NZ709976B2 - Human anti-tau antibodies - Google Patents
Human anti-tau antibodies Download PDFInfo
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- NZ709976B2 NZ709976B2 NZ709976A NZ70997613A NZ709976B2 NZ 709976 B2 NZ709976 B2 NZ 709976B2 NZ 709976 A NZ709976 A NZ 709976A NZ 70997613 A NZ70997613 A NZ 70997613A NZ 709976 B2 NZ709976 B2 NZ 709976B2
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Abstract
Provided are novel human tau-specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto. Assays, kits, and solid supports related to antibodies specific for tau are also disclosed. The antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for tau targeted immunotherapy and diagnosis, respectively. derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for tau targeted immunotherapy and diagnosis, respectively.
Description
- l -
HUMAN ANTi-TAU ANTIBODIES
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention generally relates to novel tau-specific binding molecules,
ularly human antibodies as well as fragments, derivatives and variants thereof
that recognize the tau protein, including pathologically phosphorylated tau and
aggregated forms of tau. In addition, the present invention relates to pharmaceutical
and diagnostic compositions sing such binding molecules, antibodies and
mimics thereof valuable both as a diagnostic tool to identify tau and toxic tau species
in plasma and CSF and also in passive vaccination gies for ng
neurodegenerative thies such as mer’s disease (AD), amyotrophic lateral
sclerosis/parkinsonism—dementia complex (ALS-PDC), argyrophilic grain dementia
(AGD), British type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal
degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse
neurofibrillary tangles with cation, Down’s me, temporal dementia,
frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17),
frontotemporal lobar degeneration, Gerstmann—Straussler-Scheinker disease,
Hallervorden-Spatz disease, ion body is, multiple system atrophy,
myotonic dystrophy, Niemann-Pick disease type C mP-C), non-Guamanian motor
neuron disease With neurofibrillary tangles, Pick’s disease (PiD), postencephalitic
parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical
gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis,
tangle only dementia, infarct dementia and ischemic stroke.
Background Art
[0002] n accumulation, modifications and aggregation are pathological aspects of
numerous neurodegenerative diseases. Pathologically modified and aggregated tau
including hyperphosphorylated tau mers are an invariant rk of tauopathies
and correlate with disease severity.
Tau is a microtubule-associated protein expressed in the central nervous system
with a primary function to stabilize microtubules. There are six major isoforms of tau
' 2 '
expressed mainly in the adult human brain, which are derived from a single gene by
alternative splicing. Under pathological conditions, the tau n becomes
hyperphosphorylated, resulting in a loss of tubulin binding and destabilization of
ubules followed by the aggregation and deposition of tau in pathogenic
neurofibrillary tangles. Disorders related to tau - collectively referred to as
neurodegenerative tauopathies - are part of a group of protein misfolding disorders
including Alzheimer’s disease (AD), progressive uclear palsy, Pick’s disease,
corticobasal degeneration, FTDP—l7 among others. More than 40 mutations in tau gene
have been reported to be ated with tary frontotemporal dementia
demonstrating that tau gene mutations are ent to trigger neurodegeneration
(Cairns et al., Am. J. Pathol. 171 (2007), 227-40). Studies in transgenic mice and cell
culture indicate that in AD, tau pathology can be caused by a pathological cascade in
which AB lies upstream of tau (Gotz et al., e 293 (2001), 1491—1495). Other
finding however point to a dual-pathway model where both cascades function
independently of each other (van de Nes et al., Acta Neuropathol. 111 (2006), 126-
138). Immunotherapies targeting the beta-amyloid peptide in AD have produced
encouraging results in animal models and shown promise in clinical trials. More recent
autopsy data from a small number of subjects suggests that clearance of beta—amyloid
plaques in patients with ssed AD may not be sufficient to halt cognitive
oration, izing the need for additional therapeutic strategies for AD
(Holmes et al., Lancet 372 (2008), 216-223; Boche et al., Acta Neuropathol. 120
(2010), 13-20). In the wake of the s of Abetaebased immunization therapy in
transgenic animal models, the concept of active immunotherapy was expanded to the
tau protein. Active ation of wild type mice using the tau protein was however
found to induce the ion of neurofibrillary tangles, axonal damage and
mononuclear infiltrates in the central nervous system, anied by neurologic
deficits (Rosenmann et (1]., Arch Neurol. 63 (2006), 1459-1467). Subsequent studies in
transgenic mouse lines using active vaccination with phosphorylated tau peptides
revealed reduced brain levels of tau aggregates in the brain and slowed progressior: of
behavior impairments (Sigurdsson, J. Alzheimers. Dis. 15 (2008), 157-168; Boimel et
(11., Exp. Neurol. 224 (2010), 472-485). These findings highlight the potential benefit
but also the tremendous risks associated with active immunotherapy approaches
' 3 '
targeting tau. Novel therapeutic strategies are ly needed addressing ogical
tau proteins with efficacious and safe therapy.
Passive immunization with human antibodies derived from healthy human
subjects which are evolutionarily optimized and affinity matured by the human
immune system would provide a ing new therapeutic avenue with a high
ility for excellent efficacy and safety.
BRIEF SUMlVlARY OF THE INVENTION
The present invention makes use of the tau-specific immune response of healthy
human ts for the isolation of natural anti-tau specific human monoclonal
antibodies. In particular, experiments performed in accordance with the t
invention were successfiJl in the isolation of monoclonal ecific antibodies from a
pool of y human subjects with no signs of a egenerative tauopathy.
[0006} The present invention is thus directed to human antibodies, antigen-binding
fragments and similar antigen-binding molecules which are capable of specifically
recognizing tau. By "specifically recognizing tau", "antibody specific to/for tau" and
"anti—tau antibody" is meant specifically, generally, and collectively, antibodies to the
native form of tau, or aggregated or pathologically modified tau isoforms. ed
herein are human antibodies ive for full-length, pathologically phosphorylated
and aggregated forms.
[0007] In a particular embodiment of the present invention, the human antibody or
antigen-binding fragment f demonstrates the immunological binding
characteristics of an antibody characterized by the variable regions VH and/or VL as set
forth in Fig. 7.
The antigen-binding fragment of the antibody can be a single chain FV fragment,
an F(ab') nt, an F(ab) fragment, and an F(ab')2 fragment, or any other antigen-
binding fragment. In a specific embodiment, infra, the antibody or fragment thereof is
a human IgG isotype antibody. Alternatively, the antibody is a chimeric human-murine
or murinized dy, the latter being particularly useful for diagnostic methods and
studies in animals.
[0009] Furthermore, the present invention relates to compositions comprising the
antibody of the present invention or active fragments thereof, or agonists and cognate
' 4 '
molecules, or alternately, antagonists of the same and to immunotherapeutic and
immunodiagnostic s using such compositions in the prevention, diagnosis or
ent of a tauopathy, wherein an effective amount of the composition is
administered to a patient in need thereof.
Naturally, the t invention extends to the immortalized human B memory
lymphocyte and B cell, respectively, that produces the antibody having the distinct and
unique characteristics as defined below.
The t invention also relates to polynucleotides encoding at least a variable
region of an immunoglobulin chain of the antibody of the invention. In one
embodiment, said variable region comprises at least one complementarity determining
region (CDR) of the VH and/or VL of the variable region as set forth in Figure 7.
Accordingly, the present invention also encompasses vectors comprising said
polynucleotides and host cells transformed therewith as well as their use for the
tion of an antibody and equivalent binding molecules which are specific for tau.
Means and methods for the recombinant tion of antibodies and mimics thereof
as well as methods of screening for ing binding molecules, e.g., antibodies,
known in the art. However, as bed herein, in particular with respect to
therapeutic applications in human the antibody of the present invention is a human
dy in the sense that application of said antibody is substantially free of an
immune response directed t such antibody otherwise observed for chimeric and
even humanized antibodies.
Furthermore, disclosed herein are compositions and methods that can be used to
identify tau in samples. The disclosed anti—tau antibodies can be used to screen human
blood, CSF, and urine for the presence of tau in samples, for example, by using
ELISA-based or surface adapted assay. The methods and compositions disclosed
herein can aid in neurodegenerative thies such as Alzheimer's disease diagnosis
and can be used to r e progression and therapeutic efficacy.
Hence, it is a particular object of the present invention to provide methods for
treating, diagnosing or preventing a neurodegenerative tauopathy such as Alzheimer’s
disease, amyotrophic lateral sis/parkinsonism—dementia complex, argyrophilic
grain dementia, British type amyloid angiopathy, cerebral amyloid angiopathy,
corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse
neurofibrillary s with calcification, Down’s syndrome, frontotemporal dementia,
frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal
lobar degeneration, Gerstmann-Sträussler-Scheinker e, Hallervorden-Spatz
e, inclusion body myositis, multiple system atrophy, myotonic dystrophy,
Niemann-Pick disease type C, non-Guamanian motor neuron disease with
neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein
cerebral d angiopathy, progressive subcortical gliosis, progressive supranuclear
palsy, subacute sing panencephalitis, tangle only dementia, multi-infarct dementia
and ischemic stroke. The methods comprise administering an effective concentration of
a human antibody or antibody derivative to the subject where the antibody targets tau.
[0015] Further embodiments of the present ion will be apparent from the
ption and Examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
Amino acid and nucleotide sequences of the variable region, i.e. heavy
chain and lambda light chain of human antibodies NI-105.4E4 (A), NI-105.24B2 (B)
and NI-105.4A3 (C). Framework (FR) and complementarity determining regions
(CDRs) are indicated with the CDRs being underlined. Due to the cloning strategy the
amino acid sequence at the N-terminus of the heavy chain and light chain may
potentially contain primer-induced alterations in FR1, which however do not
substantially affect the ical activity of the antibody. In order to provide a
consensus human antibody, the nucleotide and amino acid sequences of the original
clone were aligned with and tuned in accordance with the pertinent human germ line
variable region sequences in the database; see, e.g., Vbase //vbase.mrccpe.cam.ac.uk
/) hosted by the MRC Centre for Protein ering (Cambridge, UK).
Those amino acids, which are considered to potentially deviate from the consensus germ
line sequence due to the PCR primer and thus have been replaced in the amino acid
sequence, are indicated in bold.
NI-105.4E4 binds to neurofibrillary tangles (NFT), dystrophic es
and neuropil threads in AD brain and human TauP301L expressing mice. .4E4
staining identifies NFTs and neuropil threads in AD brain (A), with no significant
binding to tau in the brain of healthy l subject (B). In TauP301L transgenic
" 6 '
NFT (E, F
mouse (E—I) NI-105.4E4 binds strongly to the pathological tau resembling
and H), neuropil threads (E and G) and dystrophic neurites (E and H). In addition, NI-
105.4E4 also identifies tau aggregates at pre-tangle stage (I). NI—105.4E4 binds to
NFT, dystrophic neurites and neuropil threads in transgenic mouse expressing human
APP with the Swedish and the Arctic on and TauP301L; the arrow marks a beta-
amyloid plaque, nded by dystrophic neurites recognized by NI-105.4E4 (J).
Secondary dy only does not give signal both in human AD (C) and healthy
control (D).
Tissue amyloid plaque immunoreactivity (TAPIR) assay. Neurofibrillary
tangles were d wéth either the anti-phospho-tau antibody AT100 or sera isolated
from healthy elderly subjects.
Schematic representation of the Nl-105.4E4 and NI—105.4A3 epitopes and
epitopes of commonly used commercially available mouse monoclonal tau dies
that comprises two
are shown. Human antibody NI-105.4E4 s a unique e
linear polypeptides, one of which is located in the microtubule binding domain (R4) of
tau which is masked in physiological microtubule-associated tau. Tau-12 (Covance,
California, USA), HT7, AT8, AT180 (Thermo Scientific, U.S.A.); PHFl (Lewis et
al., Science 293 (2001), 1487-1491).
Human IgG levels in the plasma of mice following intraperitoneal
administration of 30 mg/kg NI-105.4E4 or NI—105.4A3 human anti-tau antibody.
Human IgG levels in brain homogenate of mice following intraperitoneal
stration of 30 mg/kg .4E4 or NI-105.4A3 human anti-tau antibody.
Amino acid ce of heavy chain and light chain variable regions of
(A) NI-105.17C1, (B) NI-105.6C5, (C) NI—105.29G10, (D) NI-105.6L9, (E) NI—
105.40E8, (F) NI-105.48E5, (G) NI-105.6E3, (H) NI-105.22E1, (I) NI—105.26B12, (J)
NI-105.12E12, (K) NI—105.60E7, (L) NI-105.14E2, (M) Nl-105.39E2, (N) NI-
105.19C6, and (O) NI-105.9C4 human anti-tau antibodies. Complementarity
determining regions (CDRs) are underlined.
and chl7Cl(N3lQ)
: (A) Binding of ch17C1, chl7Cl(N3lQ) mIgG2a
mIgGl Agly to recombinant Tau in an ELISA assay. (B) Comparison of recombinant
Tau binding by chl 7C1(N3 IQ) mIgG2a and chl 7C1(N31Q, I48V) mIgG2a in an
ELISA assay.
' 7 '
Comparison of recombinant Tau binding by NI-105.40E8 hIgGl and NI-
105.40E8(R1 04W) hIgGl in an ELISA assay.
. Binding of NI-105.40E8, NI-105.48E5, NI-105.6C5 and NI-
105.17C1(I48V) human anti-tau antibodies to pathologically aggregated tau in AD
(J: brain and in the brain of transgenic mouse model of tauopathy. Representative images
of human anti-tau antibody g to pathological tau aggregates in the brain of
Alzheimer’s disease (AD) and in the brain of transgenic mouse of thy (Tg).
Control tissue samples were obtained from mentally healthy subject (Ctr) or wild type
mouse brain (Wt).
[0026] . Brain penetration of NI-105.6C5 or NI-105.6E3 human anti-tau
antibodies in TauP30lL mice. "tg" indicates representative sections from transgenic
s either treated or untreated, and "wt" indicates an untreated non-transgenic
animal. Scale bar: 50 pm.
. Effects of chronic treatment of 1L mice with ch4E4(N30Q) and
ch17C1(N31Q). Total human tau (A), human pSl99 tau (B), human pT231 tau (C)
and human pT181 tau (D) levels in soluble, and insoluble fraction of brain protein
ts were quantified with commercial ELISA.
. Soluble and insoluble human tau in TauP301L mice treated with
ch17C1(N31Q) and N30Q) detected by Western blots.
[0029] . Average plasma drug concentrations for ch17C1(N31Q) and
ch4E4(N30Q) treated animals 24 h after the i.p. administration of the last dose.
Average plasma drug concentrations for ch17C1(N31Q) and N30Q) were 145
and 200 ug/ml, respectively.
