3xpT82, pT3xhT43, and hT7xpT82 assays were performed at Janssen Neuroscience Biomarkers, La Jolla. Individuals were confirmed as amyloid-positive or amyloid-negative based on the AB42 / 40 cutoff ratio of 0.09 (i.e., individuals with a ratio <0.09 = amyloid-positive = AD, while those >0.09 = amyloid-negative = dementia of non-AD cause). 27 of the 235 individuals were determined to be amyloid-negative; therefore, each group was analyzed separately.

[0239] The signal from raw CSF measurements again revealed a good correlation between the two pT3 assays and the tTau assay (Figures 18A and 18B), but not with NFL (Figure 18C), a suspected marker of general neurodegeneration, suggesting that the pT3 assay may recognize a specific form or stage of neurodegeneration.

[0240] Assays based on pT3 again showed a higher signal in amyloid-positive versus amyloid-negative individuals (Figures 18D-18E).

[0241] pT3-based assays showed a modest correlation with several cognition scores (ADAS-COG, MMSE, NTB, CDR.SOB, Figures 18F-18M), corroborating the finding in cohort 4 (Figure 16C). Interestingly, the baseline pT3-based assay signal correlated modestly with changes in cognition scores over the 18-month follow-up period, also suggesting the ability to predict cognitive decline (Figures 18N-18P).

[0242] The pT3- and tTau-based signal ratio (tau p217_ / tTau) produced similar results, data not shown.

[0243] Correlations with cognition and changes in cognition were seen in both amyloid-positive and amyloid-negative groups; however, the latter group was a small sample set. If confirmed, this suggests that the connection between p217+ and cognition may not be specific to Petition 870260042583, dated 06 / 05 / 2026, page 99 / 334 91 / 98 FROM THE. Example 9. Quantification of free versus antibody-bound tau p217+

[0244] The assays described in Example 3 were performed as follows. Assay 1: Quantification of free versus bound tau p217+ in biological fluid via immunocapture / depletion followed by rpHPLC.

[0245] The assay was tested by boosting with an antibody in CSF samples. Pooled DA CSF was boosted with 10 µg of pT3 mAb, humanized pT3 mAb, msIgG, or a comparable volume of PBS (sham) and incubated at 4°C for 24 h followed by immunocapture. Samples, as well as parental CSF not subjected to immunocapture, were fractionated by rpHPLC, and each fraction was measured using the pT3xhT43 assay to assess the amount of total and bound p217+ tau. Substantial signal was observed at a main peak, similar to that observed in Example 7 and Figure 7, in the parental sample (total p217+ tau) and the pT3 mAb or humanized pT3 mAb (bound p217+ tau) immunocaptures, but not in the IgG or sham immunocaptures (Figure 19).

[0246] Pooled DA CSF was boosted with titers of humanized pT3 mAb, incubated at 22°C for 2 h, followed by immunocapture, rpHPLC, and pT3xhT43 assay to assess bound p217+ tau (Figure 20A). IgG-depleted supernatant was also fractionated and measured to assess free p217+ tau (Figure 20B). Boosting with humanized pT3 mAb increased the measured amount of bound p217+ tau and decreased the amount of free p217+ tau in a dose-dependent manner.

[0247] Taken together, the results show that this method, which is a direct measurement of target engagement, is specific for antibodies that are directed to the tau p217+ epitope (Figure 19) and is dependent on the dose of targeted antibody (Figure 20). Assay 2: Quantification of free versus bound tau p217+ in biological fluid through selective antibody denaturation. Petition 870260042583, dated 06 / 05 / 2026, page 100 / 334 92 / 98

[0248] Biological samples (e.g., CSF) were heated for 4 minutes to near boiling, followed by cooling on ice and subsequent measurement with the pT3xhT43 and / or pT3xpT82 assays. The precise timing of this process was determined to irreversibly damage the antibodies in the sample so that they could not interfere with the assay (Figure 21) nor affect the tau p217+ signal itself (Figure 21). This is believed to be due to the lack of tertiary structure, in particular, in the tau protein, allowing it to be particularly stable at high temperatures. This sample was termed total tau p217+, while the parallel measurement of a sample that had not been subjected to heat treatment was termed free tau p217+. Subtracting the free concentration from the total concentration yielded the bound tau p217+ measurement.

