Note: Descriptions are shown in the official language in which they were submitted.
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ANTIBODY-BASED METHODS OF DETECTING AND TREATING ALZHEIMER'S
DISEASE
FIELD
[1] This application claims priority to U.S. Application No. 62/649,208
filed March
28, 2018, U.S. Application No. 62/664,662 filed April 30, 2018, and U.S.
Application
No.62/703,299 filed July 25, 2018, each of which is incorporated herein by
reference
in its entirety.
[2] Disclosed herein are antibodies, compositions, kits, and methods for
the
detection, monitoring, prevention, and/or treatment of Alzheimer's disease and
other
tauopathies.
BACKGROUND AND SUMMARY
[3] Alzheimer's disease (AD) is a progressive neurodegenerative disorder
that is
associated with the destruction of higher brain structures, such as those
involved in
memory and cognition. The disease leads to deficits in cognitive function and
declines in memory, learning, language, and in the ability to perform
intentional and
purposeful movements. AD is also accompanied by concomitant behavioral,
emotional, interpersonal, and social deterioration. These cognitive and
behavioral
deficits render living difficult (Burns et al., Alzheimer's disease, The
Lancet, vol. 360,
Jul. 13, 2002). Late-stage AD patients are often unable to speak, comprehend
language, and handle their own basic personal care, eventually requiring full-
time
care and supervision, and are often dependent on family members and nursing
homes. AD is the leading cause of senile dementia, and is predicted to
increase in
prevalence as the proportion of elderly persons in the population grows. The
total
number of persons with AD is predicted to increase at least threefold between
2000
and 2050, rendering AD a world-wide public health problem (Sloane et al., The
Public Health Impact of Alzheimer's Disease, 2000-2050: Potential Implication
of
Treatment Advances, Annu. Rev. Public Health, 23:213-31, 2002). Clinical
detection,
management, and treatment of AD remains largely inadequate. There is still an
unmet need for effective methods to detect and treat AD.
[4] AD has histologically been characterized pathologically by analyzing
brain
sections to identify the presence of extraneuronal plaques and intracellular
and
1
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extracellular neurofibrillary tangles in the brain. Plaques are composed
mainly of 13
amyloid (A13), whereas tangles comprise pathological forms of tau, such as tau
conformers and their aggregates. The relationship between plaques and tangles
and
the disease process remains unclear, although studies suggest a link between
amyloid and tau pathogenesis (Hardy et al., Genetic dissection of Alzheimer's
disease and related dementias: amyloid and its relationship to tau, Nature
Neuroscience, vol 1, No. 5, 1998; Oddo et al., A13 lmmunotherapy Leads to
Clearance of Early, but Not Late, Hyperphosphorylated Tau Aggregates via the
Proteasome, Neuron, 43: 321-332, 2004; Rapoport et al., 2002; Roberson, et
al.,
Reducing Endogenous Tau Ameliorates Amyloid 13-Induced Deficits in an
Alzheimer's Disease Mouse Model, Science, 316:750, 2007; Shipton et al., Tau
Protein Is Required for Amyloid 13-Induced Impairment of Hippocampal Long-Term
Potentiation, J. Neuroscience, 31(5):1688-1692, 2011). A central role for A13
in AD
pathology was initially proposed in a hypothesis called the "A13 cascade,"
wherein A13
deposition is followed by tau phosphorylation and tangle formation, and then
neuronal death (Hardy and Al!sop, Amyloid deposition as the central event in
the
aetiology of Alzheimer's disease, TiPS, vol 12, 1991; Hardy and Selkoe, The
Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road
to
Therapeutics, Science, vol 297, 2002; for a review see, Walsh and Selkoe,
Deciphering the Molecular Basis of Memory Failure in Alzheimer's Disease,
Neuron,
44:181-193, 2004; also see Seabrook et al., Beyond Amyloid the Next Generation
of
Alzheimer's Disease Therapeutics, Molecular Intervention, 7(5), 2007).
[5] Accordingly, therapeutic approaches for AD often focus on targeting A13
and
tau, and many studies on these targets continue today. The most advanced
disease-
targeting therapies undergoing clinical trials in AD patients include passive
immunotherapies such as BAN2401, ADUCANUMAB, GANTENERUMAB and
CRENEZUMAB for A13 removal; C2N 8E12, RO 7105705 and BIIB092 targeting tau
protein; and active vaccine such as CAD106, Lu AF20513, ABvac 40, for A13
therapy
or ACI-35 and AADvac1 to target disease modified tau.
[6] Recent work has led to a number of therapeutic approaches that directly
or
indirectly target the tau cascade in particular (for review articles, see,
e.g. Dickey and
Petrucelli, Pharmacologic reductions of total tau levels; implications for the
role of
microtubule dynamics in regulating tau expression, Molecular Neurode
generation,
2
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1:6, 2006; Schneider and Mandelkow, Tau-Based Treatment Strategies in
Neurodegenerative Disease, Neurotherapeutics, 5:443-457, 2008; Zilka et al.,
Chaperone-like Antibodies Targeting Misfolded Tau Protein: New Vistas in the
lmmunotherapy of Neurodegenerative Foldopathies, Journal of Alzheimer's
Disease,
15:169-177, 2008), including compounds that prevent or reverse tau aggregation
(Wischik et al., Selective inhibition of Alzheimer's disease-like tau
aggregation by
phenothiazines, Proc. NatL Acad. Sci. USA, 93:11213-11218, 1996; Necula et
al.,
Cyanine Dye N744 Inhibits Tau Fibrillization by Blocking Filament Extension:
Implications for the Treatment of Tauopathic Neurodegenerative Diseases,
Biochemistry, 44:10227-10237, 2005; Pickhardt et al., Screening for Inhibitors
of Tau
Polymerization, Current Alzheimer Research, 2:219-226, 2005; Taniguchi et al.,
Inhibition of Heparin-induced Tau Filament Formation by Phenothiazines,
Polyphenols, and Porphyrins, The Journal of Biological Chemistry, 280:9, 7614-
7623, 2005; Larbig et al., Screening for Inhibitors of Tau Protein Aggregation
into
Alzheimer Paired Helical Filaments: A Ligand Based Approach Results in
Successful
Scaffold Hopping, Current Alzheimer Research, 4:315-323, 2007) small-molecule
type drugs that inhibit tau kinases or activate tau phosphatases (lqbal and
Grundke-
lqbal, Inhibition of Neurofibrillary Degeneration: A Promising Approach to
Alzheimer's Disease and Other Tauopathies, Current Drug Targets, 5:495-502,
2004; Noble et al., Inhibition of glycogen synthase kinase-3 by lithium
correlates with
reduced tauopathy and degeneration in vivo, PNAS, 102:19, 6990-6995, 2005;
lqbal
and Grundke-lqbal, Developing pharmacological therapies for Alzheimer disease,
Cell. Mol. Life Sci., 64:2234-2244, 2007), microtubule stabilizing drugs
(Zhang et al.,
Microtubule-binding drugs offset tau sequestration by stabilizing microtubules
and
reversing fast axonal transport deficits in a tauopathy model, PNAS, 102(1):
227-
231, 2005), drugs that facilitate the proteolytic degradation of misfolded tau
proteins
(Dickey et al., Development of a High Throughput Drug Screening Assay for the
Detection of Changes in Tau Levels-Proof of Concept with HSP90 inhibitors,
Current
Alzheimer Research, 2:231-238, 2005, Dickey et al., Pharmacologic reductions
of
total tau levels; implications for the role of microtubule dynamics in
regulating tau
expression, Molecular Neurodegeneration, 1:6, 2006), and immunosuppresive
drugs
(Zilka et al., Chaperon-like Antibodies Targeting Misfolded Tau Protein: New
Vistas
in the lmmunotherapy of Neurodegenerative Foldopathies, Journal of Alzheimer's
Disease, 15:169-177, 2008), as well as immunotherapeutic strategies including
3
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active and passive immunization (Schneider and Mandelkow et al., Tau-Based
Treatment Strategies in Neu rodegenerative Diseases, Neurotherapeutics, 5:443-
457, 2008; Zilka et al., Chaperon-like Antibodies Targeting Misfolded Tau
Protein:
New Vistas in the lmmunotherapy of Neurodegenerative Foldopathies, Journal of
Alzheimer's Disease, 15:169-177, 2008, Tabira, T. Immunization Therapy for
Alzheimer disease: A Comprehensive Review of Active Immunization Strategies.
Tohoku J. Exp. Med., 220: 95-106 (2010)). In addition, researchers have
focused on
novel monoclonal antibody (mAbs) therapies to treat AD. See, e.g., Citron et
al.,
Alzheimer's disease: strategies for disease modification, Nature Reviews,
9:397,
2010, and Asuni et al., lmmunotherapy Targeting Pathological Tau Conformers In
a
Tangle Mouse Model Reduces Brain Pathology with Associated Functional
Improvements, The Journal of Neuroscience, 27(34):9115-9129, 2007, for review.
Therapeutic antibodies targeting disease forms of tau represent a promising
approach for treatment and/or diagnosis of AD and other tauopathies (WO
2004/007547, U52008/0050383).
[7] Despite this growing body of research on therapeutic strategies for
treating
AD and other tauopathies, there remains an unmet need for diagnostic tools
that can
accurately detect and distinguish AD and other tauopathies from other brain
pathologies in patients presenting with dementia in order to ensure
appropriately
targeted treatment decisions are made, particularly at early stages of the
disease
processes (Blennow and Hampel, CSF markers for incipient Alzheimer's disease,
Lancet NeuroL, 2:605-613, 2003; Blennow, CSF markers for incipient Alzheimer's
disease, Lancet NeuroL, 2:605-613, 2005). Significant efforts have been made
in the
last two decades to identify in vivo brain indicators and sample-based
biomarkers for
preclinical AD, all without much success. Research has focused mainly on
cerebrospinal fluid (CSF) and blood, but no definitive markers have been found
that
accurately detect and distinguish different types of dementia. Several CSF and
blood
biomarkers, for example, have been extensively studied without adequate
improvements in detection accuracy. These include biomarkers of
neurodegeneration (t-tau, NFL, NSE, VLP-1), APP metabolism (A1342, A1340,
A1338,
sAPPa, and sAPP[3), tangle pathology (p-tau), and glial activation (YKL-40,
MCP-1,
and GFAP) (Olsson et al., CSF and blood biomarkers for the diagnosis of
Alzheimer's disease: a systematic review and meta-analysis, Lancet Neurol,
7:673-
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84, 2016). Three CSF biomarkers have been reported for use in prodromal mild
cognitive impairment (MCI and AD dementia: CSF A131-42 (A131-42), also
expressed
as A[31-42 : A[31-40 ratio, t-tau, and p-tau Thr181 & Thr231 proteins (Cavedo
et al.,
The Road Ahead to Cure Alzheimer's Disease: Development of Biological Markers
and Neuroimaging Methods for Prevention Trials Across all Stages and Target
Populations, J. Prey. Alzheimer's Dis., 3:181-202, 2014). Nevertheless,
accurate
detection of early stage AD and methods to distinguish AD from other
tauopathies or
other causes of dementia in patients remains a challenge, particularly for CSF
based
assays.
[8] Research has suggested that different phosphorylated epitopes of tau,
including threonine 181, threonine 181 and 231, threonine 231, threonine 231
and
235, serine 199, serine 396 and 404, may correlate with AD, although with some
contradictory reports. (Andreasen et al., Cerebrospinal fluid levels of total-
tau,
phospho-tau and A beta 42 predicts development of Alzheimer's disease in
patients
with mild cognitive impairment, Acta Neurol. Scand. Suppl, 179:47-51, 2003;
Formichi et al., Cerebrospinal fluid tau, A beta, and phosphorylated tau
protein for
the diagnosis of Alzheimer's disease, J. Cell Physiol, 208:39-46, 2006;
Blennow and
Hampel, CSF markers for incipient Alzheimer's disease, Lancet Neurol., 2:605-
613,
2003). For instance, it has been suggested that repeated assessment of CSF p-
tau181 did not provide a useful clinical biomarker because it was insensitive
to
disease progression over a 2-year period (Bouwman et al., Longtitudinal
changes of
CSF biomarkers in memory clinic patients, Neurology, 69:1006-1011, 2007). The
predictive power of CSF tau and A13 biomarkers, and their dynamics of over
time,
therefore remains controversial. Limited ability to even detect these
biomarkers in
CSF further compounds the challenges in diagnosing AD. Several studies, for
instance, described longitudinal changes of A1342, t-tau, and p-tau levels in
CSF in
AD patients (Andersson et al., Neurobiol Aging, 29:1466-1473, 2008; Blomberg
et
al., Neurosci. Lett., 214:163-166, 1996; Arai et al., JAGS, 45:1228-31, 1997;
Kanai et
al., Ann. Neurol., 44:17-26; Kanai et al., Neurosci. Lett., 267: 65-68, 1999;
Hampel et
al., Ann Neurol., 49:545-546, 2001; Hoglund et al., Dement. Geriatr. Cogn.
Disord.,
19:256-265, 2005), whereas others reported no significant changes in these CSF
biomarkers over time (Nishimura et al., Methods Find. Exp. Clin. Pharmacol.,
20:227-235, 1998; Andreasen et al., Arch. Neurol, 56:673-680, 1999; Sunderland
et
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al., BioL Psychiatry, 46:750-755, 1999; Tapiola et al., Neurosci. Lett.,
280:119-122,
2000; deLeon et al., NeurobioL Aging, 27:394-401, 2006; Mollenhauer et al., J.
NeuraL Transm., 112:933-948, 2005; Zetterberg et al., Alzheimers Res. Ther.,
5(2):9, 2007; Mattsson et al., J. Alzheimers Dis., 30(4):767-778, 2012; Toledo
et al.,
Acta Neuropathol 125(5):659-70, 2013). The reason why CSF biomarkers do not
seem to reflect disease progression in some patients over time is not known.
One
possibility is that brain derived proteins are diluted in the CSF compartment
(deLeon
et al., Longitudinal cerebrospinal fluid tau load increases in mild cognitive
impairment, Neurosci Lett., 333:183-186, 2004). Another potential explanation
is that
older non-demented individuals (over 65) tend to have higher p-tau levels than
younger, and so elevation of the biomarker with disease progression is masked
in
the older non-demented individuals (Bouwman et al., CSF biomarker levels in
early
and late onset Alzheimer's disease, Neurobiol Aging, 30:1895-1901, 2008).
These
and other factors contribute to the lack of an effective CSF-based assay.
[9] The available assays used to detect CSF biomarkers focus almost
exclusively
on immunoassays involving a classical ELISA format, and among them are
INNOTEST hTAU, INNOTEST phospho-Tau (recognizing phospho-Threonine 181)
and INNOTEST A1342. Specificity and sensitivity of those assays, however, are
generally not sufficient to predict Alzheimer's disease, especially in its
preclinical
stages (Wennstrom et al., The Inflammatory Marker YKL-40 Is Elevated in
Cerebrospinal Fluid from Patients with Alzheimer's but Not Parkinson's Disease
or
Dementia with Lewy Bodies, 10(8): e0135458, PlosOne, 2015; Wang et al.,
Analysis
of Cerebrospinal Fluid and [11C]PlB PET Biomarkers for Alzheimer's Disease
with
Updated Protocols, 52(4):1403-13, JAD 2016).
[10] Beyond sample-based diagnostic assays, advances in molecular imaging in
recent years have led to work on potential tau-specific tracers for positron
emission
tomography (PET). Three families of radiotracers have been developed as tau
PET
tracers: the aryquinoline derivatives THK5117 and THK5351, the pyrido-indole
derivative AV-1451 (also known as T807 and Flortaucipir), and the
phenyl/pyridinyl-
butadienyl benzothiazole/ benzothiazolium derivative PBB3 (Saint-Aubert et
al., Tau
PET imaging: present and future directions, Mol. Neurodegener., 12:19, 2017).
Since
tau PET tracers displayed off target binding, mostly recognizing enzyme MAO B
or
neuromelanin (Barrio et al., The Irony of PET Tau Probe Specificity, J. NucL
Med.,
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59(1):115-116, 2018; Lemoine et al., Comparative binding properties of the tau
PET
tracers THK5117, THK5351, PBB3, and T807 in postmortem Alzheimer brains,
Alzheimers Res. Ther., 9(1):96, 2017; Ng et al., Monoamine oxidase B
inhibitor,
selegiline, reduces 18F-THK5351 uptake in the human brain, Alzheimers Res.
Ther.,
9(1):25, 2017), 2nd generation of tau PET tracers have been developed: PI-
2620,
MK-6240, GTP1 and R06958948 (www.alzforum.org). However, nonspecific binding
to multiple tissue targets is still present in some newly developed tracers,
suggesting
room for improved techniques (The 12th Human Amyloid Imaging, Miami, Florida,
USA 2018).
[11] Results from several neuropathological studies have shown that the amount
of tau pathology in the brain of patients is strongly correlated with the
progression of
AD. The pattern of anatomical localization of tau lesions may correspond to
the
domains of cognition affected over the course of Alzheimer's disease, and the
pattern and degree of brain atrophy (Braak and Braak, Neuropathological
stageing of
Alzheimer-related changes, Acta Neuropathol, 82:239-59, 1991; Murray et al.,
Neuropathologically defined subtypes of Alzheimer's disease with distinct
clinical
characteristics: a retrospective study, Lancet Neurol, (9):785-96, 2011;
Nelson et al.,
Correlation of Alzheimer disease neuropathologic changes with cognitive
status: a
review of the literature, J. Neuropathol. Exp. Neurol., 71:362-81, 2012).
Similarly,
several independent imaging studies revealed that the distribution of tau PET
signal
can correlated with cognitive decline (Ossenkoppele et al., Tau PET patterns
mirror
clinical and neuroanatomical variability in Alzheimer's disease, Brain,
139:1551-67,
2016; Bejanin et al., Tau pathology and neurodegeneration contribute to
cognitive
impairment in Alzheimer's disease, Brain, 140(12):3286-3300, 2017; Mattsson et
al.,
AV-1451 and CSF T-tau and P-tau as biomarkers in Alzheimer's disease, EMBO
Mol. Med., 9:1212-1223, 2017; Mattsson et al., Comparing 18F-AV-1451 with CSF
t-
tau and p-tau for diagnosis of Alzheimer disease, Neurology, 5:e388-e395,
2018).
Accordingly, the development of improved reagents and methods for PET imaging
of
tau in the brain could help enhance diagnostic accuracy and monitoring of AD
patients over time. While CSF tau biomarkers are primarily useful as disease
state
biomarker, tau PET imaging may also be useful in monitoring AD progression,
such
as the transition from prodromal stage to dementia (Mattsson et al., Comparing
18F-
AV-1451 with CSF t-tau and p-tau for diagnosis of Alzheimer disease,
Neurology,
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5:e388-e395, 2018). But given the invasive and radioactive nature of brain
imaging
agents, a non-invasive CSF-based assay with improved accuracy and specificity
for
AD would be beneficial.
[12] The currently available biomarkers, however, display several limitations
in
clinical use. It has been shown that CSF levels of total tau, p-tau and A1342
considerably overlap between diseased and control cases (Hulstaert et al.,
Improved
discrimination of AD patients using beta-amyloid(1-42) and tau levels in CSF,
Neurology, 52:1555-1562, 1999; Hu et al., Levels of nonphosphorylated and
phosphorylated tau in cerebrospinal fluid Alzheimer's disease patients: an
ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay,
Am.
J. Pathol., 160:1269-1278, 2002). Disease heterogeneity is considered to be
the
major cause of the overlap in CSF biomarkers (lqbal et al., Subgroups of
Alzheimer's
disease based on cerebrospinal fluid molecular markers, 58:748-757, 2005). In
tau
PET imaging, the critical challenge for development of newly tau-directed
radiotracers is to overcome nonspecific binding (Barrio et al., The Irony of
PET Tau
Probe Specificity, J. NucL Med., 59:115-116, 2018; Lemoine et al. Comparative
binding properties of the tau PET tracers THK5117, THK5351, PBB3, and T807 in
postmortem Alzheimer brains, Alzheimers Res. Ther., 9(1):96 , 2017; Ng et al.,
Monoamine oxidase B inhibitor, selegiline, reduces 18F-THK5351 uptake in the
human brain, Alzheimers Res. Ther., 9(1):25. 2017). Further, recent reports
showed
that pathological tau conformations are diverse depending on various human
tauopathies, and tau ligands have differential binding affinity to the various
tau
pathological entities (Choi et al., Development of tau PET Imaging Ligands and
their
Utility in Preclinical and Clinical Studies, 52(1):24-30, 2018). Thus, another
challenge
is to improve the binding potency to various tau pathological lesions in order
to utilize
them for diagnostics of multiple human tauopathies.
[13] The recent development of more powerful treatments targeting AD
emphasizes the need to accurately detect AD at an early stage and distinguish
it
from other forms of dementia in patients. Effective biomarkers, if available,
would
also be useful for patient stratification and longitudinal monitoring of
disease
progression, since the relative amount of plaques and tangles may show a
marked
difference between AD patients at different stages in the disease.
Stratification of
patients based on biomarker data may also be a way to identify subgroups of
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patients more prone to respond to therapy. (Hampel et al., Perspective on
future role
of biological markers in clinical therapy trials of Alzheimer's disease: a
long-range
point of view beyond 2020, Biochem. Pharmacol., 88(4):426-46, 2014).
[14] Accordingly, disclosed herein are antibodies, compositions, kits, and
methods
that provide for improved detection, monitoring, prevention, and/or treatment
of
Alzheimer's disease and other tauopathies.
[15] In various embodiments, the present disclosure provides an antibody or
antigen binding fragment thereof capable of binding tau. In some embodiments,
the
antibody or antigen binding fragment thereof can bind a phosphorylated epitope
in
tau. In some embodiments, tau species with that phosphorylated epitope is
present
in a sample (e.g., blood or CSF) from a patient with AD in a greater
concentration
than in a patient with another tauopathy or in a healthy subject.
[16] In some embodiments, the antibody or antigen binding fragment comprises a
heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region
comprises three light chain complementarity determining regions (LCDR1, LCDR2,
and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1,
HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3
comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6. In certain embodiments, the heavy chain variable region
comprises
an amino acids sequence of SEQ ID NO: 7 and the light chain variable region
comprises an amino acid sequence of SEQ ID NO 8.
[17] In various embodiments, an antibody or antigen binding fragment thereof
disclosed herein can bind to an epitope on tau protein 2N4R (SEQ ID NO: 9),
wherein the epitope is phosphorylated. The epitope may comprise one or more of
residues 188-227 of tau protein 2N4R (SEQ ID NO: 10). In some embodiments, the
epitope comprises one or more of residues 210-221 of tau protein 2N4R (SEQ ID
NO: 11). In certain embodiments, the epitope comprises at least one
phosphorylated
residue. In certain embodiments, the epitope comprises more than one
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phosphorylated residue. The phosphorylated residue(s) may comprise a phospho-
threonine at position 217 of tau protein 2N4R (SEQ ID NO: 9). In some
embodiments, the epitope also comprises a phosphorylated serine at position
210,
threonine at position 212, serine at position 214, or threonine at position
220 of tau
protein 2N4R, or any combination thereof. The antibody or antigen binding
fragment
thereof may bind to an epitope comprising or consisting of SRTPSLPpTPPTR (SEQ
ID NO: 12).
[18] In various embodiments, an antibody or antigen binding fragment thereof
disclosed herein can bind to an epitope on tau comprising one or more of
residues
188-227 of tau protein 2N4R (SEQ ID NO: 10). In some embodiments, the epitope
comprises one or more of residues 210-221 of tau protein 2N4R (SEQ ID NO: 11).
In
certain embodiments, the epitope comprises at least one phosphorylated
residue,
wherein the at least one phosphorylated residue may be a phospho-threonine at
position 217 of tau protein 2N4R (SEQ ID NO: 9). In some embodiments, the
epitope
also comprises a phosphorylated serine at position 210, threonine at position
212,
serine at position 214, or threonine at position 220 of tau protein 2N4R, or
any
combination thereof. In some embodiments, the antibody or antigen binding
fragment thereof can bind to an epitope comprising or consisting of
SRTPSLPpTPPTR (SEQ ID NO: 12).
[19] In various embodiments, an antibody or antigen binding fragment thereof
disclosed herein can bind to an epitope on tau comprising one or more of
residues
151-188 of tau protein 2N4R (SEQ ID NO: 13). In certain embodiments, the
antibody
or antigen binding fragment thereof may can bind to an epitope comprising one
or
more of residues 163-172 of tau protein 2N4R (SEQ ID NO: 14). The epitope may
comprise KGQANATRIP (sequence of SEQ ID NO: 14). In another embodiment, the
antibody is DC2E7 or an antigen binding fragment thereof, wherein DC2E7 is an
antibody produced by a hybridoma deposited under American Type Culture
Collection Patent Deposit No. PTA-124992.
[20] In another embodiment, disclosed herein is antibody DC2E2 or an antigen
binding fragment thereof, wherein DC2E2 is an antibody produced by a hybridoma
deposited under American Type Culture Collection Patent Deposit No. PTA-
124991.
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[21] In various embodiments, an antibody or antigen binding fragment disclosed
herein is conjugated to a second agent. In some embodiments, the agent is at
least
one detectable label or at least one therapeutic agent for AD or another
tauopathy. In
certain embodiments, the agent is a radiolabel.
[22] Also disclosed herein are methods of treating, delaying progression, or
preventing the progression of AD or another tauopathy in a subject. In some
embodiments, the method comprises administering to the subject an effective
amount of at least one antibody according to any one of previously disclosed
embodiments. In some embodiments, the method comprises detecting AD using
one or more of the disclosed antibodies or antigen binding fragments before
administering or recommending administration of a suitable AD treatment. In
some
embodiments, the method comprises administering an AD treatment to a patient
who
has been diagnosed as having AD using an antibody or antigen binding fragment
disclosed herein.
[23] In various embodiments, a method of detecting a tauopathy in a subject
comprises: obtaining a biological sample from the subject; contacting the
sample
from the subject with an effective amount of a molecule that is capable of
forming a
complex with tau (e.g., using at least one antibody or antigen binding
fragment
disclosed herein that is capable of binding tau to form a tau-antibody
complex);
detecting the presence and/or amount of the tau-molecule complex using an
antibody or antigen binding fragment described herein, wherein the presence
and/or
amount of tau-molecule complex indicates a tauopathy in the subject. In some
embodiments, the molecule that forms a tau-molecule complex is a first
antibody that
can bind tau to form a tau-antibody complex, and wherein the presence of the
tau-
antibody complex is detected using a second anti-tau antibody or antigen
binding
fragment, which can bind a different epitope on tau than the first antibody.
In certain
embodiments, the first or second antibody or antigen binding fragment is
linked (e.g.,
covalently or non-covalently coated on) to a solid surface or particle. In
some
embodiments, the first or second antibody is conjugated to a detectable label.
The
disclosed method may comprise a classic ELISA, digital ELISA assay, or other
ELISA assay formats, e.g., using any one or more of the antibodies or
fragments
disclosed herein that bind to phosphorylated tau, and may detect the presence
and/or amount of phosphorylated tau in the sample, wherein an increased level
of
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phosphorylated tau in the sample indicates the subject has Alzheimer's disease
rather than another tauopathy. In certain embodiments, the biological sample
is
cerebrospinal fluid. In certain embodiments, the biological sample is serum
and/or
plasma.
[24] In various embodiments, a method of distinguishing Alzheimer's disease
from
another tauopathy or another cause of dementia in a subject comprises:
obtaining a
cerebrospinal fluid or blood sample from a subject, contacting the sample with
an
anti-tau antibody or antigen binding fragment thereof disclosed herein, and
detecting
the presence and/or amount of phosphorylated tau complexed with the antibody
or
antigen binding fragment in the same sample, wherein the presence and/or an
elevated level of phosphorylated tau in the sample relative to the level in a
sample
from a healthy control subject or wherein an elevated level of phosphorylated
tau
above a threshold indicates the subject has Alzheimer's disease rather than
another
tauopathy or an alternative cause (i.e., another form of) dementia or another
neurodegenerative disorder. In some embodiments, the anti-tau antibody or
antigen
binding fragment comprises a heavy chain variable region and a light chain
variable
region, wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6 that is capable of binding to
phosphorylated
tau to form a phosphorylated tau-antibody complex. In some embodiments, the
antibody or antigen binding fragment comprises a heavy chain variable region
and a
light chain variable region, wherein the heavy chain variable region comprises
amino
acid sequence of SEQ ID NO: 7 and the light chain variable region comprises an
amino acid sequence of SEQ ID NO:8.
[25] In various embodiments, a method of detecting Alzheimer's disease (AD) or
mild cognitive impairment (MCI) in a subject comprises: contacting a
biological
sample from the subject with an effective amount of a molecule that is capable
of
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forming a complex with tau (e.g., using at least one antibody or antigen
binding
fragment disclosed herein that is capable of binding tau to form a tau-
antibody
complex); detecting the presence and/or amount of the tau-antibody complex;
and
comparing the presence/amount of tau bound to the antibody in the sample to
the
amount in a control sample or a threshold, wherein the presence and/or an
increased amount of tau complexed with the antibody relative to the control
sample
or threshold indicates AD or MCI in the subject. In some embodiments, MCI is a
precursor of AD in a patient. In some embodiments, the method distinguishes
MCI
and/or AD from other neurological diseases. In some embodiments the other
neurological diseases are selected from Parkinson's disease, Multiple
sclerosis,
amyotrophic lateral sclerosis, and/or frontotemporal dementia. In some
embodiments, the biological sample comprises cerebrospinal fluid (CSF). In
some
embodiments, the biological sample comprises blood. In some embodiments, the
biological sample comprises plasma and/or serum fractions. In some
embodiments,
the threshold is about 9.3 pg/ml of tau or about 5.3 pg/ml of tau. In some
embodiments, the threshold is between about 100-600 pg/ml. In some
embodiments,
the threshold is about 300 pg/ml.
[26] In various embodiments, a method of detecting Alzheimer's disease (AD) or
mild cognitive impairment (MCI) in a subject comprises: obtaining a biological
sample; detecting the presence and/or amount of tau protein 2N4R
phosphorylated
at least at position threonine 217 in the biological sample; and comparing the
presence/amount of tau protein 2N4R phosphorylated at threonine 217 to the
amount in a control sample or a threshold, wherein the presence and/or an
increased amount of tau protein 2N4R phosphorylated at threonine 217 relative
to
the control sample or threshold indicates AD or MCI in the subject. In various
embodiments, the MCI is a precursor of AD in a patient. In some embodiments,
the
method distinguishes MCI and/or AD from other neurological diseases. In some
embodiments, the other neurological disease is selected from Parkinson's
disease,
Multiple sclerosis, amyotrophic lateral sclerosis, and/or frontotemporal
dementia. In
some embodiments, the biological sample comprises cerebrospinal fluid (CSF).
In
some embodiments, the biological sample comprises blood. In some embodiments,
the biological sample comprises plasma and/or serum fractions. In some
embodiments, the threshold is about 9.3 pg/ml of tau. In some embodiments, the
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threshold is about 9.3 pg/ml of tau or about 5.3 pg/ml of tau. In some
embodiments,
the threshold is between about 100-600 pg/ml. In some embodiments, the
threshold
is about 300 pg/ml.
[27] In various embodiments, a method of distinguishing Alzheimer's disease
and/or mild cognitive impairment from Parkinson's disease, multiple sclerosis,
amyotrophic lateral sclerosis, and/or frontotemporal dementia in a subject
comprises:
contacting a biological sample from the subject with an effective amount of a
molecule that is capable of forming a complex with tau (e.g., using at least
one
antibody or antigen binding fragment disclosed herein that is capable of
binding tau
to form a tau-antibody complex); and detecting the presence and/or amount of
tau
complexed with the antibody or antigen binding fragment in the same sample;
and
comparing the presence/amount of tau bound to the antibody in the sample to
the
amount in a control sample or a threshold, wherein the presense and/or an
increased amount of tau complexed with the antibody relative to the control
sample
or threshold indicates Alzheimer's disease and/or mild cognitive impairment
(MCI) in
the subject. In some embodiments, the biological sample comprises
cerebrospinal
fluid (CSF). In some embodiments, the biological sample comprises blood. In
some
embodiments, the biological sample comprises plasma and/or serum fractions. In
some embodiments, the threshold is about 9.3 pg/ml of tau. In some
embodiments,
the threshold is between about 100-600 pg/ml. In some embodiments, the
threshold
is about 300 pg/ml.
[28] In various embodiments, a method of distinguishing Alzheimer's disease
and/or mild cognitive impairment from Parkinson's disease, multiple sclerosis,
amyotrophic lateral sclerosis, and/or frontotemporal dementia in a subject
comprises:
obtaining a biological sample from the subject; detecting the presence and/or
amount of tau protein 2N4R phosphorylated at least at position threonine 217
in the
biological sample; comparing the presence/amount of tau protein 2N4R
phosphorylated at threonine 217 to the amount in a control sample or a
threshold,
wherein the presence and/or an increased amount of tau protein 2N4R
phoshphorylated at threonine 217 relative to the control sample or threshold
indicates Alzheimer's disease or mild cognitive impairment in the subject. In
some
embodiments, the biological sample comprises cerebrospinal fluid (CSF). In
some
embodiments, the biological sample comprises blood. In some embodiments, the
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biological sample comprises plasma and/or serum fractions. In some
embodiments,
the threshold is about 9.3 pg/ml of tau. In some embodiments, the threshold is
about
9.3 pg/ml of tau or about 5.3 pg/ml. In some embodiments, the threshold is
between
about 100-600 pg/ml. In some embodiments, the threshold is about 300 pg/ml.
[29] In various embodiments, a method of predicting the likelihood that a
patient
with mild cognitive impairment will develop Alzheimer's disease comprises:
contacting a biological sample from the subject with an effective amount of a
molecule that is capable of forming a complex with tau (e.g., using at least
one
antibody or antigen binding fragment disclosed herein that is capable of
binding tau
to form a tau-antibody complex); and detecting the presence and/or amount of
tau
complexed with the antibody or antigen binding fragment in the same sample;
and
comparing the presence/amount of tau bound to the antibody in the sample to
the
amount in a control sample or threshold, wherein the presense and/or an
increased
amount of tau complexed with the antibody relative to the control sample or
threshold indicates an increased likelihood that the patient will develop
Alzheimer's
disease. In some embodiments, the biological sample comprises cerebrospinal
fluid
(CSF). In some embodiments, the biological sample comprises blood. In some
embodiments, the biological sample comprises plasma and/or serum fractions. In
some embodiments, the threshold is about 9.3 pg/ml of tau. In some
embodiments,
the threshold is between about 100-600 pg/ml. In some embodiments, the
threshold
is about 300 pg/ml.
[30] In various embodiments, a method of predicting the likelihood that a
patient
with mild cognitive impairment will develop Alzheimer's disease comprises:
obtaining
a biological sample from the subject; detecting the presence and/or amount of
tau
protein 2N4R phosphorylated at least at position threonine 217 in the
biological
sample; comparing the presence/amount of tau protein 2N4R phosphorylated at
threonine 217 to the amount in a control sample or a threshold, wherein the
presence and/or an increased amount of tau protein 2N4R phoshphorylated at
threonine 217 relative to the control sample or threshold indicates an
increased
likelihood that the patient will develop Alzheimer's disease. In some
embodiments,
the biological sample comprises cerebrospinal fluid (CSF). In some
embodiments,
the biological sample comprises blood. In some embodiments, the biological
sample
comprises plasma and/or serum fractions. In some embodiments, the threshold is
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about 9.3 pg/ml of tau. In some embodiments, the threshold is between about
100-
600 pg/ml. In some embodiments, the threshold is about 300 pg/ml.
BRIEF DESCRIPTION OF THE DRAWINGS
[31] The accompanying drawings, which are incorporated in and constitute a
part
of this specification, illustrate several non-limiting embodiments of the
invention and
together with the description, serve to explain the principles of the
disclosure.
[32] Fig. 1. lsotype determination for monoclonal antibody DC2E2 using ELISA.
[33] Figs. 2A-2D. Epitope mapping of antibody DC2E2 using tau deletion mutants
and tau peptides in ELISA. (Fig. 2A) Schematic illustration of tau deletion
mutants
and peptides used to evaluate the DC2E2 binding site on tau protein. (Fig. 2B)
lmmunoreactivity of DC2E2 to human six isoforms by ELISA. (Fig. 20)
Determination
of DC2E2 binding site using tau deletion mutants by ELISA. (Fig. 2D)
Determination
of DC2E2 binding site using tau derived peptides by competitive ELISA.