. Spatial working memory in lL mice treated with ch17C1(N31Q)
and ch4E4(N30Q) was assessed by two-trial Y-maze.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Neurodegenerative tauopathies are a diverse group of neurodegenerative disorders
that share pathologic lesion consisting of intracellular aggregates of
a common
abnormal ts that are mainly composed of ogically hyperphosphorylated
tau in neurons and/or glial cells. Clinical features of the tauopathies are heterogeneous
‘ 8 “
and characterized by dementia and/or motor syndrnmes, The: pragressive accumulation
of fiiamentous tau inclusions neuronal degeneration in
may cause and glial
combination with other deposits as, e. g., beta-amyloid in Alzheimer’s disease or as a
sole pathogenic entity as illustrated by mutations in the tau gene that are associated
with familial forms of frontotemporal dementia and parkinsonism linked to
chromosome 17 (FTDP-17). Because of the heterogeneity of their clinical
manifestations a potentially non-exhaustive list of tauopathic diseases can be provided
ing Alzheimer’s disease, amyotrophic lateral sclerosis/parkinsonism—dementia
complex, argyrophilic grain dementia, British type amyloid angiopatléy, cerebral
amyloid angiopathy, corticobasal ration, Creutzfeldt-Jakob disease, ia
pugilistica, diffuse neurofibrillary s with calcification, Down’s syndrome,
frontotemporal dementia, frontotemporal dementia with parkinsonism linked to
chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker
disease, Hallelyorden-Spatz disease, inclusion body myositis, multiple system atrophy,
myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron
disease with neurofibrillary tangles, Pick’s disease, postencephalitic sonism,
prion protein cerebral amyloid angiopathy, progressive tical gliosis, ssive
supranuclear palsy, subacute sclerosing panencephalitis, tangle only ia, multi-
infarct dementia and ic stroke; see for a review, e. g., Lee et al., Annu. Rev.
Neurosci. 24 (2001), 1121-1159 in which Table l catalogs the unique members of
tauopathies or Sergeant et al., Bioch. Biophy. Acta 1739 (2005), 179—97, with a list in
Figure 2 therein.
In this specification, the terms "tau", is used hangeable to specifically refer
to the native monomer form of tau. The term "tau" is also used to generally identify
other conformers of tau, for example, oligomers or aggregates of tau. The term "tau" is
also used to refer collectively to all types and forms of tau. Due to alternative ng
6 tau ms are t in the human brair; The protein ces for these
isoforms are:
Isoform Fetal-tau of 352aa
MAEFRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGD
TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK
GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSESRTPSLPTPPTR
WO 00600 ' 9 "
EPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKV
QIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEEEVKSEKLDFKDRVQSKIGSLDNIT
HVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGS
IDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:1)
Isoform Tau-B of 381aa
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPT
EDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTG
SDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKS
GDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPV
PMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPG
GGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTD
HGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL
(SEQ ID N022)
Isoform Tau-C of 410aa
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPT
EDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGI
GDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPFG
ATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTP
PTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGG
KVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLD
NITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSS
TGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID N023)
Isoform Tau-D of 383aa
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGEETDAGLKAEEAGIGD
TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK
GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTR
EPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKV
QIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHH
VEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKA
KTDHGAEIVYKSPVVSG-DTSPRHiSNVSSTGSIDMVDSPQLATLADEVSASLAKQ
GL (SEQ ID N024)
W0 2014/100600 ' 10 ' 2013/076952
Isoform Tau-E of 412aa
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPT
EDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTG
SDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKS
GDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPV
PMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPG
GGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSL
DNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVS
STGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID N025)
Isoform Tau-F of 441aa
MAEPRQEFEVMEDHAGTYGLGDRKEEQGGYTMHQDQEGDTDAGLKESPLQTPT
PGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGI
GDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPG
QKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTP
PTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMT’DLKNVKSKIGSTENLKHQPGGG
KKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNI
HHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENA
KAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLA
KQGL (SEQ ID NO:6)
[0033] The "wild type" tau amino acid sequence is represented by isoform Tau-F of
44laa (SEQ ID NO:6) further also referenced to as "hTau40", "TauF", " or
"full-length tau". The amino acid sequence of tau can be retrieved from the literature
and pertinent databases; see Goedert et al., Proc. Natl. Acad. Sci. USA 85 (1988),
4051—4055, Goedert et al., EMBO J. ), 393-399, Goedert et al., EMBO J. 9
(1990), 4225-4230 and GenBank UniProtKB/swissprot: locus TAU_HUMAN,
accession numbers -2 (Fetal-tau) and P10636-4 to -8 (Isof’orms B to F).
{883$} Another striking feature of tau protein is phosphorylation, which occurs at about
of 79 potential serine (Ser) and threonine (Thr) phosphorylation sites. Tau is highly
phosphorylated during the brain development. The degree of phosphorylation declines
it adulthood. Some of the phosphorylation sites are located Wéthin the microtubule
binding domains of tau, and it has been shown that an increase of tau phosphorylation
negatively regulates the g of microtubules. For example, Ser262 and Ser396,
W0 2014/100600 ' 11 '
which lie within or adjacent to microtubule binding motifs, are hyperphosphorylated in
the tau proteins of the abnormal paired helical filaments (PHFs), a major component of
the brillary tangles (NFTs) in the brain of AD patients. PHFs are filamentous
aggregates of tau proteins which are abnormally hyperphosphorylated and can be
stained with specific anti-tau antibodies and detected by light microscopy. The same
holds true for so called straight tau filaments. PHFs form twisted ribbons consisting of
two filaments twisted around one another with a periodicity of about 80nm. These
pathological features are commonly referred to as "tau-pathology", “tauopathology” or
"tau-related pathology". For a more detailed description of neuropathological features
of tauopathies refer to Lee et al., Annu. Rev. Neurosci. 24 (2001), 1121-1159 and
Gotz, Brain. Res. Rev. 35 (2001), 266-286, the disclosure content of which is
incorporated herein by reference. Physiological tau protein stabilizes ubules in
neurons. Pathological phyosphorylation leads to abnormal tau localization and
aggregation, which causes ilization of microtubules and impaired cellular
ort. Aggregated tau is neurotoxic in vitro (Khlistunova et al., J. Biol. Chem. 281
(2006), 1205-1214). The exact neurotoxic s s unclear, however, as do the
mechanism(s) by which they lead to neuronal death. Aggregates of tau can be observed
such as
as the main component of neurofibrillary tangles (NFT) in many tauopathies,
Alzheimer’s disease (AD), Frontotemporal dementias, supranuclear palsy, Pick’s
disease, Argyrophilic grain disease (AGD), corticobasal degeneration, FTDP-17,
Parkinson’s disease, Dementia pugilistica (Reviewed in n and Petrucelli, M01.
Neurodegener. 4:13 (2009)). Besides these observations, evidence emerges that tau-
ed neuronal death can occur ever: in the absence of tangle formation. Soluble
phospho—tau species are present in CSF (Aluise et al., Biochim. s. Acta. 1782
(2008), 549—558). Tau ates can it a misfolded state from the outside to
the inside of a cell and transfer between co-cultured cells (Frost et al., J. Biol. Chem.
284 (2009), 12845-12852).
In addition to the involvement in neurodegenerative thies, ed
alterations in tau phosphorylation during and after ischemia/reperfusion suggest tau
playing a crucial role in neuronal damage and clinical pathophysiology of
neurovascular disorders sucla as ischemic stroke (Zheng et al., J. Cell. m. 109
(2010), 26-29).
W0 2014/100600
The human au antibodies sed herein specifically bind tau and epitopes
thereof and to various conformations of tau and epitopes thereof. For example,
disclosed herein are antibodies that specifically bind tau, tau in its full-length,
pathologically modified tau isoforms and tau aggregates. As used herein, reference to
binds" tau
an antibody that "specifically binds", "selectively binds", or "preferentially
refers to an antibody that does not bind other unrelated proteins. In one example, a tau
antibody sed herein can bind tau or an e thereof and show no binding
above about 1.5 times background for other proteins. An antibody that "specifically
binds" or tively binds" a tau conformer refers to an antibody that does not bind
all conformations of tau, i.e., does" not bind at least one other tau conformer. For
example, sed herein are antibodies that can preferentially bind to aggregated
forms of tau in AD tissue. Since the human au antibodies of the present invention
have been ed from a pool of healthy human subjects exhibiting an tau-specific
immune response the tau antibodies of the present invention can also be called "human
auto-antibodies" in order to emphasize that those antibodies were indeed expressed by
the subjects and have not been isolated from, for example a human immunoglobulin
expressing phage library, which hitherto represented one common method for trying to
provide human-like antibodies.
It is to be noted that the term "a" or "an" entity refers to one or more of that entity;
for example, "an antibody," is understood to represent one or more antibodies. As
such, the terms "a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
As used herein, the term "polypeptide" is intended to encompass a singular
"polypeptide" as well as plural "polypeptides," and refers to a le ed of
monomers (amino acids) linearly linked by amide bonds (also known as e
bonds). The term "polypeptide" refers to any chain or chains of two or more amino
acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides,
tripeptides, eptides, "protein," "amino acid chain," or any other term used to
refer to a chain or chains of two or more amino acids, are included within the
definition of "polypeptide," and the term "polypeptide" can be used instead of, or
interchangeably with any of these terms.
The term "polypeptide" is also intended to refer to the products of post-expression
modifications of the polypeptide, including without limitation glycosylation,
W0 2014/100600 PCT/USZOl3/076952
acetylation, phosphorylation, amidation, derivatization by known protecting/blocking
amino acids.
groups, proteolytic cleavage, or modification by non-naturally occurring
A polypeptide can be derived from a natural biological source or produced by
recombinant technology, but is not necessarily translated from a designated nucleic
acid sequence. It can be generated in any manner, including by al synthesis.
A ptide of the invention can be of a size of about 3 or more, 5 or more, 10
or more,
or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200
500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a
defined three-dimensional structure, gh they do not necessarily have such
structure. Polypeptides with a defined three-dimensional structure are referred to as
, and ptides which do not possess a defened three-dimensional structure,
but rather can adopt a large number of different conformations, and are referred to as
unfolded. As used herein, the term glycoprotein refers to a n d to at least
or a
one carbohydrate moiety that is attached to the protein via an oxygen-containing
nitrogen-containing side chain of an amino acid residue, e. g., a serine residue or an
asparagine residue.
By an "isolated" polypeptide or a fragment, variant, or tive thereof is
intended a polypeptide that is not in its natural milieu. No particular level of
purification is required. For example, an isolated polypeptide can be removed from its
native or natural environment. Recombinantly produced polypeptides and proteins
expressed in host cells are considered isolated for purposed of the invention, as are
native or recombinant polypeptides which have been separated, fractionated, or
partially or substantially purified by any suitable technique.
Also ed as polypeptides of the present invention are fragments, derivatives,
analogs or variants of the foregoing polypeptides, and any combination f. The
terms "fragment," "variant," "derivative" and "analog" when referring to antibodies or
dy polypeptides of the present invention include any polypeptides which retain
at least some of the antigen-binding properties of the corresponding native binding
molecule, antibody, or polypeptide. Fragments of polypeptides of the preSent invention
include proteolytic fragments, as well as on fragments, in addition to specific
antibody nts discussed elsewhere . Variants of antibodies and antibody
polypeptides of the present invention e fragments as described above, and also
polypeptides with altered amino acid ces due to amino acid substitutions,
W0 2014/100600 PCT/USZOl3/076952
deletions, or insertions. ts can occur naturally or be non-naturally ing.
Non-naturally occurring variants can be produced using art-known mutagenesis
techniques. t polypeptides can comprise conservative or non—conservative amino
acid substitutions, deletions or additions. Derivatives of tau specific binding molecules,
are polypeptides
e. g., antibodies and antibody ptides of the present invention,
which have been altered so as to exhibit additional features not found on the native
polypeptide. Examples include fusion ns. Variant polypeptides can also be
referred to herein as "polypeptide analogs". As used herein a "derivative" of a binding
molecule or fragment thereof, an antibody, or an antibody polypeptide refers to a
subject polypeptide having one or more es chemically derivatized by reaction of
functional side Also included as atives" are those
a group. peptides which
contain one or more naturally occurring amino acid derivatives of the twenty standard
amino acids. For example, 4-hydroxyproline can be substituted for e; 5-
hydroxylysine can be substituted for ; 3-methylhistidine can be substituted for
histidine; homoserine can be substituted for serine; and ornithine can be substituted for
lysine.
The term "polynucleotide" is intended to encompass a singular nucleic acid as
well isolated molecule or
as plural nucleic acids, and refers to an nucleic acid
uct, A polynucleotide
e. g., ger RNA (mRNA) or plasmid DNA (pDNA).
bond (e.g. , an
can comprise a conventional phosphodiester bond or a non-conventional
amide bond, such as found in peptide c acids (PNA)). The term "nucleic acid"
refers to any one or more nucleic acid ts, e.g., DNA or RNA fragments, present
in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native nment.
For example, a recombinant polynucleotide encoding an antibody contained in a vector
is considered isolated for the purposes of the present invention. r examples of an
isolated polynucleotide include recombinant polynucleotides maintained in
heterologous host cells purified (partially or substantially) polynucleotides in
solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of
acids
polynucleotides of the present invention. Isolated polynucleotides or nucleic
according to the present invention further include such molecules produced
synthetically. In addition, polynucleotide or a nucleic acid can be or can include a
W0 2014/100600 PCT/USZOl3/076952
tory element such as a promoter, ribosome binding site, or a transcription
terminator.
As used herein, a "coding region" is a portion of nucleic acid which consists of
codons translated into amirao acids. Although a "stop codon" (TAG, TGA, or TAA) is
not translated into an amino acid, it can be considered to be part of a coding region, but
ribosome binding sites, transcriptional
any flanking sequences, for example ers,
tern’zinators, introns, and the like, are not part of a coding region. Two or more coding
regions of the present invention can be present in a single polynucleotide uct,
e.g., on a single vector, or in separate polynucleotide ucts, e. g., on separate
(different) vectors. Furthermore, any vector can contain a single coding region, or can
se two or more coding regions, e.g., a single vector can tely encode an
immunoglobulin heavy chain variable region and an immunoglobulin light chain
variable region. In addition, a vector, cleotide, or nucleic acid of the invention
can encode heterologous coding regions, either fused or unfused to a nucleic acid
encoding a binding molecule, an dy, or fragment, variant, or derivative thereof.
Heterologous coding regions include without limitation specialized elements or motifs,
such as a secretory signal peptide or a logous onal domain.
In certain embodiments, the polynucleotide or raucleic acid is DNA. In the case of
DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide
normally can e a promoter and/or other transcription or translation control
elements operably associated with one or more coding regions. An operable
association is when a coding region for a gene product, e. g., a polypeptide, is
associated with one or more regulatory sequences in such a way as to place expression
of the gene product under the influence or control of the regulatory ce(s). Two
DNA fragments (such as a polypeptide coding region and a promoter associated
therewith) are "operably associated" or "operably linked" if induction of er
function results in the transcription ofmRNA encoding the desired gene product and if
the nature of the linkage between the two DNA fragments does not interfere with the
ability of the expression regulatory sequences to direct the expression of the gene
product or interfere with the ability of the DNA template to be ribed. Thus, a
promoter region would be operably ated with a nucleic acid encoding a
polypeptide if the promoter was capable of effecting transcréption of that nucleic acid.
The promoter can be a cell-specific promoter that directs substantial transcription of
W0 2014/100600 PCT/USZOl3/076952
the DNA only in predetermined cells. Other transcription control elements, s a
ation
promoter, for example enhancers, ors, repressors, and transcription
signals, can be operably associated with the polynucleotide to direct cell-specific
transcription. Suitable promoters and other transcription control regions are disclosed
herein.
A y of ription control regions are known to those skilled in the art.