[0249] The impact of heat on the test was determined, as set out below.

[0250] The impact of heat on a mixture of humanized pT3 mAb / CSF: Pooled DA CSF aliquots were boosted with pT3 mAb at 1 pg / ml, incubated for 2 ha at 22°C, heated for 95 minutes at 0-20°C, cooled to 4°C, then measured using the pT3xpT82 assay at a 1:10 dilution (Figure 21A). The p217+ tau signal was low at ~2 minutes of heat treatment, then returned to levels observed in unboosted CSF and was stable at ~10 minutes of heat before decreasing.

[0251] Impact of heat on untreated CSF: Pooled DA CSF aliquots were heated for 0-20 minutes at 95°C, then cooled to 4°C, before measurement using the pT3xpT82 assay at a 1:10 dilution (Figure 21B). The p217+ tau signal was stable for ~10 minutes of heat before dropping.

[0252] Impact of heat on the ability of humanized pT3 mAb to interfere in the pT3xpT82 assay: Aliquots of humanized pT3 mAb at 10 pg / ml in PBS were heated for 0-20 minutes at 95°C, then Petition 870260042583, dated 06 / 05 / 2026, page 101 / 334 93 / 98 cooled to 4°C. These samples were then mixed with pooled DA CSF (at a final concentration of humanized pT3 mAb) and incubated at 22°C for 2 h before measurement using a pT3xpT82 assay at a 1:10 dilution (Figure 21C). The p217+ tau signal was low ~2 minutes of JNJ heat treatment, then returned to levels observed in unboosted CSF (see Figure 21B) and remained stable for at least 20 minutes of heat.

[0253] Parallel aliquots of pooled DA CSF were titrated with humanized pT3 mAb, incubated for 2 ha at 22°C, then subjected to heat denaturation (with 4 minutes of heat) (Figure 122A) or immunocapture / rpHPLC (Figure 22B) before measurement using the pT3xpT82 assay. Both methods showed a dose-dependent increase in bound humanized pT3 mAb, a decrease in free form, and no change in total p217+ tau signal. Additionally, the heat-mediated denaturation method provided dose-dependent humanized pT3 mAb and relative measurements of total vs. free vs. bound p217+ comparable to those obtained with the more laborious immunocapture / rpHPLC method of Assay 1 (Figure 22C). Therefore, the thermal method is recommended for standard sample analysis. Example 10. p217+ tau sign in preclinical animal models

[0254] To support clinical studies, untreated samples from several common laboratory animals were evaluated using pT3-based and / or sequence-aligned assays to predict cross-reactivity. Cynomolgus monkey

[0255] CSFs from two cynomolgus monkeys were measured at various dilutions using pT3-based and hT7-based assays (Figure 23). For comparability, the same detection antibodies were paired with each of the two capture antibodies. In some cases, the two individual CSFs were tested separately (Cyno 1 or Petition 870260042583, dated 06 / 05 / 2026, page 102 / 334 94 / 98 Cino 2), and in other cases, CSF samples were pooled to save volume. Substantial signal (AEB) was seen in all assays using hT7 as the capture antibody, regardless of the detection antibody, but no signal was detected in any of the assays using pT3 as the capture antibody. Additionally, pT3-based plate assays showed that even in cynomolgus monkey brain homogenates, there is very little or no pT3-based signal, despite the large signal in human DA brain (data not shown). This suggests that despite high tau levels, the pT3 epitope is not preserved in this species. In fact, published protein sequence analysis suggests that one amino acid is different between humans and cynomolgus monkeys in the main pT3 epitope, and structural modeling, based on the crystal structure of tau-humanized pT3 mAb, suggests that this change could abolish pT3 binding (data not shown).Common marmoset.

[0256] Common marmoset CSF was tested using pT3-based and hT7-based assays (Figure 24). CSF from three common marmosets was measured at various dilutions using pT3xhT43, pT3xpT82, and hT7xpT82 assays. For comparison, pooled cynomolgus monkey CSF (negative control) and human DA CSF (positive control) were tested simultaneously. Substantial signal (AEB) was seen in marmoset CSF using the pT3xpT82 (Figure 24B) and hT7xpT82 (Figure 20C) assays, but not with the pT3xhT43 assay (Figure 24A).