[34] Fig. 3. lmmunoreactivity of DC2E2 to different tau proteins. Lane 1:
sarcosyl-
insoluble tau isolated from human Alzheimer's disease brain tissue; lane 2:
tau151-
391; lane 3: phosphorylated tau151-391; lane 4: tau 2N4R; lane 5:
phosphorylated
tau 2N4R.
[35] Fig. 4. lsotype determination of monoclonal antibody D02E7 using ELISA.
[36] Fig. 5A-D. The nucleotide and amino-acid sequences of the variable
regions
in antibody D02E2. Fig. 5A shows the nucleotide sequence encoding the light
chain
variable region. Fig. 5B shows the amino acid sequence of the light chain
variable
region, with the CDR sequences shown in bold and underlined. Fig. 50 shows the
nucleotide sequence encoding the heavy chain variable region. Fig. 5D shows
the
amino acid sequence of the heavy chain variable region, with CDR sequences in
bold and underlined. Complementarity determining regions (CDRs) were
identified
according to the IMGT numbering system.
[37] Fig. 6A-D: The nucleotide and amino-acid sequences of the variable
regions
in antibody DC2E7. Fig. 6A shows the nucleotide sequence encoding the light
chain
variable region. Fig. 6B shows the amino acid sequence of the light chain
variable
region, with CDR sequences in bold and underlined. Fig. 60 shows the
nucleotide
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sequence encoding the heavy chain variable region. Fig. 6D shows the amino
acid
sequence of the heavy chain variable region, with CDR sequences in bold and
underlined. Complementarity determining regions (CDRs) were identified
according
to the IMGT numbering system.
[38] Fig. 7. lmmunoreactivity of DC2E7 to different tau proteins. Lane 1:
tau
2N4R; lane 2: 2N4R in vitro phosphorylated; lane 3: fetal tau; lane 4:
sarcosyl-
insoluble tau isolated from human Alzheimer's disease brain tissue. D025, a
pan tau
monoclonal antibody recognizing all forms of tau proteins, was used as
control.
[39] Fig. 8. Schematic illustration of tau deletion mutants used for
evaluating the
DC2E7 binding site on tau protein.
[40] Fig. 9. Determination of DC2E7 binding site on tau protein by
immunoblotting
using tau deletion mutants.
[41] Fig. 10. Schematic illustration of potential phosphorylation sites on
tau in
region 188-227.
[42] Fig. 11. Determination of DC2E7 binding site on tau protein by
immunobloting
using tau point mutants. Mab D025 was used as a control to measure the extent
of
phosphorylation of point mutations.
[43] Fig. 12. Determination of DC2E7 epitope on tau protein by competitive
ELISA
using tau-derived peptides.
[44] Fig. 13. Binding of antibodies DC2E7 and DC2E2 in Alzheimer's disease
(hippocampus CA1), FTD ¨ Pick's disease (dentate gyrus, hippocampus), CBD
(nucleus caudatus) and PSP (putamen/nucleus caudatus) brain sections.
Monoclonal antibody AT8 was used as a control. Tool bar 100 m.
[45] Fig. 14. Binding of antibodies DC2E7 and DC2E2 in Braak stage 1, Braak
stage 3, and Braak stage 6 brain sections. Tool bar 100 m.
[46] Fig. 15. Binding of antibodies DC2E7 and DC2E2 in brain sections from
Alzheimer's disease (hippocampus CA1), Pick's bodies in FTD ¨ Pick's disease
(dentate gyrus, hippocampus), and coiled bodies and astrocytic pathology in
CBD
(nucleus caudatus). Tool bar 20 m.
[47] Fig. 16. Exemplary calibration curve for DC2E7 digital ELISA assay.
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[48] Fig. 17A-B. Spike recovery experiment using three human CSF from healthy
individuals. (Fig. 17A) Estimated concentrations of spiked DC2E7 calibrator in
human CSF. (Fig. 17B) Recovery in % of spiked DC2E7 calibrator in human CSF.
[49] Fig. 18. DC2E7 digital ELISA results from control and AD samples.
[50] Fig. 19. DC2E7 digital ELISA results from AD and other tauopathy samples.
[51] Fig. 20: DC2E7 binding to insoluble tau species in AD and other human
tauopathies.
[52] Fig. 21: A comparison of CSF samples from AD and FTD subjects using
pT217 tau and pT181 tau assays.
[53] Fig. 22: A comparison of CSF samples from AD and control subjects using
pT217 tau and pT181 tau assays.
[54] Fig. 23A-B: A comparison of CSF samples from control, MCI, and AD
subjects using pT217 tau assays (Fig. 23A). A comparison of CSF samples from
control, AD, PD, MS, ALS, and FTD subjects using pT217 tau assays (Fig. 23B).
[55] Fig. 24: Absorbance of isolated scFV antibody fragments derived from
DC2E7.
[56] Fig. 25A-D: Alignment of amino acid sequences of scFV antibody fragments
derived from DC2E7. Fig. 25A shows scFV antibody light chain variable domains
compared to the DC2E7 sequence. Fig. 25B shows scFV antibody heavy chain
variable domains compared to the DC2E7 sequence. Fig. 25C shows the variable
light chain domains of the scFV antibody fragments which exhibited higher
affinity as
compared to DC2E7. Fig. 25D shows the variable heavy chain domains of the scFV
antibody fragments which exhibited higher affinity as compared to DC2E7.
Residues
identical to the sequence of DC2E7 are represented by dots. CDRs were
identified
according to the IMGT numbering system.
[57] Fig. 26: Comparison of the affinity of the DC2E7 and DC149 antibodies for
tau
derived peptide 2E7pep.
[58] Fig. 27A-B: Alignment of amino acid sequences of DC2E7 and DC149. Fig.
27A shows the DC149 variable light chain domain in comparison to the DC2E7
light
chian sequence. Fig. 27B shows the DC149 variable heavy chain domain in
comparison to the DC2E7 heavy chain sequence. Residues identical to the
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sequence of DC2E7 are represented by dots. CDRs were identified according to
the
IMGT numbering system.
[59] Fig. 28A-B: Alignment of amino acid sequences of DC2E7 and D0807. Fig.
28A shows the D0807 variable light chain domain in comparison to the DC2E7
light
chain sequence. Fig. 28B shows the D0807 variable heavy chain domain in
comparison to the DC2E7 heavy chain sequence. Residues identical to the
sequence of DC2E7 are represented by dots. CDRs were identified according to
the
IMGT numbering system.
[60] Fig. 29: The distribution of pT217 tau as measured by a pT217 tau digital
ELISA assay in samples from Alzheimer's disease, other tauopathies, and
control
individuals using a phosphorylated tau calibrator (left panel) and 2E7 peptide
calibrator (2E7pep) (right panel).
DETAILED DESCRIPTION
Definitions
In order to better understand the disclosure, certain definitions are provided
first.
[61] The term "affinity" refers to the strength of the sum total of
noncovalent
interactions between a single binding site of a molecule (e.g., an antibody)
and its
binding partner (e.g., an antigen). The affinity of a molecule X for its
partner Y can
generally be represented by the equilibrium dissociation constant (KD) (or its
inverse
equilibrium association constant, KA). Affinity can be measured by common
methods
known in the art, including those described herein. See, for example, Pope
M.E.,
Soste M.V., Eyford B.A., Anderson N.L., Pearson T.W., (2009) J. lmmunol.
Methods.
341(1-2):86-96 and methods described herein.
[62] The term "amino acid" refers to naturally occurring, modified, and
synthetic
amino acids, as well as amino acid analogs and amino acid mimetics that
function in
a manner similar to the naturally occurring amino acids. Naturally occurring
amino
acids are those encoded by the genetic code, as well as those amino acids that
are
later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and 0-
phosphoserine. Amino acid analogs refer to compounds that have the same basic
chemical structure as a naturally occurring amino acid, i.e., an alpha carbon
that is
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bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,
homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
Such
analogs have modified R groups (e.g., norleucine) or modified peptide
backbones,
but retain the same basic chemical structure as a naturally occurring amino
acid.
Amino acid mimetics refers to chemical compounds that have a structure that is
different from the general chemical structure of an amino acid, but that
function in a
manner similar to a naturally occurring amino acid. Suitable amino acids
include,
without limitation, both D- and L-isomers of the 20 common naturally occurring
amino
acids found in peptides as well as the naturally occurring and unnaturally
occurring
amino acids prepared by organic synthesis or other metabolic routes. Examples
of
such unnaturally occurring amino acids include, but are not limited to, N-
acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and 0-
phosphotyrosine. Modified amino acids include, but are not limited to,
hydroxyproline, pyroglutamate, gamma-carboxyglutamate, 0-phosphoserine,
azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-
alanine,
aminoproprionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic
acid,
2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-
aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid,
desmosine, 2,2'-
diaminopimelic acid, 2,3-diaminoproprionic acid, N-ethylglycine, N-
methylglycine, N-
ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-
hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-
methylglycine,
N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine,
norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and
thioproline. The
term amino acid also includes naturally occurring amino acids that are
metabolites in
certain organisms but are not encoded by the genetic code for incorporation
into
proteins. Such amino acids include, but are not limited to, ornithine, D-
ornithine, and
D-arginine.
[63] The term "antibody" refers to an immunoglobulin, whether genetically
engineered, natural, or wholly or partially synthetically or recombinantly
produced.
Intact antibodies typically comprise a heavy chain and a light chain, each
comprised
of a variable domain forming the binding pocket for an antigen and a constant
domain that contributes to effector function. The antibody, by virtue of its
chosen
heavy chain, can be a member of any immunoglobulin class and subclass,
including
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any of the human classes: IgG, IgM, IgA, IgD, and IgE, or a derivative or
fragment
thereof. Likewise, the light chain of the antibody may derive from any
species, such
as a human kappa (K) or lambda (A) light chain, determined based on the amino
acid
sequences of the constant domain.
The basic antibody structural unit typically comprises a tetramer. In various
embodiments, the tetramer comprises two identical pairs of polypeptide chains,
each
pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70
kDa). The
amino-terminal portion of each chain includes a variable region of about 100
to 110
or more amino acids primarily responsible for antigen recognition. The carboxy-
terminal portion of each chain defines a constant region primarily responsible
for
effector function. Human light chains are classified as kappa and lambda light
chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon,
and
define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within
light and
heavy chains, the variable and constant regions are joined by a "J" region of
about
12 or more amino acids, with the heavy chain also including a "D" region of
about 10
or more amino acids. See generally, Fundamental Immunology Ch 7. (Paul, W.,
2nd
ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for
all
purposes). The variable regions of each light/heavy chain pair form the
antibody
binding site. Thus, an intact antibody typically has two binding sites. Except
in
bifunctional or bispecific antibodies, the two binding sites are the same. The
chains
all exhibit the same general structure of relatively conserved framework
regions (FR)
joined by three hypervariable regions, also called complementarity determining
regions or CDRs. The CDRs from the two chains of each pair are aligned by the
framework regions, enabling binding to a specific epitope. From N-terminal to
C-
terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2,
CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain may
be done in accordance with the IMGT numbering system. Alternative definitions
are
also known to know of ordinary skill in the art. See, e.g., Kabat Sequences of
Proteins of Immunological Interest (National Institutes of Health, Bethesda,
Md.
(1987 and 1991)), or Clothia & Lesk J. MoL BioL 196:901-917 (1987); Clothia et
al.
Nature 342:878-883 (1989).
[64] "Antibody fragment" or "antigen binding fragment" comprise a portion of a
full
length antibody, generally at least the antigen binding portion/domain or the
variable
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region thereof. The term antibody fragment is a subset of the term antibody
discussed above. Examples of antibody fragments or antigen binding fragments
include: Fab, Fab', F(ab')2, Fd, scFv, (scFv)2, scFv-Fc, Fv fragment,
diabodies,
single-chain antibody molecules, immunotoxins, and multi-specific antibodies
formed
from antibody fragments. In addition, antibody fragments comprise single chain
polypeptides having the characteristics of a VH chain binding pathological
tau,
namely being able to assemble together with a VL chain or of a VL chain
binding to
pathological tau, namely being able to assemble together with a VH chain to
form a
functional antigen binding pocket and thereby providing the property of
binding to
tau. The terms also comprise fragments that per se are not able to provide
effector
functions (e.g., Antibody-dependent cell-mediate cytotoxicity ("ADCC") or
complement dependent cytotoxicity ("CDC") but provide this function after
being
combined with the appropriate antibody constant domain(s).
[65] An antibody or binding fragment thereof "capable of binding tau," as used
herein, refers to an antibody or binding fragment that preferentially binds to
tau over
other antigen targets. The term is interchangable with an "anti-tau" antibody
or an
"antibody that binds tau." In some embodiments, the antibody or binding
fragment
capable of binding to tau can do so with higher affinity for that antigen than
others.
In some embodiments, the antibody or binding fragment capable of binding tau
can
bind to that antigen with a KD of at least about 10-1, 10', 10-3, 10-4, 10-5,
10-6, 10-7,
10-8, 10-3, 10-11, 10-12 or greater (or any value in between), e.g., as
measured by
surface plasmon resonance or other methods known to the skilled artisan.
[66] The term "chimeric" antibodies refers to antibodies in which a portion of
the
heavy and/or light chain is identical with or homologous to corresponding
sequences
in antibodies derived from a particular species or belonging to a particular
antibody
class or subclass (e.g., chimeric humanized, class-switched antibodies), while
the
remainder of the chain(s) is identical with or homologous to corresponding
sequences in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such antibodies, so long
as they
exhibit desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et
al., Proc.
Natl. Acad. Sci. USA, 81:6851-6855(1984)).
[67] In one embodiment, the term "chimeric antibody" refers to a monoclonal
antibody comprising a variable region from one source or species and at least
a
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portion of a constant region derived from a different source or species,
usually
prepared by recombinant DNA techniques. In some embodiments, chimeric
antibodies comprise a murine variable region and a human constant region. Such
murine/human chimeric antibodies may be produced by expressing immunoglobulin
genes comprising DNA segments encoding murine immunoglobulin variable regions
and DNA segments encoding human immunoglobulin constant regions. Other forms
of "chimeric antibodies" may be those in which the class or subclass has been
modified or changed from that of the original antibody. Such "chimeric"
antibodies
are also referred to as "class-switched antibodies." Methods for producing
chimeric
antibodies involve conventional recombinant DNA and gene transfection
techniques
now known in the art. See, e.g., Morrison, S. L., et al., Proc. Natl. Acad
Sci. USA 81
(1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[68] "Competitive binding" may be determined in an assay in which the
immunoglobulin/antibody/binding fragment under testing inhibits specific
binding of a
reference antibody to a common antigen, such as tau (e.g., tau SEQ ID No 12).
Numerous types of competitive binding assays are known, for example: solid
phase
direct or indirect radioimmunoassay (RIA), solid phase direct or indirect
enzyme
immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in
Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland
et al., J.
lmmunol 137:3614 (1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1125
label (see
Morel et al., Mol. Immunol. 25(1):7 (1988): solid phase biotin-avidin EIA
(Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al.,
Scand. J.
Immunol. 32:77 (1990)). In some embodiments, such an assay involves the use of
purified antigen bound to a solid surface or cells bearing either of these, an
unlabeled test immunoglobulin and a labeled reference immunoglobulin.
Competitive
inhibition may be measured by determining the amount of label bound to the
solid
surface or cells in the presence of the test immunoglobulin. In some
embodiments,
the test immunoglobulin is present in excess. Usually, when a competing
antibody is
present in excess, it will inhibit specific binding of a reference antibody to
a common
antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.
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[69] The term "conjugated," as used herein, refers to a bond or chemical
moiety
formed from a chemical reaction between a functional group of a first molecule
(e.g.,
an antibody) with a functional group of a second molecule (e.g., a detectable
signor
or therapeutic agent or drug). Such bonds include, but are not limited to,
covalent
linkages and non-covalent linkages, while such chemical moieties include, but
are
not limited to, esters, carbonates, imines phosphate esters, hydrazones,
acetals,
orthoesters, peptide linkages, and oligonucleotide linkages.
[70] "Delaying progression" and "preventing the progression" refers to
administration of a therapeutic agent to a patient susceptible to, or
otherwise at risk
of, a particular disease, such as AD. Prevention encompasses prophylactic
administration to a subject at risk for AD. Anyone in the general population
is at risk
for AD. Some individuals have an increased risk for AD. Some individuals have
an
increased, genetic risk for AD. Delaying progression and preventing the
progression
can eliminate or reduce the risk or delay the onset of disease. Delay of AD
onset or
progression can be measured by comparing to standard disease progression
timelines in similar populations or individuals. See Ostrowitzki et al.,
Alzherimers Res
Ther., 9(1):95, 2017. Specific illustrative and exemplary embodiments for
delaying
progression are described below.
[71] The term "epitope" refers to a site on an antigen to which an
immunoglobulin
or antibody (or antigen binding fragment thereof) can specifically bind. Thus,
an
epitope on tau is the site on tau where an immunoglobulin or antibody (or
antigen
binding fragment thereof) can specifically bind. Specific binding refers, in
some
embodiments, to binding that is measurably different from a non-specific
interaction.
Specific binding can be measured, for example, by determining binding of a
molecule compared to binding of a control molecule, which generally is a
molecule of
similar structure that does not have binding activity, or by competition assay
with a
control molecule that shares similar binding affinity but is unlabeled. In
this case,
specific binding is indicated if the binding of the labeled target to a probe
is
competitively inhibited by excess unlabeled target. The term can be exhibited,
for
example, by a molecule having a Kd for the target of at least about 10' M,
alternatively at least about 10' M, alternatively at least about 10-6M,
alternatively at
least about 10' M, alternatively at least about 10-8M, alternatively at least
about
10' M, alternatively at least about 10-10M, alternatively at least about 10-
11M,
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alternatively at least about 10-12M, or greater. In one embodiment, the term
"specific
binding" refers to binding where a molecule binds to a particular polypeptide
or
epitope on a particular polypeptide without substantially binding to any other
polypeptide or epitope.
[72] An epitope, as the term is used herein, can be formed from either
contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a
protein.
An epitope may include additional stretches of amino acids around a core
region
bound by an antibody, e.g., 1, 2, 3, 4, 5, 10, 15, 20, or more amino acids on
the N
and/or C terminus of a core epitope peptide. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing solvents whereas
epitopes formed by tertiary folding are typically lost on 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, often in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, alanine scanning, x-ray
crystallography, and 2-dimensional nuclear magnetic resonance. See, e.g.,
Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
(1996). Exemplary methods are discussed and used herein for the disclosed
antibodies. A "conformational epitope" is an epitope to which the antibody or
tau-
binding fragment thereof binds in a conformational-specific manner. In the
case of
protein-based epitopes, the binding can depend on the epitope-carrying-
protein's
secondary, tertiary, or quaternary structure. In other words, the antibody
binds in a
structure specific manner, or a quaternary-structure-specific manner.
[73] An epitope on tau, as discussed herein, is identified with reference to
amino
acid residues on tau protein 2N4R. One of skill in the art could readily
identify
corresponding amino acid residues on other tau isoforms and fragments, e.g,
via
alignment programs such as BLAST . Unless otherwise indicated, a reference to
a
particular amino acid residue on tau refers to tau protein 2N4R and should be
understood to encompass the corresponding positions on other tau isoforms and
fragments.
[74] "Fab" fragments can be produced by papain digestion of antibodies,
each
with a single antigen-binding site, and a residual "Fc" fragment, whose name
reflects
its ability to crystallize readily (fragment crystalizable). Pepsin treatment
yields an
F(ab')2 fragment that has two antigen-binding sites and is still capable of
cross-
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linking antigen. "Fv" is the variable region portion of the heavy chain that
is included
in the Fab fragment. Any of these fragments can also be produced
recombinantly.
The Fc portion of an antibody is associated with the antibody's effector
functions,
including antibody-dependent cellular cytotoxicity (ADCC) and complement-
dependent cytotoxicity or phagocytosis. Alterations (e.g., mutations or
glycosylation
changes) in the Fc region of an antibody can be used to modulate any of its
effector
functions as well as increase its serum half-life and other pharmacokinetic
properties.
[75] The Fab fragment also contains the constant domain of the light chain and
the
first constant domain (CH1) of the heavy chain. Fab' fragments typically
differ from
Fab fragments by the addition of a few residues at the carboxy terminus of the
heavy
chain CH1 domain, including one or more cysteines from the antibody hinge
region.
Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of
the
constant domains bear at least on free thiol group. F(ab')2 antibody fragments
originally were produced as pairs of Fab' fragments which have hinge cysteines
between them. Other chemical couplings of antibody fragments are also known.
[76] The term "Fc", as used herein, includes the polypeptide comprising the
constant region of an antibody excluding the first constant region
immunoglobulin
domain. Thus Fc refers to the last two constant region immunoglobulin domains
of
IgA, IgD, and IgG, and the last three constant region immunoglobulin domains
of IgE
and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM,
Fc
may include the J chain. For IgG, Fc comprises immunoglobulin domains C gamma
2 and C gamma 3 (Cgamma2 and Cgamma3) and the hinge between C gamma
1(Cgamma1) and C gamma 2 (Cgamma2). Although the boundaries of the Fc region
may vary, the human IgG heavy chain Fc region is usually defined to comprise
residues 0226 or P230 to its carboxyl-terminus, wherein the numbering is
according
to the EU numbering system (Edelman G. M. et al., (1969) Proc. Natl. Acad.
Sci.
USA, 63(1); 78-85). The C-terminal lysine (residue 447 according to the EU
numbering system) of the Fc region may be removed, for example, during
production
or purification of the antibody, or by recombinantly engineering the nucleic
acid
encoding a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues removed,
antibody populations with no K447 residues removed, and antibody populations
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having a mixture of antibodies with and without the K447 residue. Fc may refer
to
this region in isolation or this region in the context of an Fc polypeptide,
for example
an antibody. The Fc may be a native sequence Fc or a variant Fc. Replacements
of
amino acid residues in the Fc portion to alter antibody effector function are
known in
the art (see, e.g., Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821).
One
suitable Fc, described in PCT application WO 93/10151 (hereby incorporated by
reference), is a single chain polypeptide extending from the N-terminal hinge
region
to the native C-terminus of the Fc region of a human IgG1 antibody. Another
useful
Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and in
Baum et
al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is
identical to that of the native Fc sequence presented in WO 93/10151, except
that
amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed
from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The
mutein
may exhibit reduced affinity for Fc receptors.
[77] "Fv" refers to the minimum antibody fragment which contains a complete
antigen-recognition and antigen-binding site. This region consists of a dimer
of one
heavy chain and one light chain variable domain in tight, non-covalent
association. It
is in this configuration that the three hypervariable regions of each variable
domain
interact to define an antigen-binding site on the surface of the VH-VL dimer.
Collectively, the six hypervariable regions confer antigen-binding specificity
to the
antibody. However, even a single variable domain (or half of an Fv comprising
only
three hypervariable regions specific for an antigen) may have the ability to
recognize
and bind an antigen, although at a lower affinity than the entire binding
site.
[78] As used herein, a humanized antibody that comprises a heavy or light
chain
variable "framework region" from a particular human germline immunoglobulin
sequence may contain amino acid differences as compared to the heavy or light
chain variable framework region of the particular germline sequence, due to,
for
example, naturally-occurring somatic mutations or intentional introduction of
site-
directed mutation. In various embodiments, a selected humanized antibody can
be at
least 90% identical in amino acid sequence of the heavy or light chain
variable
framework region to an amino acid sequence encoded by the heavy or light chain
variable framework region of a human germline immunoglobulin gene and contains
amino acid residues that identify the humanized antibody as being derived from
the
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human when compared to the germline immunoglobulin amino acid sequences of
other species (e.g., murine germline sequences). In certain cases, a humanized
antibody may share at least 90%, 95%, 96%3 97%3 98%3 99% or greater amino acid
sequence identity with the heavy or light chain variable framework region
encoded
by the germline immunoglobulin gene. In some embodiments, the heavy or light
chain variable framework region of a humanized antibody derived from a
particular
human germline sequence will display no more than 11 amino acid, preferably no
more than 5, or even more preferably no more than 4, 3, 2, or 1 differences
from the
amino acid sequence of the heavy or light chain variable framework region
encoded
by the human germline immunoglobulin gene.
[79] "Framework Region" or "FR" residues are those variable domain residues
other than the hypervariable region residues as herein defined. They bracket
the
hypervariable regions in the varialble domain. The FR residues can be
identified
according to a standard numbering system, e.g., the Kabat, Chothia, or
modified
Chotia numbering schemes. In the IMGT unique numbering system, the conserved
amino acids always have the same position, for instance cysteine 23 (1st-CYS),
tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-
CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). See, e.g., Lefranc M.
P.,
Immunology Today 18, 509 (1997); Lefranc M. P., The Immunologist, 7, 132-136
(1999); Lefranc, M. P., Pommie, C., Ruiz, M., Guidicelli, V., Foulquier, E.,
Truong, L.,
Thouvenin-Contet, V. and Lefranc, Dev. Comp. lmmunol., 27, 55-77 (2003). In
another embodiment, the IMGT unique numbering provides a standardized
delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT:
39-
55, FR3-IMGT: 66-104 and FR4-IMGT: 118 to 128) and of the complementarity
determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT:
105 to 117. The IMGT unique numbering can be used in 2D graphical
representations, designated as IMGT Colliers de Peries. See, e.g., Ruiz, M.
and
Lefranc, M. P., lmmunogenetics, 53, 857-883 (2002); Kaas, Q. and Lefranc, M.
P.,
Current Bioinformatics, 2, 21-30 (2007). It may also used for representing 3D
structures. See, e.g., IMGT/3Dstructure-DB Kaas, Q., Ruiz, M. and Lefranc, M.
P., T
cell receptor and MHC structural data. NucL Acids. Res., 32, D208-D210 (2004).
[80] The term "hinge" or "hinge region" or "antibody hinge region" herein
includes
the flexible polypeptide comprising the amino acids between the first and
second
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constant domains of an antibody or tau-binding fragment thereof. The "hinge
region"
as referred to herein may be a sequence of 6-62 amino acids in length, only
present
in IgA, IgD, and IgG, which encompasses the cysteine residues that bridge the
two
heavy chains.
[81] The term "human antibody," as used herein, is intended to include
antibodies
having variable and constant regions derived from human germline
immunoglobulin
sequences and may be, e.g., isolated from a human or produced recombinantly.
The
constant regions of the antibody can be, for example, the constant regions of
a
human IgG1 type antibody. Such regions can be allotypic and are described by,
e.g.,
Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218 and the
databases referenced therein.
[82] The term "humanized antibody" refers to antibodies in which the framework
regions (FR) and/or the complementarity determining regions (CDR) have been
modified to comprise amino acid residues of an immunoglobulin from a human as
compared to that of the parent immunoglobulin (e.g., parent mouse
immunoglobulin
residues). In one embodiment, a murine CDR is grafted into the framework
region of
a human antibody to prepare the "humanized antibody." In another embodiment,
human frameworks are "grafted" or spliced into mouse antibodies, preserving
the
CDRs of the mouse antibody and replacing its frameworks with frameworks of
human origin. Grafting and splicing can be done by various recombinant DNA
technologies, including PCR and mutagenesis. Various humanization methods
exist
in the art (e.g., CDR grafting, reshaping, transgenic animals, combinatorial
libraries).
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; Neuberger, M.S.,
et al.,
Nature 314 (1985) 268-270; Sastry L, Alting-Mess M, Huse WD, Short JM, Sorge
JA,
Hay BN, Janda KD, Benkoviv SJ, Lerner RA (1989) Cloning of the immunological
repertoire for generation of monoclonal catalytic antibodies: construction of
a heavy
chain variable region-specific cDNA library. Proc Natl Acad Sci USA 86, 5728-
5732;
and Huse WD, Sastry S, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkoviv
SJ, Lerner RA (1989) Generation of a large combinatorial library of the
immunoglobulin repertoire in phage lambda. Science 246, 1275-1281. In some
embodiments, humanized antibodies are more human-like while retaining their
original antigen-binding properties. Presta, L.G. Engineering of therapeutic
antibodies to minimize immunogenicity and optimize function. Advanced Drug
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Delivery Reviews, Volume 58, Issues 5-6: 640-656 (2006). In some embodiments,
the sequence of the variable domain of a rodent antibody is screened against a
library of known human variable-domain sequences or a library of human
germline
sequences. The human sequence that is closest to that of the rodent may then
be
accepted as the human framework region for the humanized antibody (Sims et
al., J.
ImmunoL 1993; 151:2296 et seq.; Chothia et al, Chothia and Lesk, J. MoL Biol.
1987; 196901-917). In another embodiment, humanization involves using a
particular
framework region derived from the consensus sequence of all human antibodies
of a
particular subgroup of light or heavy chains. The same framework may be used
for
several different humanized antibodies (Carter et al., PNAS USA, 1992; 89:4285
et
seq.; Presta et al., J lmmunol 1993; 151:2623 et seq.). Other methods designed
to
reduce the immunogenicity of the antibody molecule in a human patient include
veneered antibodies (see, e.g., U.S. Pat. No. 6,797,492 and U.S. patent
application
publications 20020034765 and 20040253645) and antibodies that have been
modified by T-cell epitope analysis and removal (see, e.g., U.S. patent
application
publications 20030153043 and U.S. Pat. No. 5,712,120). In various embodiments,
CDRs of the humanized antibodies described herein correspond to the CDR
sequences of the mouse monoclonal DC2E7 antibody, namely SEQ ID Nos. 1-6.
[83] The term "hypervariable region" when used herein refers to the amino
residues of an antibody that contribute most directly to antigen-binding. The
term is
synonymous with the term "complementarity determining region" (or "CDR"). In
one
embodiment, according to IMGT, the hypervariable region generally comprises
amino acids in three stretches on each of the heavy and light chain variable
domains
(e.g., residues 27-32 (LCDR1), 49-51 (LCDR2), and 88-96 (LCDR3) in the light
chain
variable domain and 26-33 (HCDR1), 51-58 (HCDR2), and 97-102 (HCDR3) in the
heavy chain variable domain.
[84] The term "insoluble tau" refers to aggregates of tau (such as
neurofibrillary
tangles, neuropil threads, Pick's bodies, coiled bodies etc.), which may be
identified,
e.g., by its characteristic pattern in the brain or in solution. For example,
tau
aggregates can be separated by centrifugation of homogenized brain samples and
evaluated by, e.g., Western blot or ELISA assays. See, e.g., Lasagna-Reeves et
al.,
FASEB J. 26:1946-59 (2012). Tau aggregates may be composed of tau monomers,
tau dimers, trimers, or oligomers, such as granular tau oligomers (GTO) and
various
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types of filaments, including Paired Helical Filaments (PHF) and Straight
Filaments
(SF), among others. Cowan, C.M. and Mudher, A., Are tau aggregates toxic or
protective in tauopathies?, Frontiers in Neurology, 4:114 (2013).
[85] The term "isolated nucleic acid" as used herein means a polynucleotide of
genomic, cDNA, or synthetic origin or some combination thereof, which by
virtue of
its origin either (1) is not associated with all or a portion of a
polynucleotide in which
the "isolated nucleic acid" is found in nature, (2) is operably linked to a
polynucleotide which is not liked to in nature, or (3) does not occur in
nature as part
of a larger sequence.
[86] "Linked" refers to attachment of a moiety to a peptide, antibody,
compound, or
solid particle. The term embraces instances where a moiety is coupled, or
complexed, or covalently or non-covalently attached to a peptide, antibody,
compound, or solid particle. For example, an antibody or antigen binding
fragment
disclosed herein can be covalently or non-covalently coated on a bead or other
sold
particle. The moiety can be chemically crosslinked or expressed or synthesized
as a
fusion with the peptide or antibody.
[87] As used herein, a "molecule that is capable of forming a tau-molecule
complex" is any agent that preferentially can bind to a tau species to form a
complex.
The term encompasses antibodies, e.g., the anti-tau antibodies disclosed
herein, as
well as antigen receptors, Fc-conjugated receptors, receptor fragments,
complement
factors, and any other suitable binding moieties capable of forming a complex
with
tau.
[88] The term "nucleic acid" as used herein, is intended to include DNA
molecules
and RNA molecules. A nucleic acid molecule may be single-stranded our double
stranded.
[89] "Pathological tau" includes pathological tau conformers and structures
and
encompasses all of the following: disease modified tau (e.g.,
hyperphosphorylated
tau, truncated tau, etc.), misordered tau, misdisordered tau, misdisordered
soluble
tau, insoluble tau, tau oligomers and filaments, extracellular and
intracellular tau
aggregates such as neurofibrillary tangles, neuropil threads, neuritic
plaques, ghost
tangles and axonal spheroids, Pick's bodies, coiled bodies, tuft-shape
astrocytes,
astrocytic plaques, or any other form of tau associated with AD or another
tauopathy.
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[90] A "sample" or "biological sample" as used herein refers to any portion of
tissue or bodily fluid (e.g., blood, CSF, etc.) obtained from a subject for
testing
purposes. The sample may be directly isolated from the subject or further
processed
prior to testing (e.g., by dilution, fixation, denaturation, division,
separation,
centrifugation, immune complex dissociation, and the like). Portions of the
sample
used for diagnostic or monitoring purpose are also encompassed by the terms.
The
sample may also be further processed during or after testing (e.g., by
immunoprecipitation, purification, partition, etc.). The terms "sample" and
"biological
sample" apply equally to the tissue or bodily fluid before, during, and/or
after these
processing steps. The term "control sample" as used here in refers to a sample
obtained from a healthy patient or a patient with another diagnosed tauopathy
or a
patient with a known level of a tau species in a biologic fluid, e.g., CSF.
[91] Various types of tau (i.e., tau protein species) can exist in a
subject, e.g., in
brain tissue or samples such as CSF or blood. Details on these types of tau
are
known and have been described, e.g., in W02004/007547 A2 and U.S. 9,518,101,
which are incorporated herein by reference in their entirety. The term "tau,"
when
used without further isoform specification (e.g.,tau 2N4R), encompasses all
forms of
tau as well as tau fragments unless otherwise specified.
[92] The term "pharmaceutically acceptable" means biologically or
pharmacologically compatible for in vivo use in animals or humans, and
preferably
means approved by a regulatory agency of the Federal or a state government or
listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for
use
in animals, and more particularly in humans.
[93] The term "phosphorylated tau" refers to any tau isoform comprising one or
more phosphorylated amino acids. The longest form of adult human brain tau has
80
serine or threonine residues and 5 tyrosine residues; therefore, almost 80% of
the
molecule has the potential to be phosphorylated. See Goedert, M. et al,
Multiple
isoforms of human microtubule-associated protein tau: sequences and
localization in
neurofibrillary tangles of Alzheimer's disease. Neuron 3, 519-526 (1989). The
term
phosphorylated tau also refers to any combination of phosphorylated tau amino
acids. Tau plays a key role in regulating microtubule dynamics, axonal
transport and
neurite outgrowth. All of these functions of tau may be modulated by site-
specific
phosphorylation and alteration of normal phosphorylation events (e.g.,
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hyperphosphorylation, abnormal phosphorylation) may contribute to
neurodegenerative diseases, such as AD. Johnson GV et al., Tau phosphorylation
in
neuronal cell function and dysfunction. J. Cell Sci. 2004 Nov 15;117(Pt 24):
5721-9.
[94] In some embodiments, phosphorylated tau refers to a tau protein having
any
combination of one or more phosphorylated residues between amino acids 188 and
227 (as numbered in tau protein 2N4R or the equivalent residues in another tau
fragment or isoform). In another embodiment, phosphorylated tau refers to tau
phosphorylated at least at threonine 217 (as numbered in tau protein 2N4R or
the
equivalent residues in another tau fragment or isoform). In some embodiments,
the
tau protein is phosphorylated as in the phosphorylated tau proteins detected
in the
Examples.
[95] "Physiological tau" refers to a native, unfolded tau protein found in
the brain of
health individuals, which can be of varying length. Such tau proteins are
typically
highly soluble and generally show less tendency for aggregation. See Wang et
al.,
Tau in physiology and pathology, Nature Reviews, 17:22-35, 2016.