These include, t limitation, transcription control regions which function in
vertebrate cells, such as, but not limited to, promoter and enhancer segments from
cytorriegaloviruses (the immediate early promoter, in conjunction with intron-A),
simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
Other transcréption control regions include those d from vertebrate genes such as
actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other
expression in eukaryotic cells. Additional
sequences capable of controlling gene
suitable ription control regions include tissue-specific ers and enhancers
interferons or
as well as lymphokine-inducible promoters (e.g., ers inducible by
interleukins).
{9347; Similarly, a variety of translation control elements are known to those of ordinary
skill in the art. These include, but are not d to ribosome binding sites, translation
initiation and termination codons, and elements d from picornaviruses
(particularly an internal me entry site, or IRES, also referred to as a CITE
sequence).
In other embodiments, a polynucleotide of the present invention is RNA, for
example, in the form of messenger RNA (mRNA).
Polynucleotide and nucleic acid coding regions of the present invention can be
associated with additional coding regions which encode secretory or signal peptides,
which direct the secretion of a polypeptide encoded by a polynucleotide of the present
invention. According to the signal hypothesis, proteins secreted by mammalian cells
have a signal peptide or secretory leader sequence which is cleaved from the mature
protein once export of the growing protein chain across the rough endoplasmic
lum has been initiated. Those of ordinary skill in the art are aware that
polypeptides secreted by vertebrate cells lly have a signal peptide fused to the
N-terminus of the polypeptide, which is cleaved from the complete or "full—length"
polypeptide to produce a secreted or "mature" form of the polypeptide. In certain
' 17 '
embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light
chain signal peptide is used, or a functional derivative of that ce that retains the
ability to direct the secretion of the polypeptide that is operably ated with it.
Alternatively, a heterologous mammalian signal peptide, or a functional tive
thereof, can be used. For example, the wild-type leader ce can be substituted
with the leader sequence of human tissue plasminogen activator (TPA) or mouse B-
glucuronidase.
Unless stated otherwise, the terms "disorder" and “disease" are used
interchangeably herein.
[0051] A "binding le" as used in the context of the present invention relates
prémarily to antibodies, and nts thereof, but can also refer to other non-antibody
molecules that bind to tau including but not limited to hormones, receptors, ligands,
major histocompatibility complex (MHC) molecules, chaperones such as heat shock
proteins (HSPs) as well as cell-cell adhesion molecules such as members of the
cadherin, intergrin, C-type lectin and immunoglobulin (lg) superfamilies. Thus, for the
sake of clarity only and without restricting the scope of the present invention most of
the following embodiments are discussed with respect to antibodies and dy-like
molecules which represent a specific embodiment. of binding molecules for the
development of eutic and diagnostic .
[0052] The terms "antibody" and "immunoglobulin" are used hangeably herein. An
antibody or globulin is a tau-binding molecule which comprises at least the
variable domain of a heavy chain, and ly comprises at least the variable
domains of a heavy chain and a light chain. Basic immunoglobulin structures in
vertebrate systems are relatively well understood; see, e. g., Harlow et 61]., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
As will be discussed in more detail below, the term "immunoglobulin" comprises
var’éous broad classes of polypeptides that can be distinguished biochemically. Those
skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha,
delta, or epsilon, (y, u, or, 8, a) with some subclasses among them (e.g., 71-74). It is the
nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG,
or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2,
IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional
W0 2014/100600 PCT/USZOl3/076952
specialization. Modified versions of each of these classes and isotypes are readily
discernible to the d artisan in View of the instant disclosure and, accordingly, are
within the instant invention. All immunoglobulin classes scope of the are clearly
within the scope of the t invention, the following discussion will generally be
directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard
immunoglobulin molecule ses two cal light chain polypeptides of
molecular weight approximately 23,000 Daltons, and two cal heavy chain
polypeptides of molecular weight 53,000-70,000. The four chains are typically joined
by de bonds in a "Y" configuration n the light chains bracket the leeavy
chains starting at the mouth of the "Y" and continuing through the variable region.
Light chains are classified as either kappa or lambda (K, A). Each heavy chain
class can be bound with either a kappa or lambda light chain. In general, the light and
heavy chains are covalently bonded to each other, and the "tail“ portions of the two
heavy chains are bonded to each other by covalent disulfide linkages or non-covalent
linkages when the immunoglobulins are ted either by hybridomas, B cells or
genetically engineered host cells. In the heavy chain, the amino acid sequences run
from an N—terminus at the forked ends of the Y configuration to the C-terrninus at the
bottom of each chain.
Both the light and heavy chains are divided into regions of structural and
functional homology. The terms "constan " and "variable" are used functionally. In this
, it will be appreciated that the variable domains of both the light (VL) and heavy
(VH) chain portions determine antigen recognition and specificity. Conversely, the
constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)
confer important biological properties such as secretion, transplacental mobility, Fc
receptor g, complement binding, and the like. By convention the numbering
the coristant region domains increases as they become more distal from the antigen-
binding site or amino-terminus of the antibody. The N-terminal portion is a variable
region and at the C—terminal portion is a constant region; the CH3 and CL domains
actually comprise the carboxy-terminus of the heavy and light chain, respectively.
[0056] As ted above, the variable region allows the antibody to selectively
recognize and specifically bind epitopes on antigens. That is, the VL domain and VH
domain, or subset of the complementarity ining regions (CDRs), of an antibody
W0 2014/100600 PCT/USZOl3/076952
combine to form the variable region that defines a three dimensional antigen-binding
site. This quaternary antibody structure forms the antigen—binding site present at the
end of each arm of the Y. More cally, the antigen-binding site is defined by
three CDRs on each of the VH and VL chains. Any antibody or immunoglobulin
fragment which contains sufficient ure to specifically bind to tau is denoted
herein interchangeably as a "binding fragment" or an "immunospecific fragmen ."
In naturally occurring antibodies, an antibody comprises six hypervariable
regions, mes called "complementarity determining regions" or "CDRs" present
in each antigen-binding domain, which are short, non-contiguous sequences of amino
acids that are specifically positioned to form the antigen-binding domain as the
antibody assumes its three ional configuration in an aqueous environment. The
"CDRs" are flanked by four relatively conserved "framework" regions or "FRs" which
show less inter-molecular variability. The framework regions largely adopt a B-sheet
mation and the CDRs form loops which t, and in some cases form part of,
the B-sheet structure. Thus, ork regions act to form a scaffold that es for
positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
The antigen-binding domain formed by the oned CDRs defines a surface
complementary to the epitope on the reactive n. This complementary
surface promotes the non-covalent binding of the antibody to its cognate epitope. The
amino acids comprésing the CDRs and the framework regions, respectively, can be
readily identified for any given heavy or light chain variable region by one of ordinary
skill in the art, since they have been precisely defined; see, "Sequences of Proteins of
Immunological Interest," Kabat, E., et al., US. Department of Health and Human
Services, ; and a and Lesk, J. Mol. Biol., 196 (1987), 901—917, which are
incorporated herein by reference in their entireties.
In the case where there are two or more definitions of a term which is used and/or
accepted within the art, the definition of the term as used herein is intended to include
all such meanings unless explicitly stated to the contrary. A specific example is the use
of the term ”complementarity determining region" ("CDR") to describe the non—
contiguous antigen combining sites found within the variable region of both heavy and
light chain polypeptides. This particular region has been described by Kabat et al.,
US. Dept. of Health and Human Services, "Sequences of ns of Immunological
W0 00600
Interest" (1983) and by Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which
nce, where the definitions include overlapping or are incorporated herein by
subsets of amino acid residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody or variants thereof is
intended to be within the scope of the term as defined and used herein. The appropriate
amino acid residues which encompass the CDRs as defined by each of the above cited
references are set forth below in Table 1 as a comparison. The exact residue numbers
which encompass a particular CDR will vary depending on the sequence and size of
the CDR. Those skilled in the art can ely determine which es comprise a
particular hypervariable region or CDR of the human IgG e of antibody given
the variable region amino acid sequence of the antibody.
TabEe 1: CDR Definitionsl
“WT KabatWrChothia
Vm“flusw‘26-32
VH CDR2 W5065 5258
VHCDR3WW95102 “W95102W
VLCDRl W3434W2632
‘ “W__w.“
iNumbe‘iiffé‘SiEii’EDR defiii‘i‘iiSfiE‘iii‘i‘able 1 is éEESEEiifiE‘to the nufiiBEingEBhventic?fi§“‘§et
forth by Kabat et al. (see below).
[$059] Kabat et al. also defined a numbering system for le domain sequences that
is applicable to any antibody. One of ordinary skill in the art can unambiguously assign
this system of "Kabat numbering" to any variable domain sequence, without reliance
on any experimental data beyond the itself. As used herein, "Kabat
numbering" refers to the numbering system set forth by Kabat et al., US. Dept. of
Health and Human Services, ”Sequence of Proteins of Immunological Interest" (1983).
Unless otherwise specified, references to the numbering of specific amino acid residue
positions in an antibody or antigen-binding fragment, variant, or derévative f of
the present invention are according to the Kabat numbering , which however is
W0 2014/100600 PCT/USZOl3/076952
theoretical and may not equally apply every antibody of the present invention. In one
embodiment, ing on the position of the first CDR the following CDRs can
shifted in either direction.
Antibodies or antigen-binding fragments, immunospecific fragments, variants, or
derivatives thereof of the invention include, but are not limited to, polyclonal,
monoclonal, multispecific, human, humanized, primatized, zed or
antibodies, single chain antibodies, e-binding fragments, e. g., Fab, Fab' and
F(ab')2, Fd, Fvs, single—chain Fvs , single-chain antibodies, disulfide-linked
(dev), fragments comprising either a VL or VH domain, fragments produced by a
anti-Id
expression library, and anti-idiotypic (anti-Id) antibodies (including, e. g.,
and are
antibodies to antibodies disclosed herein). ScFv molecules are known in the art
described, les of the
e. g., in US patent 5,892,019. Immunoglobulin or antibody
invention car: be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g.,
molecule.
IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or ss of immunoglobulin
In one embodiment, the dy of the present invention is not IgM or a
derivative thereof with a pentavalent structure. Particular, in specific applications
the present invention, ally therapeutic use, Ing are less useful than IgG
due to their
other bivalent antibodies or corresponding binding molecules since Ing
pentavalent structure and lack of affinity maturation often show ific cross-
reactivities and very low affinity.
[e062] In a particular embodiment, the antibody of the present invention is not a
polyclonal antibody, i. e. it substantially consists of one particular antibody species
rather than being a mixture ed from a plasma immimoglobulin sample.
Antibody fragments, including single-chain dies, can comprise the variable
region(s) alone or in combination with the entirety or a portion of the ing: hinge
, CH1, CH2, and CH3 domains. Also included in the invention are tau-birading
fragments comprising any combination of variable region(s) with a hinge region, CH1,
thereof of the CH2, and CH3 domains. dies or immunospecific fragments
birds and mammals. In one
present invention can be from any animal origin including
embodiment, the antibodies are human, murine, donkey, rabbit, goat, guinea pig,
camel, llama, horse, or chicken antibodies. In another embodiment, the variable region
can be condricthoid in origin (e.g. , from sharks).
W0 2014/100600 PCT/USZOl3/076952
In one aspect, the antibody of the present invention is a human monoclonal
antibody isolated from a human. Optionally, the framework region of the human
antibody is d and adopted in ance with the ent human germ line
variable region ces in the database; see, e. g., Vbase (http://vbase.mrc-
m.ac.uk/E . by the MRC Centre for Protein Engineeréng (Cambridge, UK).
line
For example, amino acids considered to potentially deviate from the true germ
the cloning
sequence could be due to the PCR primer sequences incorporated during
human-like antibodies such as single chain
process. Compared to artificially generated
antibody fragments ) from a phage displayed antibody library or xenogeneic
mice the human monoclonal atetibody of the present invention is characterized by (i)
being obtained using the human immune response rather than that of animal
surrogates, i. e. the antibody has been generated in response to natural tau in its relevant
conformatiOn in the human body, (ii) having protected the individual or is at least
cant for the presence of tau, and (iii) since the antibody is of human origin the
risks of cross-reactivity against self-antigens is minimized. Thus, in accordance with
the present ion the terms "human monoclonal antibody”, "human monoclonal
autoantibody", "human antibody" and the like are used to denote a tau binding
molecule which is of human origin, i.e. which has been ed from a human cell
such as a B cell or hybridoma thereof or the cDNA of which has been directly cloned
from mRNA of a human cell, for example a human memory B cell. A human antibody
is still " even if amino acid substitutions are made in the dy, e. g., to
improve binding characteristics.
animals
Antibodies derived from human immunoglobulin librariées or from
transgenic for and not
one or more human immunoglobulins that do express
endogenous immunoglobulins, as described infia and, for example in, US patent no
,939,598 by Kucherlapati et al., are denoted human-like antibodies in order
distinguish them from truly human antibodies of the present invention.
For example, the paring of heavy and light chains of human-like antibodies such
from phage display do not
as synthetic and semi-synthetic antibodies typically isolated
cell.
arily reflect the original paring as it occurred in the original human B
Accordingly Fab and scFv fragments obtained from recombinant expression ies
artificial with all possible
as commonly used in the prior art can be considered as being
associated effects on immunogenicity and stability.
WO 00600 ‘ 23 '
In contrast, the present invention provides isolated affinity-matured antibodies
from selected human subjects, which are characterized by their therapeutic utility and
their tolerance in man.
As used herein, the term "murinized antibody" or "murinized immunoglobulin"
refers to an antibody comprising one or more CDRS from a human antibody of the
invention; and a human framework region that contains amino acid
present
substitutions and/or deletions and/or insertions that are based on a mouse antibody
CDRS is called the "paren " or
sequence. The human immunoglobulin providing the
"acceptor" and the mouse antibody providing the framework s is called the
"donor", Constant regions need not be present, but if they are, they are usually
substantially identical to mouse antibody nt regions, 1'. e. at least about 85- 90%,
about 95%, about 96%, about 97%, about 98%, about 99% or more identical. Hence, in
some embodiments, a full—length murinized human heavy or light chain
immunoglobulin contains a mouse nt region, human CDRs, and a ntially
human framework that has a number of "rnurinizing" amino acid substitutions.
Typically, a "murinized antibody" is an antibody comprising a zed variable light
chain and/or a murinized le heavy chain. For example, a murinized antibody
would not encompass a typical chimeric antibody, e. g., because the entire le
region of a chimeric antibody is non-mouse. A modified antibody that has been
"murinized" by the s of "murinization" binds to the same antigen as the parent
dy that provides the CDRS and is usually less immunogenic in mice, as
compared to the parent antibody.
{0069] As used herein, the term "heavy chain portion" includes amino acid sequences
derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy
chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, ,
and/or lower hinge region) , a CH2 domain, a CH3 , or a variant or
fragment thereof. For example, a binding polypeptide for use in the invention can
comprise polypeptide chain comprising a CH1 domain; a polypeptide chain
comprising a CH1 , at least a portion of a hinge domain, and a CH2 domain; a
polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain
comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or
a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain,
CI-H domain, and a CH3 domain. In another embodiment, a polypeptide of the
W0 2014/100600 - 24 , PCT/USZOl3/076952
invention comprises a ptide chain comprising a CH3 domain. Further, a binding
polypeptide for use in the invention can lack at least a portion of a CH2 domain (e.g.,
all or part of a CH2 domain). As set forth above, it will be understood by one of
ordinary skill in the art that these s (e. g., the heavy chain portions) can be
modified such that they vary in amino acid sequence from the naturally occurring
immunoglobulin molecule.