[0257] This suggested that the hT43 epitope was absent in this species and, in fact, a protein sequence alignment indicates that one amino acid is different between humans and common marmosets in the hT43 epitope, while the pT3, hT7, and pT82 epitopes are preserved. Measurement of marmoset brain homogenate with the same tests confirmed that there was substantial signal with the pT3xpT82 and hT7xpT82 assays, but very little with the pT3xhT43 assay (data not shown). Petition 870260042583, dated 06 / 05 / 2026, page 103 / 334 In the 95 / 98 form, the analysis of the tau p217+ signal in marmosets was obtained using the pT3xpT82 assay. Mouse, Rat, Dog and Pig

[0258] Alignment of tau protein sequences predicted in mouse, rat, dog, or pig (NCBI accession numbers: NP_001033698.1, NP_058908.2, NP_001104271.1, and AGJ26517.1, respectively) with human sequences suggests that pT3 is 100% conserved in these species. However, the hT43 and pT82 sequences from mouse, rat, dog, and pig are not identical to those from humans, and therefore, samples from these would need to be evaluated using pT3xhT43 and pT3xpT82 assays.

[0259] Taken together, the data presented here indicate that the pT3xhT43 and pT3xpT82 assays developed on the Simoa platform for CSF measurement are highly sensitive, having sensitivity in femtograms, are precise, accurate, linear dilution, and the analyte is stable. The assays appear to correlate well with classic AD biomarkers and dementia scores, and may be superior to those measured in the identification and staging of AD individuals.

[0260] The assays can be used to measure the level of total tau p217+ in CSF, or to assess the p217+ fragment profile in CSF fractionated by rpHPLC. The assays can also be combined with preanalytical manipulation to measure the levels of tau p217+ that are bound by endogenous or exogenously administered antibodies, compared to antibody-free tau p217+. In this way, the assays can be used as predictive biomarkers to identify individuals for whom anti-tau p217+ antibody therapy will be appropriate, by identifying individuals with high levels of the target tau p217+. By measuring antibody-bound, total, and free tau p217+ levels, the assays can also be used as pharmacodynamic markers. Example 11. Tau p217+ sign in blood Petition 870260042583, dated 06 / 05 / 2026, page 104 / 334 96 / 98

[0261] Although CSF tau measurement has shown great utility in the diagnosis and positioning of neurodegenerative disorders, CSF collection has limitations (e.g., patient load, clinical site experience, collection volume, and frequency restrictions). As such, there is great interest in adapting tau for use in measurements of blood-derived products (e.g., serum, plasma). However, recent literature has indicated that tau measurements in serum or crude plasma do not exhibit optimal diagnostic performance and may be affected by matrix interference barriers and sensitivity. pT3-based assays may represent a new opportunity, however, due to their high sensitivity and specificity.

[0262] Serum from clinically defined AD individuals (n=4 each) was measured with pT3xpT82 and hT7xpT82 assays, either in the crude sample at various dilutions (crude, Figures 25A-25D), in denatured samples treated with acid (NaOAc pH 5) (boiling, Figures 26A-26B) as in D' Abramo et al. 2016 to remove most matrix interference, and after immunoprecipitation (IP) with pT3 microspheres followed by heat denaturation of the elute (pT3 IP, Figure 27).

[0263] Measurement in crude serum revealed that most samples were below the limit of quantification (LOQ), with a few samples outside the limits reporting much higher levels. However, the signal did not survive modest dilution and was considered an interference artifact. Evaluation of the highest dilution tested (Figures 25B and 25D), and thus the least affected by interference, suggested that the pT3xpT82 assay may detect a slightly higher signal in DA samples, but all are below the LOQ so they may not be precise and / or accurate.

[0264] Measurement in serum after treatment with acid (to dissociate protein-protein interactions) and heat (to denature most tau proteins) reduced the pT3xpT82 and hT7xpT82 signal to almost or below the LOQ (Figures 26A-26B). Again, the pT3xpT82 assay Petition 870260042583, dated 06 / 05 / 2026, page 105 / 334 97 / 98 can detect slightly more signal in DA samples, but all are close to LOQ, which may not be precise and / or accurate.