[96] The term "recombinant host cell" (or simply "host cell"), as used herein,
is
intended to refer to a cell into which a recombinant expression vector has
been
introduced. It should be understood that such terms are intended to refer to
not only
the particular subject cell but to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be identical to the
parent
cell, but are still included within the scope of the term "host cell" as used
herein.
[97] As used herein "specifically binds" in reference to an antibody means
that the
antibody binds to its target antigen or epitope with greater affinity than it
does to a
structurally different antigen(s) or epitope.
[98] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL
domains of an antibody, wherein these domains are present in a single
polypeptide
chain. Preferably, the Fv polypeptide further comprises a polypeptide linker
between
the VH and VL domains which enables the scFv to form the desired structure for
antigen binding. For a review of scFv see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag,
New
York, pp. 269-315 (1994).
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[99] The term "treatment" as used herein, is defined as the application or
administration of a therapeutic agent to a subject, who has a disease, a
symptom of
disease or a predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease,
one or
more symptoms of the disease, or the predisposition toward the disease.
Moreover,
as long as the compositions of the disclosure either alone or in combination
with
another therapeutic agent cure, heal, alleviate, relive, alter, remedy,
ameliorate,
improve or affect at least one symptom of Alzheimer's Disease or another
tauopathy
being treated, as compared to that symptom in the absence of treatment, the
result
should be considered a treatment of the underlying disorder regardless of
whether all
the symptoms of the disorder are cured, healed, alleviated, relieved, altered,
remedied, ameliorated, improved or affected or not. Treatment may be achieved
using an "effective amount" of a therapeutic agent, which shall be understood
to
embrace partial and complete treatment, e.g., partial or complete curing,
healing,
alleviating, relieving, altering, remedying, ameliorating, improving, or
affecting the
disease, one or more symptoms of the disease, or the predisposition toward the
disease. An "effective amount" of may be determined empirically. Likewise, a
"therapeutically effective amount" is a concentration or which is effective
for
achieving a stated therapeutic effect.
[100] "Tauopathy" refers to a disease associated with the formation of
pathological
tau. Tauopathy encompasses all neurological diseases that are accompanied by
the
appearance of abnormal forms of microtubule associated protein tau in the
brains of
patients. The term includes, but is not limited to, the following diseases:
Alzheimer's
disease, Gerstmann-Straussler-Scheinker disease, British dementia, Danish
dementia, Pick's disease, Progressive supranuclear palsy, Corticobasal
degeneration, Argyophilic grain disease, Guam Parkinsonism-dementia complex,
Tangle-only dementia, White matter tauopathy with globular glial inclusions,
Frontotemporal dementia (e.g., FTDP-17), Parkinsonism linked to chromosome 17
chronic traumatic encephalopathy, and nodding disease. See, e.g., Goedert M,
Clavaguera F and To!nay M. The propagation of prion-like protein inclusions in
neurodegenerative diseases. Trends Neurol. Sci. In some embodiments, a subject
with Frontotemporal dementia (FTD) may have Nonfluent/Agrammatic Primary
Progressive Aphasia (nfPPA), Semantic Variant Primary Progressive Aphasia
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(svPPA), Behavioral Variant Frontotemporal Dementia (bvFTD), or Amyotrophic
Lateral Sclerosis/Frontotemporal Dementia (ALS/FTD). In one embodiment, one or
more of those abnormal forms of tau is recognized by one of the antibodies or
binding fragments described herein in at least one assay. In some embodiments,
the
assay is IHC. In other embodiments, the assay is ELISA. The term "another
tauopathy" encompasses all neurological diseases (e.g., tau-based causes of
dementia) other than AD that are accompanied by the appearance of pathologic
tau,
e.g., any of the other taupathies listed above. In contrast, dementia may also
arise
from non-tau based etiologies, and these would fall outside of the term
tauopathy.
[101] The term "variable domain" refers to the fact that certain portions of
an
immunoglobulin differ extensively in sequence among antibodies and are used in
the
binding and specificity of each particular antibody for its particular
antigen. However,
the variability is often not evenly distributed throughout the variable
domains of
antibodies. It is concentrated in three segments called hypervariable regions
both in
the light chain and the heavy chain variable domains. The more highly
conserved
portions of variable domains are called the framework regions (FRs). The
variable
domains of native heavy and light chains each comprise four FRs, which often
adopt
a beta-sheet configuration, connected by three hypervariable regions, which
often
form loops connecting, and in some cases forming part of, the beta-sheet
structure.
The hypervariable regions (HVR) in each chain are held together in close
proximity
by the FRs and, with the hypervariable regions from the other chain,
contribute to the
formation of the antigen-binding site of antibodies (see Kabat, et al.,
Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service, National
Institutes
of Health, Bethesda, Md. (1991)). The "constant domains" of an antibody, in
contrast,
are not typically involved directly in binding an antibody to an antigen, but
contribute
to antibody dependent cellular cytotoxicity (ADCC).
[102] The term "vector", as used herein, is intended to refer to a nucleic
acid
molecule capable of transporting another nucleic acid to which it has been
linked.
One type of vector is a "plasmid", which refers to a circular double stranded
DNA
loop into which additional DNA segments may be ligated. Another type of vector
is a
viral vector, wherein additional DNA segments may be ligated into the viral
genome.
Certain vectors are capable of autonomous replication in a host cell into
which they
are introduced (e.g., bacterial vectors having a bacterial origin of
replication and
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episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) can be integrated into the genome of a host cell upon introduction
into the
host cell, and thereby are replicated along with the host genome. Moreover,
certain
vectors are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as "recombinant
expression
vectors" (or simply, "expression vectors"). In general, expression vectors of
utility in
recombinant DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used interchangeably as the
plasmid is
most commonly used form of vector. However, the invention is intended to
include
other forms of expression vectors, such as viral vectors (e.g., replication
defective
retrovi ruses, adenoviruses and adeno-associated viruses), which serve
equivalent
functions.
[103] As used herein, the singular forms "a" "an" and "the" are intended to
include
the plural forms as well, unless the context clearly indicates otherwise.
Furthermore,
to the extent that the terms "including," "includes," "having," "has," "with,"
or variants
thereof are used in either the detailed description and/or the claims, such
terms are
intended to be inclusive in a manner similar to the term "comprising." As used
herein,
the terms "about" and "approximately" mean within an acceptable error range
for the
particular value as determined by one of ordinary skill in the art, which will
depend in
part on how the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean from 1 to 1.5 standard
deviation(s) or from 1 to 2 standard deviations, per the practice in the art.
Alternatively, "about" can mean a range of up to and including 20%, 10%, 5%,
or 1%
of a given value. Alternatively, particularly with respect to biological
systems or
processes, the term can mean up to and including an order of magnitude, up to
and
including 5-fold, and up to and including 2-fold, of a value. Where particular
values
are described in the application and claims, unless otherwise stated the term
"about"
meaning within an acceptable error range for the particular value should be
assumed. Also, all ranges described herein include the endpoints as well as
all
points in between. The term "or" will be understood to mean "and/or" unless
the
context clearly indicates otherwise. All references cited herein are
incorporated by
reference in their entirety. To the extent anything in an incorporated
reference
36
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contradicts or is inconsistent with material provided herein, the present
disclosure
shall control.
Antibodies
[104] Described herein are novel anti-tau antibodies useful for detecting,
monitoring, treating, and preventing AD. In some embodiments, the antibodies
are
capable of binding to a phosphorylated tau. In some embodiments, the
antibodies
are capable of binding to phosphorylated tau in a biological sample (e.g., CSF
or
blood), making them useful for non-invasively distinguishing AD from other
tauopathies. In some embodiments, the antibodies can be conjugated to second
agents (e.g., agents other than antibodies or antigen binding fragments
disclosed
herein, such as radiotracers or therapeutic agents). In some embodiments, one
or
more of the antibodies or antigen binding fragments disclosed herein are
conjugated
to each other. In some embodiments, the antibodies and/or conjugated
antibodies
cross the blood-brain barrier to bind to tau in the brain, making them useful
for in vivo
detection or treatment of AD and other tauopathies in the brain.
[105] Sequences of antibody CDRs and variable domains, as well as full length
tau
protein 2N4R and portions thereof, are shown in Table 1 below. In various
embodiments, antibodies and binding fragments disclosed herein comprise one or
more of the CDR and/or variable domain sequences disclosed in the table,
and/or
bind to one or more portions of tau protein 2N4R disclosed in the table.
Table 1
e Sequence
NO
HCDR1
1 GFTFSSFG
(E7)
HCDR2
2 ISGDSNTI
(E7)
HCDR3
3 ARTLAY
(E7)
4 LCDR1 (E7) EDIYNR
LCDR2 (E7) GAS
6 LCDR3 (E7) LQYWSIPWT
37
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H variable DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
7 (E7) GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRS
EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable
8 LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E7)
YWSIPWTFGGGTKLEIKR
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT
MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA
AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
HVTQARMVSK SKDGTGSDDK KAKGADGKTK
IATPRGAAPP GQKGQANATR IPAKTPPAPK
TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS
9 tau 2N4R
RTPSLPTPPT REPKKVAVVR TPPKSPSSAK
SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK
KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY
KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK
SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE
NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN
VSSTGSIDMV DSPQLATLAD EVSASLAKQG L
tau 188-227 PPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVA
11 tau 210-221 SRTPSLPTPPTR
tau 210-221
12 SRTPSLPpTPPTR
(pT217)
13 tau 151-188 IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEP
14 tau 163-172 KGQANATRIP
HCDR1
GFNIKDYY
(E2)
HCDR2
16 LDPENDHT
(E2)
HCDR3
17 SYYKYDDY
(E2)
18 LCDR1 (E2) QSLLDSDGKTY
19 LCDR2 (E2) LVS
LCDR3 (E2) WQGTHFPLT
EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVRQRPDQ
H variable GLEWIGWLDPENDHTIYDPRFQDRANLTADTSSNTAYLQLSSLTS
21
(E2) EDTAVYYCSYYKYDDYWGQGTTLTVS
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLVQRP
L variable GQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKINRVEAEDLGV
22
(E2) YYCWQGTHFPLTFGAGTKLELKR
HCDR1
23 GFTLSSFG
(149)
HCDR2
24 ISSGSSTI
(149)
38
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HCDR3
25 (149) SRRLDY
LCDR1
26 (149) KSLLYKDGKTY
LCDR2
27 LMS
(149)
LCDR3
28 (149) QQFVEYPLT
DVQLVESGGGLVQPGGSRKLSCAASGFTLSSFGMHWVRQAPEK
H variable
29 (149) GLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNILFLQMTSLRSE
DTAIYYCSRRLDYWGQGTSVTVS
DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPG
L variable
30 (149) QSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVY
YCQQFVEYPLTFGTGTKLELQR
tau 210-221
31 (p1212, SRpTPSLPpTPPTR
pT217)
HCDR1
32 GFTFSGFG
(E7c107)
HCDR1
33 G
(E7cI18) FTFGSFG
HCDR2
34 V
(E7c106) SGDSNTI
HCDR2
35 I
(E7cI13) SGASSTI
36 HCDR2 (E7
ISGDGNTI
c117)
HCDR2
37 I
(E7cI18) SGDSNTV
HCDR2
38 ISGDSSTI
(E7cI29)
LCDR1
39 (E7cI21PD) ENIYNR
LCDR2
40 GAG
(E7cI14)
LCDR3
41 (E7c103) LQYWSTPWT
39
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42 LCDR39) LQYRSIPWT
(E7cI2
DAQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMRWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRS
43
(E7c101) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
44
(E7c101) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable
45 GLEWVTYISGDSNTIYYADTVKGRFTISRDNPKNTMFLQMTSLRS
(E7c102)
EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRATITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
46
(E7c102) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPE
H variable MGLEWVAYISGDSNTIYYADTVEGRFTISRDNPKNTLSLQMTSLR
47
(E7c103) SEDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLEPGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
48
(E7c103) YWSTPWTFGGGTKLEIKR
DVQLVESGGGLAQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTEKGRFTISRDNPKNALFLQMTSLRS
49
(E7c104) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTVTCKASEDIYNRLAWYQQTPGNAP
L variable SLLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCL
(E7c104) QYWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
51 H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRE
(E7c105) DTAIYYCARTLAYWGQGALVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGDAPS
52 L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E7c105) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYVSGDSNTIYYADTVKDRFTISRDNPKNTLFLQMASLRS
53
(E7c106) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
54
(E7c106) YWSIPWTFSGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSGFGMHWVRQAPE
H variable KGLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLSLQMTSLR
(E7c107) SEDTAIYYCARTLAYWGQGTLVTVS
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DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
56
(E7c107) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable
57 GLEWVAYISGDSNTIYYADTVKGRLTISRDNPKNTLFLQMTSLRSE
(E7c107PD)
DTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQIPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
58
(E7c107PD) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRNNPKNTLFLQMTSLRS
59
(E7c108) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSLSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E7c108) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWARQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRS
61
(E7cI10) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGSAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
62
(E7cI10) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLLLQMTSLRSE
63
(E7cI11) DTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSLSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
64 L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E7cI11) YWSIPWTFGGGTKLGIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWIAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSE
(E7cI12) DTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
66
(E7cI12) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGASSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSE
67
(E7cI13) DTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
68
(E7cI13) YWSIPWTFGGGTKLEIKR
H l
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWARQAPEK
variab
69 GLEWVAYISGDSNTIYYADTVKGRLTISRDNPKNTLFLQTTSLRSE
I14) (E e 7c
DTAICYCARTLAYWGQGTLVTVS
41
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DIQMTQSSSSFSVSLGDRVTITCKTSEDIYNRLAWYQQTPGNAPS
L variable LLIPGAGGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E7cI14) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWVRQAPG
H variable KGLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLR
71
(E7cI15) SEDTAIYYCARTLAYWGQGTLVTVP
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
72
(E7cI15) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLLLQMTSLRSE
73
(E7cI16) DTAIYYCARTLAYWGQGTLVTVS
DIRMTQSSSSFSVSLGVRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
74
(E7cI16) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRRLSCAASGFTFSSFGMHWARQAPEK
H variable GLEWVAYISGDGNTIYYADTVKGRFTTSRDNPKNTLFLQMTSLRS
(E7cI17) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSLSVSLGDRVTITCKASEDIYNRLAWYQQTPGSAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
76
(E7cI17) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFGSFGMHWVRQAPE
H variable KGLEWVAYISGDSNTVYYADTVKGRFTISRDNPKNTLFLQMTSLR
77
(E7cI18) SEDTAIYYCARTLAYWGQGTLVTVS
DIQMTQPSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
78 L variable LLISGASGLETGVPSRFSGSGSGGDYTLSITSLQTEDVATYYCLQ
(E7cI18) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRS
79
(E7cI19) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSSSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E7cI19) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTAKGRFAISRDNPKNTLFLQMTSLRS
81
(E7cI20) EDTAIYYCARTLAYWGQGTLVTVS
L l
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGSAPS
variab
82 LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
(E e 7cI20)
YWSIPWTFGGGTKLEIKR
42
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DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKSTLFLQMTSLRSE
83
(E7cI21PD) DTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSLSVSLGDRVAITCKASENIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
84
(E7cI21PD) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWARQAPEK
H variable GLEWVAYISGDSNTIYYADAVKGRFTISRDNPKNTLSLQMTSLRS
(E7cI22) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSLSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
86
(E7cI22) YWSIPWTFGGGTKLEIKR
DVQLVESGRGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYAGTVKGRFTISRDNPKNTLFLQMTSLRS
87
(E7cI23) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSSSVPLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
88
(E7cI23) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWARQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRS
89
(E7cI26) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable
LLISGASGLETGVPSRFSGSGSGRDYTLSVTSLQTEDVATYYCLQ
(E7d26)
YWSIPWTFGGGTKLEIKR
GVRLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable
91 GLEWVAYISGDSNTIYYADTVKGRFTISRDNPESTLFLQMTSLRSE
(E7d27)
DTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
92
(E7cI27) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLFLQMTSLRS
93
(E7cI28) EDTAIYYCARTLAYWGQGTLVTVS
DIQMTQSSSSLSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
94
(E7cI28) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRPE
(E7cI29) DTAIYYGARTLAYWGQGTLVTVS
43
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DIQMTQSSSSLSVSLGDRVTITCEASEDIYNRLAWYQQTPGDAPS
L variable LLISGASGLETGVPSRFSGSGSGRDHTLSITSLQTEDVATYYCLQ
96 (E7cI29) YRSIPWTFGGGTKLEVKR
DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEK
H variable GLEWVAYISGDSNTIYYADTVKGRFTISRDNPKNTLHLQMTSLRS
97 (E7cI30) EDTAIYYCARTLAYWGQGTPVTVS
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQTPGNAPS
L variable LLISGASGLETGVPSRFSGSGSGRDYTLSITSLQTEDVATYYCLQ
98 (E7cI30) YWSIPWTFGGGTKLEIKR
DVQLVESGGGLVQPGGSRKLSCAASGFTLSSFGMHWVRQAPEK
H variable
99 GLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNILFLQMTSLRSE
(DC149) DTAIYYCSRRLDYWGQGTSVTVS
L l e
DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPG
variab
100 QSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVY
(D C149) YCQQFVEYPLTFGTGTKLELQR
HCDR1
101 (D0807) GFTFSNYA
HCDR2
102 ISSGGTI
(DC807)
HCDR3
103 ARVNYDYGYAMDY
(DC807)
LCDR1
104 QSLENSNGNTY
(DC807)
1 LCDR2 RV
S
05
(DC807)
LCDR3
106 LQVTHVPWT
(DC807)
EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQNPEK
H variable RLEWVASISSGGTIYSPDSVKGRFTISRDNGRNILYLQMSSLRSE
107 (D0807) DTAMYSCARVNYDYGYAMDYWGQGTSVTVS
DAVMTQTPLSLPVSLGDQASISCRSSQSLENSNGNTYLNWYLQK
L variable PGQSPQLLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLG
108 (D0807) VYFCLQVTHVPWTFGGGTKLEIKR
[106] In various embodiments, antibodies or antigen binding fragments thereof
capable of binding tau disclosed herein comprise one or more of the amino acid
sequences disclosed in Table 1 above. In some embodiments, antibodies
disclosed
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herein comprise variants of one or more sequences disclosed in Table 1 while
retaining the ability to bind tau, e.g., tau phosphorylated at position 217.
[107] For example, in some embodiments an antibody or antigen binding fragment
thereof capable of binding tau comprises a heavy chain variable region and a
light
chain variable region, wherein the heavy chain variable region comprises three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the
amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 1 with a substitution at
one or
more of position 5 and 6, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, or SEQ ID NO: 2 with a substitution at one or more of position 1, 4, 5,
6, and
8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, and/or wherein
LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 with
a substitution at position 2, LCDR2 comprises the amino acid sequence of SEQ
ID
NO: 5, or SEQ ID NO: 5 with a substitution at position 3 and LCDR3 comprises
the
amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 6 with a substitution at
one or
more of position 4 and 6.
[108] A substitution at a particular position may be determined by counting
from left
to right (amino to carboxy terminal) in an amino acid sequence, e.g., a
sequence
listed in Table 1 beginning at the amino terminal end of the peptide sequence
(or the
left end in the table).
[109] In various embodiments, the substitution at position 5 in HCDR1 is
glycine,
the substitution at position 6 in HCDR1 is glycine. In some embodiments, the
substitution at position 1 in HCDR2 is valine, the substitution at position 4
in HCDR2
is alanine, the substitution at position 5 in HCDR2 is glycine, the
substitution at
position 6 in HCDR2 is serine, and/or the substitution at position 8 in HCDR2
is
valine. In some embodiments, the substitution at position 2 in LCDR1 is
asparagine.
In some embodiments, the substitution at position 3 in LCDR2 is glycine. In
some
embodiments, the substitution at position 4 of LCDR3 is arginine, and/or the
substitution at position 6 in LCDR3 is threonine.
[110] In various embodiments, HCDR1 comprises the amino acid sequence of SEQ
ID NO: 1 with a substitution at position 5, HCDR2 comprises the amino acid
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sequence of SEQ ID NO: 2 with a substitution at 8 position 8, and/or HCDR3
comprises the amino acid sequence of SEQ ID NO: 3, and/or wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 4 with a substitution at
position
2, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and/or LCDR3
comprises the amino acid sequence of SEQ ID NO: 6.
[111] In various embodiments, the substitution at position 5 in HCDR1 is
glycine,
and the substitution at position 8 in HCDR2 is valine, and the substitution at
position
2 in LCDR1 is asparagine.
[112] In various embodiments, the antibody or antigen binding fragment that
comprises variant sequences of those disclosed in Table 1 comprises any of the
antibody CDRs (e.g., all six CDRs) and/or full heavy and/or light chain
variable
domain sequences (e.g., paired sets of heavy and light chain variable domain
sequences of a particular antibody clone) selected from those shown in Figures
24-
27B.
[113] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau is disclosed, comprising a heavy chain variable region
and a
light chain variable region, wherein the heavy chain variable region comprises
three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
determining regions (LCDR1, LCDR2, and LCDR3). In varios embodiments, the
CDRs and/or variable domain sequences are selected from those listed in Table
1.
In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID
NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3
comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6.
[114] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau is disclosed, wherein the the heavy chain variable
region
comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 with a
substitution at one or more of position 1, 2, 3, 9, 12, 19, 30, 31, 35, 37,
42, 43, 48,
49, 51, 54, 55, 56, 58, 62, 63, 64, 65, 66, 68, 69, 70, 73, 76, 77, 78, 79,
80, 83, 84,
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88, 94, 96, 107, 108, and 112, and/or the light chain variable region
comprises the
amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with a substitution at one
or
more of position 3, 7, 11, 14, 17, 19, 20, 21, 24, 25, 28, 39, 42, 49, 52, 56,
69, 71,
75, 92, 94, 99,105, and 106.
[115] In some embodiments, the heavy chain variable region comprises the amino
acid sequence of SEQ ID NO: 7 with a substitution at one or more of position
1, 2, 3,
9, 12, 19, 30, 31, 35, 37, 42, 43, 48, 49, 51, 54, 55, 56, 58, 62, 63, 64, 65,
66, 68, 69,
70, 73, 76, 77, 78, 79, 80, 83, 84, 88, 94, 96, 107, 108, and 112, wherein the
substitution in the heavy chain variable region at position 1 is glycine, at
position 2 is
alanine, at position 3 is arginine, at position 9 is arginine, at position 12
is alanine, at
position 19 is arginine, at position 30 is glycine, at position 31 is glycine,
at position
35 is arginine, at position 37 is alanine, at position 42 is glycine, at
position 43 is
methionine, at position 48 is isoleucine, at position 49 is threonine, at
position 51 is
valine, at position 54 alanine, at position 55 is glycine, at position 56 is
serine, at
position 58 is valine, at position 62 is glycine, at position 63 is alanine,
at position 64
is selected from alanine and glutamic acid, at position 65 is glutamic acid,
at position
66 is aspartic acid, at position 68 is leucine, at position 69 is alanine, at
position 70 is
threonine, at position 73 is asparagine, at position 76 is glutamic acid, at
position 77
is serine, at position 78 is alanine, at position 79 is methionine, at
position 80 is
selected from serine, leucine, and histidine, at position 83 is threonine, at
position 84
is alanine, at position 88 is proline, at position 94 is cysteine, at position
95 is
glycine, at position 107 is alanine, at position 108 is proline, and/or at
position 112 is
proline.
[116] In some embodiments, the light chain variable region comprises the amino
acid sequence of SEQ ID NO: 8 with a substitution at one or more of position
3, 7,
11, 14, 17, 19, 20, 21, 24, 25, 28, 39, 42, 49, 52, 56, 69, 71, 75, 92, 94,
99, 105, and
106, wherein the substitution in the light chain variable region at position 3
is
arginine, at position 7 is proline, at position 11 is selected from serine or
leucine, at
position 14 is proline, at position 17 is valine, at position 19 is alanine,
at position 20
is alanine, at position 21 is valine, at position 24 is glutamic acid, at
position 25 is
threonine, at position 28 asparagine, at position 39 isoleucine, at position
42 is
selected from serine and aspartic acid, at position 49 is proline, at position
52 is
glycine, at position 56 is proline, at position 69 is glycine, at position 71
is histidine,
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at position 75 is valine, at position 92 is arginine, at position 94 is
threonine, at
position 99 is serine, at position 105 is glycine, and/or at position 106 is
valine.
[117] In various embodiments, the heavy chain variable region comprises the
amino
acid sequence of SEQ ID NO: 7 with a substitution at one or more of position
1, 3,
30, 37, 48, 58, 63, 68, 76, 77, and 80, and/or the light chain variable region
comprises the amino acid sequence of SEQ ID SEQ ID NO: 8 with a substitution
at
one or more of position 7, 11, 20, 28, 39, and 69.
[118] In some embodiments, the substitution in the heavy chain variable region
at
position 2 is alanine and/or the substitution at position 35 is arginine.
[119] In some embodiments, the substitution in the heavy chain variable region
at
position 49 is threonine and/or at position 79 methionine, and/or the
substitution in
the light chain variable region at position 19 is alanine.
[120] In some embodiments, the substitution in the heavy chain variable region
at
position 43 is methionine, at position 65 is glutamic acid, and/or at position
80 is
serine, and/or the substitution in the light chain variable region at position
56 is
proline and at position 94 is threonine.
[121] In some embodiments, the substitution in the heavy chain variable region
at
position 12 is alanine, at position 64 is glutamic acid, and at position 78 is
alanine,
and/or the substitution in the light chain variable region at position 21 is
valine.
[122] In some embodiments, the substitution in the heavy chain variable region
at
position 107 is alanine, and/or the substitution in the light chain variable
region at
position 42 is aspartic acid.
[123] In some embodiments, the substitution in the heavy chain variable region
at
position 19 is arginine, at position 51 is valine, at position 66 is aspartic
acid, and at
position 84 is alanine, and/or the substitution in the light chain variable
region at
position 99 is serine.
[124] In some embodiments, the substitution in the heavy chain variable region
at
position 31 is glycine and/or at position 80 is serine.
[125] In some embodiments, the substitution in the heavy chain variable region
at
position 73 is asparagine, and/or the substitution in the light chain variable
region at
position 11 is leucine.
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[126] In some embodiments, the substitution in the heavy chain variable region
at
position 37 is alanine, and/or the substitution in the light chain variable
region at
position 42 is serine.
[127] In some embodiments, the substitution in the heavy chain variable region
at
position 80 is leucine, and/or the substitution in the light chain variable
region at
position 11 is leucine and at position 105 is glycine.
[128] In some embodiments, the substitution in the heavy chain variable region
at
position 54 is alanine and/or at position 56 is serine.
[129] In some embodiments, the substitution in the heavy chain variable region
at
position 37 is alanine, at position 68 is leucine, at position 83 is
threonine, at position
99 is cysteine, the substation in the light chain variable region at position
25 is
threonine, at position 49 is proline, and/or at position 52 is glycine.
[130] In some embodiments, the substitution at position in the heavy chain
variable
region at position 19 is arginine, at position 42 is glycine, and/or at
position 112 is
proline.
[131] In some embodiments, the substitution in heavy chain variable region at
position 80 is leucine, and the substitution in the light chain variable
region at
position 3 is arginine and/or at position 17 is valine.
[132] In some embodiments, the substitution in the heavy chain variable region
at
position 19 is arginine, at position 37 is alanine, at position 55 is glycine,
at position
70 is threonine, the substitution in the light chain variable region at
position 11 is
leucine and/or at position 42 is serine.
[133] In some embodiments, the substitution in the heavy chain variable region
at
position 64 is alanine, the substitution at position 69 is alanine, and/or the
substitution in the light chain variable region at position 42 is serine.
[134] In some embodiments, the substitution in the heavy chain variable region
at
position 9 is arginine and/or at position 62 is glycine, and/or the
substitution in the
light chain variable region at position 11 is serine and/or at position 14 is
proline.
[135] In some embodiments, the substitution in the heavy chain variable region
at
position 37 is alanine, and/or the substitution in the light chain variable
region at
position 75 is valine.
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[136] In some embodiments, the substitution at position in the heavy chain
variable
region at position 56 is serine, at position 88 is proline, and/or at position
96 is
glycine, and/or the substitution in the light chain variable region at
position 11 is
leucine, at position 24 is glutamic acid, at position 42 is aspartic acid, at
position 71
is histidine, at position 92 is arginine, and/or at position 106 is valine.
[137] In some embodiments, the the substitution in the heavy chain variable
region
at position 80 is histidine and/or at position 108 is proline.
[138] In some embodiments, the antibody or antigen binding fragment thereof
capable of binding tau, comprises a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 7 with a substitution at one or more of
position
1, 3, 30, 37, 48, 58, 63, 68, 76, 77, and 80, and a light chain variable
region
comprises the amino acid sequence of SEQ ID SEQ ID NO: 8 with a substitution
at
one or more of position 7, 11, 20, 28, 39, and 69. In various embodiments, the
substitution in the heavy chain variable region at position 1 is glycine, at
position 3 is
arginine, at position 30 is glycine, at position 37 is alanine, at position 48
is
isoleucine, at position 58 is valine, at position 63 is alanine, at position
68 is leucine,
at position 76 is glutamic acid, at position 77 is serine, and/or at position
80 is serine,
and the substitution in the light chain variable region at position 7 is
proline, at
position 11 is selected from leucine and serine, at position 20 is alanine, at
position
28 is asparagine, at position 39 is isoleucine, and/or at position 69 is
glycine.
[139] In various embodiments, the substitution in the heavy chain variable
region at
position 68 is leucine, and/or the substitution in the light chain variable
region at
position 39 is isoleucine.
[140] In various embodiments, the substitution in the heavy chain variable
region at
position 48 is isoleucine.
[141] In various embodiments, the substitution in the heavy chain variable
region at
position 30 is glycine and/or at position 58 is valine, and the substitution
in the light
chain variable region at position 7 is proline and/or at position 69 is
glycine.
[142] In various embodiments, the substitution in the light chain variable
region at
position 11 is serine.
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[143] In various embodiments, the substitution in the heavy chain variable
region at
position 77 is serine, and/or the substitution in the light chain variable
region at
position 11 is leucine, at position 20 is alanine, and/or at position 28 is
asparagine.
[144] In various embodiments, the substitution in the heavy chain variable
region at
position 37 is alanine, at position 63 is alanine, and/or position 80 is
serine, and/or
the substitution in the light chain variable region at position 11 is leucine.
[145] In various embodiments, the substitution in the heavy chain variable
region at
position 1 is glycine, at position 3 is arginine, at position 76 is glutamic
acid, and/or at
position 77 is serine.
[146] In various embodiments, the substitution in the light chain variable
region at
position 11 is leucine.
[147] In some embodiments, the heavy chain variable region comprises an amino
acid sequence of SEQ ID NO: 7 and the light chain variable region comprises an
amino acid sequence of SEQ ID NO: 8.
[148] In some embodiments, an antibody or antigen binding fragment thereof
capable of binding tau, comprising a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the
light chain variable region comprises three light chain complementarity
determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 24, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 25; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 26, LCDR2
comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 28. In some embodiments, the heavy chain
variable region comprises an amino acid sequence of SEQ ID NO: 29 and the
light
chain variable region comprises an amino acid sequence of SEQ ID NO: 30.
[149] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau is disclosed, comprising a heavy chain variable region
and a
light chain variable region, wherein the heavy chain variable region comprises
three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
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determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the
amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 1 with a substitution at
one or
more of position 5 and 6, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, or SEQ ID NO: 2 with a substitution at one or more of position 1, 4, 5,
6, and
8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises
the amino acid sequence of SEQ ID NO: 4, or SEQ ID NO: 4 with a substitution
at
position 2, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, or SEQ ID
NO: 5 with a substitution at position 3, and LCDR3 comprises the amino acid
sequence of SEQ ID NO: 6, or SEQ ID NO: 6 with a substitution at one or more
of
position 4 and 6. In various embodiments, the substitution at position 5 in
HCDR1 is
glycine, the substitution at position 6 in HCDR1 is glycine, the substitution
at position
1 in HCDR2 is valine, the substitution at position 4 in HCDR2 is alanine, the
substitution at position 5 in HCDR2 is glycine, the substitution at position 6
in
HCDR2 is serine, the substitution at position 8 in HCDR2 is valine, the
substitution at
position 2 in LCDR1 is asparagine, the substitution at position 3 in LCDR2 is
glycine,
and/or the substitution at position 4 of LCDR3 is arginine, and/or the
substitution at
position 6 in LCDR3 is threonine.
[150] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau wherein HCDR1 comprises the amino acid sequence of SEQ
ID NO: 1 with a substitution at position 5, HCDR2 comprises the amino acid
sequence of SEQ ID NO: 2 with a substitution at position 8, HCDR3 comprises
the
amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence
of SEQ ID NO: 4 with a substitution at position 2, LCDR2 comprises the amino
acid
sequence of SEQ ID NO: 5, and/or LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6.
[151] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau wherein the substitution at position 5 in HCDR1 is
glycine, the
substitution at position 8 in HCDR2 is valine, and/or the substitution at
position 2 in
LCDR1 is asparagine.
[152] In various embodiment, the antibody or antigen binding fragment thereof
capable of binding tau comprises an HCDR1 comprises the amino acid sequence of
SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and
HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
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comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 41; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO 34, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprise the amino acid sequence of SEQ
ID NO: 5, and LCDR3 comprise the amino acid sequence of SEQ ID NO 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 32, HCDR2
comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 35, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 40, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;
or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 33, HCDR2
comprises the amino acid sequence of SEQ ID NO: 37, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
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acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 39, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 38, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 42;
or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 33, HCDR2
comprises the amino acid sequence of SEQ ID NO: 37, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or
an HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 39, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.
[153] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau comprising a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises the amino
acid
sequence of SEQ ID NO: 43 and the light chain variable region comprises the
amino
acid sequence of SEQ ID NO: 44; or the heavy chain variable region comprises
the
amino acid sequence of SEQ ID NO: 45 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 46; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 47 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 48; or the
heavy
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chain variable region comprises the amino acid sequence of SEQ ID NO: 49 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
50; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
51 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 52; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
53 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 54; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 55 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 56; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 57 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 58; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 59 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 60; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 61 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
62; or
[154] the heavy chain variable region comprises the amino acid sequence of SEQ
ID NO: 63 and the light chain variable region comprises the amino acid
sequence of
SEQ ID NO: 64; or the heavy chain variable region comprises the amino acid
sequence of SEQ ID NO: 65 and the light chain variable region comprises the
amino
acid sequence of SEQ ID NO: 66; or the heavy chain variable region comprises
the
amino acid sequence of SEQ ID NO: 67 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 68; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 69 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 70; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 71 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
72; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
73 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 74; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 75 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 76; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 77 and the light chain variable region
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comprises the amino acid sequence of SEQ ID NO: 78; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 79 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 80; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 81 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
82; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
83 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 84; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 85 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 86; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 87 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 88; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 89 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 90; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 91 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
92; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
93 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 94; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 95 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 96; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 97 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 98; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 57 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 58; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 65 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
66; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
77 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 78; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 79 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 80; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 83 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 84; or the heavy chain
variable
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region comprises the amino acid sequence of SEQ ID NO: 85 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 86; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 91 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
92; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
93 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 94.
[155] Also disclosed herein, in various embodiments, are antibody or antigen
binding fragments that can compete for binding with an antibody or antigen
binding
fragment thereof defined by sequence, as disclosed in this paragraph. In
another
embodiment, an antibody or antigen binding fragment thereof disclosed herein
can
bind to the same epitope on tau protein 2N4R (SEQ ID NO: 9) as that of an
antibody
or antigen binding fragment thereof defined by sequence, as disclosed in this
paragraph.