. In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof
disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are
identical to those on a second polypeptide chain of the multimer. Alternatively, heavy
chain portion-containing monomers of the invention are not identical. For e,
each r can comprise a different target binding site, forming, for example, a
bispecific antibody or diabody.
In another embodiment, the dies, or antigen-binding fragments, variants, or
derivatives thereof disclosed herein are composed of a single polypeptide chain such as
scFvs and to be expressed intracellularly (intrabodies) for potential in viva
therapeutic and diagnostic ations.
The heavy chain portions of a binding polypeptide for use in the diagnostic and
treatment s disclosed herein can be derived from different immunoglobulin
molecules. For example, a heavy chain portion of a polypeptide can comprise a CH1
domain derived from an IgGl molecule and a hinge region derived from an IgG3
molecule. In another example, a heavy chain portion can comprise a hinge region
derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In
another example, a heavy chain n can comprise a chimeric hinge d, in part,
from an IgGl molecule and, in part, from an lgG4 molecule.
[0073] As used , the term "light chain portion" includes amino acid sequences
derived from an immunoglobulin light chain. In one embodiment, the light chain
portion coneprises at least one of a VL or CL domain.
The minimum size of a e or polypeptide epitope for an antibody is thought
to be about four to five amino acids. Peptide or polypeptide epitopes can contain
least seven, at least nine or between at least about 15 to about 30 amino acids. Since a
CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino
acids comprising an e need not be contiguous, and in some cases, may not even
be on the same peptide chain. In the present invention, a peptide or polypeptide epitope
W0 00600 ' 25 ' PCT/USZOl3/076952
recognized by antibodies of the present invention contains a sequence of at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at
least 25, or between about 5 to about 30, ab0ut 10 to about 30 or about 15 to about 30
contiguous or non-contiguous amino acids of tau.
[0075] By "specifically binding", or "specifically recognizing", used interchangeably
herein, it is generally meant that a binding molecule, e. g., an antibody binds to an
epitope via its antigen-binding domain, and that the binding entails some
complementaréty between the n-binding domain and the epitope. According to
this definition, an antibody is said to fically bind" to an epitope when it binds to
that e, via its antigen—binding domain more readily than it would bind to a
random, ted epitope. A skilled artisan understands that an antibody can
specifically bind to, or specifically recognize an isolated polypeptide comprising, or
consisting of, amino acid residues corresponding to a linear portion of a non-
contiguous e. The term "specificity" is used herein to qualify the relative y
by which a certain antibody binds to a certain epitope. For example, antibody "A" can
be deemed to have a higher specificity for a given epitope than antibody "B," or
antibody "A" can be said to bind to epitope "C" with a higher specificity than it has for
related epitope "D“.
Where present, the term "immunological binding characteristics," or other binding
characteristics of an antibody wéth an antigen, in all of its tical forms, refers to
the specificity, affinity, cross-reactivity, and other binding characteristics of an
antibody.
By "preferentially binding", it is meant that the binding molecule, e. g., antibody
specifically binds to an epitope more readily than it would bind to a related, similar,
homologous, or analogous epitope. Thus, an dy which "preferentially binds" to a
given epitope would more likely bind to that e than to a d e, even
though such an antibody can cross-react with the related epitope.
By way of non-limiting example, a binding molecule, e. g., an antibody can be
considered to bind a first epitope preferentially if it binds said first epitope with a
dissociation constant (K13) that is less than the antibody’s KD for the second epitope. In
another non-limiting example, an antibody can be considered to bind a first n
preferentially if it binds the first epitope with an y that is at least one order of
magnitude less than the antibody’s K1) for the second epitope, In another non-limiting
W0 2014/100600 PCT/USZOl3/076952
example, an antibody can be considered to bind a first epitope preferentially if it binds
the first epitope with an affinity that is at least two orders of magnitude less than the
antibody’s K1) for the second epitope.
In another non-limiting example, a binding molecule, e. g., an antibody can be
considered to bind a first epitope preferentially if it binds the first epitope with an off
rate (k(off)) that is less than the antibody’s k(off) for the second e. In another
non-limiting example, an antibody can be considered to bind a first epitope
preferentially if it binds the first epitope with an y that is at least one order of
ude less than the antibody’s k(off) for the second epitope. In another non-
limiting example, an antibody can be considered to bind a first epitope preferentially if
it binds the first epitope with an affinity that is at least two orders of magnitude less
than the antibody’s k(off) for the second epitope.
A binding molecule, e. g., an antibody or antigen-binding fragment, variant, or
derivative disclosed herein can be said to bind a tau or a fragment or t thereof
with an off rate (k(off)) of less than or equal to 5 x 10'2 sec], 10'2 sec'l, 5 x10'3 sec'1 or
'3 sec'lln one embodiment, an antibody of the invention can be said to bind tau or a
104 sec],
fragment or variant thereof with an off rate (k(off)) less than or equal to 5 x
'4 sec'l, 5 x 10'5 sec'l, or 10'5 sec'l, 5 x 10"5 sec'l, 10'6 sec'l, 5 x 10'7 sec'1 or 10'7 sec‘
[0081] A binding molecule, e.g., an antibody or n-binding fragment, variant, or
derivative disclosed herein can be said to bind tau or a fragment or variant thereof wéth
an on rate (k(on)) of r than or equal to 103 M'1 sec'l, 5 x 103 M'1 sec'l, 104 M‘1
sec'1 or 5 x 104 M'1 sec'lln one embodiment, an antibody of the invention can be said
to bind tau or a fragment or variant thereof with an on rate (k(on)) greater than or equal
to 105 M'1 sec'l, 5 x 105 M'1 sec'l, 106 M'1 sec], 5 x 106 M'1 sec‘1 or 107 M'1 sec'l.
A binding molecule, of a
e. g., an antibody is said to competitively inhibit g
reference antibody to a given epitope if it entially binds to that epitope to the
extent that it blocks, to some , g of the reference antibody to the epitope.
Competitive inhibition can be determined by any method known in the art, for
example, competition ELISA assays. An antibody can be said to competitively inhibit
binding of the reference antibody to a given e by at least 90%, at least 80%, at
least 70%, at least 60%, or at least 50%. A d artisan tands that the binding
cf an antibody to its epitope can also be competitively inhibited by a binding molecule
- 27 '
that is not an antibody. For example, the specific binding of an antibody described
herein to tau, e.g., hTau40, can be competitively inhibited by microtubules.
As used herein, the term "affinity" refers to a measure of the strength of the
binding of an individual epitope with the CDR of a binding molecule, e.g., an
immunoglobulin molecule; see, e. g., Harlow et al., Antibodies: A Laboratory Manual,
Cold Spring Harbor tory Press, 2nd ed. (1988) at pages 27-28. As used herein,
the term "avidity" refers to the overall stability of the complex between a population of
globulins and an antigen, that is, the functional combining strength of an
immunoglobulin mixture with the antigen; see, e. g., Harlow at pages 29-34. Avidity is
it? related to both the affinity of individual immunoglobulin les in the tion
with specific epitopes, and also the valencies of the imn’sunoglobulins and the antigen.
For example, the ction between a bivalent monoclonal antibody and an antigen
with a highly repeating epitope structure, such as a polymer, would be one of high
avidity. The affinity or avidity of an antibody for an antigen can be determined
experimentally using any suitable ; see, for example, Berzofsky et al.,
"Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed.,
Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. n and
Company New York, N Y (1992), and methods described herein. General techniques
for ing the affinity of an dy for an antigen include ELISA, RIA, and
surface n resonance. The measured affinity of a particular antibody-antigen
interaction can vary if measured under different conditions, e. g., salt concentration,
pH. Thus, measurements of affinity and other antigen-binding parameters, e.g, KD,
IC50, are preferably made with standardized solutions of antibody and antigen, and a
standardized buffer.
[0084] Binding les, e. g., antibodies or antigen-binding fragments, variants or
derivatives thereof of the invention can also be described or specified in terms of their
cross-reactivity. As used , the term "cross-reactivity" refers to the ability of an
dy, specific for one antigen, to react with a second antigen; a measure of
relatedness between two different antigenic substances. Thus, an antibody is cross
reactive if it binds to an epitope other than the one that induced its formation. The
structural
cross reactive epitope generally contains many of the same complementary
features as the inducing epitope, and in some cases, can actually fit better than the
original.-
W0 2014/100600 ' 28 '
For example, certain antibodies have some degree of cross—reactivity, in that they
bind related, but non-identical epitopes, e. g., es with at least 95%, at least 90%,
at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at
least 55%, and at least 50% identity (as calculated using methods known in the art and
described herein) to a reference epitope. An antibody can be said to have little or no
reactivity if it does not bind epitopes with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less
than 55%, and less than 50% identity (as calculated using methods known in the art
and described herein) to a reference epitope. An antibody can be deemed "highly
specific" for a certain epitope, if it does not bind any other analog, ortholog, or
homolog of that epitope.
[$5086] Binding molecules, e.g., antibodies or antigen-binding fragments, variants or
derivatives thereof of the invention can also be described or specified in terms of their
bindirag affinity to tau. In one ment, binding affinities include those with a
iation constant or Kd less than 5 X 10'2 M, 10'2 M, 5 x 10'3 M, 10'3 M, 5 x 10'4 M,
'4 M, 5 x10'5 M, 10'5 M, 5 x 10'6 M, 10'6 M, 5 x 10'7 M, 10'7 M, 5 x 10'8 M, 10'8 M, 5
x10"
x 10'9 M, 10'9 M, 5 x10'10M,10'10M,5 x 10'“ M, 10'11M, 5 x 10'12 M, 10'12M,
M, 10'13 M, 5 x 10'14M, mm, 5 x 10‘15 M, or 10'15 M.
As previously indicated, the subunit structures and three ional
ration of the constant regions of the various immunoglobulin classes are well
known. As used herein, the term "VH domain" includes the amino terminal variable
domain of an globulin heavy chain and the term "CH1 domain" includes the
first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge
region of an immunoglobulin heavy chain molecule.
As used herein the term "CH2 domain" includes the portion of a heavy chain
molecule that extends, e. g. , from about residue 244 to residue 360 of an antibody using
conventional numbering schemes (residues 244 to 360, Kabat numbering system; and
es 231-340, EU numbering system; see Kabat EA et al. op. cit). The CH2
domain is unique in that it is not closely paired with another . Rather, two N-
linked branched ydrate chains are interposed n the two CH2 domains of
an intact native IgG molecule. It is also well documented that the CH3 domain extends
— 29 -
from the CH2 domain to the C-terminal of the IgG molecule and comprises
approximately 108 residues.
chain
As used herein, the term "hinge region" includes the portion of a heavy
le that joins the CH1 domain to the CH2 domain. This hinge region comprises
approximately 25 residues and is flexible, thus allowing the two N—terminal antigen-
into three
binding regions to move independently. Hinge regions can be subdivided
ct domains: upper, middle, and lower hinge domains; see Roux et al., J.
Immunol. 161 (1998), 4083.
As used herein the term "disulfide bond" includes the covalent bond formed
that can
between two sulfur atoms. The amino acid cysteine ses a thiol group
form a de bond or bridge with a second thiol group. In most naturally occurring
the two
IgG molecules, the CH1 and CL regions are linked by a disulfide bond and
239 and
heavy chains are linked by two disulfide bonds at positions corresponding to
242 using the Kabat numbering system (position 226 or 229, EU numbering ).
[0091] As used herein, the terms "linked", "fused" or "fusion" are used interchangeably.
These terms refer to the joining together of two more elements or components, by
"in—frame
whatever means including chemical conjugation or recombinant means. An
fusion" refers to the joining of two or more polynucleotide open reading frames
(ORFs) correct
to form a continuous longer ORF, in a manner that maintains the
translational reading frame of the original ORFs. Thus, a recombinant fusion n
a single protein ning two or more segments
that correspond to polypeptides
encoded by the original ORFs (which segments are not normally so joined in nature).
Although the reading frame is thus made continuous hout the fused segments,
linker
the segments can be physically or spatially separated by, for example, me
the CDRS of an immunoglobulin
sequence. For example, cleotides encoding
variable region can be fused, in-frame, but be separated by'a polynucleotide encoding
at least one immunoglobulin framework region or additional CDR regions, as long
the "fused" CDRs are co-translated as part of a continuous polypeptide.
The term "expression" as used herein refers to a process by which a gene produces
a biochemical, for example, an RNA or polypeptide. The process includes any
manifestation of the functional presence of the gene within the cell including, without
tion, gene knockdown as well as both ent expression and stable sion.
It includes without limitation transcription of the gene into messenger RNA (mRNA),
W0 2014/100600 - 3O - PCT/USZOl3/076952
transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA)
mRNA into polypeptide(s). If
or any other RNA product, and the translation of such
the final desired product is a biochemical, expression es the creation of that
biochemical and any precursors. Expression of a gene produces a "gene product." As
used , a gene product can be either a nucleic acid, e. g., a messenger RNA
produced by transcription of a gene, or a polypeptide which is translated from a
transcript. Gene products bed herein further include nucleic acids with post
transcriptional modifications, e. g., polyadenylation, or polypeptides with post
translational modifications, e. g., methylation, glycosylation, the addition of lipids,
association with other protein subunits, proteolytic cleavage, and the like.
As used herein, the term "sample" refers to any biological material obtained from
fluid
a subject or patient. In one aspect, a sample can comprise blood, cerebrospinal
("CSF"), or urine. In other aspects, a sample can comprise whole blood, plasma, B
cells enriched from blood samples, and cultured cells (e. g., B cells from a subject). A
sample can also include a biopsy or tissue sample ing neural tissue. In still other
cells. Blood samples
aspects, a sample can comprise whole cells and/or a lysate of the
In one the can be collected by methods known in the art. aspect, pellet can be
ended by vortexing at 4°C in 200 pl buffer (20 mM Tris, pH. 7.5, 0.5% t,
1 mM EDTA, 1 mM PMSF, 0.1M NaCl, IX Sigma se Inhibitor, and IX Sigma
Phosphatase Inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes with
intermittent vortexing. After spinning at 15,000 X g for 5 minutes at about 4°C,
aliquots of supernatant can be Stored at about -70°C.
As used herein, the terms "treat" or "treatment" refer to both therapeutic treatneent
slow
and prophylactic or preventative measures, wherein the object. is to t or
down (lessen) an undesired physiological change or disorder, such as the development
of Parkinsonism. Beneficial or desired clinical results e, but are not limited to,
alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not
ing) state of disease, delay or slowing of e progression, amelioration or
palliation of the disease state, and remission (whether partial or total), r
survival
detectable or undetectable. "Treatment“ can also mean prolonging as
ed to expected al if not receiving treatment. Those in need of treatment
include those already with the condition or disorder as well as those prone to have the
W0 2014/100600 ZOl3/076952
disorder is
condition or disorder or those in which the manifestation of the condition or
to be prevented.
is meant any
By "subject" or "individual" or l" or nt" or ,,marnmal,“
subject, particularly a mammalian subject, e. g., a human patient, for whom diagnosis,
prognosis, prevention, or therapy is desired.