[0265] Measurement in serum after denaturation and IP of pT3 to remove most interfering substances and concentrate tau p217+, revealed much higher levels in DA samples than in VS samples (Figure 27). p217+ levels were ~4x higher than crude or boiling measurements and such VS samples were now at the LOQ and DA samples were now all in the linear range.

[0266] These results indicated that the pT3-based assays described here may be useful as a blood-based measurement of pathological tau, particularly when paired with an enrichment strategy such as IP.

[0267] Although the invention has been described in detail and with reference to specific embodiments thereof, it will be evident to those skilled in the art that various alterations and modifications can be made to it without departing from the spirit and scope of the invention. References Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008 Almagro, Mol Recognit. 17:132-43, 2004 Barthelemy et al., J Alzheimers Dis. 51(4):1033-43, 2016 Butner and Kirschner, J Cell Biol. 115(3):717-30, 1991 Chothia and Lesk, J Mol Biol. 196:901-17, 1987 Chothia et al., J. Mol. Biol. 227: 799-817, 1992 Clavaguera et al., Nat Cell Biol. 11:909-13, 2009 D'Abramo et al Neurobiol Aging. 37:58-65, 2016 Epitope Mapping Protocols in Molecular Biology Methods, Vol. 66, GE Morris, Ed. (1996) Fishwild et al., Nat Biotechnol. 14:845-51, 1996 Frost et al., J Biol Chem. 284:12845-52, 2009 Hanger et al. J Biol Chem. 282(32):23645-54, 2007 Petition 870260042583, of 06 / 05 / 2026, p. 106 / 334 98 / 98 Hanger et al., Trends Mol Med. 15:112-9, 2009 Iqbal et al., Curr Alzheimer Res. 7(8): 656-664, 2010 Knappik et al., J Mol Biol. 296:57-86, 2000 Kohler and Milstein, Nature. 256:495-7, 1975 Krebs et al., J Immunol Methods. 254:67-84, 2001 Lefranc et al., Dev Comp Immunol. 27:55-77, 2003 Lonberg et al., Nature. 368:856-9, 1994 Mendez et al., Nat Genet. 15:146-56, 1997 Meredith et al. PLoS One. 8(10):e76523, 2013 Morris et al., Neuron, 70:410-26, 2011 Russell et al., J Alzheimers Dis. 55(1):303-313, 2017 Shi et al., J Mol Biol. 397:385-96, 2010 Tramontano et al., J. Mol. Biol. 215:175-182, 1990 Wu and Kabat, J Exp Med. 132:211-50, 1970 Petition 870260042583, dated 06 / 05 / 2026, p. 107 / 334

Claims

1 / 9 CLAIMS 1. A method for measuring tau p217+ peptides in a sample, characterized by comprising: (i) bringing the sample into contact with a capture antibody directed against a tau p217+ epitope to capture the tau p217+ peptides in the sample, and (ii) bringing the captured tau p217+ peptides into contact with at least one of a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein and a second detection antibody directed against an epitope containing amino acid residues 7 to 20 of the tau protein, thereby measuring at least one of a quantity of tau p217+ peptides and a quantity of long tau p217+ peptides, respectively, wherein the amino acid numbering is with reference to the amino acid sequence presented in SEQ ID NO: 1 2. A method according to claim 1, characterized by comprising placing captured tau p217+ peptides in contact with the first detection antibody and the second detection antibody in order to measure the amount of tau p217+ peptides and the amount of long tau p217+ peptides, respectively, and optionally determining a ratio between the amount of long tau p217+ peptides and the amount of tau p217+ peptides.

3. Method according to claim 2, characterized by further comprising: (i) determining an amount of short tau p217+ peptides by subtracting the amount of long tau p217+ peptides from the amount of tau p217+ peptides, and (ii) optionally determining a ratio between the amount of short tau p217+ peptides and the amount of tau p217+ peptides or a ratio between the amount of long tau p217+ peptides and the amount of short tau p217+ peptides.