[156] In various embodiments, an antibody or antigen binding fragment thereof
that
is disclosed herein may bind to an epitope on tau comprising one or more of
residues 188-227 of tau protein 2N4R (SEQ ID NO: 10). In some embodiments, the
epitope comprises one or more of residues 210-221 of tau protein 2N4R (SEQ ID
NO: 11). In various embodiments, an antibody or antigen binding fragment
thereof
that is disclosed herein may bind to an epitope on tau comprising residues 188-
227
of tau protein 2N4R (SEQ ID NO: 10). In some embodiments, the epitope
comprises
residues 210-221 of tau protein 2N4R (SEQ ID NO: 11). The epitope may contain
at
least one, or more than one, phosphorylated residue, which may include a
phospho-
threonine at position 217 of tau protein 2N4R (SEQ ID NO: 9). In some
embodiments, the epitope also comprises a phosphorylated serine at position
210,
threonine at position 212, serine at position 214, or threonine at position
220 of tau
protein 2N4R, or any combination thereof. In certain embodiments, the epitope
comprises or consists of SRTPSLPpTPPTR (sequence of SEQ ID NO: 12). In certain
embodiments, the epitope comprises or consists of a region comprising more
than
one phosphorylated residue, e.g., SRpTPSLPpTPPTR (sequence of SEQ ID NO:
31). In some embodiments, the epitope is determined by any method known to the
skilled artisan. In some embodiments, alanine scanning or deletion/mutagenesis
studies are used to determine the epitope. In some embodiments, mass
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spectrometry is used to determine the epitope. In certain embodiments, the
epitope
is determined by a crystal structure of the antibody-tau complex, where the
epitope is
defined as any contact residue on tau within 5 angstroms of the antibody
binding
pocket. In some embodiments, the antibody or antigen binding fragment
comprises
a heavy chain variable region amino acid sequence of SEQ ID NO: 7 and a light
chain variable region amino acid sequence of SEQ ID NO: 8. In some
embodiments,
the antibody or antigen binding fragment comprises a heavy chain variable
region
amino acid sequence of SEQ ID NO: 29 and a light chain variable region amino
acid
sequence of SEQ ID NO: 30. In some embodiments, the antibody or antigen
binding
fragment comprises a heavy chain variable region comprises the amino acid
sequence of SEQ ID NO: 7 with a substitution at one or more of position 1, 3,
30, 37,
48, 58, 63, 68, 76, 77, and 80, and/or light chain variable region comprises
the amino
acid sequence of SEQ ID SEQ ID NO: 8 with a substitution at one or more of
position 7, 11, 20, 28, 39, and 69. In some embodiments, the substitution in
the
heavy chain variable region at position 68 is leucine, and the substitution in
the light
chain variable region at position 39 is isoleucine. In various embodiments,
the
substitution in the heavy chain variable region at position 48 is isoleucine.
In various
embodiments, the substitution in the heavy chain variable region at position
30 is
glycine and at position 58 is valine, and the substitution in the light chain
variable
region at position 7 is proline and at position 69 is glycine. In various
embodiments,
the substitution in the light chain variable region at position 11 is serine.
In various
embodiments, the substitution in the heavy chain variable region at position
77 is
serine, and the substitution in the light chain variable region at position 11
is leucine,
at position 20 is alanine, and at position 28 is asparagine. In various
embodiments,
the substitution in the heavy chain variable region at position 37 is alanine,
at
position 63 is alanine, and position 80 is serine, and the substitution in the
light chain
variable region at position 11 is leucine. In various embodiments, the
substitution in
the heavy chain variable region at position 1 is glycine, at position 3 is
arginine, at
position 76 is glutamic acid, and at position 77 is serine. In various
embodiments, the
substitution in the light chain variable region at position 11 is leucine.
[157] In other aspects, disclosed herein are antibodies that can compete for
binding
to tau protein 2N4R (SEQ ID NO: 9) with an antibody comprising a heavy chain
variable region and a light chain variable region, wherein the heavy chain
variable
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region comprises an amino acid sequence of SEQ ID NO: 7 and the light chain
variable region comprises an amino acid sequence of SEQ ID NO: 8. In some
embodiments, the antibody or antigen binding fragment comprises a heavy chain
variable region amino acid sequence of SEQ ID NO: 29 and a light chain
variable
region amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody
or antigen binding fragment comprises a heavy chain variable region comprises
the
amino acid sequence of SEQ ID NO: 7 with a substitution at one or more of
position
1, 3, 30, 37, 48, 58, 63, 68, 76, 77, and 80, and/or light chain variable
region
comprises the amino acid sequence of SEQ ID SEQ ID NO: 8 with a substitution
at
one or more of position 7, 11, 20, 28, 39, and 69. In some embodiments, the
substitution in the heavy chain variable region at position 68 is leucine, and
the
substitution in the light chain variable region at position 39 is isoleucine.
In various
embodiments, the substitution in the heavy chain variable region at position
48 is
isoleucine. In various embodiments, the substitution in the heavy chain
variable
region at position 30 is glycine and at position 58 is valine, and the
substitution in the
light chain variable region at position 7 is proline and at position 69 is
glycine. In
various embodiments, the substitution in the light chain variable region at
position 11
is serine. In various embodiments, the substitution in the heavy chain
variable region
at position 77 is serine, and the substitution in the light chain variable
region at
position 11 is leucine, at position 20 is alanine, and at position 28 is
asparagine. In
various embodiments, the substitution in the heavy chain variable region at
position
37 is alanine, at position 63 is alanine, and position 80 is serine, and the
substitution
in the light chain variable region at position 11 is leucine. In various
embodiments,
the substitution in the heavy chain variable region at position 1 is glycine,
at position
3 is arginine, at position 76 is glutamic acid, and at position 77 is serine.
In various
embodiments, the substitution in the light chain variable region at position
11 is
leucine.
[158] In various embodiments, competition is measured by flow cytometry or
fluorescence microscopy, ELISA, HTRF and/or SPR. In one embodiment,
competition ELISA is used to measure competitive binding. In another
embodiment,
a BIACORE assay is used. In some embodiments, the antibody or antigen binding
fragment with which the disclosed antibody competes comprises a heavy chain
variable region amino acid sequence of SEQ ID NO: 7 and a light chain variable
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region amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody
or antigen binding fragment with which the disclosed antibody competes
comprises a
heavy chain variable region amino acid sequence of SEQ ID NO: 29 and a light
chain variable region amino acid sequence of SEQ ID NO: 30. In some
embodiments, the antibody or antigen binding fragment with which the disclosed
antibody competes comprises a heavy chain variable region comprises the amino
acid sequence of SEQ ID NO: 7 with a substitution at one or more of position
1, 3,
30, 37, 48, 58, 63, 68, 76, 77, and 80, and/or light chain variable region
comprises
the amino acid sequence of SEQ ID SEQ ID NO: 8 with a substitution at one or
more
of position 7, 11, 20, 28, 39, and 69. In some embodiments, the antibody or
antibody
fragment with which the disclosed antibody competes, comprises a substitution
in
the heavy chain variable region at position 68 is leucine, and the
substitution in the
light chain variable region at position 39 is isoleucine. In various
embodiments, the
substitution in the heavy chain variable region at position 48 is isoleucine.
In various
embodiments, the substitution in the heavy chain variable region at position
30 is
glycine and at position 58 is valine, and the substitution in the light chain
variable
region at position 7 is proline and at position 69 is glycine. In various
embodiments,
the substitution in the light chain variable region at position 11 is serine.
In various
embodiments, the substitution in the heavy chain variable region at position
77 is
serine, and the substitution in the light chain variable region at position 11
is leucine,
at position 20 is alanine, and at position 28 is asparagine. In various
embodiments,
the substitution in the heavy chain variable region at position 37 is alanine,
at
position 63 is alanine, and position 80 is serine, and the substitution in the
light chain
variable region at position 11 is leucine. In various embodiments, the
substitution in
the heavy chain variable region at position 1 is glycine, at position 3 is
arginine, at
position 76 is glutamic acid, and at position 77 is serine. In various
embodiments, the
substitution in the light chain variable region at position 11 is leucine. In
some
embodiments, the antibody or antigen binding fragment can bind to the same one
or
more amino acids on tau protein 2N4R (SEQ ID NO: 9) as an antibody comprising
a
heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises an amino acid sequence of SEQ ID NO: 7 and the
light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
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[159] In various embodiments, the antibody or antigen binding fragment with
which
the disclosed antibody competes can bind to an epitope on tau comprising one
or
more of residues 188-227 of tau protein 2N4R (SEQ ID NO: 10). In certain
embodiments, the epitope comprises one or more of residues 210-221 of tau
protein
2N4R (SEQ ID NO: 11). In other embodiments, the epitope comprises at least one
phosphorylated residue. In another embodiment, the at least one phosphorylated
residue comprises a phospho-threonine at position 217 of tau protein 2N4R (SEQ
ID
NO: 9), and optionally also comprise a phosphorylated serine at position 210,
threonine at position 212, serine at position 214, or threonine at position
220 of tau
protein 2N4R, or any combination thereof. In another embodiment, the epitope
comprises or consists of SRTPSLPpTPPTR (sequence of SEQ ID NO: 12). In certain
embodiments, the epitope comprises or consists of SRpTPSLPpTPPTR (sequence
of SEQ ID NO: 31).
[160] In various embodiments, an antibody or antigen binding fragment thereof
that
can bind to a region or fragment of tau protein 2N4R comprising or consisting
of
residues 188-227 of tau protein 2N4R (SEQ ID NO: 10), wherein the region or
fragment is phosphorylated. In various embodiments, the region or fragment
comprises or consists of residues 210-221 of tau protein 2N4R (SEQ ID NO: 11).
In
various embodiments, the region or fragment is phosphorylated at a position
corresponding to residue 217 of tau protein 2N4R, and optionally also at one
or more
of positions corresponding to serine 210, threonine 212, serine 214, and
threonine
220 of tau protein 2N4R (SEQ ID NO: 9). In various embodiments, the region or
fragment of tau protein 2N4R comprises or consists of SRTPSLPpTPPTR (sequence
of SEQ ID NO. 12). In certain embodiments, the epitope comprises or consists
of
SRpTPSLPpTPPTR (sequence of SEQ ID NO: 31).
[161] In various embodiments, an antibody or antigen binding fragment
disclosed
herein can bind a phosphorylated epitope on tau, e.g., as measured by flow
cytometry or fluorescence microscopy, ELISA, HTRF and/or SPR. In one
embodiment, ELISA as used to measure or check for binding to phosphorylated
and
dephosphorylated tau. In another embodiment, a BIACORE assay is used.
[162] In various embodiments, disclosed herein is an antibody or antigen
binding
fragment thereof that can bind to an epitope on tau comprising one or more of
residues 151-188 of tau protein 2N4R (SEQ ID NO: 13). In some embodiments, the
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epitope on tau comprises one or more of residues 1 63-1 72 of tau protein 2N4R
(SEQ ID NO: 14). In various embodiments, disclosed herein is an antibody or
antigen binding fragment thereof that can bind to an epitope on tau comprising
residues 151-188 of tau protein 2N4R (SEQ ID NO: 13). In some embodiments, the
epitope on tau comprises residues 163-172 of tau protein 2N4R (SEQ ID NO: 14).
In
certain embodiments, the epitope comprises or consist of KGQANATRIP (sequence
of SEQ ID NO. 14). In certain embodiments, one or more of the residues on the
epitope are phosphorylated, e.g., a phosphorylated threonine at position 169
of tau
protein 2N4R.
[163] In various embodiments, disclosed herein is an antibody or antigen
binding
fragment thereof that can bind to a portion or fragment of tau comprising
residues
151-188 of tau protein 2N4R (SEQ ID NO: 13). In some embodiments, the antibody
or antigen binding fragment thereof that can bind to a portion or fragment of
tau
comprising residues 163-172 of tau protein 2N4R (SEQ ID NO: 14).
[164] In some embodiments, the epitope of an antibody disclosed herein is
determined by any method known to the skilled artisan. In some embodiments,
alanine scanning or deletion/mutagenesis studies are used to determine the
epitope.
In some embodiments, mass spectrometry is used to determine the epitope. In
certain embodiments, the epitope is determined by a crystal structure of the
antibody-tau complex, where the epitope is defined as any contact residue on
tau
within 5 angstroms of the antibody binding pocket.
[165] In various embodiments, an antibody or antigen binding fragment thereof
capable of binding tau is disclosed, comprising a heavy chain variable region
and a
light chain variable region, wherein the heavy chain variable region comprises
three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the
amino acid sequence of SEQ ID NO: 15, HCDR2 comprises the amino acid
sequence of SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence of
SEQ ID NO: 17; and wherein LCDR1 comprises the amino acid sequence of SEQ ID
NO: 18, LCDR2 comprises the amino acid sequence of SEQ ID NO: 19, and LCDR3
comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments,
the
heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 21
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and the light chain variable region comprises an amino acid sequence of SEQ ID
NO: 22. In various embodiments, an antibody or antigen binding fragments
thereof
can compete for binding to one or more amino acids on tau protein 2N4R (SEQ ID
NO: 9) with the previously described antibody or antigen binding fragment
thereof. In
another embodiment, an antibody or antigen binding fragment thereof can bind
the
same epitope on tau protein 2N4R (SEQ ID NO: 9) bound by the antibody or
antigen
binding fragments previously disclosed in this paragraph. In an embodiment, an
antibody or antigen binding fragment thereof can bind the fragment or region
of tau
protein 2N4R (SEQ ID NO: 9) bound by the antibody or antigen binding fragments
previously disclosed in this paragraph.
[166] In various embodiments, any of the antibodies or antigen binding
fragments
described herein can comprise a heavy chain constant region and a light chain
constant region. The heavy chain constant region may be an IgG, IgM, IgA, IgD,
and
IgE isotype, or a derivative or fragment thereof that retains at least one
effector
function of the intact heavy chain. The heavy chain constant region may be a
human
IgG isotype. The heavy chain constant region may be a human IgG1 or human IgG4
isotypes. The heavy chain constant region may be a human IgG1 isotype. The
light
chain constant region may be a human kappa light chain or lambda light chain
or a
derivative or fragment thereof that retains at least one effector function of
the intact
light chain. The light chain constant region may be a human kappa light chain.
[167] In various embodiments, any of the disclosed antibodies or antigen
binding
fragments may be a rodent antibody or antigen binding fragment thereof, a
chimeric
antibody or an antigen binding fragment thereof, a CDR-grafted antibody or an
antigen binding fragment thereof, or a humanized antibody or an antigen
binding
fragment thereof. In another embodiment, any of the disclosed antibodies or
antigen
binding fragments comprises human or human-derived heavy and light chain
variable regions, including human frameworks or human frameworks with one or
more backmutations. See, e.g., the backmutation strategies outlined in U.S.
Patent
No. 7,566,771, the disclosure of which is incorporated herein by reference. In
various embodiments, any of the disclosed antibodies or antigen binding
fragments
may be a Fab, Fab', F(ab')2, Fd, scFv, (scFv)2, scFv-Fc, or Fv fragment.
[168] In various embodiments, antibody DC2E7 or a functional antigen binding
fragment thereof is disclosed, wherein DC2E7 is an antibody produced by a
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hybridoma deposited under American Type Culture Collection Patent Deposit No.
PTA-124992. In various embodiments, antibody DC2E2 or a functional antigen
binding fragment thereof is disclosed, wherein DC2E2 is an antibody produced
by a
hybridoma deposited under American Type Culture Collection Patent Deposit No.
PTA-124991. Also disclosed are antibodies and antigen binding fragments that
can
bind to the same epitope on tau protein 2N4R (SEQ ID NO: 9) or can compete for
binding to tau protein 2N4R (SEQ ID NO: 9) with either antibody DC2E7 or
antibody
DC2E2.
[169] In various embodiments, any of the disclosed antibodies or antigen
binding
fragments thereof may be conjugated to a second agent. The second agent may
be,
e.g., at least one detectable agent, including but not limited to, an enzyme,
a
radioisotope, a fluorophore, a nuclear magnetic resonance marker, or a heavy
metal.
In one embodiment, the at least one detectable label is a radioisotope. In
some
embodiments, the antibody or antigen binding fragment conjugated to a
detectable
label (e.g., a radiolabel) comprises a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the
light chain variable region comprises three light chain complementarity
determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain
variable region comprises an amino acid sequence of SEQ ID NO: 7 and the light
chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
[170] In some embodiments, the antibody or antigen binding fragment conjugated
to
a detectable label (e.g., a radiolabel) comprises a heavy chain variable
region and a
light chain variable region, wherein the heavy chain variable region comprises
three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises
amino acid sequence of SEQ ID NO: 23, HCDR2 comprises the amino acid
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sequence of SEQ ID NO: 24, and HCDR3 comprises the amino acid sequence of
SEQ ID NO: 25; and wherein LCDR1 comprises the amino acid sequence of SEQ ID
NO: 26, LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and LCDR3
comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, In
some embodiments, the heavy chain variable region comprises an amino acid
sequence of SEQ ID NO: 29 and the light chain variable region comprises an
amino
acid sequence of SEQ ID NO: 30. In some embodiments, the antibody or antigen
binding fragment thereof conjugated to a detectable label comprises a heavy
chain
variable region and a light chain variable region, wherein the heavy chain
variable
region comprises three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), In some embodiments, the antibody or antigen binding
fragment conjugated to a detectable label (e.g., a radiolabel) comprises a
heavy
chain variable region and a light chain variable region, wherein the heavy
chain
variable region comprises the amino acid sequence of SEQ ID NO: 43 and the
light
chain variable region comprises the amino acid sequence of SEQ ID NO: 44; or
the
heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 45
and the light chain variable region comprises the amino acid sequence of SEQ
ID
NO: 46; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 47 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 48; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 49 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 50; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 51 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 52; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 53 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
54; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
55 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 56; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 57 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 58; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 59 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 60; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 61 and the light chain
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variable region comprises the amino acid sequence of SEQ ID NO: 62; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 63 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
64; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
65 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 66; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 67 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 68; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 69 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 70; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 71 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 72; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 73 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
74; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
75 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 76; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 77 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 78; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 79 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 80; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 81 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 82; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 83 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
84; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
85 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 86; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 87 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 88; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 89 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 90; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 91 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 92; or the
heavy
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chain variable region comprises the amino acid sequence of SEQ ID NO: 93 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
94; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
95 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 96; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 97 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 98; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 57 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 58; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 65 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 66; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 77 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
78; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
79 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 80; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 83 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 84; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 85 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 86; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 91 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 92; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 93 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
94.
[171] In some embodiments, the antibody or antigen binding fragment conjugated
to
a detectable label (e.g., a radiolabel) may compete for binding or bind the
same
epitope as this antibody defined by sequence.
[172] Examples of suitable types of detectable labels consistent with the
present
disclosure include, but are not limited to, enzymes, radioisotopes and
radionuclides,
colloidal metals, fluorescent compounds, chemiluminescent compounds, biotinyl
groups, predetermined polypeptide epitopes recognized by a secondary reporter
(e.g., leucine zipper pair sequences, binding sites for secondary antibodies,
metal
binding domains, epitope tags), and chemi/electrochemi/bioluminescent
compounds.
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The enzymes include, but are not limited to, peroxidase (e.g., horse radish
peroxidase), luciferase, alkaline phosphatase, P-galactosidase, glucose
oxidase,
glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate
dehydrogenase, or glucose-6 phosphate dehydrogenase. Alternatively, the label
may
be biotin, digoxin-genin, or 5-bromo-desoxyuridine. Fluorescent labels can be
also
combined with the antibodies and antigen binding fragments thereof, including
rhodamine, lanthanide phosphors, fluorescein and its derivatives,
fluorochromes,
rhodamine and its derivatives, green fluorescent protein (GFP), red
fluorescent
protein (RFP), dansyl, umbelliferone, and others. Such conjugates may be
prepared
by methods known to a person skilled in the art. They can be bound with labels
directly; via a spacer group or linkage group such as polyaldehyde,
glutaraldehyde,
ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid
(DPTA); or in the presence of other binding agents such as those routinely
known in
the art.
[173] Other detectable labels may include radioactive labels such as iodine-
123,
iodine-125, iodine-126, iodine-133, iodine-131, bromine-77, technetium-99m,
indium-
113m, gallium-67, gallium-68, ruthenium-95, ruthenium-97, ruthenium-103,
ruthenium-106, mercury-203, scandium-47, tellurium-121m, tellurium-128,
thulium-
165, thulium-167, thulium-168, fluorine-18, yttrium-99, and zirconium-89.
Existing
methods known to a person skilled in the art for labelling antibodies with
radioisotypes, either directly or indirectly, e.g., via a chelating agent such
as EDTA or
DTPA, can be used.
[174] In various embodiments, any of the disclosed antibodies or antigen
binding
fragments can be conjugated to a second agent, where the second agent may be
at
least one therapeutic agent for Alzheimer's disease or another tauopathy. In
some
embodiments, the antibody or antigen binding fragment conjugated to a
therapeutic
agent for Alzheimer's disease comprises a heavy chain variable region and a
light
chain variable region, wherein the heavy chain variable region comprises three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the
amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence
of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3;
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and wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain
variable region comprises an amino acid sequence of SEQ ID NO: 7 and the light
chain variable region comprises an amino acid sequence of SEQ ID NO: 8. In
some
embodiments, the antibody or antigen binding fragment comprises any one or
more
(e.g., all six CDRs and/or a heavy and/or light chain variable domain)
sequence
selected fro those shown in Table 1. In some embodiments, the antibody or
antigen
binding fragment comprises a heavy chain variable region and a light chain
variable
region, wherein the heavy chain variable region comprises the amino acid
sequence
of SEQ ID NO: 43 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 44; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 45 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 46; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 47 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 48; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 49 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
50; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
51 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 52; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 53 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 54; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 55 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 56; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 57 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 58; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 59 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
60; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
61 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 62; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 63 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 64; or the heavy chain variable region comprises the
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amino acid sequence of SEQ ID NO: 65 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 66; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 67 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 68; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 69 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
70; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
71 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 72; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 73 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 74; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 75 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 76; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 77 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 78; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 79 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
80; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
81 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 82; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 83 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 84; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 85 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 86; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 87 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 88; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 89 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
90; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
91 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 92; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 93 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 94; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 95 and the light chain variable region
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comprises the amino acid sequence of SEQ ID NO: 96; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 97 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 98; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 57 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
58; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
65 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 66; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 77 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 78; or the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO: 79 and the light chain variable region
comprises the amino acid sequence of SEQ ID NO: 80; or the heavy chain
variable
region comprises the amino acid sequence of SEQ ID NO: 83 and the light chain
variable region comprises the amino acid sequence of SEQ ID NO: 84; or the
heavy
chain variable region comprises the amino acid sequence of SEQ ID NO: 85 and
the
light chain variable region comprises the amino acid sequence of SEQ ID NO:
86; or
the heavy chain variable region comprises the amino acid sequence of SEQ ID
NO:
91 and the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 92; or the heavy chain variable region comprises the amino acid sequence
of
SEQ ID NO: 93 and the light chain variable region comprises the amino acid
sequence of SEQ ID NO: 94.
[175] In some embodiments, the antibody or antigen binding fragment comprseis
SEQ ID NOS: 29 and 30. In some embodiments, the antibody or antigen binding
fragment conjugated to a therapeutic agent may compete for binding or bind the
same epitope as this antibody defined by sequence.
[176] In some embodiments, the therapeutic agent may a passive immunotherapy
against amyloid beta or tau, or an inhibitor of acetylcholinestarase. In some
embodiments, the therapeutic agent may be selected from one or more of:,
CAD106,
Gantenerumab, Crenezumab, IVIG, AADvac1, ACI-35, NI05-15, CHF-5074, MK-
8931, AZD 3293, LY33 14814, Elenbecestat, Tideglusib, Intranasal Humulin R,
Intransal glulisine, 5B742457 with donepezil, Azeliragon, Nivaldipine. See
Godyn et
al., Pharmacological Reports, 68:127-138, 2016. In some embodiments, the
therapeutic agent may be selected from:, Aducanumab, ALZT-OP1a + ALZT-OP1b,
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Aripiprazole, AVP-786, AZD3293 (LY3314814), Brexprprazole (OPC-34712),
CAD106, 0NP520, Elenbecestat, Insulin (humulin), Lumateperone, JNJ-54861911,
Methylphenidate, MK-4305 (suvorexant), Nabilone, Nilvadipine, Pioglitazone,
RVT-
101 (intepirdine), Sodium Oligo-mannurarate (GV-971), TRx0237, TTp488
(azeliragon), AADvac1, ABBV-8E12, AD-SVF cells, ANAVEX 2-73, Atomoxetine,
AVP-786, AZD0530 (saracatinib), BAC, BAN2401, Benfotiamine, B1409306,
Bryostatin 1, Candesartan, CB-AC-02, Cilostazol, CPC-201, CT1812, DA01B,
Dronabinol, E2609, Formoterol, hUCB-MSCs, JNJ-54861991, Levetiracetam,
Liraglutide, LY3202626, NewGam 10% IVIG, Nilotinib, ORM-12741, Pimavanserin,
Piromelatine, Posiphen, P0912, Probucol, Rasagiline, Riluzole, RVT-101,
S47445,
Sargramostim, Simvastatin + L-Arginine + Tetrahydrobiopterin (SLAT), STA-1,
SUVN-502, T-817 MA, Temisartan, UB-311, Valacyclovir, VX-745, Xanamema. See
Cummings et al., Alzheimer's disease drug development pipeline: 2017,
Alzheimer's
& Dementia, 3:367-384, 2017. In some embodiments, the anti-tau therapy
comprises
a small molecule or peptide vaccine therapy, or an anti-tau antibody therapy.
See
U.S. Patent No. 9,518,101, which is incorporated by reference in its entirety.
[177] In further embodiments, an immunoconjugate is provided having the
formula
(A)-(L)-(C), wherein: (A) is an antibody or antigen binding fragment thereof
disclosed
herein; (L) is an optional linker; and (C) is a second agent (e.g., a
detectable label or
a therapeutic agent for treating AD or another tauopathy); wherein the linker
(L) joins
(A) to (C). In some embodiments, (C) is a therapeutic agent, an imaging agent,
a
detectable agent, or a diagnostic agent. In some embodiments, these conjugates
are
referred to herein as antibody-drug-conjugates (ADCs).
[178] Optional linker (L), as used herein, may be present or absent. When
present,
(L) is a molecule that is used to join the (A) to (C). In some embodiments,
the linker
is capable of forming covalent bonds to both the antibody and to the second
agent.
Suitable linkers are well known to those of skill in the art and include, but
are not
limited to, straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or
peptide linkers. Where the antibody and second agent are polypeptides, the
linkers
may be joined to the constituent amino acids through their side groups (e.g.,
through
a disulfide linkage to cysteine). However, in another embodiment, the linkers
may be
joined to the alpha carbon amino and carboxyl groups of the terminal amino
acids.
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[179] In some circumstances, it is desirable to free the second agent from the
antibody when the immunoconjugate has reached its target site. Therefore, in
these
circumstances, immunoconjugates may comprise linkages which are cleavable in
the
vicinity of the target site. Cleavage of the linker to release the second
agent from the
antibody may be prompted by enzymatic activity or conditions to which the
immunoconjugate is subjected either inside the target cell or in the vicinity
of the
target site. In yet other embodiments, the linker unit is not cleavable and
the drug is
not released or is released, for example, by antibody degradation.
[180] A number of different reactions are available for covalent attachment of
drugs
and/or linkers to antibodies or antigen binding fragments thereof. This is
often
accomplished by reaction of the amino acid residues of the antibody molecule,
including the amine groups of lysine, the free carboxylic acid groups of
glutamic and
aspartic acid, the sulfhydryl groups of cysteine and various moieties of the
aromatic
amino acids. One of the most commonly used non-specific methods of covalent
attachment is the carbodiimide reaction to link a carboxy (or amino) group of
a
compound to amino (or carboxy) groups of the antibody. Additionally,
bifunctional
agents such as dialdehydes or imidoesters have been used to link the amino
group
of a compound to amino groups of an antibody molecule. Also available for
attachment of drugs to antibodies is the Schiff base reaction. This method
involves
the periodate oxidation of a drug that contains glycol or hydroxyl groups,
thus
forming an aldehyde which is then reacted with the binding agent. Attachment
occurs
via formation of a Schiff base with amino groups of the binding agent.
lsothiocyanates can also be used as coupling agents for covalently attaching
drugs
to binding agents.
[181] In some embodiments, the linker is cleavable by a cleaving agent that is
present in the intracellular environment (e.g., within a lysosome or endosome
or
caveolea). The linker can be, e.g., a peptidyl linker that is cleaved by an
intracellular
peptidase or protease enzyme, including, but not limited to, a lysosomal or
endosomal protease. See, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics
83:67-123. Other examples of such linkers are described, e.g., in U.S. Pat.
No.
6,214,345. In other embodiments, the cleavable linker is pH-sensitive, i.e.,
sensitive
to hydrolysis at certain pH values. Typically, the pH sensitive linker is
hydrolysable
under acidic conditions. For example, an acid-labile linker that is
hydrolysable in the
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lysosome (e.g., a hydrozone, semicarbazone, thiosemicarbazone, cis-aconitic
aminde, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.
Pat. Nos.
5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm.
Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) In
yet
other embodiments, the linker is cleavable under reducing conditions (e.g., a
disulfide linker). A variety of disulfide linkers are known in the art,
including, for
example, those that can be formed using SATA (N-succinimidyl-S-
acetylthioacetate),
SPDP (N succinimidy1-3-(2-pyridyldithio)propionate), SPDB (N-succinimidy1-3-(2-
pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-
alpha(2
pyridyl-dithio)toluene)-, SPDB and SMPT. (See, e.g., Thorpe et al., 1987,
Cancer
Res. 47:5924-5931; Wawrzynczak et al., In lmmunoconjugates: Antibody
Conjugates
in RadioImagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987.
See also U.S. Pat. No. 4,880,935.) In another embodiment, the linker is a
malonate
linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl
linker
(Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog
(Laur et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).
[182] In another embodiment, the linker unit is not cleavable and the drug is
released by antibody degradation. (See U.S. Publication No. 2005/0238649). In
one
embodiment, the linker is not substantially sensitive to the extracellular
environment.
As used herein, "not substantially sensitive to the extracellular
environment," in the
context of a linker, means that no more than about 20%, about 15%, about 10%,
about 5%, about 3%, or no more than 1% of the linkers, in a sample of antibody-
drug
conjugate compound, are cleaved when the antibody-drug conjugate compound
presents in an extracellular environment (e.g., in plasma). Whether a linker
is not
substantially sensitive to the extracellular environment can be determined,
for
example, by incubating with plasma the antibody-drug conjugate compound for a
predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and quantifying the
amount
of free drug present in the plasma.
[183] In some embodiments, (L) can also comprise a spacer group or a linkage
group such as polyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid
(EDTA) or diethylenetriaminepentaacetic acid (DPTA).
[184] In some embodiments, more than one second agent may be attached, either
directly or by a linker, to an antibody or antigen binding fragment disclosed
herein.
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In certain embodiments, the ratio of second agent to antibody can range on
average
from about 1:1 to about 1:8. See U.S. Pat No. 7,498,298. The loading
(drug/antibody ratio) of an ADC may be controlled in different ways. See
W02006/034488.
Nucleic Acids, Vectors, Host Cells
[185] Also disclosed are isolated nucleic acid(s) encoding at least one
variable
region of an immunoglobulin chain of any of the antibodies or antigen binding
fragments described herein. In certain embodiments, an isolated nucleic
acid(s)
encodes the antibody or antigen binding fragment of any one of the previously
described antibodies or antigen binding fragments. Other embodiments provide
an
isolated vector comprising the nucleic acids. Another embodiment includes an
isolated host cell comprising any of the nucleic acids or vectors.
[186] The polynucleotides or nucleic acids encoding the antibodies or antigen
binding fragments described herein can be, e.g., DNA, cDNA, RNA or
synthetically
produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule
comprising any of those polynucleotides either alone or in combination. In
some
embodiments, the polynucleotide is part of a vector. Such vectors can comprise
further genes such as marker genes and/or control elements, allowing for the
selection and/or expression of the vector in a suitable host cell and under
suitable
conditions.
[187] In some embodiments, the polynucleotide is operatively linked to one or
more
expression control sequences, allowing expression in prokaryotic or eukaryotic
cells.
Expression of the polynucleotide may comprise transcription of the
polynucleotide
into translatable mRNA. Regulatory elements ensuring expression in eukaryotic
cells, for example mammalian cells, are known to those skilled in the art.
They
usually comprise regulatory sequences ensuring initiation of transcription and
optionally poly-A signals ensuring termination of transcription and
stabilization of the
transcript. Additional regulatory elements can include transcriptional as well
as
translational enhancers, and/or naturally associated or heterologous promoter
regions.
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[188] In this respect, the person skilled in the art will appreciate that the
polynucleotides encoding at least the CDRs and/or variable domain of the light
and/or heavy chain can encode the variable domains of both immunoglobulin
chains
or only one. Likewise, the polynucleotides can be under the control of the
same
promoter or can be separately controlled for expression. Possible regulatory
elements permitting expression in prokaryotic host cells comprise, e.g., the
PL, lac,
trp or tac promoter in E. coli, and examples for regulatory elements
permitting
expression in eukaryotic host cells are the AOXI of GAL1 promoter in yeast or
the
CMV-, SV40-, RSV-promoter, CMV-enhancer, SV40-enhancer or a globin intron in
mammalian and other animal cells.
[189] Beside elements that are responsible for the initiation of
transcription, such
regulatory elements can also comprise transcription termination signals, such
as the
SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
Furthermore, depending on the expression system used, leader sequences capable
of directing the polypeptide to a cellular compartment or secreting it into
the medium
can be added to the coding sequence of the polynucleotides and are known in
the
art. When used, the leader sequence(s) may be assembled in appropriate phase
with translation, initiation and termination sequences, and optionally, a
leader
sequence capable of directing secretion of translated protein, or a portion
thereof,
into the periplasmic space or extracellular medium. In some embodiments, the
heterologous sequence can encode a fusion protein including a C- or N-terminal
identification peptide imparting desired characteristics, e.g., stabilization
or simplified
purification of expressed recombinant product. In this context, suitable
expression
vectors are known in the art, and include, without limitation, the Okayama-
Berg
cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, PcDNA1, PcDNA3
(lnvitrogen), and pSPORT1 (GIBCO BRL).
[190] In some embodiments, the expression control sequences can be eukaryotic
promoter systems in vectors capable of transforming or transfecting eukaryotic
host
cells, but control sequences for prokaryotic hosts can also be used. Once the
vector
has been incorporated into the appropriate host, the host is maintained under
conditions suitable for high level expression of nucleotide sequences, and, as
desired, the collection and purification of the immunoglobulin light chains,
heavy
chains, light/heavy dimers or intact antibodies, binding fragments or other
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immunoglobulin forms can follow. See, e.g., Beychok, Cells of lmmunoglobulin
Synthesis, Academic Press, N.Y., (1979).
[191] In various embodiments, vectors, particularly plasmids, cosmids,
viruses, and
bacteriophages can be used that comprise a polynucleotide encoding a variable
domain of an immunoglobulin chain of an antibody of the present disclosure;
optionally in combination with another polynucleotide that encodes the
variable
domain of the other immunoglobulin chain of an antibody of the present
disclosure.
In some embodiments, the vector is an expression vector and/or gene transfer
or
targeting vector. Expression vectors derived from viruses such as retrovirus,
vaccina
virus, adeno-associated virus, herpes virus, or bovine papilloma virus, can be
used
for delivery of the polynucleotides or vector of the present disclosure into
targeted
cell populations. Any methods that are known to those skilled in the art can
be used
to construct recombinant viral vectors. Alternatively, the polynucleotides and
vectors
provided by the invention can be reconstituted into liposomes for deliver to
target
cells. The vector(s) containing the polynucleotides of the present disclosure
(e.g., the
heavy and/or light variable domain(s) of the immunoglobulin chains encoding
sequences) can be transferred into a host cell by known methods, which vary
depending on the type of cellular host. For example, calcium chloride
transfection is
commonly utilized for prokaryotic cells, whereas calcium phosphate treatment
or
electroporation can be used for other cellular hosts.
[192] In some embodiments, a host cell is transformed with a polynucleotide or
vector described herein. The host cell can be a prokaryotic or eukaryotic
cell. The
poly nucleotide or vector that is present in the host cell can either be
integrated into
the genome of the host cell or it can be maintained extrachromosomally. The
host
cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect,
fungal,
plant, animal or human cell. Preferred fungal cells are, for example, those of
the
genus Saccharomyces, such as S. cerevisiae. Depending upon the host employed
in
a recombinant production procedure, the antibodies or antigen binging
fragments
thereof encoded by the polynucleotide can be glycosylated. Certain antibodies
or
antigen binding fragments thereof consistent with the present disclosure can
also
include an initial methionine amino acid residue. A polynucleotide disclosed
herein
can be used to transform or transfect the host using any of the techniques
commonly
known to those of ordinary skill in the art. Furthermore, methods for
preparing fused,
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operably linked genes and expressing them in, e.g., mammalian cells and
bacteria
are well-known in the art. In general, expression vectors containing promoter
sequences which facilitate the efficient transcription of the inserted
polynucleotide
are use in connection with a host. The expression vector typically contains an
origin
of replication, a promoter, and a terminator, as well as specific genes which
are
capable of providing phenotypic selection of the transformed cells. Suitable
source
cells for DNA sequences and host cells for immunoglobulin expression and
secretion
can be obtained from a number of sources, such as the American Type Culture
Collection ("Catalogue of Cell Lines and Hybridomas," Fifth edition (1985)
Manassas,
Va., U.S.A., and other available version, incorporated herein by reference).