II. Antibodies
The present invention generally relates to human anti-tau antibodies and antigen-
invention
binding fragments thereof. In one embodiment, an antibody of the present
demonstrates the immunological binding characteristics and/or biological ties as
outlined for the antibodies illustrated in the Examples. In accordance with the present
invention human monoclonal antibodies specific for tau were cloned from a pool
healthy human subjects.
In the course of the experiments performed in accordance with the present
ion l attempts failed to clone tau specific antibodies but almost always
resulted in false-positive clones. In order to circumvent this problem, antibodies
conditioned media of human memory B cell cultures were screened in parallel for
control
binding to recombinant tau protein, PHFTau extracted from AD brain, healthy
brain extracts and bovine serum n (BSA). Only B—cell cultures that were
BSA were
positive for inant tau and/or PHFTau but not control brain t or
subjected to antibody cloning.
l attempts to isolating to specific antibodies were focused at pools of healthy
levels
human subjects with high plasnéa binding activity to tau, suggestive of elevated
of circulating tau antibodies plasma. Unexpectedly, these attempts failed to e
c human memory B cells and the antibodies described in the current
invention were isolated from pools of healthy human subjects that were not preselected
for high tau plasma reactivity or had low plasma reactivity to tau.
[0099'] Due to this measure, several dies could be isolated. Selected antibodies
were flirther analyzed for class and light chain subclass determination. Selected
relevant antibody messages from memory B cell es are then transcribed by
PCR, cloned and combined into expression vectors for recombinant production; see
appended Examples. Recombinant expression of the human antibodies in HEK293 or
towards
CHO cells and the. subsequent characterization of their binding specificities
full-length tau, pathologically modified forms thereof on Western Blot and their
distinctive binding to pathologically aggregated tau confirmed that for the first time
human antibodies have been cloned that are highly specific for tau and recognize
distinctive the ogically modified forms of tau protein.
[@100] Thus, the present invention generally relates to an isolated naturally occurring
human monoclonal anti—tau antibody and binding fragments, derivatives and variants
thereof. In one embodiment of the ion, the antibody is capable of specifically
binding fiall-length recombinant tau and/or the pathologically ated and/or
phosphorylated form (PHFTau) isolated from AD brain under denaturing conditions on
Western Blot.
In one embodiment, the present invention is directed to an anti-tau antibody, or
antigen-binding fragment, varéant or derivatives thereof, Where the antibody cally
binds to the same epitope of tau as a reference antibody selected from the group
consisting of Nl-105.17C1, NI-105.6C5, NI-105.29G10, NI-105.6L9, NI-105.40E8, NI-
105.48E5, NI-105.6E3, NI-105.22E1, NI-105.26B12, NI-105.12E12, NI-105.60E7,
105.14E2, NI-105.39E2, .19C6, or NI-105.9C4.
Additional human anti-tau dies are disclosed in U.S. Patent Application
Publication No. 087861, the content of which is incorporated herein by reference
its entirety.
[0103] In one embodiment, an dy described herein specifically binds to tau at an
epitope comprising the amino acid residues selected from the group consisting of:
es corresponding to residues 125-131, 397—441, 226-244, 217-227, 37-55, 387—406,
421-427, 9, 1-158, 197-207, 57-67, 1, 313-319, 309-319, and 221-231 of
hTau4O (SEQ ID NO:6). In a r embodiment, an antibody described herein
specifically binds to tau at an epitope sing the amino acid residues corresponding
to es 37-55 and 387-406 of hTau4O (SEQ ID NO:6). In a specific embodiment, tau
is hTau40 (SEQ ID NO:6).
In one embodiment, an dy described herein binds to tau at an epitope
comprising the microtubule binding domain of tau. In a specific embodiment, an antibody
described herein binds to tau at an epitope comprising amino acid residues from the R4
region of tau as depicted in Figure 4. In one embodiment, an antibody described herein
competes with microtubules for specific binding to tau. In another embodiment, an
antibody described herein has reduced binding affinity to microtubule associated tau
W0 2014/100600 PCT/USZOl3/076952
compared to the antibodies binding affinity to tau no associated with microtubules. In a
flirther embodiment, an dy bed herein does not bind, or substantially does not
bind to tau associated with ubules. In specific ments, the tau protein can
native tau protein or recombinant tau protein. In a specific ment, tau is hTau40.
[0105] In one embodiment, a human anti-tau antibody of the present ion can
specifically bind pathologically aggregated tau and not substantially bind tau in the
physiological form in brain tissue. In addition, a human au antibody of the present
invention can be further characterized by its ability to recognize tau at the pre-tangle
and/or dystrophic neurites in the
stage, in neurofibrillary tangles (NFT), neutropil threads
brain. Hence, the present invention provides a set of human tau antibodies with binding
specificities, which are thus particularly useful for diagnostic and eutic purposes.
In on, or alternatively, an anti—tau antibody of the present invention
preferentially recognizes ogically aggregated tau rather than physiological forms,
particular when analyzed according to Examples 4 and 18. In addition, or alternatively, an
anti-tau antibody of the present invention binds to disease g mutants of human tau,
in particular those described in Example 4. In this context, the binding specificities can
in the range of having half maximal effective concentrations (ECSO) of about 100 pM to
100 nM, or an ECSO of about 100 pM to 10nM for ype tau.
Hence, to
an anti-tau antibody of the present invention binds preferentially
pathological modified forms of tau in brain, e. g. pathological aggregates of tau as
exemplified by immunohistochemical staining described in Examples 4 and 18. In
binds to
another embodiment an anti-tau antibody of the present invention preferentially
both recombinant tau and pathologically modified forens of tau as exemplified in Example
2 by n Blot.
[0108] The present ion is also drawn to an antibody, or antigen-binding fragment,
variant or derivatives thereof, where the antibody comprises an antigen-binding domain
identical to that of an antibody selected from the group consisting of NI—105.17C1, NI-
105.17C1(N31Q), NI-105.6C5, NI-105.29G10, 5.6L9, NI—105.40E8, NI-105.48E5,
NI-105.6E3, NI-105.22E1, NI-105.26B12, NI-105.12E12, NI-105.60E7, NI-105.14E2,
NI-105.39E2, NI—105.19C6, and NI-105.9C4.
The present invention further exemplifies l such binding molecules, e. g.
antibodies and binding fragments thereof, which can be characterized by comprising
their variable region, e. g, binding domain at least one complementarity determining
region (CDR) of the VH and/or VL variable region comprising any one of the amino acid
sequences ed in Fig. 7. The corresponding nucleotide sequences encoding the
above-identified variable regions are set forth in Table 4 below. An exemplary set of
CDRs of the above amino acid sequences of the VH and/or VL region as depicted in Fig.
7. However, as discussed in the following the person skilled in the art is well aware of the
fact that in addition or alternatively CDRs can be used, which differ in their amino acid
three or even more amino acids in
sequence from those set forth in Fig. 7 by one, two,
case of CDR2 and CDR3.
Table 2. Amino acid sequences of the VH region, VH CDR1,VH CDR2, VH CDR2, VL
region VL CDR2, VL CDR2, and VL CDR3 of cific antibodies.
2vH/vL A A 2CDRl CDR2 AACDR3
NI-10517C1 SEQ IDN081
I SEQ ID No45ASEQ ID NO.79 SEQ ID NO:80
VLSEQ ID NO46
‘ SEQ ID N0:822SEQ IDN083 SEQID NO:84
NI-105AiA6AD‘5AAAAAAAA A
2SEQ IDN0482SEQ ID N0:85 NO:86AAA SEQ IDNo87
2 {VL2 SEQIDNo 49 EQIDNO88 SEQIDNO89 SEQIDNO90
NI 10 2 vH2SEQIDN0:50 {SEQ IDNO9 {SEQ ID N9.92 SEQ ID NO:93
2 2 2SEQIDN095 aSEQIDNo96 2
NI-105.6L9 MW]SEQ ID No98 SEQ ID NO99
| SEQ ID S{EQ ID N0102 =
:
2 N0:I01 2
NI-105.40E8 vH SEQ ID N0 54 SEQ ID SEQID 8130 IDND;105
2 N0:103 .330104
VL SEQ ID N0 55 SEQ ID 2 SEQ ID SEQ ID NO:108
{N02106 -
2 2N0:107
NI-105.48E5 vH SEQID N0 56 SEQ ID SEQ ID SEQ ID ND111
‘ N0:109 2 N02110
vL SEQ ID N0:57 SEQ ID SEQ ID 2SEQID NO:114A
. N0:112 NO:113
NI-105.6E3 vH SEQ ID N0 58 SEQ ID I SEQ ID SEQ “3 N011”
% 2N02115 NO:116
2 vL SEQ ID N0 59 2 SEQ ID SEQ ID SEQ ID NO:120
2 2 2 2N0:118 2NO:119
NI-105.22E1 VH SEQ ID N0:60 SEQ ID 5SEQ ID 2
‘ N0;121 N0I22 2 SEQ ID N0;123
vL SEQ ID N0;61 {SEQ ID 2SEQID SEQ ID NO:126
.“Wux~“u..4..._._....._...W~._~t«v»--»“unun“
IUXE‘tibody vH NL ICDRl ICDR2 513113
I 1N0124 N0:125
: ‘ I
_________ I ‘SEQ
.26B12 VHSEQIDN062ISEQID ID ISEQIAADAN0129I
N0127 N0:128
" NQ’EISEME
W~v~ Wxx‘vvr>1»:__»..u....‘“
SEQIDN0:64 gSEQ ID ~
SEQ ID
INO‘130 NO:131
ISEQIQNQIEE
A.i2E12 vH gSEQ ID SEQ ID ISEQAID NO:135
. N0133 IN02134
IvL ISEQAIDMNQTEE: SEQ ID '\
W.~~\““.V.rfi...n,,....““W~~“““==»---»n--"n“I
SEQ ID ISEQIDNQSI38
NO:13_Q NO:137 g
NI—105.66EI..__....§.V...1 SEQIDNQ:67 ESEQID ESEQ ID SEQ IDNQEI4I“
§2N0139 §N02140
...,“......;.»u+.w-:-M—.—..a...“.mm \‘“—»»:
VL SEQID SEQ ID NO:144
u “EAAISEQIDINQSEEISEQ ID
INO:143 I
N0142
“‘W..Ww.mmm,3.4,“WW._M‘.,.,...,..........mm:
N10514E2 VHIWS’EQIBNQEBSEQ ID SEQ ID SEQIDNo:147
NQ145 INO:146 i
I: SEQIQNolso
I SEQIDANOflo
VL SEQID ISEQID
§N0I48 ;=N0:149 3
INITIAISE‘EET VH Ali-“SEQ ID N071 ISEQiii" ISEQIISW SEQIDEBHsa‘
NO151 NO:152 i
“W““K‘u..».1uu..“n‘
““W x
IvL SEQIDN0172 IEQ ID ISEQ ID SEQIDNQSIEM
NO: 154 NO:155
«“-r4.54..»u-.-n.“_‘x
gNI-105.19C6 IVHEEQIDN073 SEQIDWSEQIDWSEQIDN0159
::N0 157 NO:158 I
I .,...._.A.._...n‘qu“\wv.r““2““n..nu4W4v4u4-n»»Au-nu
;vL SEQ IDNQ:I4ISEQID“ ISEQ ID SEQ ID N0:162§
= NO160 gN0:161
I VH ISEQID N0:75 EEQ .“‘“—.44—» ..._.n ~ MW“‘— N
W...~uu—.—.»u_»“unu
INI-105.9C4 %SEQ ID EEEQIDNOIQ?
I I I No:_16_3 IN0:164 E
LI...:“““WNN‘...,,»......“Hum W.~_m ‘.:..........u...“W._»w,,.,.,:_..:.“m“
VL SEQ ID NO:76 SEQID I SEQ ID I SEQ ID NQEIEEAA"
IzNO166 NO:167 =‘
~~x .9...»w - -§-
SEQ ID NO45 ISEQID NO79ISEQ ID NO:80 SEQ ID NO:81
1 W “112.:
s NI-105.17C1 I SEQ IDNQ221 ISEQ ID gSEQ IDN083 SEQ ID NO:84
ng I
(N31Q)
I NO:224
I I
IMNN“..gum...“unnuwww“.:.............“mW___m“......_._
least one
[0i10] In one embodiment, an antibody of the present invention comprises at
CDR comprising, or consisting of an amino acid sequence selected from the group
consisting of SEQ IDE? 0: 79-168 and 224.
WO 00600 PCT/U82013/076952
In one embodiment, an dy of the present invention comprises one, two,
three, four, five or six CDRs comprising, or consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO: 79-168 and 224.
VH CDRl,
In one embodiment, an antibody of the t invention comprises a
the amino acid
VH CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 comprising
97-102, 103-108, 109—114,
sequences, respectively SEQ ID NO: 79-84, 85-90, 91-96,
163-168.
115—120, 121—126, 127-132, 133-138, 139-144, 145-150, 151—156, 157-162, or
VH CDRl, VH
In one embodiment, an antibody of the present invention comprises a
amino acid
CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 comprising the
and 84.
sequences, respectively, SEQ ID N05: 79, 80, 81, 224, 83,
three VH
In one embodiment, an antibody of the invention comprises one, two, or
CDRs comprising, or consisting of an amino acid sequence selected from the group
121—
consisting of SEQ ID NO: 79—81, 85-87, 91-93, 97—99, 103-105, 109-111, 115-117,
123,127-129,133-135,139-141,145-147,151-153,157-159, and 163-165.
[0114] In one embodiment, an antibody of the invention comprises a VH CDRl,
ID NO:
CDR2, and VH CDR3 comprising the amino acid sequences, respectively, SEQ
79-81, 85-87, 91-93, 97-99, 103-105, 109—111, 115-117, 121-123, 127-129, 133-135,
139-141,145-147,151—153,157-159, or 5.
three VL
In one embodiment, an antibody of the invention ses one, two, or
CDRs comprising, or consisting of an amino acid sequence selected from the group
consisting of SEQ ID NO: 82-84, 88-90, 94—96, 100-102, 106-108, 112-114, 0,
224.
124-126, 130-132, 136-138, 142-144,148-150, 154-156,160-162, 8, and
In one embodiment, an antibody of the invention comprises a VL CDRl,
ID NO:
CDR2, and VL CDR3 comprising the amino acid sequences, respectively, SEQ
82-84, 88-90, 94-96, 100—102, 106-108, 112-114, 118-120, 124-126, 2, 136-138,
4, 148—150, 154-156, 160-162, or 166-168. In one embodiment, a VL CDRl, VL
ID NO:
CDR2, and VL CDR3 comprising the amino acid ces, respectively, SEQ
83, 84, and 224.
ing to one embodiment, an antibody of the invention comprises a heavy
chain le region comprising a VH CDRl of SEQ ID NO: 79, 85, 91, 97, 103, 109,
115,121,127,133,139,145,151,157, or 163; a VH CDR2 of SEQ ID NO: 80, 86, 92,
CDR3 of SEQ ID
98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; or aVH
165.