4. Method for measuring tau p217+ peptides in a sample, characterized by comprising: (i) bringing the sample into contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in the sample, and bringing the sample into contact with a phosphorylation-independent capture antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein, to capture total tau peptides in the sample; (ii) performing at least one of: a.a. place the captured p217+ tau peptides in contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein in order to measure a quantity of p217+ tau peptides, place the captured total tau peptides in contact with the first detection antibody in order to measure a quantity of total tau peptides, and determine a ratio between the quantity of p217+ tau peptides and the quantity of total tau peptides; and b.The captured p217+ tau peptides were brought into contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein, thus measuring the quantity of long p217+ tau peptides, and the total captured tau peptides were brought into contact with the second detection antibody, thus measuring the quantity of total long tau peptides, and determining a ratio between the quantity of long p217+ tau peptides and the quantity of total long tau peptides, with the amino acid numbering being referenced to the amino acid sequence presented in SEQ ID NO:

1.

5. Method according to claim 4, characterized by Petition 870260042583, dated 06 / 05 / 2026, page 109 / 334 3 / 9, further comprising determining an amount of short p217+ tau peptides by subtracting the amount of long p217+ tau peptides from the amount of p217+ tau peptides, determining an amount of total short tau peptides by subtracting the amount of total long tau peptides from the amount of total tau peptides, and determining the ratio between the amount of short p217+ tau peptides and the amount of total short tau peptides.

6. A method, according to any one of claims 1 to 5, characterized in that the sample is a biological sample from an individual selected from the group, consisting of blood, brain homogenate, or cerebrospinal fluid (CSF) from the individual.

7. Method according to claim 6, characterized in that the biological sample is blood.

8. Method according to claim 6, characterized in that the biological sample is CSF.

9. Method, according to claim 6, characterized in that the biological sample has been fractionated using reversed-phase high-performance liquid chromatography (rpHPLC).

10. Method according to claim 6, characterized by further comprising: a. determining whether the individual suffers from a tauopathy or is at risk of developing a tauopathy; b. determining whether the individual is suitable for treatment with an anti-tau p217+ antibody; c. determining the efficacy of a tauopathy treatment in the individual; or d. monitoring treatment with an anti-tau p217+ antibody in an individual, wherein the determination or monitoring comprises comparing at least one of the following: the amount of tau p217+ peptides, the amount of long tau p217+ peptides, the amount of short tau p217+ peptides, and the ratios of these amounts in the individual to a corresponding baseline value.

11. A method according to claim 10, comprising monitoring treatment with an anti-tau p217+ antibody in an individual, the method being characterized by comprising: (i) obtaining a biological sample from the individual; (ii) separating the biological sample into a first sample containing tau p217+ peptides free of the anti-tau p217+ antibody and a second sample containing tau p217+ peptides bound to the anti-tau p217+ antibody; (iii) separating the second sample, preferably via rpHPLC, to obtain a third sample containing tau p217+ peptides free of the anti-tau p217+ antibody; (iv) placing each of the first and third samples in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in each of the first and third samples;(v) perform at least one of (a) bringing the captured tau p217+ peptides into contact with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein in order to measure the amount of tau p217+ peptides in each of the first and third samples, and (b) bringing the captured tau p217+ peptides into contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure the amount of long tau p217+ peptides in each of the first and third samples, optionally (c) determining the amount of short tau p217+ peptides by subtracting the amount of long tau p217+ peptides from the amount of tau p217+ peptides in each of the first and third samples; (vi) monitor treatment with the anti-tau p217+ antibody with Petition 870260042583, dated 06 / 05 / 2026,p. 111 / 334 5 / 9 based on at least one of the following: the amount of tau p217+ peptides, the amount of long tau p217+ peptides, the amount of short tau p217+ peptides, and the ratios of these amounts, in each of the first and third samples.