Furthermore, transgenic animals, for example mammals, comprising cells of the
invention can be used for large scale production of the antibodies or antigen
binding
fragments disclosed herein.
[193] In another embodiment, a hybridoma producing antibody DC2E7 is
disclosed.
The hybridoma has been deposited at the American Type Culture Collection at
Patent Deposit No. PTA-124992.
[194] In another embodiment, a hybridoma producing antibody DC2E2 is
disclosed.
The hybridoma has been deposited at the American Type Culture Collection at
Patent Deposit No. PTA-124991.
Compositions, Formulations and Combinations
[195] In some exemplary embodiments, an antibody or antigen binding
fragment thereof described herein may be co-formulated and/or co-administered
with
one or more additional compounds that are also useful in the detection,
prevention,
and/or treatment of AD or another tauopathy.
[196] In some embodiments, an antibody or antigen binding fragment thereof
described herein is formulated for use in an assay to detect phosphorylated
tau in a
biologic sample from a human subject (e.g., CSF or blood). In certain
embodiments,
the antibody or antigen binding fragment thereof is conjugated to a detectable
label,
e.g., an enzyme, a radioisotope, a fluorophore, a nuclear magnetic resonance
marker, or a heavy metal. In some embodiments, two or more (e.g., 3, 4, 5,
etc.)
antibodies or antigen binding fragments described herein are formulated
suitable for
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use in the assay. In certain embodiments, the antibodies or antigen binding
fragments comprise antibody clones DC2E7 or DC2E2 and/or antibodies or
fragments comprising the CDRs or variable domains from those clones, alone or
conjugated to suitable detectable labels. In some embodiments, the
formulations
further comprise additional elements and reagents (e.g., solid supports or
particles
such as magnetic beads) suitable for use in diagnostic assays such as the
classic
and digital ELISA assays described below.
[197] In some embodiments, the antibodies and antigen binding fragments
described herein are prepared for use in methods of detecting phosphorylated
tau in
a biologic sample. For instance, the antibodies can be prepared and formulated
as
discussed in Example 3 below. In some embodiments, the antibodies or antigen
binding fragments, e.g., DC2E7, DC149, D0807, and/or DC2E2 or antigen binding
fragments or variants thereof, may be purified from serum-free hybridoma
supernatant, for example using a Protein G affinity column. The purified
supernatant
can then be eluted, e.g, with 0.1 M Glycine-HCI, pH 2.7 and neutralized with
1M Tris-
HCI pH 9Ø Pooled fractions can be diazlied in a buffer solution such as
phosphate
buffered saline (PBS). In some embodiments, the buffered antibody solution can
be
concentrated, e.g., by ultrafiltration. In some embodiments, antibody
concentration
is determined by measuring absorbance at 280 nm, using the formula c(mg/m1) =
A280nm/1.43.
[198] DC2E7 or an antigen binding fragment, when used as the capture
antibody in the digital ELISA, may be prepared in a suitable buffer solution.
In some
embodiments, DC2E7 or an antigen binding fragment is immobilized on a solid
support (e.g., a solid surface or ELISA capture bead). In some embodiments,
DC2E7 or an antigen binding fragment is joined to a solid surface, e.g., as
described
according to Example 10 below. Briefly, DC2E7 may be coupled to a bead, e.g, a
magnetic bead (Quanterix). In some embodiments, DC2E7 is coupled to the bead
at
a concentration of about 0.1-5.0 mg/mL (e.g., about 1.0 mg/mL). In some
embodiments, DC2E2 or an antigen binding fragment is used as a detector
antibody
and may be conjugated to a detectable label. For instance, the antibody or
antigen
binding fragment may be biotinylated for detection purposes, e.g., by exposure
to 1-
200 fold (e.g., about 120 fold) excess of biotin relative to the antibody
concentration.
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[199] In some embodiments, an antibody or antigen binding fragment thereof
described herein may be co-formulated and/or co-administered with a detectable
label. In some embodiments, the detectable label is an enzyme, a radioisotope,
a
fluorophore, a nuclear magnetic resonance marker, or a heavy metal. In some
embodiments, the detectable label is in association (e.g., covalent or non-
covalent)
association with the antibody or antigen binding fragment. Suitable additives
and
formulation conditions for antibody administration may be used, e.g., those
known in
the art for formulating antibodies for parenteral administration (e.g.,
intravenous,
subcutaneous, intraperitoneal, intramuscular), or intravenous, intramuscular
or
subcutaneous injection.
[200] In some embodiments, an antibody or antigen binding fragment thereof
is formulated in combination with additional compounds that are also useful in
the
prevention and/or treatment of AD or another tauopathy. These include, without
limitation, compounds that are useful in active and passive immunotherapies
for AD,
such as beta-amyloid peptides (e.g., N-terminal amyloid beta peptides) and tau
peptides which might or might not be conjugated to other compounds, such as
mutated diphtheria toxin, KLH or other carriers. Other options include
antibodies
against beta-amyloid, such as bapineuzumab, solaneuzumab, gantenerumab,
crenezumab, ponezumab, and IVIG immunoglobulin, other immunization therapies
targeting Abeta oligomers, other tau antibodies, compounds preventing the
hyperphosphorylation of tau, and other active and passive immunization
therapies
targeting tau aggregates.
[201] Other drugs that may be helpful in combination therapy with the
antibodies and tau-binding fragments described herein include amyloid-beta
aggregation inhibitors (e.g., Tramiprosate), gamma-secretase inhibitors (e.g.,
semagacestat), and gamma-secretase modulators (tarenflurbil). Furthermore, one
or
more of the novel antibodies disclosed herein may be used or formulated in
combination with two or more of the foregoing therapeutic agents. At early
stages of
the disease, combination therapies can be advantageous. Combination therapies
are
also advantageous at later stages of the disease, such as combination of hAb
and
growth factors and other biologically active molecules inducing neuronal
plasticity
and regeneration. Such combination therapies may advantageously utilize lower
dosages of the administered therapeutic agents, thus avoiding possible
toxicities or
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complications associated with the various monotherapies. According to a
related
embodiment, an antibody or antigen binding fragment thereof described herein
can
be used in combination with at least one combination agent chosen from
acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine, galantamine,
tacrine,
nutritive supplements), N-Methyl-D-aspartate (NMDA) receptor antagonists
(e.g.,
memantine), inhibitors of DNA repair (e.g., pirenzepine or a metabolite
thereof),
transition metal chelators, growth factors, hormones, non-steroidal anti-
inflammatory
drugs (NSAID), antioxidants, lipid lowering agents, selective
phosphodiesterase
inhibitors, inhibitors of tau aggregation, inhibitors of protein kinases,
inhibitors of anti-
mitochondrial dysfunction drugs, neurotrophins, inhibitors of heat shock
proteins,
inhibitors of Lipoprotein-associated phospholipase A2, and any
pharmaceutically
acceptable salts thereof. In one embodiment, a disclosed antibody and/or tau-
binding fragment thereof is combined with a cholinesterase inhibitor (ChEl)
and/or
memantine. In one embodiment, the combination agent is selected from the group
consisting of an anti-apoptotic compound, a metal chelator, an inhibitor of
DNA
repair, 3-amino-I -propanesulfonic acid (3APS), 1,3-propanedisulfonate (1
,3PDS), a
secretase activator, a beta-secretase inhibitor, a gamma-secretase inhibitor,
a beta-
amyloid peptide, a beta-amyloid antibody, a neurotransmitter, a beta-sheet
breaker,
an anti-inflammatory molecule, and a cholinesterase inhibitor. In one
embodiment,
the cholinesterase inhibitor is, rivastigmine, donepezil, galantamine, or a
nutritive
supplement. In another embodiment, the additional agent is selected from BACE
inhibitors; muscarinic antagonists; cholinesterase inhibitors; gamma secretase
inhibitors; gamma secretase modulators; HMG-CoA reductase inhibitors;
nonsteroidal anti-inflammatory agents; N-methyl-D-aspartate receptor
antagonists;
anti-amyloid antibodies; vitamin E; nicotinic acetylcholine receptor agonists;
CBI
receptor inverse agonists or CBI receptor antagonists; an antibiotic; growth
hormone
secretagogues; histamine H3 antagonists; AMPA agonists; PDE4 inhibitors; GABAA
inverse agonists; inhibitors of amyloid aggregation; glycogen synthase kinase
beta
inhibitors; promotors of alpha secretase activity; PDE-I 0 inhibitors and
cholesterol
absorption inhibitors.
[202] Other compounds that may be used in combination with the antibody and
antigen binding fragment described herein include the therapeutic antibodies
and
peptides described in U.S. 9,518,101, which is incorporated herein by
reference in its
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entirety. Also useful are the compounds described in WO 2004/058258, which is
incorporated herein by reference in its entirety (see especially pages 16 and
17)
including therapeutic drug targets (page 36-39), alkanesulfonic acids and
alkanolsulfuric acids (pages 39-51), cholinesterase inhibitors (pages 51-56),
NMDA
receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal
anti-
inflammatory drugs (pages 60-61), antioxidants (pages 61-62), peroxisome
proliferators-activated receptor (PPAR) agonists (pages 63-67), cholesterol-
lowering
agents (pages 68-75); amyloid inhibitors (pages 75-77), amyloid formation
inhibitors
(pages 77-78), metal chelators (pages 78-79), antipsychotics and anti-
depressants
(pages 80-82), nutritional supplements (pages 83- 89) and compounds increasing
the availability of biologically active substances in the brain (see pages 89-
93) and
prodrugs (pages 93 and 94). Other compounds that may be used in combinations
include those described in Cummings et al., Alzheimer's disease drug
development
pipeline: 2017, Alzheimer's & Dementia: Translational Research & Clinical
Interventions 3 (2017) 367-384.
[203] Also provided herein are compositions comprising an antibody or an
antigen
binding fragment thereof, as described herein, and another component, such as
a
carrier. Also provided are compositions/formulations comprising a humanized
antibody or antigen binding fragment thereof, as described herein, and a
carrier, e.g.,
a carrier suitable for diagnostic or therapeutic uses.
[204] In one embodiment, formulations and compositions of the antibodies used
in
accordance with the present disclosure are prepared for storage and/or
administration by mixing an antibody or antigen binding fragment thereof
having the
desired degree of purity with optional carriers, e.g., pharmaceutically
acceptable
carriers, diluents, excipients or stabilizers (Remington's Pharmaceutical
Sciences
21st edition, Mohr, M. Ed. (2006)), in the form of lyophilized formulations or
aqueous
solutions. In one embodiment, acceptable carriers, excipients, or stabilizers
are
nontoxic to recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids; antioxidants
including
ascorbic acid and methionine; preservatives (such as octadecyldimethylbezyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium
chloride, phenol, butyl or benyl alcholol; alkyl parabens such as methyl or
propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
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molecular weight (less than about 10 residues) polypeptides; proteins, such as
serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
histidine,
arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions
such as
sodium, metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants
such as TWEEN, PLURONICS or polyethylene glycol (PEG). Examples of
lyophilized antibody formulations are described in WO 97/04801, expressly
incorporated herein by reference.
[205] In one embodiment, the antibodies and antigen binding fragments thereof
can
be incorporated into pharmaceutical compositions suitable for administration
to a
subject. In certain embodiments, the pharmaceutical composition comprises an
antibody or antigen binding fragment thereof as disclosed herein and a
pharmaceutically acceptable carrier. In one embodiment, "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the
like that are physiologically compatible. Additional examples of
pharmaceutically
acceptable carriers include one or more of water, saline, phosphate buffered
saline,
dextrose, glycerol, ethanol and the like, as well as combinations thereof. In
many
cases, the composition will include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary substances
such as wetting or emulsifying agents, preservatives or buffers, which enhance
the
shelf life or effectiveness of the antibody or antigen binding fragments
thereof.
[206] The compositions described herein, comprising the disclosed antibodies
or
antigen binding fragments, may be in a variety of forms. These include, for
example,
liquid, semi-liquid, semi-solid and solid dosage forms, such as liquid
solutions (e.g.,
injectable and infusible solutions), dispersions or suspensions, tablets,
pills,
powders, liposomes and suppositories. In some embodiments, such compositions
may also comprise buffers (e.g., neutral buffered saline or phosphate buffered
saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol,
proteins, polypeptides or amino acids such as glycine, antioxidants, chelating
agents
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such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or
preservatives. Alternatively, compositions of the present invention may be
formulated
as a lyophilizate. Antibodies and antigen binding fragments thereof may also
be
encapsulated within liposomes.
[207] Dosage forms suitable for use in diagnostic assays or for internal
administration generally contain from about 0.1 milligram to about 500
milligrams of
antibody or antigen binding fragment thereof per unit or container. In these
compositions, the active ingredient will ordinarily be present in an amount of
about
0.5-99.999% by weight based on total weight of the composition. The preferred
dosage form depends on the intended use and/or mode of administration.
[208] In certain embodiments, the antibodies or antigen binding fragments
disclosed
herein can traverse the blood-brain barrier or are formulated to traverse the
blood-
brain barrier. Certain neurodegenerative diseases, including AD and related
tauopathies, are associated with an increase in permeability of the blood-
brain
barrier, such that the antibody or antigen binding fragment thereof can be
readily
introduced into the brain. When the blood-brain barrier remains intact,
several art-
known approaches exist for transporting molecules across it, including, but
not
limited to, physical methods, lipid-based methods, and receptor and channel-
based
methods. Methods for circumventing the blood-brain barrier include, but are
not
limited to, direct injection into the brain (see, e.g., Papanastassiou et al.,
Gene
Therapy 9: 398-406 (2002)) and implanting a delivery device in the brain (see,
e.g.,
Gill et al., Nature Med. 9: 589-595 (2003): and Gliadel Wafers, T.M.,
Guildford
Pharmaceutical). Methods of creating openings in the barrier include, but are
not
limited to, ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086),
osmotic
pressure (e.g., by administration of hypertonic mannitol (Neuwelt, E. A.,
Implication
of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press,
N.Y.
(1989))), permeabilization by, e.g., bradykinin or permeabilizer A-7 (see,
e.g., U.S.
Pat. Nos. 5,112,596,5,268,164, 5,506,206, and 5,686,416), and transfection of
neurons that straddle the blood-brain barrier with vectors containing genes
encoding
the antibody or antigen binding fragment thereof (see, e.g., U.S. Patent
Publication
No. 2003/0083299). Lipid-based methods of transporting the antibody or antigen
binding fragment thereof across the blood-brain barrier include, but are not
limited to,
encapsulating the antibody or antigen binding fragment thereof in liposomes
that are
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coupled to active fragments thereof that bind to receptors on the vascular
endothelium of the blood-brain barrier (see, e.g., U.S. Patent Application
Publication
No. 20020025313), and coating the antibody or antigen binding fragment thereof
in
low-density lipoprotein particles (see, e.g., U.S. Patent Application
Publication No.
20040204354) or apolipoprotein E (see, e.g., U.S. Patent Application
Publication No.
20040131692).
[209] In various embodiments, a composition can comprise any one or more
antibodies or antigen binding fragments described herein and a carrier and/or
diluent. In some embodiments, the antibody or antigen binding fragment
comprises
a heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region
comprises three light chain complementarity determining regions (LCDR1, LCDR2,
and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:
1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3
comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6. In some embodiments, the antibody or antigen binding fragment
comprises any one or more (e.g., all six CDRs and/or a heavy and/or light
chain
variable domain) sequence selected fro those shown in Table 1. In some
embodiments, the antibody or antigen binding fragment comprises any of the
sequences shown in table 1, e.g., a set of six CDRs and or paired heavy and
light
chain variable domain sequences listed in Table 1. In some embodiments, the
antibody or antigen binding fragment comprseis SEQ ID NOS: 29 and 30. In some
embodiments, the composition is suitable for use in a diagnostic assay, e.g.,
classic
or digital ELISA. In some embodiments, the composition is a pharmaceutical
composition and comprises a pharmaceutically-acceptable carrier.
[210] In certain embodiments, a composition comprises any two antibodies or
antigen binding fragments as previously described. In certain embodiments, a
composition comprises at least one further antibody or antigen binding
fragment,
and/or at least one additional agent. In an exemplary embodiment, one antibody
or
antigen binding fragment comprises a heavy chain variable region and a light
chain
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variable region, wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the
light chain variable region comprises three light chain complementarity
determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6; and the other antibody or antigen binding
fragment comprises a heavy chain variable region and a light chain variable
region,
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 15, HCDR2 comprises the amino acid sequence of SEQ
ID NO: 16, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 17; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 18, LCDR2
comprises the amino acid sequence of SEQ ID NO: 19, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 20.
[211] In some embodiments, the first antibody or antigen binding fragment
comprises any one or more (e.g., all six CDRs and/or a heavy and/or light
chain
variable domain) sequence selected fro those shown in Table 1. In some
embodiments, the antibody or antigen binding fragment comprises any of the
sequences shown in table 1, e.g., a set of six CDRs and or paired heavy and
light
chain variable domain sequences listed in Table 1. In some embodiments, the
antibody or antigen binding fragment comprseis SEQ ID NOS: 29 and 30. In some
embodiments, the second antibody or antigen binding fragment comprises any one
or more (e.g., all six CDRs and/or a heavy and/or light chain variable domain)
sequence selected fro those shown in Table 1. In some embodiments, the
antibody
or antigen binding fragment comprises any of the sequences shown in table 1,
e.g.,
a set of six CDRs and or paired heavy and light chain variable domain
sequences
listed in Table 1. In some embodiments, the antibody or antigen binding
fragment
comprseis SEQ ID NOS: 29 and 30.
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[212] In some embodiments, the composition is suitable for use in a diagnostic
assay, e.g., classic or digital ELISA. In some embodiments, the composition is
a
pharmaceutical composition and comprises a pharmaceutically-acceptable
carrier.
The pharmaceutical composition may further comprise at least one additional
therapeutic agent for treating Alzheimer's disease or another tauopathy.
Methods of Treatment
[213] The antibodies and compositions described herein can be used for various
methods of treatment and prevention of AD and other tauopathies. In some
embodiments, the antibodies and compositions can be used to detect patients
suitable for anti-tau based treatments, to inform treatment selection, or to
monitor the
progress of treatment over time. In other embodiments, the antibodies and
compositions disclosed herein can be used as direct treatments or preventions
for
AD and other tauopathies by administering one or more antibodies or
compositions
to a patient in need thereof.
[214] In preventative (i.e., prophylactic) applications, the antibodies and
pharmaceutical compositions disclosed herein (e.g., antibody DC2E7 or an
antibody
or antigen binding fragment that can bind the same epitope as, or comprises
the
CDRs or variable domains from, antibody DC2E7) may be administered to a
patient
susceptible to, or otherwise at risk of, Alzheimer's disease or another
tauopathy. In
some embodiments, the antibody or pharmaceutical composition is administered
in
an amount sufficient to eliminate or reduce the risk, lessen the severity, or
delay the
outset of the disease, including biochemical, histologic and/or behavioral
symptoms
of the disease, its complications and intermediate pathological phenotypes
presenting during development of the disease. In therapeutic applications,
compositions are administered to a patient suspected of, diagnosed with,
and/or
already suffering from such a disease in an amount sufficient to cure, or at
least
partially reduce, arrest, or reverse the symptoms of the disease (biochemical,
histologic, and/or behavioral), including its complications and intermediate
pathological phenotypes in development of the disease.
[215] In some embodiments, treating Alzheimer's disease refers to decreasing
or
preventing behavioral, functional, and cognitive deterioration over time. In
some
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embodiments, behavioral, functional, and cognitive aspects of Alzheimer's
Disease
can be evaluated by any one or more of a series of standardized tests known to
persons of ordinary skill in the art including, but not limited to,
neuropsychological
testing, the Mini-Mental State Exam, Mini-cog exam, Neuropsychiatric
Inventory,
Blessed Roth Dementia Rating Scale, Spanish and English Neuropsychoiogical
Assessment Scales (SENAS), Psychiatric Behavioral Assessment, Functional
Assessment, Clock Drawing Test, Boston Naming Test. California Verbal Learning
Test, Cognitive Symptoms Checklist, Continuous Performance Test, Controlled
Oral
Word Association Test, Cognistat, d2 Test of Attention, Delis-Kaplan Executive
Function System, Dementia Rating Scale, Digit Vigilance Test, Figural Fluency
Test,
Finger Tapping Test, Halstead Category Test, Halstead-Reitan
Neuropsychological
Battery, Hooper Visual Organization Test, Kaplan Baycrest Neurocognitive
Assessment, Kaufman Short Neuropsychological Assessment, Luria-Nebraska
Neuropsychological Battery, Memory Assessment Scales, Quick Neurological
Screening Test, Repeatable Battery for the Assessment of Neuropsychological
Status, Stroop Test, Symbol Digit Modalities Test, Tactual Performance Test,
Thematic Apperception Test, Tower of London, Trail Making Tests A and B,
Verbal
(Word) Fluency Tests, and Wisconsin Card Sort Test. Additional tests for
depression, anxiety, aphasia, agitation, and behavioral parameters known to
persons
of ordinary skill in the art are also used. In addition, in some embodiments,
patients
may be diagnosed or monitored for treatment efficacy using any of the
detection
methods disclosed herein involving one or more of the disclosed antibodies or
antigen binding fragments thereof.
[216] In some embodiments, the antibodies and pharmaceutical compositions
disclosed herein (e.g., antibody DC2E7 or an antibody or antigen binding
fragment
that can bind the same epitope as, or comprises the CDRs or variable domains
from,
antibody DC2E7) may be administered to treat AD or another tauopathy in a
patient.
In some embodiments, the efficacy of an Alzheimer's disease treatment is
determined by the improvement, or lack of deterioration, or a reduction in the
rate of
deterioration in at least one assessment selected from the group consisting of
Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog), the
Clinical
Dementia Rating Sum of Boxes (CDR-sb), the Alzheimer's Disease Cooperative
Study Activities of Daily Living Scale (ADCS-ADL), the Neuropsychiatric
inventory
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(NPI), the Progressive Deterioration Scale (PDS), Amsterdam Instrumental
Activities
of Daily Living (IADL), the Clinical Dementia Rating Scale (CDR), the
Disability
Assessment for Dementia Scale (DAD), and the Mini-Mental State Evaluation
(MMSE). In some embodiments, the treatment results in a reduction in the rate
of
deterioration in ADAS-cog scores. In other embodiments, the treatment results
in a
median reduction in the rate of deterioration of ADAS-cog scores of two to
five
points.
[217] In some embodiments, a method of delaying the progression of Alzheimer's
disease is provided, comprising administering one or more of the disclosed
antibodies, e.g., antibody DC2E7 or an antibody or antigen binding fragment
that
binds the same epitope as, or comprises the CDRs or variable domains from,
antibody DC2E7. In one embodiment, "delaying" progression of AD means to
defer,
hinder, slow, retard, stabilize, and/or postpone development of the disease.
This
delay can be for a varying length of time, depending on the history of the
disease
and/or individual being treated. As is evident to one skilled in the art, a
sufficient or
significant delay can, in effect, encompass prevention, in that the individual
does not
develop the disease. In one embodiment, a method that "delays" progression of
AD
or another tauopathy is a method that reduces the probability of disease
development in a given time frame and/or reduces extent of the disease in a
given
time frame, when compared to not using the method. Such comparisons are
typically
based on clinical studies, using a statistically significant number of
subjects.
[218] In certain embodiments, AD or another tauopathy may be delayed by days,
months, or years. For example, the method may delay progression of AD or
another
tauopathy by one or more weeks, months, or years.
[219] Patients, subjects, or individuals include mammals, such as human,
bovine,
equine, canine, feline, porcine, and ovine animals. The subject may be a
human, and
may or may not be afflicted with disease or presently show symptoms. In the
case of
AD, virtually anyone is at risk of suffering from AD if he or she lives long
enough.
Therefore, the present methods may be administered prophylactically to the
general
population without the need for any assessment of the risk of the subject
patient. In
other embodiments, the subject is assessed for AD using a detection method
disclosed herein.
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[220] In one embodiment, the patient herein is optionally subjected to a
diagnostic
test prior to therapy, which may include the diagnostic methods disclosed
herein. In
one embodiment, the disclosed methods are useful for individuals who have a
known
genetic risk of AD. Such individuals include those having relatives who have
experienced this disease and those whose risk is determined by analysis of
genetic
or biochemical markers. Genetic markers of risk towards AD include mutations
in the
APP gene, particularly mutations at position 717 and positions 670 and 671
referred
to as the Hardy and Swedish mutations, respectively. See Hardy (1997) Trends
Neurosci. 20:154-9). Other markers of risk are mutations in the presenilin
genes,
P51 PS2, and ApoE4, family history of AD, hypercholesterolemia or
atherosclerosis.
Individuals presently suffering from AD may also be identified from behavioral
characteristics. In asymptomatic patients, treatment can begin at any age
(e.g., 10,
20, 30). In some patients, it may not be necessary to begin treatment and/or
monitoring until a patient reaches an older age, e.g., 40, 50, 60, or 70, or
later, or
any time period in between. Treatment may entail multiple dosages over a
period of
time. Treatment can be monitored in various ways, including by using the AD
detection methods disclosed herein.
[221] Periodic use of one or more of these tests can advise a physician or
other
medical professional as to the progression, or regression of Alzheimer's
disease and
related tauopathies and the need for further treatment. The choice of test and
the
determination of success of treatment are within the expertise of medical
professionals in the Alzheimer's disease field. An improved score in one or
more
tests is an indication of decrease in severity of AD in that subject.
[222] In various embodiments, a method of treating, delaying progression, or
preventing the progression of Alzheimer's disease of another tauopathy in a
subject
is disclosed, comprising administering to the subject an effective amount of
at least
one antibody or antigen binding fragment described herein. This method may
result
in reducing motor impairment, improving motor function, reducing cognitive
impairment, improving cognitive function, or a combination thereof. The use of
any
antibody described herein may be used in treating, delaying progression, or
preventing Alzheimer's disease by administering the antibody or antigen
binding
fragment to a subject in need thereof.
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Methods of detecting and monitoring progression of AD and other tauopathies
[223] The disclosure herein focuses, in part, on the discovery of certain
epitope
residues on tau, particularly those containing one or more phosphorylated
resiudes
on tau, that can be detected in certain biological samples (e.g., blood or
CSF) and
used to identify and distinguish AD, other tauopathies, and other forms of
dementia.
For example, the useful epitope residues may comprise one or more of residues
188-227 of tau protein 2N4R (SEQ ID NO: 10). In certain embodiments, the
epitope
comprises one or more of residues 210-221 of tau protein 2N4R (SEQ ID NO: 11).
In
certain embodiments, the epitope comprises at least one phosphorylated
residue,
e.g., phospho-threonine at position 217 of tau protein 2N4R (SEQ ID NO: 9),
and
optionally also comprises a phosphorylated serine at position 210, threonine
at
position 212, serine at position 214, or threonine at position 220 of tau
protein 2N4R,
or any combination thereof. In another embodiment, the epitope comprises or
consists of SRTPSLPpTPPTR (sequence of SEQ ID NO: 12) or that stretch of amino
acid residues with one or more additional phophorylated positions in it. In
another
embodiment, the epitope comprises or consists of SRpTPSLPpTPPTR (sequence of
SEQ ID NO: 31). The disclosure is based in part on the surprising finding that
the
amount of phosphorylated tau species in these samples can be used to
distinguish
AD from other tauopathies and from subjects with other forms of dementia, and
thereby diagnose, monitor, and/or guide treatment decisions for AD or for
another
tauopathy or for another cause of dementia based on the results of the assay.
Without being bound by theory, the discovery that the level of these
phosphorylated
epitopes (e.g., at position 217) can be used to distinguish AD, other
tauopathies, and
other forms of dementia in certain samples is particularly unexpected given
the fact
that the phosphorylated epitopes can often be detected in the brain across
different
tauopathies.
[224] The disclosure provided herein also focuses, in part, on antibodies
(e.g., any
antibodies comprising one or more sequences shown in Table 1, e.g., all six
CDRs
and/or a heavy and/or light chain variable domain selected from those shown in
Table 1) that are capable of binding to these particular phosphorylated tau
epitopes
in certain types of biological samples (e.g., CSF or blood) and are
particularly useful
for identifying and distinguishing AD, other tauopathies, and other forms of
dementia
based on the level of binding in the samples. The disclosure is based in part
on the
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surprising finding that the amount of phosphorylated tau species bound by the
disclosed antibodies in these samples can be used to distinguish AD from other
tauopathies and from subjects with other forms of dementia, and thereby
diagnose,
monitor, and/or guide treatment decisions for AD or for another tauopathy or
for
another cause of dementia based on the results of the assay.
[225] In addition to AD, other tauopathies are known in the art, including
frontotemporal dementia (FTD), corticobasal disease (CBD), and progressive
supranuclear palsy (PSP). Murray et al., Clinicopathologic assessment and
imaging
of tauopathies in neurodegenerative dementias, Alzheimer's Research & Therapy,
6:1, 2014. Numerous other causes of dementia are also known. Without being
bound
by theory, the elevated amounts of the phosphorylated tau species in certain
biological samples from AD patients and patients with other tauopathies (e.g.,
CSF
or blood) that are bound by the antibodies or antigen binding fragments
disclosed
herein may contribute to their usefulness in non-invasively detecting and
distinguishing AD from other causes of dementia or other tauopathies in these
bodily
fluids.
[226] Numerous tau species are present in certain types of samples (e.g.,
blood
and/or CSF) from both healthy subjects and those with dementia. Despite these
many potential targets, antibodies that can bind to particular tau species in
these
samples such that they can provide diagnostic power (e.g., antibodies that
bind to
tau species only present in samples from subject with AD and/or other
tauopathies or
present in samples from those subjects at elevated and/or distinct levels)
have not
previously been identified. Without being bound by theory, in some embodiments
an
antibody or antigen binding fragment disclosed herein provides an improvement
over
the art because of its ability to bind a phosphorylated epitope on tau that is
present in
certain types of samples (e.g., blood or CSF) from a patient with AD at a
greater
level than in a comparable sample from a patient with another tauopathy, other
neurologic disease, or in a healthy subject. This difference can be used, in
some
embodiments, to detect whether a subject presenting with dementia has AD,
another
tauopathy, or another cause of dementia, and/or to monitor the course of AD
treatment. For example, distinct thresholds or fold differences in the amount
of tau
bound by the antibody (e.g., as compared to levels in healthy subjects or as
compared to subjects with known causes of dementia) can be detected and
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correlated with AD, another tauopathy, or another cause of dementia in a
subject. In
some embodiments, this difference can be used to detect whether a subject
presenting with dementia has AD or another tauopathy. In some embodiments,
this
difference can be used to detect whether a subject presenting with dementia
has AD
or some other cause of dementia. In some embodiments, this difference can be
used
to distinguish whether a subject presenting with dementia has AD or another
tauopathy. In some embodiments, this difference can be used to distinguish
whether
a subject presenting with dementia has AD or some other cause of dementia.
[227] In various embodiment, a method of detecting a tauopathy in a subject is
disclosed, comprising: obtaining a biological sample from the subject;
contacting the
sample from the subject with an effective amount of a molecule that is capable
of
forming a tau-molecule complex (e.g., at least one receptor, antibody, or
antigen
binding fragment disclosed hererin that is capable of binding tau to form a
tau-
antibody complex); detecting the presence and/or amount of the tau-molecule
complex using an antibody or antigen binding fragment disclosed herein;
wherein the
presence and/or amount of tau-molecule complex indicates a tauopathy in the
subject. In various embodiments a method of detecting a tauopathy in a subject
comprises: contacting a biological sample from the subject with an effective
amount
of at least one antibody or antigen binding fragment disclosed herein that is
capable
of binding tau to form a tau-antibody complex, wherein the presence and/or
amount
of tau-antibody complex indicates a tauopathy in the subject. In some
embodiments,
detection is by an immunomagnetic reduction bio-assay, e.g., using a bioassay
device from MagQu Co. Ltd.
[228] In some embodiments, the tau detected in the sample is a phosphorylated
tau. In various embodiments, the at least one antibody or antigen binding
fragment
can bind an epitope on tau comprising one or more of residues 188-227 of tau
protein 2N4R (SEQ ID NO:10). In various embodiments, the at least one antibody
or
antigen binding fragment can bind an epitope on tau comprising one or more of
residues 210-221 of tau protein 2N4R (SEQ ID NO:11). In some embodiments, the
antibody or antigen binding fragments used in the methods comprises a heavy
chain
variable region and a light chain variable region, wherein the heavy chain
variable
region comprises three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), and the light chain variable region comprises three light
chain
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complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein
HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises
the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid
sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.
[229] In certain embodiments, the antibody or antigen binding fragment used in
the
methods comprise a heavy chain variable region and a light chain variable
region,
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1 with a substitution at position 2, HCDR2 comprises
the
amino acid sequence of SEQ ID NO: 2 with a substitution at one or more of
position
2 and 12, and/or HCDR3 comprises the amino acid sequence of SEQ ID NO: 3,
and/or wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 with a
substitution at position 2, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 5, and/or LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In
various embodiments, the the substitution at position 2 in HCDR1 is glycine,
the
substitution at position 2 in HCDR2 is isoleucine, the substitution at
position 12 in
HCDR2 is valine, and/or the substitution at position 2 in LCDR1 is asparagine.
[230] In various embodiments, the antibody or antigen binding fragment
comprises
any of the sequences shown in table 1, e.g., a set of six CDRs and or paired
heavy
and light chain variable domain sequences listed in Table 1.
[231] In certain embodiments, the antibody or antigen binding fragment used in
the
methods comprise a heavy chain variable region and a light chain variable
region,
wherein the heavy chain variable region comprises an amino acid sequence of
SEQ
ID NO: 7 and the light chain variable region comprises an amino acid sequence
of
SEQ ID NO: 8. The antibody or antigen binding fragment used in the disclosed
methods may, in various embodiments, comprise DC2E7 or an antigen binding
fragment thereof or any of the variants disclosed herein that are capable of
binding
to tau. In various embodiments, the antigen or antigen binding fragment
further
comprises a detectable label.
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[232] In some embodiments, the heavy chain variable region comprises the amino
acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 with a substitution at one or
more
of position 1, 2, 3, 9, 12, 19, 30, 31, 35, 37, 42, 43, 48, 49, 51, 54, 55,
56, 58, 62, 63,
64, 65, 66, 68, 69, 70, 73, 76, 77, 78, 79, 80, 83, 84, 88, 94, 96, 107, 108,
and 112,
and/or the light chain variable region comprises the amino acid sequence of
SEQ ID
NO: 8 or SEQ ID NO: 8 with a substitution at one or more of position 3, 7, 11,
14, 17,
19, 20, 21, 24, 25, 28, 39, 42, 49, 52, 56, 69, 71, 75, 92, 94, 99, 105, and
106. In
some embodiments, the substitution in the heavy chain variable region at
position 1
is glycine, at position 2 is alanine, at position 3 is arginine, at position 9
is arginine, at
position 12 is alanine, at position 19 is arginine, at position 30 is glycine,
at position
31 is glycine, at position 35 is arginine, at position 37 is alanine, at
position 42 is
glycine, at position 43 is methionine, at position 48 is isoleucine, at
position 49 is
threonine, at position 51 is valine, at position 54 alanine, at position 55 is
glycine, at
position 56 is serine, at position 58 is valine, at position 62 is glycine, at
position 63
is alanine, at position 64 is selected from alanine and glutamic acid, at
position 65 is
glutamic acid, at position 66 is aspartic acid, at position 68 is leucine, at
position 69
is alanine, at position 70 is threonine, at position 73 is asparagine, at
position 76 is
glutamic acid, at position 77 is serine, at position 78 is alanine, at
position 79 is
methionine, at position 80 is selected from serine, leucine, and histidine, at
position
83 is threonine, at position 84 is alanine, at position 88 is proline, at
position 94 is
cysteine, at position 95 is glycine, at position 107 is alanine, at position
108 is
proline, and/or at position 112 is proline, and/or the substitution in the
light chain
variable region at position 3 is arginine, at position 7 is proline, at
position 11 is
selected from serine and leucine, at position 14 is proline, at position 17 is
valine, at
position 19 is alanine, at position 20 is alanine, at position 21 is valine,
at position 24
is glutamic acid, at position 25 is threonine, at position 28 asparagine, at
position 39
isoleucine, at position 42 is selected from serine and aspartic acid, at
position 49 is
proline, at position 52 is glycine, at position 56 is proline, at position 69
is glycine, at
position 71 is histidine, at position 75 is valine, at position 92 is
arginine, at position
94 is threonine, at position 99 is serine, at position 105 is glycine, and/or
at position
106 is valine.