NO: 81, 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or According
W0 2014/100600 ZOl3/076952
to another embodiment, an dy comprises a light chain variable region comprésing a
VL CDRl of SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154,
160,166 or 224; a VL CDR2 of SEQ ID NO: 83, 89, 95,101,107,113,119,125,131,
137,143, 149, 155, 161, or 167; or a VL CDR3 of SEQ ID NO: 84, 90, 96, 102, 108,114,
UI 120, 126, 132, 138, 144, 150, 156, 162, or 168. In another eréibodiment, the antibody
comprises a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 79, 85,
91, 97,103,109,115,121,127,133,139,145,151,157, or 163; aVH CDR2 ofSEQ ID
NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; or a VH
CDR3 ofSEQ ID NO: 81, 87, 93, 99, 105, 111,117, 123, 129, 135, 141,147, 153,159,
CDRl of
or 165, and further comprises a light chain variable region comprising a VL
SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or
224; aVL CDR2 of SEQ ID NO: 83, 89, 95, 101, 107,113,119, 125, 131, 137,143,149,
1, or 167; or aVL CDR3 ofSEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126,132,
138,144,150,156,162, or168.
[0118] According to one embodiment, an antibody of the invention comprises a heavy
chain variable region comprising a VH CDRl of SEQ ID NO: 79, 85, 91, 97, 103, 109,
115,121,127,133,139,145,151,157, or 163; aVH CDR2 of SEQ ID NO: 80, 86, 92,
98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and aVH CDR3 of SEQ ID
NO: 81, 8'1”, 93, 99,105, 111,117,123, 129, 135, 141,147,153, 159, or 165. According
to another embodiment, an dy comprises a light chain variable region comprising a
VL CDRl of SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154,
160, 166, or 224; a VL CDR2 of SEQ ID NO: 83, 89, 95,101, 107, 113, 119, 125,131,
137, 143, 149, 155, 161, or 167; and a VL CDR3 of SEQ ID NO: 84, 90, 96, 102, 108,
114, 120, 126, 132, 138, 144, 150, 156, 162, or 168. In another embodiment, the
antibody comprises a heavy chain variable region comprising a VH CDRl of SEQ ID
NO: 79, 85, 91, 97, 103, 109, 115,121, 127, 133, 139, 1,157, or 163; aVH CDR2
of SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164;
and aVH CDR3 of SEQ ID NO: 81, 87, 93, 99, 105,111,117,123, 129, 135, 141,147,
153, 159, or 165, and r comprises a light chain variable region comprising a VL
CDRl ofSEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160,
166, or 224; a VL CDR2 of SEQ ID NO: 83, 89, 95, 101, 107, 113, 119, 125, 131,137,
143, 149, 155, 161, or 167; and a VL CDR3 of SEQ ID NO: 84, 90, 96, 102, 108, 114,
120, 126, 132, 138, 144, 150,156,162, or 168.
' 38 '
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO:
80, and VH CDR3 of SEQ ID NO: 81, and can flirther se a light chain le
region comprising a VL CDRI of SEQ ID NO: 82, a VL CDR2 of SEQ ID NO: 83, and a
VL CDR3 of SEQ ID NO: 84.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region sing a VH CDRl of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO:
80, and VH CDR3 of SEQ ID NO: 81, and can further comprise a light chain variable
region comprising a VL CDRl of SEQ ID NO: 224, a VL CDR2 of SEQ ID NO: 83, and
a VL CDR3 of SEQ ID NO: 84.
In one embodiment, an dy of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 85, a VH CDR2 of SEQ ID NO:
86, and VH CDR3 of SEQ ID NO: 87, and can further comprise a light chain variable
region comprising a VL CDRI of SEQ ID NO: 88, a VL CDR2 of SEQ ID NO: 89, and a
VL CDR3 of SEQ ID NO: 90.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 91, a VH CDR2 of SEQ ID NO:
92, and VH CDR3 of SEQ ID NO: 93, and can filrther comprise a light chain le
region comprising a VL CDRl of SEQ ID NO: 94, a VL CDR2 of SEQ ID NO: 95, and a
VL CDR3 of SEQ ID NO: 96.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region sing a VH CDRI of SEQ ID NO: 97, a VH CDR2 of SEQ ID NO:
98, and VH CDR3 of SEQ ID NO: 99, and can further comprése a light chain variable
region comprising a VL CDRI of SEQ ID NO: 100, a VL CDR2 of SEQ ID NO: 101,
and a VL CDR3 of SEQ ID NO: 102.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 103, a VH CDR2 of SEQ ID
NO: 104, and VH CDR3 of SEQ ID NO: 105, and can further se a light chain
variable region comprising a VL CDRl of SEQ ID NO: 106, a VL CDR2 of SEQ ID NO:
107, and a VL CDR3 of SEQ ID NO: 108.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 109, a VH CDR2 of SEQ ID
NO: 110, and VH CDR3 of SEQ ID NO: 111, and can further comprise a light chain
variable region comprising a VL CDRl of SEQ ID NO: 112, a VL CDR2 of SEQ ID NO:
113, and aVL CDR3 of SEQ ID NO: 114.
In one ment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 115, a VH CDR2 of SEQ ID
NO: 116, and VH CDR3 of SEQ ID NO: 117, and can further se a light chain
variable region comprising a VL CDR1 of SEQ ID NO: 118, a VL CDR2 of SEQ ID NO:
119, and a VL CDR3 of SEQ ID NO: 120.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 121, a VH CDR2 of SEQ ID
NO: 122, and VH CDR3 of SEQ ID NO: 123, and can further comprise a light chain
variable region comprising a VL CDRl of SEQ ID NO: 124, a VL CDR2 of SEQ ID NO:
125, and a VL CDR3 of SEQ ID NO: 126.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 127, a VH CDR2 of SEQ H}
NO: 128, and VH CDR3 of SEQ ID NO: 129, and can further comprise a light chain
le region comprising a VL CDRl of SEQ ID NO: 130, a VL CDR2 of SEQ ID NO:
131, and a VL CDR3 of SEQ ID NO: 132.
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDRl of SEQ ID NO: 133, a VH CDR2 of SEQ ID
NO: 134, and VH CDR3 of SEQ ID NO: 135, and can further comprise a light chain
le region comprising a VL CDRl of SEQ ID NO: 136, a VL CDR2 of SEQ ID NO:
137, and a VL CDR3 of SEQ ID NO: 138.
In one embodiment, an antibody of the invention can comprise a heavy chain
variéable region comprising a VH CDRl of SEQ ID NO: 139, a VH CDR2 of SEQ ID
NO: 140, and VH CDR3 of SEQ ID NO: 141, and can further comprise a light chain
variable region comprising a VL CDRl of SEQ ID NO: 142, a VL CDR2 of SEQ ID NO:
143, and a VL CDR3 of SEQ ID NO: 144.
In one ment, an antibody of the invention can comprise a beavy chain
variable region comprising a VH CDRl of SEQ ID NO: 145, a VH CDR2 of SEQ ID
NO: 146, and VH CDR3 of SEQ ID NO: 147, and can further comprise a light chain
le region comprising a VL CDRl of SEQ ID NO: 148, a VL CDR2 of SEQ ID NO:
149,, and a VL CDR3 of SEQ ID NO: 150.
' 40 '
In one embodiment, an antibody of the invention can comprise a heavy chain
variable region comprising a VH CDR1 of SEQ ID NO: 151, a VH CDR2 of SEQ ID
NO: 152, and VH CDR3 of SEQ ID NO: 153, and can further comprise a light chain
variable region comprising a VL CDR1 of SEQ ID NO: 154, a VL CDR2 of SEQ ID? NO:
155, and a VL CDR3 of SEQ ID NO: 156.
In one embodiment, an antibody of the inveration can comprise a heavy chain
variable region comprising a VH CDR1 of SEQ ID NO: 157, a VH CDR2 of SEQ ID
NO: 158, and VH CDR3 of SEQ ID NO: 159, and can further comprise a light chain
variable region comprising a VL CDR1 of SEQ ID NO: 166, a VL CDR2 of SEQ ID NO:
161, and a VL CDR3 of SEQ ID NO: 162.
In one embodiment, an antibody of the invention can comprise a heavy chain
le region comprising a VH CDR1 of SEQ ID NO: 163, a VH CDR2 of SEQ ID
NO: 164, and VH CDR3 of SEQ ID NO: 165, and can further comprise a light chain
le region sing a VL CDR1 of SEQ ID NO: 166, a VL CDR2 of SEQ ID NO:
167, and a VL CDR3 of SEQ ID NO: 168.
In one embodiment, the antibody of the present invention is any one of the
dies comprising an amino acid sequence of the VH and/or VL region as depicted in
Fig. 7 and Table 3. In one embodiment, the antibody of the present invention is
characterized by the vation of the cognate pairing of the heavy and light chain as
was present in the human B-cell.
Table 3: Amino acid sequences of the VB and VL region of tau
specific antibodies. BG — before germlining
“ff,dqulhvy(VH)d4 Antbody
le light (VL) chains
1
“““W'~
BG VH SWEQMTDMNOM
““Wr
VH SEQ. Ifi‘f‘iii6i‘2i3“m
NI—105.17C1 V, SEEjT‘iETfiEEZEM
SEQTIB’I’NEEEI‘
“ ‘ H
: N31Q, I48V VL SEQTID. NO:222
BG VH SEQ: ID. NO:47
NI-105.6C5 ‘Vé WQIDNO48
W\\\‘t\‘-->4
E‘Antibo5§""""W“ “XEifilé‘Zcid 5666;328:251variab‘161162v"§1v11) and?
.___
varlable light (VL)1chains .1
VL‘ SEQ‘1DNo‘49 W...»
WWVHWSEQ“IDN‘0‘-‘50“W“
NI—105.29G10 WVW
SEQ ID. No.51
.‘,‘W~«‘-.-.444.._.“".‘WA
WW... SEQ”113“‘N052
“405-6” “ s
N‘QEEA
ENI-105.40E8 R164W VHW SWDNOZZHW
E “
EVL ‘ ‘SEQ.1D‘.‘N0:55
“"““V SEQID“N0‘5‘6“““‘“ ;E
NI—105.48E5 ..
ESEQIDNQ“5‘1‘“
E “‘““““VH SEQ“113N6:58
E-NI 105.6E3
1 SEQ113.N059
EN110522E1E $EQ113“No60vv‘E‘SEQID NO;61W“““““““““E
SEQ113‘. NO62
‘N1-10526B12 EEGVL E SEQ11).NO63“W“1‘"..___.
VL ESEQ“.‘IDNO“64““
E ‘ ““ SEQ.1D.N0;65““““““
N1-105.12E12
E EVLWSEQ113N066W“““E
.“Wka‘u‘a-r-finu..__.“W~x“‘....»_n.......“,W_.“«m Faun-"nuE SEQ“616?:
i 1
05 '60E7
E: NI—l va‘m’m“““W“winESEQID: NO:68m
FWVHWSEQIDNC“):63““““NI—105.14E2 VLWESEQ. 113.“N070 :E
NI-105.39E2
“‘W‘u‘a-.4.......““
E NI-lOS 19C6 WEVH NO:73 E
VL SEQ113“ND;W721“
“ EBCEVH SEQ. ID. No.75
VH SEQ113‘N076
“HOS-9‘34 BGVWSEQwNowW
EQ‘.“1“13‘.“NE3‘§1§W
WO 00600 ' 42 -
In one embodiment, an antibody of the t invention comprises a heavy chain
variable region (VH) comprising, or ting of an amino acid sequence selected from
the group consisting of SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69,
71, 73, 75, 76, and 220. In one embodiment, an antibody of the present invention
comprises a light chain variable region (VL) comprising, or consisting of an amino acid
sequence selected from the group consisting of SEQ ID NO: 46, 49, 51, 53, 55, 57, 59,
61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, and 222. In one embodiment, an antibody of
the present ion comprises a heavy chain variable region (VH) comprising, or
consisting of an amino acid sequence ed from the group consisting of SEQ ID NO:
44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, and 220, and further
comprises a light chain variable region (VL) comprising, or consisting of an amino acid
sequence ed from the group ting of SEQ ID NO: 46, 49, 51, 53, 55, 57, 59,
61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, and 222. In a specific embodiment, the
antibody comprises a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46; or a VH of
SEQ ID NO: 45 and a VL of SEQ ID NO: 221; or a VH of SEQ ID NO: 45 and a VL of
SEQ ID NO: 222; or a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 49; or a VH of
SEQ ID NO: 50 and a VL of SEQ ID NO: 51; or a VH of SEQ ID NO: 52 and a VL of
SEQ ID NO: 53; or a VH of SEQ ID NO: 54 and a VL of SEQ ID NO: 55; or a VH of
SEQ ID NO: 220 and a VL of SEQ ID NO: 55; or a VH of SEQ ID NO: 56 and a VL of
2O SEQ ID NO: 57; or a VH of SEQ ID NO: 58 and a VL of SEQ ID NO: 59; or a VH of
SEQ ID NO: 60 and a VL of SEQ ID NO: 61; or a VH of SEQ ID NO: 62 and a VL of
SEQ ID NO: 64; or a VH of SEQ ID NO: 65 and a VL of SEQ ID NO: 66; or a VH of
SEQ ID NO: 67 and a VL of SEQ ID NO: 68; or a VH of SEQ ID NO: 69 and a VL of
SEQ ID NO: 70; or a VH of SEQ ID NO: 71 and a VL of SEQ ID NO: 72; or a VH of
SEQ ID NO: 73 and a VL of SEQ ID NO: 74; or a VH of SEQ ID NO: 76 and a VL of
SEQ ID NO: 78; or a VH of SEQ ID NO: 44 and a VL of SEQ ID NO: 46; or a VH of
SEQ ID NO: 47 and a VL of SEQ ID NO: 49; or a VH of SEQ ID NO: 62 and a VL of
SEQ ID NO: 63; or a VH of SEQ ID- NO: 75 and a VL of SEQ ID NO: 77.
Alternatively, the antébody of the present invention is an antibody or antigen-
binding fragment, derivative or variant thereof, which competes for binding to tau, such
as, for example, hTau40, with at least one of the antibodies having the VH and/or VL
region as depicted in Fig. 7 and Table 3. In one embodiment, an antibody of the present
invention competes for specific binding to hTau40 with NI-105.17C1, NI-105.6C5, NI-
‘ 43 '
105.29G10, .6L9, NI-105.40E8, NI-105.48E5, NI-105.6E3, NI—105.22E1, NI-
105.26E:;12, NI-105.12E12, NI-105.60E7, NI-105.14E2, NI-105.39E2, NI-105.19C6, or
NI-105.9C4. Those antibodies can be human as well, in particular for therapeutic
ations. Alternatively, the antibody is a murine, murénized and chimeric murine-
human antibody, which are particularly useful for diagnostic methods and studies in
In one embodiment the antibody of the present invention is provided by cultures
of single or oligoclonal B—cells that are cultured and the supernatant of the culture, which
contains antibodies produced by said B-cells is screened for presence and affinity of anti-
tissue
tau dies therein. The screening process comprises the steps of a sensitive
d plaque immunoreactivity (TAPIR) assay such as described in international
application W02004/09503l, the disclosure content of which is incorporated herein by
reference; screen on brain sections for binding to PHFTau; screening for binding of a
peptide derived from tau of the amino acid sequence ented by SEQ lD NO:6 with
phosphate groups on amino acids Ser-202 and Thr-205; on amino acid Thr-231; and/or on
amino acids Ser—396 and Ser-404 of said sequence; a screen for g of recombinant
human tau of the amino acid sequence represented by SEQ ID N026 and isolating the
antibody for which binding is detected or the cell ing said antibody.
