Patent Application
20220187322
The present invention relates to the use of anti-tau antibodies in an assay to differentiate tau species in different tau pathologies. The assays according to the invention can be used i.e. to diagnose patients with tauopathies such as Alzheimer's disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy and globular glial tauopathy.
The term tauopathy defines a group of pathological diseases characterized by deposition of the microtubule-associated protein tau. The deposited tau is phosphorylated abnormally and accumulates as intracellular inclusions. There are a number of specific tauopathies, each of which vary by the distribution and morphological appearances of the protein-containing inclusions, as well as the relative burden of pathology affecting neurons and neuronal processes versus glial and glial processes (Dickson et al., 2011). The most common tauopathies are progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease, and globular glial tauphathy (GGT) and chronic traumatic encephalopathy (CTE). All but Pick's disease are commonly associated with movement disorders (Keith A. Josephs, Chapter IX, in Movement Disorders (Second Edition), 2015). Tauopathies are also denoted Fronto Temporal Dementia (FTD), Fronto Temporal Lobar Degeneration (FTLD). Most cases of FTD are associated with genetic mutations in MAPT (tau) or GRN (granulin). FTD is associated with accumulation of tau protein.
In order to diagnose and find the right treatment for these patients it is important to have a method that can diagnose these patients and differentiate tau pathology that is characteristic for each disease. The inventors of the present invention have provided a number of assays that can help in the diagnosis of these diseases.
The present invention relates to an in vitro assay for measuring phosphorylated tau in a sample, said assay comprises the use of 2 antibodies i) a capture antibody specific for the phosphorylated(p) serine(S) residue 396 (pS396) on tau and ii) a detection antibody binding tau on a different epitope than the capture antibody. The detector antibody may bind a non-phosphorylated residue on tau as disclosed further herein.
In a second aspect the invention relates to a method for measuring phosphorylated tau in a sample, which method comprises the steps of
(A) The Tau12-pS396 assay, also known as the full-length pS396 (or FL pS396) assay, uses the anti-pS396 antibody as the capture antibody and Tau12 (epitope at amino acids 6-18) as the detection antibody.
(B) The HT7-pS396 (mid-region pS396 or MR pS396) assay measures pS396 on tau species that stretch from the mid-region (epitope 159-168). This assay uses the pS396 antibody as the capture and HT7 the detection antibody.
(C) The pS396-Tau46 (C-terminus pS396 or CT pS396) assay, which uses the anti-pS396 antibody as capture and Tau46 (epitope 404-441) as detection antibody, is specific for pS396 phosphorylated tau that contains the extreme C-terminus region (amino acids 404-441).
All three assays measure tau pS396 in human tauopathy brain samples. The results indicate that the populations of pS396 tau in different tauopathies significantly vary with respect to the tau species present, suggesting that the combined measurement of pS396 and tau fragmentation is a promising approach to separating between these diseases.
CT pS396 is present in the CSF and plasma of rTg4510 transgenic tau mouse animals. The CT pS396 assay is an important tool for studying molecular changes in CT pS396 processing in this model and for preclinical evaluation of drug efficacy.
CT pS396 is not measurable in untreated human CSF. However, pre-analytical processing by spin filtration and SEC enriches CT pS396 signal, with the highest concentrations eluting at 12.0-13.5 ml. Consistent results have been recorded using spin filters of different capacities and models (Table 1), by changing the CSF starting volume (Table 1), and by varying the SEC elution volumes collected per fraction.
The sequential treatment of CSF samples by spin filtration followed by SEC fractionation is necessary for the CT pS396 signal enrichment because omitting either step leads to much reduced or undetectable amounts.
Traditional ELISA has been used for many years in analysing molecules of different kinds in samples. The present invention is directed to the use of the single molecule array assay (Simoa) ELISA technology to detect tau species in samples from patients diagnosed with tauopathies such as Alzheimer's disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy, globular glial tauopathy and chronic traumatic encephalopathy
In the present invention tau is human tau of the following sequence, Methionine being number 1
In the first step of the single-molecule immunoassay capture antibodies are attached to the surface of paramagnetic beads (˜2.7 um diameter) that will be used to concentrate a dilute solution of tau molecules in a sample. A biotinylated detection antibody is added to the mixture, and the capture and detector antibodies are allowed to react to the tau in the sample. To remove non-specific protein binding the mixture may be washed. Subsequently beta-galactosidase-labelled streptavidin is added, followed by an optional washing step, and resorufin beta-D-galctopyranoside is added. The reaction mixture is allowed to react and generate a fluorescent product which may be read and analysed in an appropriate machine.