[233] In various embodiments, the substitution in the heavy chain variable
region at
position 68 is leucine, and the substitution in the light chain variable
region at
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position 39 is isoleucine. In various embodiments, the substitution in the
heavy
chain variable region at position 48 is isoleucine. In various embodiments,
the
substitution in the heavy chain variable region at position 30 is glycine and
at
position 58 is valine, and the substitution in the light chain variable region
at position
7 is proline and at position 69 is glycine. In various embodiments, the
substitution in
the light chain variable region at position 11 is serine. In various
embodiments, the
substitution in the heavy chain variable region at position 77 is serine, and
the
substitution in the light chain variable region at position 11 is leucine, at
position 20 is
alanine, and at position 28 is asparagine. In various embodiments, the
substitution in
the heavy chain variable region at position 37 is alanine, at position 63 is
alanine,
and position 80 is serine, and the substitution in the light chain variable
region at
position 11 is leucine. In various embodiments, the substitution in the heavy
chain
variable region at position 1 is glycine, at position 3 is arginine, at
position 76 is
glutamic acid, and at position 77 is serine. In various embodiments, the
substitution
in the light chain variable region at position 11 is leucine.
[234] In various embodiments, the antibody or antigen binding fragment
comprises
any of the sequences shown in table 1, e.g., a set of six CDRs and or paired
heavy
and light chain variable domain sequences listed in Table 1.
[235] In some embodiments, the method comprises contacting a biological sample
from a subject (e.g., blood or CSF) with at least one capture antibody and at
least
one detection antibody. In some embodiments, more than one capture antibody
and/or more than one detection antibody is used. In some embodiments, the
capture
and detection antibodies are the same (e.g., when detecting tau oligomers in a
sample). In some embodiments, a first antibody or antigen binding fragment
(e.g., a
detection antibody) capable of forming a tau-antibody complex, and wherein the
presence of the tau-antibody complex is detecting using a second anti-tau
antibody
or antigen binding fragment (e.g., a detection antibody) disclosed herein. In
some
embodiments, the second antibody or antigen binding fragment binds a different
epitope on tau than the first antibody.
[236] In some embodiments, the first antibody or antigen binding fragment can
bind
an epitope on tau comprising one or more of residues 188-227 of tau protein
2N4R
(SEQ ID NO: 10) or one or more of residues 210-221 of tau protein 2N4R (SEQ ID
NO: 11). The epitope may comprise at least one phosphorylated residue at
position
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217 of tau protein 2N4R (SEQ ID NO: 9) and optionally also may comprise a
phosphorylated serine at position 210, threonine at position 212, serine at
position
214, or threonine at position 220 of tau protein 2N4R, or any combination
thereof. In
certain embodiments, the epitope on tau comprises or consists of SRTPSLPpTPPTR
(SEQ ID NO: 12). In certain embodiments, the epitope on tau comprises or
consists
of SRpTPSLPIDTPIDTR (SEQ ID NO 31). In some embodiments, the first antibody or
antigen binding fragment comprises a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the
light chain variable region comprises three light chain complementarity
determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In certain embodiments, the first
antibody or
antigen binding fragment comprises a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises an amino
acid
sequence of SEQ ID NO: 7 and the light chain variable region comprises an
amino
acid sequence of SEQ ID NO: 8. In an embodiment, the first antibody or antigen
binding fragment comprises antibody DC2E7 or an antigen binding fragment
thereof.
[237] In another embodiment, the first antibody or antigen binding fragment
comprises a heavy chain variable region and a light chain variable region,
wherein
the heavy chain variable region comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable
region comprises three light chain complementarity determining regions (LCDR1,
LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ
ID NO: 23, HCDR2 comprises the amino acid sequence of SEQ ID NO: 24, and
HCDR3 comprises the amino acid sequence of SEQ ID NO: 25; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence
of SEQ ID NO: 28. In some embodiments, the heavy chain variable region
comprises
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an amino acid sequence of SEQ ID NO: 29 and the light chain variable region
comprises an amino acid sequence of SEQ ID NO: 30.
[238] In various embodiments, the second antibody or antigen binding fragment
may bind to an epitope on tau comprising one or more of residues 151-188 of
tau
protein 2N4R (SEQ ID NO: 13). In some embodiments, the epitope comprises one
or
more of residues 163-172 of tau protein 2N4R (SEQ ID NO: 14). In certain
embodiments, one or more of the residues on the epitope are phosphorylated,
e.g., a
phosphorylated threonine at position 169 of tau protein 2N4R. In some
embodiments, the second antibody or antigen binding fragment comprises a heavy
chain variable region and a light chain variable region, wherein the heavy
chain
variable region comprises three heavy chain complementarity determining
regions
(HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three
light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, HCDR2
comprises the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 17; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 19, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 20.
In certain embodiments, the second antibody or antigen binding fragment
comprises
a heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises an amino acid sequence of SEQ ID NO: 21 and
the
light chain variable region comprises an amino acid sequence of SEQ ID NO: 22.
In
an embodiment, the second antibody or antigen binding fragment comprises
antibody DC2E2 or an antigen binding fragment thereof.
[239] In other embodiments, the first and second antibodies are reversed,
and/or
more than one first and second antibody are used (which may be the same or
different antibodies).
[240] In some of the reversed embodiments, the first antibody or antigen
binding
fragment may bind to an epitope on tau comprising one or more of residues 1 51-
1 88
of tau protein 2N4R (SEQ ID NO: 13). In some embodiments, the epitope
comprises
one or more of residues 163-172 of tau protein 2N4R (SEQ ID NO: 14). In
certain
embodiments, one or more of the residues on the epitope are phosphorylated,
e.g., a
phosphorylated threonine at position 169 of tau protein 2N4R. In some
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embodiments, the first antibody or antigen binding fragment comprises any of
the
sequences shown in table 1, e.g., a set of six CDRs and or paired heavy and
light
chain variable domain sequences listed in Table 1.
[241] In some embodiments, the first antibody or antigen binding fragment
comprises a heavy chain variable region and a light chain variable region,
wherein
the heavy chain variable region comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable
region comprises three light chain complementarity determining regions (LCDR1,
LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ
ID NO: 15, HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, and
HCDR3 comprises the amino acid sequence of SEQ ID NO: 17; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 18, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 19, and LCDR3 comprises the amino acid sequence
of SEQ ID NO: 20. In an embodiment, the first antibody or antigen binding
fragment
comprises a heavy chain variable region comprising an amino acid sequence of
SEQ
ID NO: 21 and a light chain variable region comprising an amino acid sequence
of
SEQ ID NO: 22. In an embodiment, the first antibody or antigen binding
fragment
comprises antibody DC2E2 or an antigen binding fragment thereof.
[242] In some of the reversed embodiments, the second antibody or antigen
binding
fragment can bind an epitope on tau comprising one or more of residues 188-227
of
tau protein 2N4R (SEQ ID NO: 10) or one or more of residues 210-221 of tau
protein
2N4R (SEQ ID NO: 11). The epitope bound by the second antibody or antigen
binding fragment may comprise at least one phosphorylated residue at position
217
of tau protein 2N4R (SEQ ID NO: 9). In some embodiments, the epitope also
comprises a phosphorylated serine at position 210, threonine at position 212,
serine
at position 214, or threonine at position 220 of tau protein 2N4R, or any
combination
thereof. In certain embodiments, the second antibody may bind an epitope on
tau
comprising SRTPSLPpTPPTR (SEQ ID NO: 12). In some embodiments, the second
antibody or antigen binding fragment comprises any of the sequences shown in
table
1, e.g., a set of six CDRs and/or paired heavy and light chain variable domain
sequences listed in Table 1.
[243] In some embodiments, the second antibody or antigen binding fragment
comprises a heavy chain variable region and a light chain variable region,
wherein
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the heavy chain variable region comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable
region comprises three light chain complementarity determining regions (LCDR1,
LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ
ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and
HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6. In certain embodiments, the second antibody or antigen binding
fragment comprises a heavy chain variable region and a light chain variable
region,
wherein the heavy chain variable region comprises an amino acid sequence of
SEQ
ID NO: 7 and the light chain variable region comprises an amino acid sequence
of
SEQ ID NO: 8. In an embodiment, the second antibody or antigen binding
fragment
comprises antibody DC2E7 or an antigen binding fragment thereof.
[244] In various embodiments, the first or second antibody or antigen binding
fragment is linked and/or coated onto a solid surface or particle. The solid
surface
may be the surface of a microtiter plate. A microtiter plate or multiwell
plate typically
has, e.g., 6, 12, 24, 48, 96, 384, or 1536 or more sample wells arranged in a
2:3
rectangular matrix. Each well typically holds somewhere between tens of
nanoliters
to several milliliters of liquid. If a solid particle is used in place of or
in addition to a
solid surface, it may be a bead. The particle may be a magnetic bead. The bead
may
be a plastic or synthetic polymer bed comprising: polyethylene, polypropylene,
polystyrene, polyamide, polyurethane, phenolic polymer, nitrocellulose,
naturally
derived polymer, latex rubber, polysaccharide, polypeptide, composite
material,
ceramic, silica or silica-based material, carbon, metal or metal compound,
gold,
silver, steel, aluminum, copper, inorganic glass, or silica material, or a
combination
thereof. The bead may have a spherical, disk, ring, or cube-like shape.
[245] In certain embodiments, the first and/or second antibody is conjugated
to a
detectable label. The label may comprise an enzyme, a radioisotope, biotin, a
nuclear magnetic resonance marker, a heavy metal, or a combination thereof.
The
method may further comprise detecting a signal from the detectable label. In
one
embodiment, detecting the label is achieved by detection of a fluorescent
signal or
the intensity of that signal from a labeled antibody following binding to tau.
In some
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embodiments, the detectable label is biotin and it is detected by contacting
the
sample with streptavidin conjugated to an enzyme, preferably horse radish
peroxidase, alkaline phosphatase, or 6-galactosidase.
[246] In certain embodiments, the tau detection methods disclosed herein
comprises a classic/conventional ELISA assay (i.e., analog readout systems).
Additionally or alternatively, the methods may comprise a digital ELISA (i.e.,
digital
readout systems which enable concentrations to be determined digitally rather
than
by measurement of the total analog signal down to a single immunocomplex,
although, at some concentrations, these methods can also be used to read
analog
signals). See Rissin, D.M., et al., Single-molecule enzyme-linked
immunosorbent
assay detects serum proteins at subfemto molar concentrations. Nat.
Biotechnol.,
2010. 28(6), 555-559. For example, a single-molecule array (simoa) may be
used,
which is a digital ELISA in which, after the formation of a sandwich complex
on
magnetic microbeads, the beads are transferred, in substrate solution, to an
array of
micro wells, e.g., femtoliter-sized micro wells. In some embodiments, these
wells
accommodate only one bead each. In some embodiments, after the addition of a
fluorogenic substrate for the enzyme with which the detection antibody is
labeled, an
oil film is then applied to seal the wells to a small volume, e.g., confining
the reaction
volume to 50 fL. In some embodiments, this small volume allows for a readable
signal to be detected even if only one sandwich complex is present on the
bead. As
a reporter, the enzyme 6-galactosidase and the substrate resorufin-6-D-
galactopyranoside may be used and the wells having a detectable signal are
counted as are all the wells containing a bead. The ratio between these counts
can
provide an average enzyme output per bead (AEB). When the AEB is low (<0.1),
Poisson statistics may be used to show that either a bead has only one or less
complex on its surface. When the AEB signal gets higher, increasing the
probability
of more than one complex per bead, a transition to light intensity
calculations may be
used, which allows for a usable AEB even at signals Ai . The algorithm for the
transition may be implemented using software for a Simoa instrument, e.g.,
software
which samples and calibrates, e.g., from a 96-well microtiter plate or
separate vials.
[247] Other immunoassays are known in the art and may be used with the
disclosed antibodies and labeled antibodies to detect phosphorylated tau in a
sample. For instance, a single-molecule counting (SMC) platform may be used,
e.g.,
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one in which antibodies with and without fluorescent labels form sandwich
complexes with tau, either on beads or plates, the complexes are then broken
up
and the molecules of fluorescently labeled (e.g., Alexa Fluor) detection
antibody are
drawn into a capillary tube and counted as they pass a laser beam that excites
the
fluorophore. A digital event can be counted if the fluorescence reaches above
a
background threshold. At higher concentrations, the total sum of all emitted
photons
may be used as readout for the signal, allowing for a high dynamic range. In
some
embodiments, detection is by single-molecule counting, e.g., using a single-
molecule
counting device from Merck Millipore (developed by Singulex). Another high
sensitive immunoassay that may be used involves attaching magnetic
nanoparticles
to an antibody disclosed herein, and detecting an alteration in the
oscillation of the
nanoparticles in an alternating magnetic field in a concentration-dependent
manner
after binding to the analyte, e.g., detecting immunomagnetic reduction (IMR).
In
some embodiments, detection is by an immunomagnetic reduction bio-assay, e.g.,
using bio-assay from MagQu Co. Ltd.
[248] In various embodiments, a sample may be diluted prior to being contacted
with an antibody or antigen binding fragment thereof. In certain situations,
circulating
antigens can be masked by naturally existing antibodies against those antigens
that
also circulate in the blood. The HIV protein p24 is a well-known example. In
addition,
naturally existing tau/anti-tau antibody complexes have been reported in the
literature. Wu J, Li L. Autoantibodies in Alzheimer's disease: potential
biomarkers,
pathogenic roles, and therapeutic implications. Journal of Biomedical
Research.
2016;30(5):361-372. The presence of those complexes may interfere with the
detection assays disclosed herein. Accordingly, in various embodiments, the
sample
may be subjected to immune complex dissociation (ICD) (e.g., dissociating
naturally
existing tau-antibody/tau-molecule complex in the sample such that tau is no
longer
bound to the naturally existing antibody/molecule and is thus free to be
detected by
the methods of the disclosure) prior to being contacted with an antibody or
antigen
binding fragment of the disclosure. In some embodiments, immune complex
dissociation is achieved by applying heat and/or acid to the sample or any
other
known method of achieving ICD.
[249] In any of the preceding embodiments, the biological sample can comprise
cerebrospinal fluid (CSF). CSF can be obtained from a patient, e.g., by a
lumbar
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puncture performed in a medical setting. Alternatively, the biological sample
can
comprise blood plasma and/or or serum.
[250] In various embodiments, the methods disclosed herein detect the presence
and/or amount of a phosphorylated tau in a sample, which is compared to the
amount of phosphorylated tau in the sample to the level in a control sample
from a
healthy individual, and wherein an increase in the level of phosphorylated tau
in the
sample over the control indicates a tauopathy. In another embodiment, the
detected
tauopathy is Alzheimer's disease. In certain embodiments, an increase in the
level of
phosphorylated tau in a sample from a subject over a sample from a healthy
control
and/or a patient with a known non-AD tauopathy sample indicates the subject
has
Alzheimer's disease rather than another tauopathy or another cause of
dementia.
[251] For instance, a 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,
5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more fold increase in the
level of tau
detected by an antibody or antigen binding fragment (or any fold increase in
between) in a sample from a subject with dementia as compared to the level in
a
control sample from a healthy subject may indicate the presence of AD or
another
tauopathy. In some embodiments, the fold increase is between about 1-100 fold,
or
about 2-3 fold. In some embodiments, the fold increase is between about 1-50,
1-25,
1-10, or 1-5 fold. In some embodiments, a level of bound tau in a sample from
a
subject with dementia greater than a threshold (e.g., 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7,
0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 9.3 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000
pg/ml,
or any value in between) observed in a control sample from a healthy subject
may
indicate the presence of AD or another tauopathy. In some embodiments, the
threshold is about 9.3 pg/ml of tau. In some embodiments, the threshold is any
value
between 0.93 and 93 pg/ml of tau. In some embodiments, the threshold is about
5.37
pg/ml. In some embodiments, the threshold is about 305 pg/ml. In some
embodiments, the threshold is about 100-600 pg/ml. In some embodiments, a 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40,
45, 50, or more fold increase in the level of tau detected by an antibody or
antigen
binding fragment (or any fold increase in between) in a sample from a subject
with
dementia as compared to the level in a control sample from a patient with a
known
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tauopathy other than AD (e.g., FTD, CBD, or PSP), or a control sample from a
patient with another form of dementia, may indicate the presence of AD in the
subject with dementia. In some embodiments, the fold increase is between about
1-
100 fold, or about 1-50 fold, or about 1-25 fold, or about 1-5 fold, or about
2-3 fold. In
some embodiments, a level of bound tau in a sample from a subject with
dementia
greater than a threshold (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1, 1.5, 2, 2.5,
3, 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 12,13, 14, 15,16, 17, 18, 19, 20, 25, 30,
35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 205, 210, 215,
220, 225,
230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 300, 305,
310, 315,
320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390,
395, 400,
410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,
560, 570,
580, 590, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pg/ml, or any value
in
between) observed in a control sample from a patient with a known tauopathy
other
than AD (e.g., FTD, CBD, or PSP), or a control sample from a patient with
another
form of dementia, may indicate the presence of AD in the subject with
dementia. In
various embodiments, the level of tau detected by antibody binding is
quantified
using routine methods, e.g., by measuring fluorescence intensity, by Western
blot,
mass spectrometry, classic ELISA, digital ELISA, or other methods known to the
skilled artisan.
[252] In some embodiments, the threshold is about 0.1-10 pg/ml when the
detection
assay is calibrated using a phosphorylated tau protein, i.e., a full-length
tau protein
comprising one or more phosphorylated positions. In some embodiments, the
threshold is about 100-600 pg/ml when the detection assay is calibrated using
a 2E7
synthetic peptide (2E7pep) with the following sequence:
GQKGQANATRIPAKGGGSGGGSGGGSSRTPSLPpTPPTREPK.
[253] In various embodiments, a method for distinguishing Alzheimer's disease
from
another tauopathy or another cause of dementia in a subject is disclosed,
comprising: obtaining a biological sample from the subject; contacting the
sample
from the subject with an effective amount of a molecule that is capable of
forming a
tau-molecule complex (e.g., at least one receptor, or antibody, or antigen
binding
fragment disclosed hererin that is capable of binding tau to form a tau-
antibody
complex); detecting the presence and/or amount of the tau-molecule complex
using
an antibody or antigen binding fragment disclosed herein; wherein the presence
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and/or an elevated level of phosphorylated tau bound to the molecule in the
sample
relative to the level in a sample from a healthy control subject and/or
relative to the
level in a sample from a subject with a known tauopathy other than AD (e.g.,
FTD,
CBD, or PSP) indicates the subject has Alzheimer's disease rather than another
tauopathy or an alternative cause of dementia. In various embodiments, the
subject
with Frontotemporal dementia (FTD) may have Nonfluent/Agrammatic Primary
Progressive Aphasia (nfPPA), Semantic Variant Primary Progressive Aphasia
(svPPA), Behavioral Variant Frontotemporal Dementia (bvFTD), or Amyotrophic
Lateral Sclerosis/Frontotemporal Dementia (ALS/FTD).
[254] In various embodiments, a method for distinguishing Alzheimer's disease
from
another tauopathy or another cause of dementia in a subject is disclosed,
comprising: obtaining a cerebrospinal fluid or blood sample from a subject;
contacting the sample with an anti-tau antibody or antigen binding fragment
thereof
disclosed herein; and detecting the presence and/or amount of the tau-antibody
complex using an antibody or antigen binding fragment disclosed herein,
wherein the
presence and/or an elevated level of phosphorylated tau bound to the antibody
in the
sample relative to the level in a sample from a healthy control subject and/or
relative
to the level in a sample from a subject with a known tauopathy other than AD
(e.g.,
FTD, CBD, or PSP) indicates the subject has Alzheimer's disease rather than
another tauopathy or another form of dementia. In some embodiments, the method
uses an antibody or antigen binding fragment comprising a heavy chain variable
region and a light chain variable region, wherein the heavy chain variable
region
comprises three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), and the light chain variable region comprises three light
chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein
HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises
the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid
sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, wherein
the antibody or antigen binding fragment is capable of binding to
phosphorylated tau
to form a phosphorylated tau-antibody complex; and detecting the presence
and/or
amount of phosphorylated tau complexed with the antibody or antigen binding
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fragment in the sample, wherein the presence and/or an elevated level of
phosphorylated tau in the sample relative to the level in a sample from a
healthy
control subject indicates the subject has Alzheimer's disease rather than
another
tauopathy or an alternative cause of dementia. In certain embodiments, the
antibody
or antigen binding fragment comprises a heavy chain variable region and a
light
chain variable region, wherein the heavy chain variable region comprises an
amino
acid sequence of SEQ ID NO: 7 and the light chain variable region comprises an
amino acid sequence of SEQ ID NO: 8. In an embodiment, the antibody or binding
fragment comprises antibody DC2E7 or an antigen binding fragment thereof.
[255] In various embodiments, the antibody-tau complex is detected with a
second
antibody or antigen binding fragment thereof capable of binding tau. In some
embodiments, the second antibody or antigen binding fragment comprises a heavy
chain variable region and a light chain variable region, wherein the heavy
chain
variable region comprises three heavy chain complementarity determining
regions
(HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three
light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, HCDR2
comprises the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 17; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 19, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 20.
In certain embodiments, the second antibody or antigen binding fragment
comprises
a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:
21
and a light chain variable region comprising an amino acid sequence of SEQ ID
NO:
22. In various embodiments, second antibody or antigen binding fragment
comprises
antibody DC2E2 or an antigen binding fragment thereof. In some embodiments,
the
detection and capture antibodies are reversed.
[256] In various embodiments, a method of treatment is disclosed, comprising
administering a therapeutic agent for Alzheimer's disease to a subject
suffering from
Alzheimer's disease, wherein the subject has been identified as having
Alzheimer's
disease according to any of the preceding methods. Treatment may comprise
administering an antibody, therapeutic peptide, or small molecule that treats
AD,
e.g., any of the treatments mentioned herein. In various embodiments,
therapeutic
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agents may include one or more of the antibodies or therapeutic peptides
disclosed
in U.S. Patent No. 9,518,101 and/or WO 2016/079597, which are hereby
incorporated by reference in their entirety. In certain embodiments, the
therapeutic
agent may be antibody DC2E7 or an antigen binding fragment thereof and/or
DC2E2, or an antigen binding fragment thereof. In an embodiment, the
therapeutic
agent may be an antibody or an antigen binding fragment thereof that binds to
the
same epitope as DC2E7 and/or DC2E2.
[257] Also disclosed herein, in certain embodiments, are uses of the disclosed
antibodies and antigen binding fragments conjugated to detectable labels
(e.g.,
radiolabels) that can be administered (e.g., intravenously) to a subject to
detect the
pattern of phosphorylated tau in the brain and thereby diagnose AD or another
tauopathy.
[258] In various embodiments, a method of detecting Alzheimer's disease or
another tauopathy in a human subject is disclosed, comprising administering to
the
subject the antibody or antigen binding fragment disclosed herein conjugated
to a
detectable label such as a radioisotope and detecting a signal from the
radioisotope
or other detectable label in the brain of the patient, wherein the detection
of a signal
indicates the subject has Alzheimer's disease or another tauopathy. In some
embodiments, the brain distribution of tau species bound by the disclosed
antibodies
and antigen binding fragments conjugated to detectable labels may be used to
deteremine whether a subject has AD or another tauopathy. For instance,
antibody
binding may differ in AD (where the tau species may be more prominent in
hippocampus CA1) as compared to other tauopathies such as FTD ¨ Pick's disease
(where the tau species may be more prominent in dentate gyrus, and, to some
extent, hippocampus), CBD (where the tau species may be more prominent in
nucleus caudatus) and PSP (where the tau species may be more prominent in
putamen/nucleus caudatus). In some embodiments, a subject is administered an
antibody and antigen binding fragment conjugated to a detectable label (e.g.,
via
intravenous administration) and then their brain is imaged (e.g., via PET) to
generate
a map of tau bound by the labeled antibody and antigen binding fragment in the
brain. The map may be analyzed against known maps for AD and other tauopathies
to determine whether the patient has AD or another tauopathy.
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[259] In certain embodiments, the detection is done by positron emission
tomography. The distribution of the signal in the brain may indicate whether
the
subject has Alzheimer's disease or another tauopathy. See Murray et al.,
Clinicopathologic assessment and imaging of tauopathies in neurodegenerative
dementias, Alzheimer's Research & Therapy, 6:1, 2014. In certain embodiments,
the
antibody or antigen binding fragment comprises a heavy chain variable region
and a
light chain variable region, wherein the heavy chain variable region comprises
three
heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3),
and the light chain variable region comprises three light chain
complementarity
determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the
amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence
of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3;
and wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In an embodiment, the heavy chain
variable
region comprises an amino acid sequence of SEQ ID NO: 7 and the light chain
variable region comprises an amino acid sequence of SEQ ID NO: 8. In an
embodiment, the antibody or binding fragment comprises antibody DC2E7 or an
antigen binding fragment thereof. In an embodiment, the antibody or binding
fragment comprises an antibody or antigen binding fragment that competes for
binding or binds the same epitope as antibody DC2E7 or an antigen binding
fragment thereof.
[260] In various embodiments, an antibody or antigen binding fragment thereof
conjugated to a radioisotope may be administered to a subject as described
herein.
The radiosignal may be detected in the brain by positron emission tomography.
Three dimensional images of the antibody conjugate concentration within the
body
may then be constructed by computer analysis. The concentrations of signal
throughout the brain can be used to correlate with distinct patterns
associated with
AD or other tauopathies, or to determine whether a patient presenting with
dementia
has or does not have AD, another tauopathy, or another cause of their
dementia.
These signal patterns may be interpreted by one skilled in the art to
determine if a
subject has AD or another tauopathy. See Murray et al., Clinicopathologic
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assessment and imaging of tauopathies in neurodegenerative dementias,
Alzheimer's Research & Therapy, 6:1, 2014.
[261] In various embodiments, a human subject presenting with symptoms of
dementia is first subjected to any one or more of the methods of detecting
Alzheimer's disease or another tauopathy disclosed herein before a treatment
decision is made. In certain embodiments, a sample (e.g., CSF or blood) is
taken
from a human subject presenting with symptoms of dementia and analyzed
according to the methods described above, and/or the human subject is
administered antibody DC2E7 conjugated with a radioisotope and a signal is
detected in the subject's brain to determine if the subject has Alzheimer's
disease or
another tauopathy. In an embodiment, a human subject who has been determined
to
have Alzheimer's disease or another tauopathy may be administered a
pharmaceutical composition that treats AD or another tauopathy. In an
embodiment,
the pharmaceutical composition comprises an antibody or antigen binding
fragment
disclosed herein. In an embodiment, the pharmaceutical composition comprises
one
or more of the antibodies or therapeutic peptides disclosed in U.S. Patent No.
9,518,101 and/or WO 2016/079597. Alternatively, if the subject is determined
not to
have AD or another tauopathy, an alternative treatment may be administered.
[262] In various embodiments, a method of determining the stage of Alzheimer's
disease in a human subject is disclosed, comprising: obtaining a cerebrospinal
fluid
or blood sample from the subject, contacting the sample with an effective
amount of
a molecule that is capable of forming a tau-molecule complex (e.g., at least
one
receptor, antibody, or antigen binding fragment disclosed hererin that is
capable of
binding tau to form a tau-antibody complex); detecting the amount of the tau-
molecule complex using an antibody or antigen binding fragment disclosed
herein;
and comparing the amount of tau complexed with the molecule to the amount in a
sample of known AD stage or a threshold, thereby identifying the stage of
Alzheimer's disease. In some embodiments, a higher amount of tau in the sample
indicates a more advanced stage of AD. In various embodiments, levels and/or
thresholds are evaluated as described above. In some embodiments, an advanced
stage is determined by comparison to the amount of phosphorylated tau detected
in
a sample of known AD stage, e.g., Braak Stages 1-VI. Braak et al.,
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"Neuropathological stageing of Alzheimer-related changes". Acta
Neuropathatogica, 82(4): 239-59 (1991).
[263] In various embodiments, a method of determining the stage of Alzheimer's
disease in a human subject is disclosed, comprising: obtaining a cerebrospinal
fluid
or blood sample from the subject, contacting the sample with an antibody or
antigen
binding fragment disclosed herein, wherein the antibody or antigen binding
fragment
is capable of binding to phosphorylated tau to form a phosphorylated tau-
antibody
complex, detecting the amount of phosphorylated tau complexed with the
antibody or
antigen binding fragment in the sample, and comparing the amount of tau
complexed
with the antibody to the amount in a sample of known AD stage or a threshold,
thereby identifying the stage of Alzheimer's disease. In some embodiments, a
higher amount of tau in the sample indicates a more advanced stage of AD. In
various embodiments, levels and/or thresholds are evaluated as described
above. In
some embodiments, an advanced stage is determined by comparison to the amount
of phosphorylated tau detected in a sample of known AD stage, e.g., Braak
Stages I-
V!. Braak et al., "Neuropathological stageing of Alzheimer-related changes".
Acta
Neuropathologica, 82(4): 239-59 (1991). In some embodiments, the antibody or
antigen binding fragment comprises a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the
light chain variable region comprises three light chain complementarity
determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antibody or
antigen binding fragment comprises a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises an amino
acid
sequence of SEQ ID NO: 7 and the light chain variable region comprises an
amino
acid sequence of SEQ ID NO: 8. In an embodiment, the antibody or binding
fragment
comprises antibody DC2E7 or an antigen binding fragment thereof. In some
embodiments, the level of tau determined in the sample is compared to the
level in a
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sample from a patient of known AD stage and/or to a control sample from a
healthy
individual to determine whether the test subject has an elevated amount of tau
in
their sample.
[264] In various embodiments, a method of determining the effectiveness of an
anti-
tau therapy for Alzheimer's disease is disclosed, comprising: obtaining a
cerebrospinal fluid or blood sample from a human subject; contacting the
sample
with an antibody or antigen binding fragment disclosed herein, wherein the
antibody
or antigen binding fragment is capable of binding to phosphorylated tau to
form a
phosphorylated tau-antibody complex; and detecting the presence and/or amount
of
phosphorylated tau complexed with the antibody or antigen binding fragment in
the
sample, wherein an elevated level of phosphorylated tau in the sample relative
to the
level in a sample from a healthy control subject or a threshold indicates the
subject is
more likely to respond to an anti-tau therapy for Alzheimer's disease. In
various
embodiments, an elevated level over a threshold or a fold increase in tau is
determined as described above.
[265] In some embodiments, the anti-tau antibody or antigen binding fragment
used
in determining the effectiveness of an anti-tau therapy for Alzheimer's
disease
comprises a heavy chain variable region and a light chain variable region,
wherein
the heavy chain variable region comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable
region comprises three light chain complementarity determining regions (LCDR1,
LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ
ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and
HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6. In certain embodiments, the antibody or antigen binding fragment
comprises a heavy chain variable region and a light chain variable region,
wherein
the heavy chain variable region comprises an amino acid sequence of SEQ ID NO:
7
and the light chain variable region comprises an amino acid sequence of SEQ ID
NO: 8. In an embodiment, the antibody or binding fragment comprises antibody
DC2E7 or an antigen binding fragment thereof.
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[266] In certain embodiments, an anti-tau therapy is administered to a subject
identified as being more likely to respond to the therapy. In an embodiment,
the anti-
tau therapy comprises administering an antibody that binds to tau and promotes
its
clearance from the brain or inhibit the spreading of tau pathology. In an
embodiment,
the antibody or antigen binding fragment comprises comprising a heavy chain
variable region and a light chain variable region, wherein the heavy chain
variable
region comprises three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), and the light chain variable region comprises three light
chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein
HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises
the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid
sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In an
embodiment, the anti-tau therapy comprises any of those disclosed herein,
and/or
any disclosed in U.S. Patent No. 9,518,101 and WO 2016/079597. In various
embodiments, the anti-tau therapy comprises a small molecule or peptide
vaccine
therapy or antibody therapy. See U.S. Patent No. 9,518,101 and WO 2016/079597,
which are incorporated by reference in their entirety.
[267] In various embodiments, a method of monitoring the effectiveness of an
anti-
tau therapy for Alzheimer's disease is disclosed, comprising: (a) obtaining
cerebrospinal fluid or blood sample from a human subject prior to treatment;
(b)
contacting the sample with an antibody or antigen binding fragment disclosed
herein,
wherein the antibody or antigen binding fragment is capable of binding to
phosphorylated tau to form a phosphorylated tau-antibody complex; (c)
detecting the
presence and/or amount of phosphorylated tau complexed with the antibody or
antigen binding fragment; (d) administering an anti-tau therapy to the
subject; (e)
repeating steps (a)-(c) after administering the anti-tau therapy, whereby a
reduction
in the level of phosphorylated tau in the sample after treatment as compared
to the
level in the sample before treatment indicates an effective therapy. In some
embodiments, the anti-tau antibody or antigen binding fragment comprises a
heavy
chain variable region and a light chain variable region, wherein the heavy
chain
variable region comprises three heavy chain complementarity determining
regions
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(HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three
light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3),
wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2
comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the
amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino
acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In
certain embodiments, the method further comprises administering the anti-tau
therapy again to a subject who has a lower level of phosphorylated tau a the
sample
obtained after treatment as compared to the level in the sample obtained
before
treatment. In an embodiment, the antibody or antigen binding fragment
comprises a
heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises an amino acid sequence of SEQ ID NO: 7 and the
light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
In
an embodiment, the antibody or binding fragment comprises antibody DC2E7 or an
antigen binding fragment thereof. In various embodiments, the anti-tau therapy
comprises a small molecule or peptide vaccine or antibody therapy. See WO
2016/079597 and U.S. Patent No. 9,518,101, which are incorporated by reference
in
their entirety. In an embodiment, the anti-tau therapy comprises administering
an
antibody that binds to tau and promotes its clearance from the brain. In
certain
embodiments, the anti-tau therapy comprises administering an anti-tau antibody
or
antigen binding fragment comprising a heavy chain variable region and a light
chain
variable region, wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the
light chain variable region comprises three light chain complementarity
determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In an embodiment, the anti-tau therapy
comprises any of those disclosed herein, and/or any disclosed in U.S. Patent
No.
9,518,101 and WO 2016/079597.
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Other methods of monitoring and/or confirming AD
[268] A number of additional measures may be used to determine, confirm,
and/or
monitor AD status, e.g., by behavioral assessment, for use in confirming an AD
diagnosis or to evaluate the effects of treatment and/or disease progression.
In some
embodiments, the measures include mean change in Alzheimer's Disease Activity
Scale-Cognitive subscale 13 (ADAS-Cog 13) scores and Mean change in
Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL)
scores. Other measures may include change in MRI volumetry, change in Clinical
Dementia Rating (CDR-SB/CDR-GS), change in neuropsychiatric behavior:
neuropsychiatric inventory (NPI) total and domain scores, and/or change in
cognition: MMSE total score. In another embodiment, the measures can include
any
of the following: time to the occurrence of death, institutionalization, loss
of ability to
perform activities of daily living, time to severe dementia, ADCS-ADL, ADAS-
cog
score, MMSE scores, cognitive performance, plasma CSF biomarkers, ADAS-total
score, Quality of life assessed by Quality of Life Alzheimer's disease scale,
behavioral test scores, and the US FDA's Clinical Dementia Rating-sum of
boxes.