As ned above, due to its generation upon a human immune response the
human monoclonal antibody of the present invention will recognize epitopes which are of
particular pathological relevance and which might not be accessible or less immunogenic
in case of immunization processes for the generation of, for example, mouse monoclonal
antibodies and in vitro screening of phage display libraries, respectively. Accordingly, it
is prudent to stipulate that the epitope of the human anti-tau dy of the present
invention is unique and no other antibody which is capable of g to the e
recognized by the human monoclonal antibody of the present invention exists. Therefore,
the present invention also extends generally to au antibodies and tau binding
molecules which compete with the human monoclonal antibody of the present invention
for specific binding to tau. The present invention is more specifically directed to an
antibody, or antigen-binding fragment, variant or derivatives f, where the antibody
specifically binds to the same epitope of tau as a reference antibody ed from the
group consisting of NI-105.17C1, .6C5, NI-105.29G10, NI-105.6L9, NI-
W0 2014/100600
105.40E8, NI—105.48E5, NI—105.6E3, NI-105.22E1, NI-105.26B12, .12E12, NI-
E7, .14E2, NI-105.39E2, .19C6, and NI-105.9C4.
Competition between antibodies is determined by an assay in which the
immunoglobulin under test inhibits specific binding of a reference antibody to a common
antigen, such tau. Numerous types of competitive binding assays are known, for as
example: solid phase direct or indirect mmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay; see Stahli et al.,
Methods in Enzymology 9 (1983), 242-253; solid phase direct biotin-avidin EIA', see
Kirkland et al., J. Immunol. 137 (1986), 619 and Cheung et al., Virology 176
(1990), 546-552; solid phase direct d assay, solid phase direct labeled sandwich
A Laboratory Manual, Cold Spring Harbor Press
assay; see Harlow and Lane, Antibodies,
(1988); solid phase direct label RIA using 1125 label; see Morel et al, Molec. Immunol. 25
(1988), 7-15 and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82. Typically,
such an assay involves the use of purified tau or aggregates thereof bound to a solid
surface or cells bearing either of these, an unlabelled test immunoglobulin and a labeled
reference immunoglobulin, i. e. the human monoclonal antibody of the present invention.
Competitive inhibition is measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. In one ment, the competitive binding assay
is performed under conditions as described for the ELISA assay in the appended
Examples. Antibodies identified by competition assay (competing antibodies) include
antibodies binding to the same epitope as the reference antibody and antibodies binding to
the reference antibody
an adjacent e sufficiently proximal to the epitope bound by
for steric hindrance to occur. Usually, when a competing antibody is present in excess,
will t specific binding of a reference antibody to a common n by at least 50%
or antigen-binding
or 75%. Hence, the present invention is further drawn to an antibody,
fragment, variant or derivatives thereof, where the antibody competitively ts a
reference antibody selected from the group consisting of NI—105.17Cl,. NI-105.6C5, NI-
105.29G10, NI-105.6L9, NI-105.40E8, NI-105.48E5, NI—105.6E3, NI-105.22E1, NI-
105.26B12, NI-105.12E12, .60E7, NI-105.14E2, NI-105.39E2, NI-105.19C6, or
NI-105.9C4 from binding to tau.
In another embodiment, the present ion provides an ed polypeptide
comprising, consisting essentially of, or ting of an immunoglobulin heavy chain
W0 2014/100600 PCT/USZOl3/076952
variable region (VH), where at least one of VH—CDRs of the heavy chain variable region
are at least 80%, 85%,
or at least two of the VH-CDRS of the heavy chain le region
90%, 95%, 96%, 97%, 98% or 99% identical to reference heavy chain VH-CDRI, VH-
CDR2 or VH-CDR3 amino acid sequences from the antibodies disclosed herein.
Alternatively, the VH-CDRl, VH-CDRZ and VH-CDR3 s of the VH are at least 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% cal to reference heavy chain VH -CDR1,
herein.
VH-CDR2 and VH—CDR3 amino acid ces from the antibodies disclosed
invention has
Thus, according to this embodiment a heavy chain variable region of the
shown
VH—CDRl, VH-CDR2 and VH~CDR3 polypeptide sequences related to the groups
in Fig. 7. While Fig. 7 shows VH-CDRs defined by the Kabat system, other CDR 10
definitions, e.g. , VH-CDRs defined by the Chothia system, are also included in the present
invention, and can be easily identified by a person of ordinary skill in the art using
data presented in Fig. 7. In one embodiment, the amino acid sequence of the reference
VH CDR1 is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151,
NO: 80, 86,
157, or 163; the amino acid sequence of the reference VH CDR2 is SEQ ID
92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and the amino acid
sequence of the reference VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123,
129,135,141,147,153,159, or 165.
In another ment, the present invention provides an isolated polypeptide
2O comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain
le region (VH) in which the VH-CERI, VH-CDR2 and 3 regions have
polypeptide sequences which are identical to the VH-CDRI, VH-CDR2 and VH-CDR3
groups shown in Fig. 7". In one embodiment, the amino acid sequence of the VH CDRI
SEQ ID NO: 79, 85, 91, 97,103,109,115,121,127,133,139,145,151,157, or 163;
amino acid sequence of the VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122,
VH CDR3 is
128, 134, 140, 146, 152, 158, or 164; and the amino acid ce of the
165.
SEQ ID NO: 81, 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or
In r embodiment, the present invention ,provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain
variable region (VH) in which the VH-CDRI, VH-CDR2 and VH-CDR3 regions have
polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and 3
for one, two, three, four, five, six, seven, eight, nine, or ten
groups shown in Fig. 7, except
amino acid
amino acid substitutions in any one VH—CDR. IE certain embodiments the
substitutions are conservative. In one embodiment, the amino acid sequence of the
CDRl is SEQ ID NO: 79, 85, 91, 97, 103,109,115,121,127,133,139,145, 151,157, or
163; the amino acid sequence of the VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110,
the VH
116, 122, 128, 134, 140, 146, 152, 158, or 164; and the amino acid sequence of
CDR3 is SEQ ID NO: 81, 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or
165.
In another embodiment, the present ion provides an isolated polypeptide
comprising, consisting essentially of, or ting of an immunoglobulin light chain
variable region (VL), where at least one of the VL-CDRs of the light chain variable region
are at least 80%, 85%,
or at least two of the VL-CDRS of the light chain variable region
90%, 95%, 96%, 97%, 98% or 99% identical to reference light chain VL-CDRI, VL-
CDR2 disclosed
or 3 amino acid sequences from antibodies
Alternatively, the VL-CDRl, VL-CDRZ and 3 regions of the VL are at least 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to nce light chain VL-CDRl,
herein.
CDR2 and VL-CDR3 amino acid sequences from antibodies disclosed Thus,
according to this embodiment a light chain variable region of the invention has VL-CDRl ,
in Fig.
VL-CDRZ and VL-CDR3 polypeptide sequences related to the polypeptides shown
7. While Fig. 7 shows VL-CDRs defined by the Kabat system, other CDR definitions,
also included in the present invention.
e. g., VL-CDRS defined by the Chothia , are
In one embodiment, the amino acid sequence of the reference VL CDRl is SEQ ID NO: 20
the amino
82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224;
acid ce of the reference VL CDR2 is SEQ ID NO: 83, 89, 95, 101, 107, 113, 119,
of the reference
125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence
VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156,
162, or 168.
In another embodiment, the present invention provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an immunoglobulin light chain
variable region (VL) in which the VL-CDRl, 2 and VL-CDR3 regions have
polypeptide sequences which are identical to the VL-CDRI, VL-CDR2 and VL-CDR3
the amino acid sequence of the VL CDRl
groups shown in Fig. 7. In one embodiment,
SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, I36, 142, 148, 154, 160, 166, or
224; the amino acid sequence of the VL CDR2 is SEQ ID NO: 83, 89, 95, 101, 107, 113,
119, 125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence ofthe VL
W0 2014/100600 ' 47 ' PCT/USZOl3/076952
CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162,
or 168.
In another ment, the present invention provides an ed ptide
comprising, consisting essentially of, or consisting of an globulin light chain
variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have
polypeptide sequences which are identical to the VL—CDRl, VL-CDR2 and VL-CDR3
groups shown in Fig. 7, except for one, two, three, four, five, six, seven, eight, nine, or
amino acid tutions in any one VL-CDR. In certain ments the amino acid
tutions are conservative. In one embodiment, the amino acid sequence of the VL
CDRI is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160,
166, or 224; the amino acid sequence of the VL CDR2 is SEQ ID NO: 83, 89, 95, 101,
107, 113, 11.9, 125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence of
the VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150,
156, 162, or 168.
[0147] In another embodiment, the invention provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain
variable region (VH) which is. identical to a reference heavy chain variable region shown
in Fig. 7 and Table 3. In one embodiment, the amino acid sequence of the reference
heavy chain variable regior; comprises SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60,
62, 65, 67, 69, 71, 73, 75, 76, or 220.
In another embodiment, the invention provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an globulin heavy chain
variable region (VH) having a polypeptide sequence which is identical to a reference
heavy chain variable region (VH) sequence shown in Fig. 7 and Table 3, except for one,
two, three, four, five, six, seven, eight, nine, or ten aneino acid substitutions. In n
embodiments the amino acid substitutions are conservative. In one embodiment, the
amino acid sequence of the reference heavy chain variable region sequence comprises
SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, or 220.
According to one embodiment, the invention provides an isolated polypeptide
comprising, ting essentially of, or consisting of an immunoglobulin light chain
variable region (VL) at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
a reference light chain variable region (VL) amino acid sequence from the antibodies
disclosed herein. Thus, according to this embodiment a light chain variable region of
' 48 '
the invention has a ptide sequence related to the light chain le regions
shown in Fig. 7 and Table 3. In one embodiment, the amino acid sequence of the
reference light chain variable region (VL) comprises SEQ ID NO: 46, 49, 51, 53, 55, 57,
59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or 222.
In another embodiment, the invention provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an immunoglobulin light chain
variable region (VL) which is identical to a reference light chain le region shown
in Fig. 7 and Table 3. In one embodiment, the amino acid ce of the reference
light chain variable region comprises SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64,
66, 68, 70, 72, 74, 77, 78, 221, or 222.
In another ment, the invention provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an immunoglobulin light chain
variable region (VL) having a polypeptide sequence which is identical to a nce
light chain variable region (VL) sequence shown in Fig. 7 and Table 3, except for one,
two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. In certain
embodiments the amino acid substitutions are conservative. In one embodiment, the
amino acid sequence of the reference light chain variable region sequence comprises
SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or
222.
[0152] An globulin or its encoding cDNA can be further modified. Thus, in a
further embodiment the method of the present invention comprises any one of the step(s)
of producing a chimeric antibody, murinized dy, single-chain antibody, Fab-
fragment, bi—specific antibody, fiision antibody, labeled antibody or an analog of any one
of those. Corresponding methods are known to the person skilled in the art and are
described, e.g., in Harlow and Lane odies, A Laboratory Manual", CSH Press,
Cold Spring Harbor (1988). When derivatives of said antibodies are ed by the
phage display que, surface plasmon resonance as employed in the BIAcore system
can be used to increase the efficiency of phage antibodies which bind to the same epitope
as that of any one of the dies described herein (Schier, Human Antibodies
Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The
production of chimeric antibodies is described, for example, in international application
WO89/09622. Methods for the production of humanized antibodies are described in, e.g.,
European application EP-Al 0 239 400 and international application WO90/07861. A
invention are so-
further source of antibodies to be utilized in accordance with the present
called xenogeneic antibodies. The general ple for the production of xenogeneic
such antibodies in mice is described in, e. g., international
antibodies as human-like
As sed
applications WO91/10741, 2602, 4096 and WO 96/33735.
besides complete
above, the antibody of the invention can exist in a variety of forms
antibodies; including, for example, FV, Fab and F(ab)2, as well as in single chains; see e.g.
international application W088/O9344.
The antibodies of the present ion or their corresponding immunoglobulin
chain(s) can be further modified using conventional techniques known
in the art, for
and/or
example, by using amino acid deletion(s), insertion(s), substitution(s), aridition(s),
recombination(s) and/or any other modification(s) known in the art either alone or in
combination. s for introducing such ations in the DNA sequence
known to the
underlying the amino acid sequence of an immunoglobulin chain are well
person skilled in the art; see, e.g, Sambrook, Molecular Cloning A Laboratory Manual,
Cold Spring Harbor Laboratory (1989) NY. and Ausubel, Current Protocols in
lar y, Green Publishing Associates and Wiley Interscience, (1994).
Modifications of the antibody of the invention include chemical and/or enzymatic
side chain
derivatizations at one or more constituent amino acids, including
modifications, ne modifications, and N— and C-terrninal modifications including
ation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or
lipid es, cofactors, and the like. Likewise, the t invention encompasses
production of chimeric proteins which comprise the described antibody or some fragment
f at the amino terminus fused to heterologous molecule such as an
immunostimulatory ligand at the carboxyl terminus; see, e. g., international application
WOOD/30680 for corresponding technical details.
onally, the t invention encompasses peptides including those
containing a binding molecule as described above, for example containing the CDR3
in particular CDR3
region of the variable region of any one of the mentioned antibodies,
of the heavy chain since it has frequently been observed that heavy chain CDR3
(HCDR3) is the region having a greater degree of variability and a predominant
participation in antigen-antibody interaction. Such peptides can easily be synthesized or
produced by recombinant means to produce a binding agent useful according to the
aneIEtiOIl. Such methods are well known to those of ordinary skill in the art. Peptides can
be synthesized for example, using automated peptide synthesizers which are
commercially available. The peptides can also be produced by recombinant techniques by
incorporating the DNA sing the peptide into an expression vector and transforming
cells with the expression vector to produce the peptide.
[0155] Hence, the t invention s to any g molecule, e. g., an antibody or
binding fragment thereof which is oriented towards the human anti—tau antibodies of the
present invention and display the ned properties, i. e. which specifically recognize
tau. Such antibodies and binding molecules can be tested for their binding specificity and
affinity by ELISA and Western Blot and immunohistochemisty as described herein, see,
e. g., the Examples. Furthermore, preliminary results of subsequent experiments
performed in accordance with the present invention revealed that in one ment, the
human ant—tau antibody of the present ion binds primarily to pathologically
aggregated tau resembling neurofibrillary tangles (NFT), neuropil threads t on
human. brain sections of patients who suffered from Alzheimer’s disease (AD) in
addition. Thus, in a particular preferred embodiment of the t invention, the human
antibody or g fragment, derivative or variant thereof recognizes tau on human AE
brain sections.
As an alternative to obtaining immunoglobulins directly from the culture of
immortalized B cells or B memory cells, the immortalized cells can be used as a source of
rearranged heavy chain and light chain loci for subsequent expression and/or c
manipulation. Rearranged antibody genes can be reverse transcribed from; appropriate
mRNAs to produce cDNA. If desired, the heavy chain constant region can be exchanged
for that of a different isotype or eliminated altogether. The variable regions can be linked
to encode single chain Fv s. Multiple Fv regions can be linked to confer binding
ability to more than one target or chimeric heavy and light chain combinations can be
employed. Once the genetic material is available, design of analogs as bed above
which retain both their ability to bind the d target is straightforward. s for
the cloning of antibody variable regions and generation of recombinant antibodies are
known to the person skilled in the art and are described, for example, Gilliland et al.,
Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.