12. A method according to claim 10, comprising monitoring treatment with an anti-tau p217+ antibody in an individual, the method being characterized by comprising: (i) obtaining a biological sample from the individual, (ii) obtaining a semi-denatured sample from the biological sample containing total tau p217+ peptides, wherein the semi-denatured sample is heated to denature the antibodies in the sample and obtaining an undenatured sample from the biological sample containing tau p217+ peptides free of anti-tau p217+ antibody, (iii) placing each semi-denatured sample and the undenatured sample in contact with a capture antibody directed against a tau p217+ epitope to capture tau p217+ peptides in each of the samples, (iv) performing at least one of (a) placing the captured tau p217+ peptides in contact with a first detection antibody directed against an epitope comprising residues of amino acids 119 to 126 of the tau protein for,thus, measure a quantity of tau p217+ peptides in each of the samples, and (b) place the captured tau p217+ peptides in contact with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein in order to measure a quantity of long tau p217+ peptides in each of the samples, optionally (c) determine a quantity of short tau p217+ peptides by subtracting the quantity of long tau p217+ peptides from the quantity of tau p217+ peptides in each of the first and third samples, and (v) monitor the treatment with the anti-tau p217+ antibody with Petition 870260042583, dated 06 / 05 / 2026, page. 112 / 334 6 / 9 based on at least one of the following: the amount of tau p217+ peptides, the amount of long tau p217+ peptides, the amount of short tau p217+ peptides, and the ratios of these amounts in each of the samples.

13. A method according to any one of claims 1 to 12, characterized in that the capture antibody is conjugated to a microsphere, and the detection antibody is biotinylated.

14. Method, according to any one of claims 1 to 13, characterized in that the lower limit of quantification of the method is about 40 fg / ml of p217+ tau peptides and the lower limit of detection of the method is about 2 fg / ml of p217+ tau peptides.

15. Method, according to any one of claims 7 to 14, characterized in that the tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, dementia with tangles only, diffuse neurofibrillary tangles with calcification, dementia with argyrophilic grains, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatza disease, inclusion body myositis, Creutzfelt-Jakob disease, multiple system atrophy, Niemann-Pick type C disease, cerebral amyloid angiopathy caused by prion protein, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, post-encephalitic parkinsonism,Chronic traumatic encephalopathy and pugilistic dementia (boxing disease), with tauopathy being, preferably, Alzheimer's disease.

16. Method, according to any one of claims 1 to 15, characterized in that the capture antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36 and 37, respectively; preferably, the capture antibody has a variable region of the heavy chain comprising the polypeptide sequence of SEQ ID NO: 28 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO:

29.

17. Method, according to any one of claims 1 to 16, characterized by the first detection antibody comprising HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively; preferably, the first detection antibody comprises a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 8 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO:

9.

18. Method, according to any one of claims 1 to 17, characterized in that the second detection antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively; preferably, the detection antibody comprises a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO:

19.

19. Kit characterized by comprising: a. a capture antibody directed against a p217+ tau epitope, optionally a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein; and b. at least one detection antibody directed against a tau protein epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein.

20. Kit, according to claim 19, characterized in that the capture antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 32, 33 and 34, respectively, and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 35, 36 and 37, respectively; preferably, the capture antibody has a variable region of the heavy chain comprising the polypeptide sequence of SEQ ID NO: 28 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 29, and the phosphorylation-independent capture antibody is directed against a tau epitope comprising amino acids 159 to 163 of the tau protein.

21. Method according to claim 19 or 20, characterized by: a. the first detection antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 2, 3 and 4, respectively; and LCDR1, LCDR2 and LCDR3 of the immunoglobulin light chain having the polypeptide sequences of SEQ ID NOs: 5, 6 and 7, respectively; preferably, the first detection antibody comprises a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 8 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO: 9; and b. the second detection antibody comprises HCDR1, HCDR2 and HCDR3 of the immunoglobulin heavy chain having the polypeptide sequences of SEQ ID NOs: 12, 13 and 14, respectively; and LCDR1, LCDR2 and LCDR3 of the light chain of Petition 870260042583, of 06 / 05 / 2026, page.115 / 334 9 / 9 immunoglobulin having the polypeptide sequences of SEQ ID NOs: 15, 16 and 17, respectively; preferably, the detection antibody comprises a variable region of the heavy chain having the polypeptide sequence of SEQ ID NO: 18 and a variable region of the light chain having the polypeptide sequence of SEQ ID NO:

19. Petition 870260042583, dated 06 / 05 / 2026, p. 116 / 334.

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