[269] In various embodiments, a method of detecting Alzheimer's diseases or
another tauopathy in a human subject comprises administering to a subject an
antibody or antigen bringing fragment disclosed herein that has been
conjugated to a
radioisotope and detecting a signal in the brain of the patient, wherein the
detected
signal pattern in the brain indicates whether the subject has Alzheimer's
disease or
another tauopathy. Detection of the signal may be done by positron emission
topography. The distribution of the signal in the brain may be used to
indicate
whether the subject has Alzheimer's disease or another tauopathy. In an
embodiment, the administered antibody or antigen binding fragment conjugated
to a
radioisotope comprises a heavy chain variable region and a light chain
variable
region, wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID
NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and
wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2
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comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the
amino acid sequence of SEQ ID NO: 6. In an embodiment, the antibody or antigen
binding fragment comprises a heavy chain variable region comprising an amino
acid
sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino
acid sequence of SEQ ID NO: 8. In an embodiment, the antibody or antigen
binding
fragment comprises DC2E7 or an antigen binding fragment thereof.
[270] The antibodies and antigen binding fragments disclosed herein for
therapeutic
or diagnostic uses can be administered by any suitable administration route,
e.g,
parenteral, topical, intradermal, intravenous, oral, subcutaneous,
intraperitoneal,
intranasal or intramuscular routes. A typical route of administration may be
subcutaneous although others may be equally effective. Another typical route
may
be intramuscular injection. This type of injection is typically performed in
the arm or
leg muscles. Intravenous injections as well as intraperitoneal injections,
intra-arterial,
intracranial, or intradermal injections may also be used. The antibodies or
antigen
binding fragments disclosed herein may be administered as injectable dosages
of a
solution or suspension of the substance in a physiologically acceptable
carrier and/or
diluent, e.g., a sterile liquid such as water, oil, saline, glycerol, or
ethanol. The
antibodies or antigen binding fragments disclosed herein may also be
administered
by drilling a small hole in the skull for administration, which may allow
crossing of the
blood brain barrier.
Kits
[271] In various embodiments, kits are disclosed herein, comprising one or
more of
the antibodies or antigen binding fragments described herein. In certain
embodiments, the kit comprises an antibody or antigen binding fragment
comprising
a heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises three heavy chain complementarity determining
regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region
comprises three light chain complementarity determining regions (LCDR1, LCDR2,
and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:
1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3
comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1
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comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino
acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 6. In certain embodiments, the antibody or antigen binding fragment
comprises a heavy chain variable region and a light chain variable region,
wherein
the heavy chain variable region comprises an amino acid sequence of SEQ ID NO:
7
and the light chain variable region comprises an amino acid sequence of SEQ ID
NO: 8. In an embodiment, the antibody or binding fragment comprises antibody
DC2E7 or an antigen binding fragment thereof.
[272] In certain embodiments, the kit comprises an antibody or antigen binding
fragment comprising a heavy chain variable region and a light chain variable
region,
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3) selected from those in Table 1, e.g., a set
of
six CDRs from those in table 1. In some embodiments, the antibody or antigen
binding fragment comprises a light chain variable domain and a heavy chain
variable
domain and a light chain variable domain selected from those in Table 1, e.g.,
paired
heavy and light chain variable domain sequences selected from those in Table
1.
[273] In certain embodiments, the kit comprises an antibody or antigen binding
fragment comprising a heavy chain variable region and a light chain variable
region,
wherein the heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity determining
regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid
sequence of SEQ ID NO: 1 with a substitution at position 2, HCDR2 comprises
the
amino acid sequence of SEQ ID NO: 2 with a substitution at one or more of
position
2 and 12, and/or HCDR3 comprises the amino acid sequence of SEQ ID NO: 3,
and/or wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 with a
substitution at position 2, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 5, and/or LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In
various embodiments, the substitution at position 2 in HCDR1 is glycine, the
substitution at position 2 in HCDR2 is isoleucine, and/or the substitution at
position
12 in HCDR2 is valine, and/or the substitution at position 2 in LCDR1 is
asparagine.
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In various embodiments, the heavy chain variable region comprises the amino
acid
sequence of SEQ ID NO: 7 with a substitution at one or more of position 1, 3,
30, 37,
48, 58, 63, 68, 76, 77, and 80, and/or the light chain variable region
comprises the
amino acid sequence of SEQ ID SEQ ID NO: 8 with a substitution at one or more
of
position 7, 11, 20, 28, 39, and 69. In various embodiments, the substitution
in the
heavy chain variable region at position 68 is leucine, and the substitution in
the light
chain variable region at position 39 is isoleucine. In various embodiments,
the
substitution in the heavy chain variable region at position 48 is isoleucine.
In various
embodiments, the substitution in the heavy chain variable region at position
30 is
glycine and at position 58 is valine, and the substitution in the light chain
variable
region at position 7 is proline and at position 69 is glycine. In various
embodiments,
the substitution in the light chain variable region at position 11 is serine.
In various
embodiments, the substitution in the heavy chain variable region at position
77 is
serine, and the substitution in the light chain variable region at position 11
is leucine,
at position 20 is alanine, and at position 28 is asparagine. In various
embodiments,
the substitution in the heavy chain variable region at position 37 is alanine,
at
position 63 is alanine, and position 80 is serine, and the substitution in the
light chain
variable region at position 11 is leucine. In various embodiments, the
substitution in
the heavy chain variable region at position 1 is glycine, at position 3 is
arginine, at
position 76 is glutamic acid, and at position 77 is serine. In various
embodiments, the
substitution in the light chain variable region at position 11 is leucine.
[274] In various embodiments, a kit comprises two or more antibody or antigen
binding fragments. In certain embodiments, a kit comprises:
a. an antibody or antigen binding fragment comprising a heavy chain
variable
region and a light chain variable region, wherein the heavy chain variable
region
comprises three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), and the light chain variable region comprises three light
chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein
HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises
the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid
sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; and
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b. another antibody or antigen binding fragment comprising a heavy chain
variable region and a light chain variable region, wherein the heavy chain
variable
region comprises three heavy chain complementarity determining regions (HCDR1,
HCDR2, and HCDR3), and the light chain variable region comprises three light
chain
complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein
HCDR1 comprises the amino acid sequence of SEQ ID NO: 15, HCDR2 comprises
the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprises the amino acid
sequence of SEQ ID NO: 17; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid sequence of SEQ ID
NO: 19, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 20.
[275] In certain embodiments, the kit comprises two or more antibodies or
antigen
binding fragments, wherein an antibody or antigen binding fragment comprises a
heavy chain variable region and a light chain variable region, wherein the
heavy
chain variable region comprises an amino acid sequence of SEQ ID NO: 7 and the
light chain variable region comprises an amino acid sequence of SEQ ID NO: 8,
and
the other antibody or antigen binding fragment comprises a heavy chain
variable
region and a light chain variable region, wherein the heavy chain variable
region
comprises an amino acid sequence of SEQ ID NO: 21 and the light chain variable
region comprises an amino acid sequence of SEQ ID NO: 22. In certain
embodiments, the kit comprises antibody or DC2E7 or an antigen binding
fragment
thereof and/or antibody or DC2E2 or an antigen binding fragment thereof.
[276] In various embodiments, a kit comprises two or more antibody or antigen
binding fragments. In certain embodiments, a kit comprises:
a. an antibody or antigen binding fragment comprising any of the
sequences shown in table 1, e.g., a set of six CDRs and or paired
heavy and light chain variable domain sequences listed in Table 1;
b. another antibody or antigen binding fragment comprising a heavy
chain variable region and a light chain variable region, wherein the
heavy chain variable region comprises three heavy chain
complementarity determining regions (HCDR1, HCDR2, and
HCDR3), and the light chain variable region comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
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LCDR3), wherein HCDR1 comprises the amino acid sequence of
SEQ ID NO: 15, HCDR2 comprises the amino acid sequence of
SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence
of SEQ ID NO: 17; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid
sequence of SEQ ID NO: 19, and LCDR3 comprises the amino acid
sequence of SEQ ID NO: 20.
[277] In various embodiments, a kit comprises two or more antibody or antigen
binding fragments. In certain embodiments, a kit comprises:
a. an antibody or antigen binding fragment comprises a heavy chain
variable region and a light chain variable region, wherein the heavy
chain variable region comprises three heavy chain complementarity
determining regions (HCDR1, HCDR2, and HCDR3), and the light
chain variable region comprises three light chain complementarity
determining regions (LCDR1, LCDR2, and LCDR3), wherein
HCDR1 comprises the amino acid sequence of SEQ ID NO: 23,
HCDR2 comprises the amino acid sequence of SEQ ID NO: 24,
and HCDR3 comprises the amino acid sequence of SEQ ID NO:
25; and wherein LCDR1 comprises the amino acid sequence of
SEQ ID NO: 26, LCDR2 comprises the amino acid sequence of
SEQ ID NO: 27, and LCDR3 comprises the amino acid sequence of
SEQ ID NO: 28;
b. and another antibody or antigen binding fragment comprising a
heavy chain variable region and a light chain variable region,
wherein the heavy chain variable region comprises three heavy
chain complementarity determining regions (HCDR1, HCDR2, and
HCDR3), and the light chain variable region comprises three light
chain complementarity determining regions (LCDR1, LCDR2, and
LCDR3), wherein HCDR1 comprises the amino acid sequence of
SEQ ID NO: 15, HCDR2 comprises the amino acid sequence of
SEQ ID NO: 16, and HCDR3 comprises the amino acid sequence
of SEQ ID NO: 17; and wherein LCDR1 comprises the amino acid
sequence of SEQ ID NO: 18, LCDR2 comprises the amino acid
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sequence of SEQ ID NO: 19, and LCDR3 comprises the amino acid
sequence of SEQ ID NO: 20.
[278] In another embodiment, any of the preceding kits may further comprise
instructions for using the one or more antibodies or antigen binding fragments
to
detect phosphorylated tau in a sample from a subject according to the methods
disclosed herein and thereby detect Alzheimer's disease in the subject. The
instructions may call for the sample to be cerebrospinal fluid or blood. The
kits may
further comprise additional components for use with a classic ELISA or a
digital
ELISA.
EXAMPLES
[279] Several of the foregoing embodiments are illustrated in the non-limiting
examples set forth below. However, other embodiments of the disclosure will be
apparent to those skilled in the art from consideration of the specification
as a whole.
It is intended that the specification and examples be considered as exemplary
only.
In addition, all references cited herein are to be considered incorporated by
reference in their entirety.
Example 1
Preparation of Recombinant Human Tau Proteins
[280] Human full-length tau 2N4R and tau deletion mutants: Recombinant tau
proteins were generated from clone 140 (Goedert et al., Multiple isoforms of
human
microtubule-associated protein tau: sequences and localization in
neurofibrillary
tangles of Alzheimer's disease, Neuron 3, 519-526 (1989), which was subcloned
into
the expression plasmid pET-17b (Novagen) and expressed in bacteria. Each tau
deletion mutant was verified by DNA-sequencing. All tau deletion mutants and
tau
peptides were numbered according to the longest human tau isoform 2N4R, which
is
441 amino acids in length and thus is also called tau441 (D'Souza, I., and
Schellenberg, G.D. (2005). Regulation of tau isoform expression and dementia.
Biochimica et biophysica acta 1739, 104-115). Production of tau proteins
involved
the following steps: a) expression of tau in bacteria; b) tau purification by
ion
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exchange chromatography; c) tau purification by gel-filtration; d)
concentration and
storage of isolated tau.
a) Bacterial expression of human full-length tau (2N4R) and recombinant tau
deletion mutants: human tau (above) expression plasmids were transformed into
Escherichia coli (E. coli), production strain BL21(DE3). Bacterial cells
containing the
appropriate expression plasmid were cultivated and induced as described in
"Molecular Cloning: A Laboratory Manual" by Sambrook and Russell (2001). A
single
colony of BL21(DE3) bacteria, transformed with pET-17b plasmid driving
expression
of a tau protein or its fragment, were grown at 37 C in 500 mL of Luria broth
medium
with 100 pg/ml ampicillin at 300 rpm and induced by the addition of isopropyl-
3-D-1-
thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. After further
incubation at 37 C for 3 hours, bacteria were collected by centrifugation at
3,000xg
for 15 min at 4 C.
b) Cation-exchange chromatography purifications of the basic and neutral tau
proteins (six tau isoforms, point mutants of tau isoform 2N4R: Ser198Ala,
Ser199Ala,
Ser202Ala, Thr205Ala, Ser208Ala, Ser210Ala, Thr212Ala, Ser214Ala, Thr217Ala,
Ser231Ala, tau221-441, tau99- 441, tau188- 44, tau31-441, tau151-391, tau127-
441, tau2N4R8(134-168), tau2N4R8(49-243) were done essentially as previously
described (Krajciova et al.,Preserving free thios of intrinsically disordered
tau protein
without use of a reducing agent, Analytical Biochemistry, 383:343-345, 2008).
After
expression, the bacterial pellets were resuspended in 10 ml of lysis buffer
(50 mM
1,4-piperazinediethanesulfonic acid (PIPES) pH 6.9, 50 mM sodium chloride
(NaCI),
1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol (DTT), 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), 5% (v/v) glycerol), quickly frozen in
liquid
nitrogen, and stored at -80 C until used for purification of tau proteins. For
tau
protein purification, the frozen bacterial suspensions were quickly thawed and
placed
on ice. Bacterial cell walls were broken by sonication on ice by using
Sonopuls HD
2200, tip TT-13 (Bandelin, Germany) set to 50% duty cycle, 50 W power output,
6
times for 30 s with 30 s pauses. The lysates were clarified by centrifugation
(21,000xg for 15 min at 4 C) and the supernatants were filtered through a 0.45
pm
membrane filter. Large-scale purification of the recombinant tau proteins was
done at
6 C using an AKTA-FPLC workstation (Amersham Biosciences, Sweden). The
filtered lysates were loaded at a 3 ml/min flow rate onto a 5-ml HiTrap SP HP
column
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(GE Healthcare, Uppsala, Sweden) equilibrated with the lysis buffer, and
washed
extensively with 60 ml of the lysis buffer until the baseline at 280 nm became
stable.
Bound tau proteins were eluted by a gradient (0-30% within 15 ml) of Buffer B
(lysis
buffer supplemented with 1 M NaCI). Individual 1 mL fractions were collected
and
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE). To remove nucleic acids, which copurify with positively charged tau
proteins,
the fractions containing tau protein were pooled and purified by a second
cation-
exchange chromatography step, using a 5-ml HiTrap SP HP column (GE Healthcare,
Uppsala, Sweden) with a less steep gradient of Buffer B (0-30% in 45 ml).
Anion-
exchange chromatography purification of the acidic tau proteins (tau1-226,
tau1-136,
tau1-242) was done as previously described (Csokova et. al, Rapid purification
of
truncated tau proteins: model approach to purification of functionally active
fragments of disordered proteins, implication for neurodegenerative diseases,
Protein Expression and Purification, 35:366-372, 2004). After expression,
bacterial
pellets were resuspended in 10 ml of histidine lysis buffer (20mM histidine,
pH 6.0,
50mM NaCI, 1mM EDTA, 5mM DTT, 0.1mM PMSF, and 5% (v/v) glycerol). Bacterial
cell walls were broken by sonication on ice by using Sonopuls HD 2200, tip TT-
13
(Bandelin, Germany) set to 50% duty cycle, 50 W power output, 6 times for 30 s
with
30 s pauses. The lysates were clarified by centrifugation (21,000xg for 15 min
at
4 C). Bacterial lysates were precipitated by 1% streptomycin sulfate
(Medexport,
Russia), incubated on ice for 5 min, clarified by centrifugation (21,000xg for
15 min at
4 C), and filtered through a 0.45 pm membrane filter. The filtered
streptomycin
precipitated lysates were loaded at 3 ml/min flow rate onto a 5m1 HiTrap
QSepharose HP column (Amersham Biosciences, Sweden) and washed extensively
with 30-50m1 histidine lysis buffer until the A280 baseline became stable. Tau
proteins were eluted with a two-step salt gradient (0.05-0.5M NaCI in 40m1
followed
by 0.5-1M NaCI in 20 ml) in histidine lysis buffer.
c) In the final gel-filtration step of purification (the same for all tau
proteins),
pooled tau protein fractions obtained by ion exchange chromatography, were
injected onto a gel-filtration column (HiLoad 26/60 Superdex 200 prep grade
column,
GE Healthcare) at 3 ml/min in either PIPES or Histidine lysis buffer for
basic/neutral
or acidic tau proteins, respectively, supplemented with 100 mM NaCI. Eluted
tau
proteins were pooled.
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d) For tau protein concentration after gel-filtration purification, pooled
fractions
were diluted with 1.5 volumes of 2.5% glycerol, and loaded again on a HiTrap
SP HP
column (basic and neutral tau proteins) or on a HiTrap Q HP column (acidic tau
proteins). The concentrated recombinant tau protein was then eluted from the
column with a 1 M NaCI step gradient. Finally, the buffer was exchanged to
phosphate-buffered saline (PBS, 8.09 mM disodium phosphate (Na2HPO4), 1.47
mM potassium dihydrogen phosphate (KH2PO4), 136.89 mM NaCI, 2.7 mM
potassium chloride (KCI)) saturated with argon, using a 5 mL HiTrap Desalting
column (GE Healthcare). Protein quantitation of purified samples was done
using
bicinchoninic acid (BOA) quantitation kits (Pierce, USA), with bovine serum
albumin
(BSA) as a standard. Tau proteins were aliquoted into working aliquots, snap-
frozen
in liquid nitrogen, and stored at -70 C.
Example 2
Preparation of Hybridoma Cell Lines Producing Monoclonal Antibodies Against
Human Tau1-242, Screening of Monoclonal Antibodies by ELISA, and Initial
Characterization of Monoclonal Antibody DC2E2
[281] Six-week-old Balb/c mice were primed subcutaneously with 50 pg of
recombinant tau1-242- (prepared as described in Example 1) in complete
Freund's
adjuvant (SIGMA) and boosted three times at four-week intervals with 50 pg of
the
same antigen in incomplete Freund's adjuvant. Three days before the fusion,
mice
were injected intravenously with 50 pg of the same antigen in PBS. Spleen
cells from
immunized mice were fused with NS/0 myeloma cells according to the method of
Kontsekova et al., The effect of postfusion cell density on establishment of
hybridomas, Folia Biol. 34, 18-22 (1988). Splenocytes were mixed with NS/0
myeloma cells (ratio 5:1) and fused for 1 minute in 1 ml of 50% polyethylene
glycol
(PEG) 1550 (Serva) in serum free Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% dimethyl sulphoxide. The fused cells were resuspended in
DMEM containing 20% horse serum, L-glutamine (2 mM), hypoxanthine (0.1 mM),
aminopterin (0.04 mM), thymidine (0.016 mM), and gentamycin (40 U/m1), at a
density of 2.5 x 105 spleen cells per well on 96-well plates.
[282] The cells were incubated for 10 days at 37 C and growing hybridomas were
screened for the production of anti-tau1-242-specific monoclonal antibodies by
an
enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated
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overnight with tau1-242 (5 pg/ml, 50 p1/well) at 37 C in PBS. The plates were
blocked with 1% nonfat dried milk to reduce nonspecific binding, washed with
PBS-
0.05% Tween 20, and incubated with 50 p1/well of hybridoma culture supernatant
for
1 hr at 37 C. Bound monoclonal antibodies were detected with sheep anti-mouse
immunoglobulin (Ig) conjugated with horse radish peroxidase (HRP, DAKO). The
reaction was developed with TMB one (Kem-En-Tec Diagnostics) as a peroxidase
substrate and stopped with 50 pl of 2 M H2504. Absorbance at 450 nm was
measured using a Powerwave HT (Bio-Tek). Readouts with an absorbance value of
at least twice the value of the negative controls (PBS) were considered
positive.
Positive hybridoma cultures were further subcloned in soft agar according to
the
procedure described in Kontsekova et al., One-step method for establishing 8-
azaguanine-resistant hybridomas suitable for preparation of triomas, J.
Immunol.
Methods, 145, 247-250 (1991).
[283] The monoclonal antibody DC2E2 (produced by the mouse hybridoma cell line
deposited with the American Type Culture Collection, with the ATCC Patent
Deposit
Designation PTA-124991.) was identified among the positive hybridoma cultures
so
produced and selected. DC2E2 was further characterized as described below. The
antibody isotype was determined to be murine IgG1 by ELISA (Fig. 1) using a
mouse
Ig isotyping kit (ISO-2, SIGMA).
Example 3
Mapping of the DC2E2 Epitope Using Recombinant Tau Deletion Mutants, Tau-
Derived Peptides and Mass Spectrometry
[284] Deletion mutants of human tau protein 2N4R were used for epitope mapping
of DC2E2 using ELISA (Fig. 2A). Recombinant human tau isoforms 2N4R, 1N4R,
2N3R, ON4R, 1N3R, ON3R and tau deletion mutants were prepared as described in
Example 1. Microtiter plates were coated overnight at 37 C with recombinant
tau
proteins (5 pg/ml in PBS, 50 p1/well). The plates were blocked with PBS-
Tween20
(0.1% v/v) to reduce nonspecific binding and were incubated with 50 p1/well of
DC2E2 hybridoma culture supernatant, for 1 hr at 37 C. Bound monoclonal
antibody
was detected with sheep anti-mouse Ig HRP-conjugated (DAKO). The reaction was
developed with TMB one solution (Kem-En-Tec Diagnostics) as a peroxidase
substrate and stopped with 50 pl of 2M H2504. Absorbance was measured at 450
nm using a Powerwave HT (Bio-Tek). Readouts with an absorbance value of at
least
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twice the value of the negative controls (PBS) were considered positive. DC2E2
recognized the following human tau proteins: six human tau isoforms (Fig. 2B),
tau
1-242, tau 31-441, tau 99-441, tau 1-226, tau151-391, tau 127-441, but failed
to
recognize the deletion mutants tau 1-136, tau 221-441 and tau 2N4F1(8.134-168)
(Fig. 20). Together, these findings suggest that DC2E2 recognizes binding
sites or
epitopes on tau, located in the proline reach region of tau between amino
acids 151-
188.
[285] To further define the epitope, competition ELISA was performed. For
competition experiments, the DC2E2 monoclonal antibody (mAb) was purified from
serum-free hybridoma supernatant on a Protein G affinity column, as follows.
The
hybridoma supernatant was adjusted to pH 7.5, the solution was precleared by
centrifugation, filtered through a 0.45 pm membrane filter, and loaded onto a
5 ml
Protein G Sepharose column. DC2E2 mAb was eluted from the column with 0.1 M
Glycine-HCI, pH 2.7. Eluted fractions were immediately neutralized with 1M
Tris-HCI
pH 9Ø Pooled fractions were dialyzed against PBS, concentrated by
ultrafiltration,
and stored at -70 C. The concentration of the antibody was determined by
measuring absorbance at 280 nm, using the formula c(mg/m1) = A280nm/1.43.
[286] For competition ELISA, peptides (tau 141-170, 161-190, 181-210, 171-200)
were synthesized by EZBiolabs with purity higher than 90%. ELISA plates (Nunc
Medisorp, Thermo Scientific, Denmark) were coated overnight at 4 C with 50
p1/well
of 0.4 pg/ml of recombinant purified tau 1-242 in PBS. The plates were washed
4
times with PBS/Tween 20 (0.1% v/v), and blocked with PBS/Tween 20 for 2 h at
25 C. Peptides were separately dissolved in PBS at a final concentration of 5
mM.
Serial dilutions (2,5-fold) of the peptides in PBS/Tween 20 were prepared in
polypropylene plates with conical well bottom (Greiner, #651201)
(concentration
range 200 pM, 32 pM, 12,8 pM, 5,1 pM, 2 pM, 0,8 pM, and 0,3 pM). 60 pl of each
dilution were added per well. Purified D02E2 was diluted to a concentration of
0.6
pg/ml in PBS/Tween 20 and 60 pl of this diluted antibody was mixed with each
serial
dilution of peptides resulting in 120 pl mixtures with 0.36 ng of
antibody/60p1
containing each respective test peptide at a concentration of 200 pM, 32 pM,
12,8
pM, 5,1 pM, 2 pM, 0,8 pM, and 0,3 pM The antibody/peptide mixtures were
incubated for 1 hr at 25 C on a rotating platform set to 250 rpm. Fifty
microliters (50
pl) of antibody/peptide mixtures were transferred from the polypropylene
plates into
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tau1-242-coated and PBS/Tween 20-blocked plates (in duplicates) and incubated
for
1 hr at 25 C on a rotating platform set to 250 rpm. The plates were washed 4x
times
with PBS/Tween 20 and incubated for 1 hr at 25 C on a rotating platform (set
to 250
rpm) with 50 pl of Polyclonal Goat Anti-Mouse lmmunoglobulins/ HRP (Dako,
#P0447) diluted 1:1000 in PBS/Tween 20. The plates were washed 4x times with
PBS/Tween and then incubated with 50 p1 /well of TMB one solution (Kem-En-Tec
Diagnostics) as a peroxidase substrate. The reaction was stopped by adding 50
pl of
2 M H2SO4 (Merck). Absorbance was measured at 450 nm using a Powerwave HT
(Bio-Tek).
[287] Competition ELISA was performed with the following peptides: tau 141-
170,
161-190, 171-200, 181-210. (Fig. 2D) Only peptide tau 161-190 competed with
tau
1-242 for the binding to DC2E2 (Fig. 2D). However, shifting of peptides to the
C-
terminus of tau (171-200, 181-210) or to the N-terminus of tau (141-170) led
to loss
of competing activity with tau 1-242 for binding to DC2E2. These results
suggest that
the epitope is located between amino acids 161-181.
[288] An LC/MALDI mass spectrometry approach was used with the aim to define
the epitope more precisely. Sequence of binding site was identified by binding
of
proteolytically digested tau proteins to DC2E2 monoclonal antibody immobilized
on
magnetic beads and subsequent identification of eluted peptides by LC/MALDI
mass
spectrometry. Tau proteins (tau 2N4R and tau151-391) were digested with the
mixture of trypsin, Glu-C, chymotrypsin at 37 C overnight or formic acid for 2
hours
at 108 C. Binding reaction was performed in 1% CHAPS in PBS for 2 hours. Non-
bound peptides were removed by three washes with 1% CHAPS in PBS. Bound
peptides were eluted by three washes with formic acid and lyophilized.
Peptides
were separated by UHPLC (Dionex, Ultimate 3000 nano-LC system), fractions were
mixed with HCCA matrix solution, and dispensed onto MTP Anchor Chip 384 MALDI
sample plate (Bruker Da!tonics). The fractionated samples were analyzed with a
MALDI TOF/TOF (Ultraflextreme, Bruker Da!tonics) instrument operated in the
positive ion mode. The collected MS and MS/MS spectra were searched against
database of tau proteins using Mascot search engine (Matrix Science).
[289] Database analysis revealed several peptide sequences, all of them
containing
KGQANATRIP. This peptide is located on tau 2N4R in the region of 163-172, a
proline rich region.
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Example 4
DC2E2 Recognizes an A68 Triplet Specific For Insoluble Tau In Alzheimer's
Disease, Phosphorylated Tau, and Unphosphorylated Tau
[290] In Alzheimer's disease, tau is hyperphosphorylated. Therefore, for
detail
characterization of binding properties of DC2E2, hyperphosphorylated PHF-tau
and
in vitro phosphorylated tau were examined.
[291] Sarcosyl insoluble tau complexes (PHF-Tau) were isolated from human AD
brains using the sarkosyl method (Greenberg and Davies, A preparation of
Alzheimer's paried helical filaments that displays distint tau proteins by
polyacrylamide gel electrophoresis, PNAS, 87:5827-31, 1990). For protein
extraction, frozen human AD brain tissue (frontal cortex, samples of Braak
stage VI
obtained from the Netherlands brain bank) was homogenized in 10 volumes of
cold
extraction buffer (10 mM Tris pH 7.4, 0.8 M NaCI, 1 mM EGTA, and 10% sucrose)
and homogenate was centrifuged for 20 min at 20,000xg. To prepare sarcosyl-
insoluble tau, supernatant was supplemented with N-lauroylsarcosine (SIGMA) to
a
final concentration of 1% and incubated for 1 h at room temperature, while
shaking.
After centrifugation at 100,000xg for lh, the resulting supernatant was
discarded,
and a pellet comprising the sarkosyl-insoluble tau fraction was resuspended in
1/50
volume of the supernatant used for the preparation of the insoluble tau.
[292] For in vitro phosphorylation of tau 2N4R and tau151-391, kinase extract
was
used. Adult rat brain (1g/2,5 ml) was homogenized in kinase buffer (10 mM TRIS-
HCI, pH 7,4; 5mM EGTA, 2mM DTT; 1mM PMSF; 2mM MgCl2, leupeptin (20 pg/ml),
pepstatin (20 pg/ml), aprotinin (20 pg/mI)) and after centrifugation at
100,000 x g for
30 min at 4 C the supernatant (kinase extract) was used for the
phosphorylation of
tau. The phosphorylation reaction was carried out at 37 C in 10 mM TRIS-HCI,
pH
7,4; 5mM EGTA, 2mM DTT; 1mM PMSF; leupeptin (20pg/m1); pepstatin (20pg/m1);
aprotinin (20pg/m1); 2mM ATP; 2mM MgCl2 and 10pM okadaic acid. The brain
kinase extract (5p1) was added to 50 pl tau solution (1 M) and reaction was
incubated at 37 C for 24h. The phosphorylation of tau was analysed by SDS-PAGE
and immunoblotting using rabbit anti-serum raised against six recombinant
human
tau isoforms (Csokova et al., Rapid purification of truncated tau proteins:
model
approach to purification of functionally active fragments of disordered
proteins,
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implication for neurodegenerative diseases, Protein Expression and
Purification,
35:366-372, 2004).
[293] Phosphorylated and unphosphorylated tau proteins 2N4R and tau151-391
and sarcosyl insoluble PHF-tau were analyzed by immunoblotting using DC2E2.
Soluble tau proteins were diluted with an equal volume of 2x SDS (sodium
dodecylsulfate) sample loading buffer (with 13-mercaptoethanol) (Laemmli,
1970) and
250 ng of proteins were loaded per lane. For insoluble PHF-tau, the pellets
were
dissolved in lx SDS-sample loading buffer, in 1/50 volume of the soluble
fraction
used for the preparation of the insoluble tau fraction. Then, equal volumes of
soluble
tau and sarkosyl-insoluble tau fractions were used for immunoblotting, which
corresponded to 15 pg of total protein in the soluble fraction (see Filipcik
et al. 2010).
Samples were heated at 95 C for 5 min, loaded onto 5-20% gradient SDS
polyacrylamide gels, and electrophoresed in a Tris-glycine-SDS buffer system
for 40
minutes at 25 mA. Proteins were transferred to a polyvinylidene fluoride
(PVDF)
membrane (1 h at 150 mA in 10 mM CAPS, pH 12). After the transfer, the
membranes were blocked in 5% non-fat dry milk in phosphate-buffered-saline
(PBS;
136.89 mM NaCI, 2.7 mM KCI, 8.09 mM Na2HPO4, 1.47 mM KH2PO4) for 1 h at RT,
and then incubated for 12 h with DC2E2 hybridoma culture supernatant, followed
by
three washes with large volumes of PBS-T (1%Tween 20). The membranes were
incubated (1 h at room temperature) with HRP conjugated goat anti-mouse Ig
(DAKO, Denmark), diluted 1:3,000 with 1% non-fat dry milk in PBS, as a
secondary
antibody. This incubation was followed by washing (three times) with 0.1%
Tween 20
in PBS. The blots were developed with SuperSignal West Pico Chemiluminescent
Substrate (Pierce, U.S.A), and the protein signals detected using a LAS3000
imaging system (FUJI Photo Film Co., Japan). The chemiluminescence signal
intensities were quantified using AIDA (Advanced Image Data Analyzer, Raytest,
Straubenhardt, Germany) software.
[294] DC2E2 recognized both forms of tau proteins, in vivo phosphorylated tau
2N4R, 151-391 and wild type (unphoshorylated) forms of tau 2N4R and tau151-391
(Fig. 3).
[295] Furthermore, it is noteworthy that DC2E2 recognized the A68 triplet, a
characteristic feature of pathological tau (PHF-tau) in AD neurofibrillary
degeneration. These results demonstrate that DC2E2 can recognize and bind
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different type of tau proteins, pathological tau isolated from AD brain,
physiological
tau (2N4R), truncated tau151-391 and its phosphorylated forms. Thus, DC2E2
recognized epitopes independent of epitope phosphorylation. Disclosed binding
properties of DC2E2 suggest the possibility of using the antibody as
diagnostic tool
for AD.
Example 5
Sequencing of DC2E2 Variable Regions
[296] Determination of the nucleotide and amino acid sequences of the light
and
heavy chain variable regions of DC2E2 (Fig. 5). The nucleotide sequence of
DC2E2
variable regions (Fig. 5A and 50) was determined by DNA sequencing of cDNA
synthesized using total RNA extracted from the mouse hybridoma cell line DC2E2
(ATCC), which expresses the DC2E2 monoclonal antibody. Total RNA was extracted
using RNeasy Mini Kit (Qiagen, Germany). Synthesis of the first strand cDNA
was
carried out using the "High capacity cDNA reverse transcription" kit according
to the
manufacturer's protocol (Applied Biosystems, USA). The composition of the
reagents
for the 2x reverse transcription master-mix was as follows (quantities per 20
pL
reaction): 2 pL of 10x RT buffer; 0.8 pL of 25x dNTP Mix (100 mM); 2 pl of 10x
RT
Random Primers (50 pM); 1 pL of MultiScribeTM Reverse Transcriptase (50 U/pL);
4.2 pL of nuclease-free H20. For reverse transcription, 10 pL of the 2x
reverse
transcription master-mix was mixed with RNA sample (1 pg/10 pL) and cDNA was
synthesized under the following conditions: 10 min at 25 C, 120 min at 37 C, 5
min
at 85 C, and final cooling to 4 C. The genes encoding the variable regions of
the
light and heavy chains were apmplified by polymerase chain reaction (PCR)
using
Phusione High-Fidelity DNA Polymerase (Thermo Fisher Scientific, USA). The
forward primers (M13-L6 5'-
TGTAAAACGACGGCCAGTATGAGGTKCYYTGYTSAGYTYCTGRGG -3' and M13-
H1 5`-TGTAAAACGACGGCCAGTATGAAATGCAGCTGGGTCATSTTCTTC -3') for
the light and heavy chain, respectively, were selected after screening a
library of
mouse immunoglobulin signal sequence forward primers. Using signal sequence
forward primers has the advantage of amplifying the whole V-gene of both light
and
heavy antibody chains, without bias introduced when the beginning of the
variable
region is being used for primer design. The reverse primers for the light and
heavy
chains (M13-KC 5'- CAGGAAACAGCTATGACCACTGGATGGTGGGAAGATGG-3'
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and M13-CG1 5'- CAGGAAACAGCTATGACCCAGTGGATAGACAGATGGGGG -3')
were derived from kappa and IgG1 chains constant regions, respectively.
[297] The PCR products were sequenced and the resulting DNA sequences of the
variable regions of the light and heavy chains of DC2E2 are shown in Figs. 5A
and
50, respectively. Complementarity determining regions (CDRs) are underlined in
the
DC2E2 light and heavy chains protein sequences (Figs. 5B and 5D,
respectively).
CDRs and framework regions (FR) were identified according to the
ImMunoGeneTics (IMGT) numbering system (see, e.g., Lefranc M.P. The IMGT
unique numbering for immunoglobulins, T-cell receptors, and lg-like domains.
The
Immunologist, 7, 132-1 36, 1999 (1999)).
Example 6
Preparation of Hybridoma Cell Lines Producing Monoclonal Antibodies Against
Insoluble Tau Species In Human Alzheimer's Disease, Screening Of Monoclonal
Antibodies By ELISA, And Initial Characterization Of Monoclonal Antibody DC2E7
[298] Sarcosyl-insoluble tau (PHF-tau) from AD brain was used as an immunogen
for immunization of Balb/c mice. Sarcosyl insoluble tau complexes were
isolated
from human AD brain (frontal cortex, Braak stage VI, Netherlands brain bank)
using
the sarkosyl method (Greenberg and Davies, A preparation of Alzheimer paired
helical filaments that diplays distinct tau proteins by polyacrylamide gel
electrophoresis, PNAS, 87:5827-31, 1990) as described in Example 4.