The assay developed by the inventors of the present invention is based on two antibodies, a capture antibody and a biotinylated detector antibody. The capture antibody is conjugated to a paramagnetic bead as described in Example 1. These are specific for the phosphorylated (p) S396 of tau. The generation of such antibodies are disclosed in for example WO2017/009308 and these antibodies are further described in Table I below. In the Examples of the present invention the pS396 specific antibody used is the antibody designated “010-2 Humanized” from patent WO2018/011073 and described in Table II below
Table II: pS396 Antibodies Disclosed in WO2018/011073
The antibodies in the below scheme are all engineered versions of the antibody designated “010-2 humanized antibody”. Differences compared to the 010-2 humanized antibody are shown specifically, otherwise grey boxes in the table are intended to indicate identical amino acids as the 010-2 humanized antibody. Thus, for example, D55E has the same CDR 1-3 of the light chain and CDR1 and 3 of the heavy chain as the 010-2 humanized antibody (grey boxes), whereas the CDR2 of the heavy chain differs (amino acid residues are given) and thus VH differs from the 010-2 humanized antibody (amino acid residues are given)
The detection/or detector (used interchangeably herein) antibodies used have been biotinylated as described in Example 2, and may bind the C-terminal, mid or N-terminal region of tau at a site different from the pS396 residue. The detector antibody may bind non-phosphorylated residues. In particular, epitopes of the C-terminus on tau are amino acids 1-20 (such as 6-18), the mid region of tau is amino acids 140-170 (such as 159-168) and N-terminus of tau is 400-441 (such as 404-421). In the Examples, the following antibodies are used:
Tau12 (#806502, BioLegend) is used and binds to the amino acids 6-18 of tau, detection antibody is HT7 (#MN1000, Invitrogen) binds mid region 159-168 amino acids, and Tau46 (#806601, BioLegend) binds the C-terminal region amino acids 404-441.
Three assays measuring pS396 on different tau species have been developed (
The method, as described in Example 4, comprises the steps of
The amount of pS396 conjugated antibody beads used are usually at least 1000 beads such as at least 10,000, 100,000 beads or more. The sample may be a CSF, plasma or bio-fluid sample from a mammal, for example a human CSF sample from a human suffering from a tauopathy such as Alzheimer's disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy or globular glial tauopathy. It may be advantageous to concentrate the sample with respect to tau for example by use of spin filtration columns and size exclusion chromatography as shown in Example 3.
The results of the assays used individually or in combination can be used to diagnose or differentiate tauopathies, such as Alzheimer's disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy and globular glial tauopathy, as shown in Example 4. For example, the CT assay can be used to diagnose Alzheimer's disease and Pick's disease. By comparing the MR/FL ratio it can be used to differentiate Alzheimer's disease over the other tauopathies and the control, and further the Pick's disease was significant different from corticobasal degeneration, progressive supranuclear palsy, globular glial tauopathy and the control. By suing the CT/FL ratio, Alzheimer's disease can be used to differentiate over the other tauopathies and the control and Pick's disease was different compared to corticobasal degeneration, progressive supranuclear palsy and globular glial tauopathy and control.
The pS396 antibody was buffer exchanged into bead conjugation buffer (BCB; 50 mM MES pH 6.2) using Ultracel 50K spin filtration columns (#UFC505096, Amicon). The filter was first rinsed with 450 ul BCB by centrifuging at 14000×g at room temperature (RT) for 5 min, and discarding the flow-through. Thereafter, 1.6 g/L antibody was buffer exchanged into BCB by centrifuging at 14000×g, RT, for 5 min. The flow-through was discarded and the filter returned to the collection tube. BCB was added to the retentate to bring the volume to 450 ul, and re-centrifuged under the same conditions. This step was repeated once after discarding the flow-through. Subsequently, the filter was rinsed with 40 ul BCB, inverted into a new collection tube and the antibody recovered by centrifuging for 2 min at 1000 xg, RT. The concentration of the antibody was estimated with Nanodrop Lite (ThermoFisher Scientific) and stored at 4° C. until use.