[299] Six-week-old Balb/c mice were primed subcutaneously with approximately
20-
30 pg of insoluble tau protein isolated from human Alzheimer's brain tissue in
complete Freund's adjuvant (SIGMA) and boosted five times at four-week
intervals
with 20-30 pg of the same antigen in incomplete Freund's adjuvant. Three days
before the fusion, mice were injected intravenously with 20-30 pg of the same
antigen in PBS. Spleen cells from immunized mice were fused with NS/0 myeloma
cells according to the method of Kontsekova et al., The effect of postfusion
cell
density on establishment of hybridomas, Folia Biol. 34, 18-22 (1988).
Splenocytes
were mixed with NS/0 myeloma cells (ratio 5:1) and fused for 1 minute in 1 ml
of
50% polyethylene glycol (PEG) 1550 (Serva) in serum free Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% dimethyl sulphoxide. The fused
cells were resuspended in DMEM containing 20% horse serum, L-glutamine (2 mM),
hypoxanthine (0.1 mM), aminopterin (0.04 mM), thymidine (0.016 mM), and
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gentamycin (40 Wm!), at a density of 2.5 x 105 spleen cells per well on 96-
well
plates.
[300] The cells were incubated for 10-14 days at 37 C and growing hybridomas
were screened for the production of anti PHF-tau-specific monoclonal
antibodies by
an enzyme-linked immunosorbent assay (ELISA). Microtiter plates were coated
overnight with PHF-tau (5 pg/ml, 50 p1/well) at 37 C in PBS. The plates were
blocked
with 1% nonfat dried milk to reduce nonspecific binding, washed with PBS-0.05%
Tween 20, and incubated with 50 p1/well of hybridoma culture supernatant for 1
hr at
37 C. Bound monoclonal antibodies were detected with sheep anti-mouse
immunoglobulin (Ig) conjugated with horse radish peroxidase (HRP, DAKO). The
reaction was developed with TMB one (Kem-En-Tec Diagnostics) as a peroxidase
substrate and stopped with 50 pl of 2 M H2504. Absorbance at 450 nm was
measured using a Powerwave HT (Bio-Tek). Readouts with an absorbance value of
at least twice the value of the negative controls (PBS) were considered
positive.
Positive hybridoma cultures were further subcloned in soft agar according to
the
procedure described in Kontsekova et al., One-step method for establishing 8-
azaguanine-resistant hybridomas suitable for preparation of triomas, J.
Immunol.
Methods, 145, 247-250, (1991).
[301] The monoclonal antibody DC2E7 (produced by the mouse hybridoma cell line
deposited with the American Type Culture Collection, with the ATCC Patent
Deposit
Designation PTA-124992) was identified among the positive hybridoma cultures.
DC2E7 was further characterized as described below. The antibody isotype was
determined to be murine IgG2a by ELISA (Fig. 4) using a mouse Ig isotyping kit
(ISO-2, SIGMA).
Example 7
Sequencing of DC2E7 Variable Regions
[302] Determination of the nucleotide and amino acid sequences of the light
and
heavy chain variable regions of DC2E7 (Fig. 6). The nucleotide sequence of
DC2E7
variable regions (Figs. 6A and 6C) was determined by DNA sequencing of cDNA
synthesized using total RNA extracted from the mouse hybridoma cell line DC2E7
(ATCC), which expresses the DC2E7 monoclonal antibody. Total RNA was extracted
using RNeasy Mini Kit (Qiagen, Germany). Synthesis of the first strand cDNA
was
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carried out using the "High capacity cDNA reverse transcription" kit according
to the
manufacturer's protocol (Applied Biosystems, USA). The composition of the
reagents
for the 2x reverse transcription master-mix was as follows (quantities per 20
pL
reaction): 2 pL of 10x RT buffer; 0.8 pL of 25x dNTP Mix (100 mM); 2 pl of 10x
RT
Random Primers (50 pM); 1 pL of MultiScribeTM Reverse Transcriptase (50 U/pL);
4.2 pL of nuclease-free H20. For reverse transcription, 10 pL of the 2x
reverse
transcription master-mix was mixed with RNA sample (1 pg/10 pL) and cDNA was
synthesized under the following conditions: 10 min at 25 C, 120 min at 37 C, 5
min
at 85 C, and final cooling to 4 C. Amplification of the genes encoding the
variable
regions of the light and heavy chains was done by polymerase chain reaction
(PCR)
using Phusione High-Fidelity DNA Polymerase (Thermo Fisher Scientific, USA).
The
forward primers (M13-L12 5'-
TGTAAAACGACGGCCAGTATGAAGTTTCCTTCTCAACTTCTGCTC-3' and M13-
H5 5'-TGTAAAACGACGGCCAGTATGGACTCCAGGCTCAAMAGTTTTCCTT-3'
were selected after screening a library of mouse immunoglobulin signal
sequence
forward primers. The usage of signal sequence forward primers has the
advantage
to amplify the whole V-gene of both light and heavy antibody chains, without
biases
introduced when the beginning of variable region is being used for primer
design.
The reverse primers for the light and heavy chains (M13-KC 5'-
CAGGAAACAGCTATGACCACTGGATGGTGGGAAGATGG-3' and M13-CG2a 5'-
CAGGAAACAGCTATGACCCAGTGGATAGACCGATGGGGC -3' were derived from
kappa and IgG2a chains constant regions, respectively.
[303] The PCR products were sequenced and the resulting DNA sequences of
variable regions of light and heavy chains of DC2E7 are shown in Figs. 6A and
6C,
respectively. Complementarity determining regions (CDRs) are underlined in the
DC2E7 light and heavy chains protein sequences (Figs. 6B and 6D,
respectively).
CDRs and framework regions (FR) were identified according to the
ImMunoGeneTics (IMGT) numbering system (see, e.g., Lefranc M.P. The IMGT
unique numbering for immunoglobulins, T-cell receptors, and lg-like domains.
The
Immunologist 7, 132-1 36, 1999 (1999)).
Example 8
Monoclonal Antibody DC2E7 Is Specific For Phosphorylated Forms Of Tau Protein
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[304] Recombinant full length tau 2N4R, phosphorylated 2N4R, PHF-tau and fetal
tau were used for further characterization of binding activity of monoclonal
antibody
DC2E7 by immunoblotting. Recombinant human tau isoform 2N4R was prepared as
described in Example 1. PHF- tau was prepared as described in Example 4.
[305] Fetal rat tau extraction and purification was done essentially as
described in
lvanovova et al., High-yield purification of fetal tau preserving its
structure and
phosphorylation, J. lmmunol. Methods, 339:17-22, 2008, using 1% perchloric
acid.
Brain tissue obtained from 1-7 day old rat pups was homogenized in ice-cold 1%
perchloric acid (1.5 g tissue per 5 ml of perchloric acid, Applichem) and
allowed to
stand on ice for 20 min. The homogenate was spun at 15,000xg for 20 min, and
the
clear supernatant was concentrated and simultaneously the buffer was changed
to
washing buffer (20 mM Tris, pH 7.4, 150 mM NaCI, 0.1% Tween 20) using an
Amicon Ultra Centrifugal Filter device (Millipore). The filtered extract were
loaded at
a flow rate of 0.2 ml/min onto a Poly-Prep column C10/10 (GE Healthcare)
packed
with Sepharose carrying immobilized pan-tau mAb DC25. Unbound proteins were
washed off with 10-15 ml washing buffer until the absorbance of the eluting
fractions
(at 280 nm) became stable. Fetal tau, bound to mAb DC25, was eluted with 0.1 M
glycine, pH 2.6. Eluted 0.5 ml fractions were immediately neutralized with 50
pl of 1M
Tris-HCI, pH 9, and assayed by SDS-PAGE. Fractions containing fetal tau were
concentrated using Amicon Ultra Centrifugal Filter devices (Millipore) with
simultaneous buffer exchange to PBS. Fetal tau purified by affinity
chromatography
was precipitated according to Chen et al., (2005) by addition of four volumes
of ice
cold acetone containing 10% trichloroacetic acid. The mixture was incubated at
-
20 C for 2 hours and centrifuged at 15,000xg for 20 min at 2 C. The
supernatant
was discarded and the precipitate was resuspended in 1 ml of ice-cold acetone,
allowed to stand on ice for 20 min and again centrifuged as above. The
resulting
pellet was dried at room temperature and dissolved in a volume of PBS equal to
that
volume before precipitation.
[306] Full length tau isoform 2N4R, sarcosyl insoluble PHF-tau and fetal tau
were
analyzed by immunoblotting using DC2E7 as described in Example 4. In
immunoblotting DC2E7 antibody recognized PHF-tau and fetal tau (Fig. 7). Both
types of tested tau were phosphorylated, but at different levels of
phosphorylation.
No immunoreactivity was observed on recombinant human tau 2N4R. On the other
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hand, the antibody bound in vitro phosphorylated versions of human tau isoform
2N4R. The results indicate that DC2E7 recognizes an epitope which is
phosphorylated, thus the antibody is specific for phosphorylated tau species.
The
epitope recognized by the antibody is present in phosphorylated PHF-tau, fetal
tau
and in vitro phosphorylated tau. Accordingly, DC2E7 is capable of
distinguishing
phosphorylated tau derived from Alzheimer's disease brain tissue (PHF-tau)
from
physiological tau (2N4R).
Example 9
Mapping Of The DC2E7 Phosphoepitope Using Recombinant Tau Deletion Mutants,
Tau Point Mutants, and Tau-Derived Peptides
[307] The immunoreactivity of DC2E7 suggested that antibody recognizes epitope
on phosphorylated tau (fetal tau, PHF- tau, in vitro phosphorylated tau, see
supra).
Therefore, to determine the epitope, phosphorylated deletion mutants of tau
protein
were used (Fig. 8). Phosphorylation of tau deletion mutants was done using
kinase
extract as described in Example 4.
[308] Phosphorylated deletion mutants of human tau protein 2N4R (tau 1-296,
tau
188-441, tau 221-441, tau151-391, tau 122-227) were used for the localization
of the
epitope of DC2E7 in immunoblotting (see Example 4). lmmunoblotting analysis
demonstrated that DC2E7 recognized all of the following deletion mutants: tau1-
296,
tau188-441, tau151-391, tau122-227, except tau protein 221-441 (Fig. 9). These
results indicate that the DC2E7 phospho-epitope lies in proline reach region,
between amino acid residues 188-227.
[309] The abovementioned mapping of the DC2E7 epitope with tau deletion
mutants suggested an epitope between Pro188-Ala227. In this region of tau
there
are 11 phosphorylation sites (Ser191, Tyr197, Ser198, Ser199, Ser202, Thr205,
Ser208, Ser210, Thr212, Ser214, Thr217) detected on PHF-tau (Hanger et al.,
2009,
Fig. 10). Four of them (Ser198, Ser199, Ser202, Thr217) were found also on
fetal
tau (Morishima-Kawashima et al.,1995, Fig. 10). Because DC2E7 recognizes also
fetal tau, the first focus was on phospho-sites that are present and confirmed
in fetal
tau (Ser198; Ser199; Ser202; Thr217).
[310] To define the exact phosphosite(s) for DC2E7, mutated forms of tau 2N4R
with single point mutations in which Serine and Threonine residues were
replaced by
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Alanine were generated. Insertion of the desired point mutations (Ser198A1a;
Ser199A1a; Ser202A1a; Thr217A1a) into human 2N4R tau protein was done using
QuickChange site directed mutagenesis kit (Agilent Technologies, USA, CA)
according the manufacturer's instructions. 5-50 g of plasmid containing
coding
sequence for wild-type 2N4R tau protein and 125 ng of each primer were used in
one 50 I reaction. Cycling parameters were as follows: 95 C for 30 seconds,
then
PCR continued with cycle which was repeated 16 times: denaturation 95 C for
30
seconds, annealing 55 C for 60 seconds, elongation 68 C for 6 min. The whole
reaction was digested with Dpn I, which is specific for methylated DNA, to
eliminate
parental plasmid. 50 I of supercompetent Escherichia coli XL-1 Blue (provided
with
the mutagenesis kit) were transformed with 1 I of the reaction and spread
onto LB-
agar plates supplemented with ampicillin. Plasmids were isolated from grown
colonies and verification of the mutagenesis was done using DNA sequencing.
Plasmids with desired mutations were used for production of recombinant
proteins
as described in Example 1.
[311] Prepared tau mutants were purified as described in Example 1,
phosphorylated by kinase extract from the rat brain and stained with DC2E7 in
immunoblotting. Phosphorylation of deletion mutants of tau induced shift in
molecular
weight, thus all proteins were phosphorylated, as shown staining with pan tau
antibody DC25 (epitope 347-353 aa, AXON Neuroscience, SE) (Fig. 11). Analysis
of
phosphorylated tau point mutant demonstrated that DC2E7 detected all single
mutations (Ser198Ala, Ser199Ala, Ser202A1a) but Thr217Ala (Fig. 11). These
results
suggested, that phospho-threonine at position 217 creates key part of the
epitope
recognized by antibody DC2E7.
[312] However, other phospho-sites which are located in proximity the phospho-
site
Thr217 could be a part of the epitope, or may be involved into creation of the
epitope. Therefore, the possible impact of phospho-sites Thr205, 5er208,
Ser210,
Thr212, 5er214, 5er231 on epitope recognized by DC2E7 was examined. Prepared
mutant tau proteins with single point mutations (Thr205Ala, Ser208Ala,
Ser210Ala,
Thr212Ala, Ser214Ala, Ser231A) were phosphorylated by kinase extract and
tested
in immunoblotting with DC2E7 (Fig. 11). The immunoblotting analysis
demonstrated
that the antibody recognized all tau proteins carrying the point mutations
(Fig. 11).
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Thus, phospho-sites located in the proximity of Thr217 did not affect the
epitope
recognized by DC2E7.
[313] These mapping experiments suggested the presence of phospho-threonine
217 within the epitope of DC2E7. In order to examine the effect of residues in
the
region of DC2E7 epitope on antibody binding, synthetic peptides of different
lengths
carrying different phospho-sites were analyzed in competitive ELISA. Peptides
(tau
210-224/pT212/pT217, 210-222/pT212/pT217, 210-221/pT212/pT217, 210-
220/pT212/pT217, 210-219/pT212/pT217, 210-218/pT212/pT217, tau 201-
230/pT212, tau 193-222/pS208, 193-222/pS214, tau 193-
231/pS208/pS210/pT212/pS214/pT217, tau 193-231/pS202/pS205/pT212/pT220,
tau 201-229), were synthesized by EZBiolabs (USA) with purity higher than 95%.
Each synthetized peptide contained phospho-site(s) located around the residue
Thr217, except of unphosphorylated peptide tau 201-229 (used as negative
control).
As positive control, in vitro phosphorylated tau151-391 was used.
[314] Next, all peptides were analyzed for their ability to compete with in
vitro
phosphorylated tau151-391 for binding to DC2E7 by competition ELISA. ELISA
plates (Nunc Medisorp, Thermo Scientific, Denmark) were coated overnight at 37
C
with 50 p1/well of PHF-tau, diluted 200 x in PBS. The coated plates were
washed 5
times with PBS/Tween 20 (0.05% v/v), and blocked with PBS/Tween 20 for 1 h at
25 C. Each of the peptides was separately dissolved in PBS at a final
concentration
of 1mM. Serial dilutions (2.5x) of peptides in PBS/Tween 20 were prepared in
polypropylene microtiter plates with conical well bottom (Greiner) within the
concentration range of 200 pM; 80 pM; 32 pM; 12.8 pM; 5.12 pM; 2.048 pM;
0.8192
pM; 0.32768 pM). The monoclonal antibody DC2E7 was diluted to a concentration
of
0.6 pg/ml in PBS and 60 pl of this diluted antibody was added into each well
to serial
dilution of peptides resulting in 120 p1/well of mixture. The antibody/peptide
mixtures
were incubated for lhr at 25 C on a rotating platform set to 230 rpm. 50
p1/well of
antibody/peptide mixtures were transferred from polypropylene plates into PHF-
tau
coated and PBS/Tween 20 blocked ELISA plates (in duplicates) and incubated for
lhr at 25 C. The plates were washed 5x times with PBS/Tween 20 and incubated
with 50 p1/well of Polyclonal goat anti-mouse immunoglobulins/HRP (Dako)
diluted
1:1000 in PBS/Tween 20 for lhr at 25 C. After washing, the plates were then
incubated with 50 p1/well of 1 mg/2 mL o-PDA (o-phenylenediamine, Sigma) in
0.1 M
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Na-Acetate pH=6.0 (Roth) supplemented with 1.5 p1/2 ml of 30% H202 (Sigma) for
20 minutes at 25 C in dark. The reaction was stopped by adding 50 p1/well of
2M
H2504 (Merck) followed by reading the plates at 492 nm (Powerwave HT, Bio-
Tek).
[315] All analyzed peptides that encompassed phosphorylated threonine 217
competed with phosphorylated tau151-391 for binding to DC2E7 (Fig.12). In
summary, the DC2E7 binds a tau epitope that includes a phosphorylated
threonine
217. No other phospho-sites appeared to impact antibody immunoreactivity. But,
notably, shortening of the C terminal part of the tested peptides from 224 to
220
amino acids (210-220/pT212/pT217, 210-219/pT212/pT217, 210-218/pT212/pT217)
led to the loss of competing activity despite the presence of phosphorylated
threonine 217. The data thus suggest that DC2E7 binds a phosphoepitope on
human phosphorylated tau of 12 amino acids which comprise the residues 210-
SRTPSLPTPPTR-221, where at least the threonine 217 is phosphorylated.
Example 10
Monoclonal Antibodies DC2E2 and DC2E7 Recognize Pathology In Human
Alzheimer's Disease Brain and Tauopathies
[316] This example shows that DC2E7 and DC2E2 recognise neurofibrillary
lesions
in Alzhemer's disease. The following brain areas were used for
immunohistochemical study: hippocampus and entorhinal cortex from Alzheimer's
disease (Braak stage 6), FTD (Pick's disease), and control brains (Braak stage
1 and
3), caudate nucleus from corticobasal degeneration (CBD) and putamen/caudate
nucleus from progressive supranuclear palsy (PSP). The brain tissue paraffin
blocks
were obtained from Amsterdam brain bank.
[317] The brains blocks embedded in paraffin were cut on a microtome (Leica
RM2255) to get 8 pm thick sections. The sections were placed on HistoBond
slides
(Marienfeld, Germany). For immunohistochemistry sections were pre-treated with
formic acid (98% for 1 min at 4 C or 80% for 1 hour) and heat (autoclave, 121
C, 20
min.), followed by overnight incubation with primary antibodies (AT8 1:1000,
DC2E7
1: 10 000, DC2E2 1:200). All sections were incubated with anti-mouse
biotinylated
secondary antibody at room temperature for 1 hour and with avidin-biotin
peroxidase-complex for 1 hour. The immunoreaction was visualised with VIP
(Vectastain Elite ABC Kit, Vector Laboratories, CA, USA) and counterstained
with
methyl green (Vector Laboratories).
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[318] lmmunohistochemical analyses revealed that both DC2E7 and DC2E2
recognized tau pathology in Alzheimer's disease and other tauopathies (Fig.
13) in a
similar way as monoclonal antibody AT8 which is considered to be golden
standard
for histopathological staining. DC2E7 and DC2E2 stained neurofibrillary
pathology in
the hippocampus of AD patient, Pick's bodies in the dentate gyrus of FTD
patient,
glial tau pathology in caudate nucleus of patients suffering either from CBD
and
PSP. DC2E7 and DC2E2 do not recognize tau pathology in normal brain fulfilling
criteria for Braak stage 1, in prodromal stage (Braak stage 3) both antibodies
identified in the hippocampus neurofibrillary pathology, in the full blown AD
antibodies visualized extensive tau pathology in the form of neurofibrillary
tangles,
neuropil threads and neuritic plaques (Fig. 14). By using higher
magnification, it was
demonstrated that the antibodies bound neurofibrillary tangles in AD, Pick's
bodies in
FTD and glial tau pathology in PSP and CBD (Fig. 15).
Example 11
DC2E7 ELISA
Preparation of standard for DC2E7 ELISA
[319] In vitro phosphorylated tau151-391 was purified by affinity
chromatography.
Two affinity columns were prepared: DC2E7 and DC190 (epitope 368-376, AXON
Neuroscience, SE). Antibodies were purified according method described in
Example 3. Antibodies were coupled to CnBr-activated Sepharose 4B (GE
Healthcare, #17-0430-01) according to the manufacturer recommendation. In
vitro
phosphorylation reaction (see Example 4) was dialyzed against PBS at 4 C (3 x
100-
fold excess). All purification steps were performed at 4 C. DC2E7 column was
washed with 3 x 5 ml of WBNP0.1 buffer (50 mM Tris pH 7.4, 150 mM NaCI and 0.1
% NP40). In vitro phosphorylation reaction was diluted 4-fold with ice cold
WBNP1
buffer (50 mM Tris pH 7.4, 150 mM NaCI and 1 % NP40) and filtered through 0.2
pm
filter. Sample was applied onto DC2E7 column. After sample entered resin
column
was washed as follows: 2 x 5 mL with WBNP1 buffer, 2 x 5 mL with WBNP0.1
buffer,
1 x 5 mL with 50 mM Tris.HCI pH 7.4, 150 mM NaCI. Bound proteins were eluted
with 3 x 2 ml of elution buffer (100 mM glycin/HCI, pH 2.8). Eluted proteins
were
immediately adjusted to pH 7-8 with 1 M Tris.HCI, pH 9, and diluted 1:1 with
WBNP0.1 buffer. Sample was applied on DC190 affinity column and purified as
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described for DC2E7 affinity purification. Eluted proteins were dialyzed
against PBS
and concentration was spectrophotometrically estimated. Purified protein was
designated "DC2E7 calibrator".
[320] DC2E7 ELISA was set up in high sensitive format, digital EL ISA, using
Simoa-
HD1 analyzer (Quanterix). Reagents for digital ELISA were prepared according
to
the Quanterix Homebrew Assay Development Guide with following details. DC2E7
antibody was used as a capture antibody and DC2E2 antibody was used as a
detector antibody. DC2E7 was coupled to magnetic beads (Quanterix) at
concentration of 0.5 mg/mL. Detector antibody was prepared by biotinylation of
DC2E2, whereby 120 ¨ fold excess of Biotin, EZ-LinkTM NHS-PEG4-Biotin (Thermo
Scientific, #21329) over antibody concentration was used. DC2E7 calibrator was
diluted in calibrator diluent (20 mM sodium phosphate pH 7.4, 137 mM NaCI, 2.7
mM
KCI, 2 % BSA and 0.01 % casein) in serial two-fold dilutions starting from 100
pg/mL,
following by 50, 25, 12.5, 6.25, 3.13, 1.56 and 0 pg/ml. Prepared calibrator
concentrations were mixed in a 3:1 ratio with sample diluent (80 mM sodium
phosphate pH 7.4, 548 mM NaCI, 10.8 mM KCI, 0.04 % casein and 0.4 % Tween
20). CSF samples from control individuals were also diluted with sample
diluent, as
previously described. Spike recovery of CSF sample was performed using spikes
of
CSF with 10, 5, 2 and 0 pg/ml of DC2E7 calibrator. Diluted calibrators and
samples
were pipetted into 96 well plate, inserted in Simoa HD1 analyzer and also
capture
antibody DC2E7 beads diluted in bead diluent, detector DC2E2 diluted in
detector
diluent to 1.2 g/ml, SBG diluted in SBG diluent to 200 pM and substrate RGP
were
inserted in analyzer (all buffers, SBG and RGP were obtained from Quanterix).
Assay was programmed in Simoa 1.5 software and analysis was performed. After
analysis evaluation was done by Graphpad Prism and/or by software included in
Simoa 1.5 software. An example of a calibration curve is shown on Fig. 16. An
example of a spike recovery experiment is shown on Fig. 17.
Example 12
DC2E7 Digital ELISA Significantly Distinguishes Between Alzheimer's Disease
Patients And Control Individuals
[321] DC2E7 digital ELISA was used for analysis of CSF samples from
Alzheimer's
disease patients (n = 20) and healthy individuals (n = 20) (Fig. 18). Analysis
demonstrated that DC2E7 digital ELISA distinguished Alzheimer's disease
patients
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from control individuals with very high significance. Area of ROC curve was
very
close to 1. At a concentration >4.43 pg/mL the assay gives 95 % sensitivity
and 89.5
% specificity and at concentration >6.19 pg/mL, sensitivity is 95% and
specificity
100%.
Example 13
DC2E7 Digital Elisa Significantly Distinguishes Between Alzheimer's Disease
Patients And Patients Suffering From Other Tauopathies
[322] DC2E7 digital ELISA was used for analysis of CSF samples from
Alzheimer's
disease patients (n = 6) and frontotemporal dementia (n = 16) (Fig.19).
Analysis
demonstrated that DC2E7 digital ELISA distinguished Alzheimer's disease
patients
from patients having other tauopathies with very high significance. Area of
ROC
curve is 0.97. At concentration > 3.89 pg/ml the assay gives 93.8 %
sensitivity and
100% specificity.
Example 14
DC2E7 Recognizes Insoluble Tau Species In Alzheimer's Disease And Human
Tauopathies
[323] Previous results showed that DC2E7 recognized an epitope which occurs in
phosphorylated tau. Therefore, the immunoreactivity of DC2E7 to phosphorylated
tau protein which occurs in other tauopathies was analyzed.
[324] Insoluble tau complexes (abnormal tau forms) were isolated from human AD
brain (Braak stage V, frontal cortex, Amsterdam Brain bank), corticobasal
degeneration (London brain bank, frontal cortex) and FTD (Amsterdam Brain
bank)
as described in Example 4. Extracted insoluble tau proteins were analyzed in
immunoblotting using DC2E7 as described in Example 4.
[325] lmmunoblot analyses of brains from patients with AD and aforementioned
tauopathies revealed that insoluble tau fractions were detectable using DC2E7.
The
results suggest that the pathological tau aggregates in tauopathies are
composed of
hyperphosphorylated tau similar to PHF-tau in AD (Fig. 20). Moreover, DC2E7
recognized three predominant PHF-tau-like bands of 60, 64, and 68 kDa (A68
triplet)
in CBD and FTD similar to that of AD.
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Example 15
pT217 Tau Digital ELISA Assay Distinguished AD Patients From FTD and Healthy
Subjects With High Sensitivity And Specificity
[326] A pT217 tau digital assay using antibody DC2E7 was used to analyze CSF
from AD patients (n = 30), FTD patients (nfPPA, n = 14; svPPA, n = 10; bvFTD,
n =
10, PSP, n = 19; and CBD, n = 15), and healthy individuals (n = 30). The assay
was
compared to an INNOTEST Phospho-Tau(181P) assay.
[327] Analysis demonstrated that the pT217 tau digital ELISA assay
distinguished
AD patients from FTD and healthy subjects. The assay better separated AD from
FTD/controls (>5.37 pg/ml, 94.1% sensitivity, 91.7% specificity, Fig. 21), as
compared to the lnnotest Phospho-Tau(181P) assay (> 52 pg/ml, 78.0%
sensitivity,
100% specificity. (Fig. 21). The assay was also slightly better at
distinguishing AD
patients from healthy subjects (>3.855 pg/ml, AUC 0.96, 95% Cl, 0.89-0.99, 95%
sensitivity, 89.7% specificity) than the p-tau 181 assay (> 52 pg/ml, AUC
0.93, 95%
Cl, 0.85-0.97, 92% sensitivity, 87.5% specificity) (Fig. 22). Comparing the
high
correlation between pT217 and pT181 assays in the AD group (P< 0.0001,
Spearman coefficient r=0.8226) to the low correlations observed in healthy
controls
(P<0.5, Spearman coefficient r=0.4032) and FTD (P=0.387, Spearman coefficient
r=0.232) suggests that the pT217 tau species in CSF may provide an AD specific
biomarker in CSF. No benefit was observed for using of ratio INNOTEST
A642/pT217 tau when compared to pT217 tau itself.
[328] High correlation was observed between pT217 tau and pT181 tau assays in
the AD group (P< 0.0001, Spearman coefficient r=0.8226) and the low
correlations
was observed in non-demented controls (P<0.5, Spearman coefficient r=0.4032).
[329] No correlation was observed between pT217 tau and pT181 tau assays in
patients from FTD (P = 0.7517, Spearman coefficient r = 0.08929); whereby
strong
correlation was observed between pT181 and HTAU assays in the same FTD
patients (P = 0.0019, Spearman coefficient r = 0.7321)
[330] All these results suggest that the pT217 tau species in CSF may provide
an
AD specific biomarker in CSF.
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Example 16
pT217 Tau Digital ELISA Assay Distinguished AD Patients From FTD And Healthy
Subjects With High Sensitivity And Specificty Using Different Calibrators
[331] The pT217 tau digital ELISA assay can distinguish between subjects with
mild
cognitive impairment (MCI) and healthy subjects. The pT217 tau concentrations
in
cerebrospinal fluid (CSF) are similar for subjects with MCI and AD (Fig. 23A).
Three
subjects with MCI later developed AD.
[332] The pT217 tau assay was used to test CSF samples from subjects with
other
neurological disorders including multiple sclerosis (MS), Parkinson's disease
(PD),
amotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), where
tau
pathology may be present (Fig. 23B). The FTD patients represent quite
heterogeneous populations, some of those patients suffered from progressive
supranuclear palsy (PSP) or corticobasal degeneration (CBD), while the others
developed symptoms typical for primary progressive aphasia (PPA). The pT217
tau
digital ELISA assay distinguishes between AD and the other neurological
disorders
(cut-off 9.3 pg/ml, specificity, 94%, sensitivity 100%).
[333] The pT217 tau digital ELISA assay can distinguish AD patients from FTD
patients and healthy subjects with similar sensitivity and specificity
regardless of the
chosen calibrator. Distribution of pT217 tau in CSF samples from AD patients,
FTD
patients and healthy subjects using either in vitro phosphorylated tau protein
or
synthetic peptide showed similar sensitivity and specificity. Using the
phosphorylated tau protein calibrator, the assay exhibited 94.1 % sensitivity
and 91.7
% specificity (>5.37 pg/ml). Using the 2E7 peptide (2E7pep), the assay
exhibited
93.9 % sensitivity and 90.0 % specificity (>305 pg/ml) (Fig. 29). 2E7pep
possessed
the following amino acid sequence:
GQKGQANATRIPAKGGGSGGGSGGGSSRTPSLPpTPPTREPK. The first
underlined sequence represents an epitope of the DC2E2 antibody, the second
underlined sequence represents an epitope of the DC2E7 antibody (first 3 amino
acids and last amino acids are derived from tau protein, while the sequence
(GGGS)3 is a linker connecting the epitopes of DC2E2 and DC2E7).
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Example 17
Optimization of Antibody DC2E7 By Recombinant Technologies
[334] The affinity of the DC2E7 antibody was optimized by ribosome display and
phage display technologies. First, cDNA encoding an scFv format of antibody
DC2E7 was generated by amplification and cloning from a DC2E7 hybridoma cell
line. The gene for the scFv DC2E7 was then mutated by error-prone PCR and a
library of mutated scFv DC2E7 fragments was affinity selected against 2E7pep
by
ribosome display (Hanes et al., 1998) and phage display technologies (Harrison
at
al., 1996)
[335] After four rounds of selection, isolated scFv's fragments were cloned
into an
expression vector such that scFvs were fused to a myc-tag and expressed in E.
coli
J M109 (Sanmark et. al., 2015). Bacterial lysates of 30 colonies containing
mutated
DC2E7 scFv were tested using a peptide competition immunoassay. The
immunoassays were performed in 96 well microtiter plates coated with 100
l/well of
0.01 g/m1 of 2E7pep in PBS at 4 C overnight. Plates were washed 4x and
blocked
in PBS-T for 30 min at room temperature. Binding was performed with 100 I of
10x
diluted bacterial lysate in PBS-T, containing anti-myc antibody diluted 10,000
x, on a
shaking platform for 1 hour at RT. The wells were washed four times with PBS-T
and
competition with a 2E7pep at a concentration of 10 g/m1 per lysate was
performed
for 2 hours at RT on a shaking platform. Then, plates were washed 4x with PBS-
T
and incubated with streptavidin-HRP conjugate diluted 1:10000 in PBS-T for 1
hour
at RT. The plates were washed 4xPBS-T and developed using a TMB substrate for
5
min before the colorimetric reaction was stopped using 1 M H2504. The
absorbance
at 450 nm was read using a microtiter plate absorbance reader.
[336] Absorbance for the scFvs generated by ribosome display and phage display
are shown in Fig. 24. scFv K+ represents unmutated DC217. All scFv showing
signal
above the line corresponding to scFv K+ are expected to have higher affinity
(labeled
with an asterisk), as compared to the original DC217 scFv.
[337] Alignments of the heavy and light chain amino acid sequences of the
scFvs
isolated by ribosome display and phage display ("PD" at the end of a clone
name
indicates it was obtained by phage display) are shown in Fig 25. VL (Fig. 25A)
and
VH (Fig. 25B) sequences are shown. Antibodies with improved binding relative
to
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DC2E7 are shown in Fig. 250 (light chain) and Fig. 25D (heavy chain). The
first line
represents the original DC2E7 antibody. Residues identical to the sequence in
DC2E7 are represented by dots. Numbering of amino acid residues in VL and VH
and the labeling of CDRs is according to the IMGT numbering system.
Example 18
Comparison of Antibodies DC2E7, DC149, and D0807
[338] Antibody DC149 was generated in a similar manner to what was described
in
Example 2, with the following difference: Mice were immunized with a double
phosphorylated peptide at positions Thr212 and Thr217, i.e.,
CSRpTPSLPpTPPTREPK (210-224 of 2N4R tau) conjugated via the first cysteine to
KLH. Screening was performed by ELISA as described in Example 2, whereby
D0149 was identified by specific binding to peptide comprising
SRTPSLPpTPPTREPK (i.e., the same peptide mono-phosphorylated at Thr217 that
DC2E7 bound) and D0807 by specific binding to the double-phosphorylated
peptide.
The D0149 and D0807 antibodies did not bind to an unphosphorylated peptide or
to
unphosphorylated tau protein, while D0807 also did not bind to mono-
phosphorylated peptides.
[339] DC2E7, and DC149 an D0807 were analyzed in a classical ELISA assay
using a similar setup to what was described in example 10, whereby 2E7pep
calibrator was used. All three antibodies bound to tau peptide phosphorylated
at
least on Thr217 with very similar affinities (Fig. 26).
[340] Amino acid sequences for antibodies DC217, DC149 and D0807 were
determined by cloning and by mass spectrometry. Alignment of the heavy and
light
chain sequences in DC2E7 and D0149 are shown in Fig. 27A-B. Alignments of the
heavy and light chain sequences in DC2E7 and D0807 are shown in Fig. 28A-B.
Residues identical to the sequence of DC2E7 are represented by dots.
Complementarity-determining regions (CDRs) of VL and VH are boxed. Numbering
of amino acid residues in VL and VH and the labeling of CDRs is according to
the
IMGT numbering system.
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Literature references:
Hanes et al., (1998). Ribosome display efficiently selects and evolves high-
affinity
antibodies in vitro from immune libraries. PNAS, Vol. 95, pp. 14130-14135.
doi.org/10.1073/pnas.95.24.14130.
Harrison et al., (1996). Screening of phage antibody libraries. vol. 26783-
109, pp. 83-
109. doi.org/10.1016/S0076-6879(96)67007-4.
Kabat, E. A., Wu, T. T., Perry, H. M., Gottesmann, K. S. & FoeIler, C. (1991)
in
Sequences of Proteins of Immunological Interest (U.S. Department of Health and
Human Services, Bethesda, MD) Vol. I, pp. 151 and 464, 5th Ed.
Example 19
Independent Validation of pT217 Assay
The protocol for pT217 detection and quantitation discussed previously was
applied
to SIMOA using an HD-1 Analyzer. The protocol was found suitable for the
measurement of pT217 in human CSF samples. The assay's sensitivity, linearity,
parallelism, and recovery were analyzed. Standard curves were measured by
spiking
2E7 pep calibrator peptide into CSF samples and into PBS. DC2E7 was used as
the
capture antibody and DC2E2 was used as the detection antibody. The observed
limit
of detection was 184.4 pg/mL. Repeatability was assessed: lntra-assay (<15%
for
values within the linear range of the standard curve), Inter-assay (<15% for
values
within the linear range of the standard curve), and Intra-plate (assay (<15%
for
values within the linear range of the standard curve). Parallelism and
recovery were
determined to be <15%.
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