Paramagnetic carboxylated singleplex beads (#103207, Quanterix) were washed thrice with bead wash buffer (BWB; 1×PBS+1% Tween 20) and then twice with BCB using a magnetic separator. The beads at a concentration of 1.4×106 beads/μL were activated by adding 0.3 g/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (#A35391, Thermo Scientific) and incubating at 4° C. for 30 min. Thereafter, the activated beads were washed once with ice-cold BCB and the supernatant discarded. The antibody (0.2 g/L) was added to the beads and the mixture incubated for 2 h at RT with shaking to allow the antibodies bind to the beads. Shaking was always performed with a HulaMixer Sample Mixer (#15920D, ThermoFisher Scientific) under the following conditions with each step lasting 5 sec: orbital=5 rpm, reciprocal=90°, vibro/pause=5°. Afterwards, the supernatant was removed and the antibody-conjugated beads washed twice with BWB. The reaction was blocked with bead blocking buffer (1% BSA in 1×PBS) for 1 h at RT with shaking. Finally, the beads were washed twice with BWB and then once with bead diluent (BD; 50 mM Tris pH 7.8, 50 mM NaCl, 10 mM EDTA, 1% BSA, 0.1% Tween20). After removing the supernatant with a magnetic separator, the beads were resuspended in BD and stored at 4° C. until use.
The detection antibodies were buffer exchanged into biotinylation reaction buffer (BRB; 100 mM PBS pH 7.4) in Ultracel 50K spin filtration columns (#UFC505096, Amicon). After cleaning the column by centrifuging 450 ul BRB at 14000 xg, RT, for 5 min, the flow-through was discarded and the antibody transferred to the filter. BRB was added to bring the volume to 450 ul and centrifuged at 14000×g at RT for 5 min. The buffer exchange was repeated two more times, at each stage by bringing the antibody volume to 450 ul with BRB and centrifuging for 5 min at 14000 xg, RT. The filter was rinsed with 40 ul BRB, inverted into a new collection tube and the antibody recovered by centrifuging for 2 min at 1000 xg, RT. The concentration of the antibody was estimated using Nanodrop Lite. Forty times excess of EZ-Link NHS-PEG4-Biotin (#21329, Thermo Scientific) was added to the antibody and incubated for 30 min at RT. Free biotin was removed by repeating the buffer exchange process performed prior to the biotin labelling. The biotin-conjugated antibodies were stored at 4° C. until use.
Appropriate concentrations of the assay calibrator (recombinant tau 441 phosphorylated in vitro by Glycogen Synthase Kinase 3β (#T08-50FN, SignalChem)) were prepared by diluting stock concentrations with the assay diluent (Tau 2.0 diluent, #101556, Quanterix) before analysis. Quality control samples include TBS-soluble human Alzheimer's disease brain extract diluted 500 and 5000 times with the assay diluent.
Tris buffered saline (TBS)-soluble human brain extracts, rTg4510 transgenic mice CSF and plasma samples were diluted with Tau 2.0 diluent to the desired concentrations indicated in
The level of pS396 tau in human CSF was enriched by concentrating samples in spin filtration columns and fractionating the retentate by size exclusion chromatography (SEC) on a Superdex S200 10/300 GL column (#17-5175-01, GE Healthcare) running on an Ethan LC system (GE Healthcare). The running buffer was 50 mM Tris pH 7.5+10% glycerol. Collected fractions were analysed directly using the Simoa pS396 assays. This method has been verified using spin filtration columns of different capacities and properties (Table 1).
Each pS396 assay uses a two-step protocol on the Simoa HD-1 instrument (Quanterix, Lexington, Mass., USA). In this assay configuration, 100 ul of the bead mixture, consisting of 1000 beads/ul each of pS396 antibody-coated beads and Helper Beads (#103208, Quanterix), is aspirated into a reaction cuvette. Thereafter, 20 ul biotinylated detection antibody (2 ug/ml) and 100 ul of the analyte of interest were added and the reaction mixture incubated for 47 cadences (1 cadence=45 sec) to allow the analyte to react with the capture and detection antibodies. The beads were subsequently washed and 100 ul of 450 pM streptavidin-conjugated β-galactosidase (SBG; #100439, Quanterix). Following another incubation for 7 cadences and a subsequent wash, 25 μl resorufin β-D-galactopyranoside (RGP; #103159, Quanterix) was added. Hydrolysis of RGP was catalysed by SBG, yielding the fluorescent product resorufin. The beads were transferred onto a disc of 200,000 wells, each only large enough to accommodate one bead. Extra beads were removed and the disc surface sealed before imaging. The fluorescent signals were converted to average enzyme per bead (AEB) and the sample concentrations extrapolated from a four-parametric logistic calibration curve generated with known protein concentrations.
Three assays measuring pS396 on different tau species have been developed (
| Number | Date | Country | Kind |
|---|---|---|---|
| PA201900377 | Mar 2019 | DK | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/058062 | 3/24/2020 | WO | 00 |