[01%7] Once the appropriate genetic material is obtained and, if desired, modified to
encode an analog, the coding sequences, including those that , at a minimum, the
variable regions of the heavy and light chain, can be inserted into expression systems
host cells. A
contained on vectors which can be transfected into standard recombinant
mammalian cells
variety of such host cells can be used; for efficient processing, however,
lines useful for this purpose include, but are
can be considered. Typical mammalian cell
not limited to, CHO cells, HEK 293 cells, or NSO cells.
[0158] The tion of the antibody or analog is then undertaken by culturing the
of the host
modified recombinant host under culture conditions appropriate for the growth
then recovered by
cells and the expression of the coding sequences. The antibodies are
include signal
isolating them from the e. The expression systems are designed to
that the resulting antibodies are secreted into the medium; however,
peptides so
intracellular production is also possible.
ion also relates to a
In accordance with the above, the present
polynucleotide encoding the antibody or equivalent binding molecule of the present
le region of an
ion. In one embodiment, the polynucleotide s at least a
immunoglobulin chain of the antibody described above. Typically, said variable region
encoded by the polynucleotide comprises at least one complementarity determining
region (CDR) of the VH and/or VL of the le region of the said antibody.
domain of the
The person skilled in the art will readily appreciate that the le
construction of
antibody having the above-described variable domain can be used for the
fimction. Thus, the
other polypeptides or antibodies of desired specificity and biological
and antibodies comprising at least one
present invention also encompasses polypeptides
have
CDR of the above-described variable domain and which advantageously
in the
substantially the same or similar binding properties as the antibody described
appended examples. The person skilled in the art knows that binding affinity can be
making amino acid tutions within the CDRs or . the
enhanced by
ariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) which
partially p with the CDRs as defined by Kabat; see, e. g., Riechmann, et al, Nature
wherein one or
332 (1988), 323-327. Thus, the t invention also relates to antibodies
or not more than two amino acid
more of the mentioned CDRs comprise one or more,
in one or both
substitutions. In one embodiment, the antibody of the invention comprises
set forth in
of its immunoglobulin chains two or all three CDRS of the variable s as
Fig. l.
Binding molecules, e. g., antibodies, or antigen-binding fragments, variants, or
skill in the
derivatives thereof of the invention, as known by those of ordinary art, can
W0 2014/100600 PCT/USZOl3/076952
comprise a constant region which mediates one or more effector functions. For example,
binding of the Cl component of complement to an antibody constant region can activate
the complement . Activation of complement is important in the opsonization and
lysis of cell pathogens. The activation of complement also stimulates the inflammatory
response and can also be involved in autoimmune hypersensitivity. Further, antibodies
bind to receptors on various cells via the Fc region, with a PC receptor binding site on the
dy Fc region binding to a PC receptor (FCR) on a cell. There are a number of Fc
receptors which are specific for different classes of dy, including IgG (gamma
receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
Binding of antibody to Fc receptors on cell surfaces triggers a number of ant and
diverse biological responses including ment and destruction of antibody-coated
particles, clearance of immune xes, lysis of antibody-coated target cells by killer
cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of
inflammatory mediators, placental transfer and control of immunoglobulin production.
[0162] Accordingly, certain embodiments of the present invention include an antibody, or
antigen-binding fragment, variant, or derivative f, in which at least a fraction of one
or more of the constant region domains has been deleted or otherwise altered so as
provide desired biochemical characteristics such as reduced effector functions, the ability
to non-covalently dimerize, increased ability to localize at the site of tau aggregation and
deposition, reduced serum half-life, or increased serum ife when compared with a
whole, unaltered dy of approximately the same immunogenicity. For example,
certain antibodies for use in the diagnostic and treatment methods described herein are
domain deleted antibodies which se a polypeptide chain similar to an
immunoglobulin heavy chain, but which lack at least a portion of one or more heavy
chain s. For instance, in certain antibodies, one entire domain of the constant
region of the modified antibody will be deleted, for example, all or part of the CH2
domain will be deleted. In other embodiments, certain antibodies for use in the diagnostic
and treatment methods bed herein have a constant region, e. g., an IgG heavy chain
nt region, which is altered to ate glycosylation, referred to elsewhere herein
as aglycosylated or "agly" antibodies. Such "agly" antibodies can be ed
enzymatically as well as by engineering the sus glycosylation site(s) in the
constant . While not being bound by theory, it is believed that "agly" antibodies can
have an improved safety and stability profile in viva. Methods of producing aglycosylated
' 53 -
antibodies, having desired effector function are found for e in international
application W02005/018572, which is incorporated by reference in its entirety.
In certain antibodies, or n—binding fragments, ts, or derivatives thereof
described herein, the Fc n can be mutated to decrease effector function using
techniques known in the art. For example, the deletion or inactivation (through point
mutations or other means) of a constant region domain can reduce Fc receptor g of
the circulating modified antibody thereby increasing tau localization. In other cases it can
be that nt region modifications consistent with the instant invention te
ment binding and thus reduce the serum half-life and nonspecific association of a
conjugated cytotoxin. Yet other modifications of the nt region can be used to
modify disulfide es or accharide moieties that allow for enhanced localization
due to increased antigen specificity or antibody flexibility. The resulting physiological
profile, bioavailability and other biochemical effects of the modifications, such as tau
localization, biodistribution and serum half-life, can easily be measured and quantified
using well know immunological techniques without undue experimentation.
In n antibodies, or antigen-binding fragments, variants, or derivatives thereof
described , the Fc portion can be mutated or ged for alternative protein
of example by enhancing
sequences to increase the cellular uptake of antibodies by way
receptor—mediated endocytosis of antibodies via Fc7 receptors, LRP, or Thyl receptors or
by ‘SuperAntibody Technology', which is said to enable antibodies to be shuttled into
living cells without harming them (Expert Opin. Biol. Ther. (2005), 237-241). For
example, the generation of fusion proteins of the antibody binding region and the cognate
protein ligands of cell surface ors or bi— or multi-specific antibodies with a specific
sequences hiding to tau as well as a cell surface receptor can be engineered using
techniques known in the art.
In certain antibodies, or antigen—binding fragments, variants, or derivatives thereof
described herein, the Fc portion can be mutated or exchanged for alternative protein
increase its blood brain barrier
sequences or the antibody can be chemically d to
penetration.
[0166] Modified forms of antibodies, or n-binding fragments, variants, or
derivatives thereof of the invention can be made from whole precursor or parent
antibodies using techniques known in the art. Exemplary techniques are discussed in
more detail herein. Antibodies, or antigen-binding fragments, variants, or derivatives
— 54 -
thereof of the invention can be made or manufactured :using techniques that are known in
the art. In certain embodiments, antibody molecules or fragments thereof are
"recombinantly produced," i.e., are produced using recombinant DNA logy.
Exemplary techniques for making antibody molecules or fragments thereof are discussed
in more detail elsewhere herein.
Antibodies, or antigen—binding fragments, variants, or derivatives f of the
invention also include derivatives that are modified, e. g., by the nt attachment of
such that covalent attachment does not prevent the
any type of molecule to the antibody
antibody from specifically binding to its cognate epitope. For example, but not by way of
limitation, the antibody derivatives include antibodies that have been modified, e. g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by
known protecting/blocking groups, proteolytic ge, linkage to a ar ligand or
known
other protein, etc. Any of numerous chemical modifications can be carried out by
techniques, inclUding, but not limited to c chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can
contain one or more non-classical amino acids.
In particular embodiments, dies, or antigen-binding fragments, ts, or
derivatives thereof of the invention will not elicit a deleterious immune response in the
animal to be treated, e. g.,
e. g., in a human. In n embodiments, binding molecules,
antibodies, or antigen-binding fragments thereof of the invention are deréved from a
t, same species from which
e. g., a human patient, and are subsequently used in the
they are d, e.g., human, alleviating or zing the occurrence of deleterious
immune responses.
De-immunization can also be used to decrease the immunogenicity of an antibody.
As used herein, the term "de-immunization" includes alteration of an antibody to modify
T cell epitopes; see, e. g, international ations WO98/52976 and WOOD/34317.
example, VH and VL sequences from the starting antibody are analyzed and a human T
cell epitope "map" from each V region g the location of epitopes in relation to
complementarity determining regions (CDRs) and other key residues within the sequence.
Individual T cell epitopes from the T cell epitope map are analyzed in order to fy
alternative amino acid substitutions with a low risk of altering activity of the final
antibody. A range of alternative VH and VL sequences are designed comprising
combinations of amino acid substitutions and these ces are subsequently
' 55 '
orated into a of binding ptides, e. g., tau-specific antibodies
range or
immunosEecific fragments thereof for use in the diagnostic and treatment methods
disclosed herein, which are then tested for function. lly, between 12 and 24 variant
antibodies are generated and tested. Complete heavy and light chain genes comprising
modified V and human C regions are then cloned into expression vectors and the
subsequent ds introduced into cell lines for the tion of whole antibody. The
antibodies are then compared in appropriate biochemical and biological assays, and the
optimal variant is identified.
Monoclonal antibodies can be prepared using a wide variety of techniques known
in the art including the use of oma, recombinant, and phage display techEologies,
or a combination thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known iE the art and taught, for example, in
Harlow et 61]., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
2nd ed. (1988); Harnmerling et al., in: onal Antibodies and T-Cell Hybridomas
er, N.Y., 563-681 (1981), said references incorporated by reference in their
entireties. The term "monoclonal antibody" as used herein is not limited to antibodies
produced through hybridoma technology. The term "monoclonal antibody" refers to an
antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or
phage clone, and not the method by which it is produced. Thus, the term "monoclonal
antibody" is not limited to antibodies produced through hybridoma technology. In certain
embodiments, dies of the present invention are derived from human B cells which
have been immortalized Via transformation with Epstein-Barr virus, as described herein.
In the well—known hybridoma process r et al., Nature 256 (1975), 495) the
relatively short-lived, or mortal, lymphocytes from a mammal, e.g., B cells derived from.
line
a human subject as described , are fused with an immortal tumor cell (e. g. ,. a
myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both
immortal and capable of producing the genetically coded dy of the B cell. The
resulting hybrids are segregated into single genetic strains by selection, dilution, and re-
growth with each individual strain comprising specific genes for the formation of a single
antibody. They produce antibodies, which are homogeneous against a d antigen
and, in nce to their pure genetic parentage, are termed "monoclonal".
Hybridoma cells thus prepared are seeded and grown in a suitable culture medium
that contain one or more substances that inhibit the growth or al of the unfused,
w 56 —
cell lines
parental myeloma cells. Those skilled in the art will iate that reagents,
and media for the formation, selection and growth of hybridomas are commercially
available from a number of sources and standardized protocols are well established.
for Generally, culture medium in which the hybridoma cells are g is assayed
production of monoclonal antibodies against the desired antigen. The binding specificity
of the monoclonal antibodies ed by hybridoma cells is determined by in vitro
assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme—linked
immunoabsorbent assay (ELISA) as described herein. Afier hybridoma cells are
identified that produce antibodies of the desired specificity, affinity and/or activity,
clones can be subcloned by ng dilution procedures and grown by standard methods;
Academic Press, pp
see, e. g., Goding, Monoclonal Antibodies: Principles and Practice,
59-103 (1986). It will further be appreciated that the monoclonal antibodies ed by
the subclones be separated from culture medium, ascites fluid or serum
can by
conventional purification procedures such as, for example, protein-A, hydroxylapatite
chromatography, gel electrophoresis, dialysis or affinity chromatography.
In another embodiment, lymphocytes can be selected by micromanipulation
the le genes isolated. For example, eral blood mononuclear
cells can be
isolated from an immunized or naturally immune , e.g., a human, and cultured
that meet the
for about 7 days in vitro. The cultures can be screened for specific IgGs
screening criteria. Cells from positive wells can be isolated. Individual Ig-producing
cells be ed by FACS or by identifying them in a
can complement-mediated
and the
hemolytic plaque assay. Ig-producing B cells can be anipulated into a tube
VH and VL genes can be amplified using, e.g., RT—PCR. The VH and VL genes can
cloned into an antibody expression vector and transfected into cells (e. g., eukaryotic or
prokaryotic cells) for expression.
Alternatively, antibody-producing cell lines can be selected and cultured using
in a variety of
techniques well known to the skilled artisan. Such techniques are described
le for use
laboratory manuals and primary publications. In this t, ques
in the invention as described below are described in Current Protocols in Immunology,
n et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley
Sons, New York (1991) which is herein incorporated by reference in its entirety,
including supplements.
PCT/USZOl3/076952
known
Antibody fragments that recognize specific epitopes can be generated by
techniques. For example, Fab and F(ab')2 fragments can be produced recombinantly or by
proteolytic cleavage of immunoglobulin les, using enzymes such as papain (to
e Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab’)2 fragments
of the
contain the variable region, the light chain constant region and the CHI domain
heavy chain. Such fragments are ent for use, for example, in immunodiagnostic
procedures ing coupling the immunospecific portions of immunoglobulins to
detecting reagents such as radioisotopes.
: Human antibodies, such as bed herein, are particularly desirable for
therapeutic use in human patients. Human antibodies of the present invention are isolated,
be suspected to be at risk
e.g., from y human subjects who because of their age may
of developing a tauopathic disorder, e.g., Alzheimer’s e, or a patient with the
disorder but with an unusually stable disease course. However, though it is prudent to
will.
expect that elderly healthy and symptom-free subjects, respectively, more regularly
latter can be used
have developed protective anti-tau antibodies than younger subjects, the
as well as source for obtaining a human antibody of the present ion. This
form of a
particularly true for younger patients who are predisposed to p a familial
tauopathic disease but remain symptom—free since their immune system functions more
efficiently than that in older adults.
[0177] In one embodiment, an antibody of the invention comprises at least one heavy or
of the
light chain CDR of an antibody le. In another embodineent, an antibody
another
invention comprises at least two CDRs from one or more antibody molecules. In
from one or
embodiment, an antibody of the invention comprises at least three CDRs
an antibody of the invention comprises
more antibody les. In another embodiment,
In another ment, an
at least four CDRs from one or more antibody molecules.
antibody of the invention comprises at least five CDRs from one or more antibody
molecules. In another embodiment, an antibody of the invention comprises at least six
CDRs from one or more dy molecules. Exemplary antibody molecules comprising
described herein.
at least one CDR that can be ed in the subject antibodies are
[0178] dies of the present invention can be produced by any method known in
recombinant
art for the synthesis of antibodies, in particular, by chemical synthesis or by
expression techniques as described herein.
' 58 ' 2013/076952
{$139} in one embodiment, an antihedy, or antigembindirtg fragment, t, er
derivative thereof of the invention comprises a synthetic constant regien wherein etie er
more ts are eartiaiiy er entirety deieted {”domairrwdeteted antibodiesft. in certain
embodiments compatible modified antibodies will se domain d constructs or
ts wherein the entire CH2 domain has been removed (ACH2 constructs). For other
embodiments a short connecting peptide can be substituted for the deleted domain to
provide flexibility and freedem ef movement fer the variabie regierr. ’i‘hcse skiiied in the
art wiii artpreeiate that such ucts are particoiariy preferred due to the reguiatcry
ereperties ef the CH3 demain on the eatahotic rate of the airtihedy, Domain deleted
ceastructs can be derived using a vecter encoding an igGg human censtant domain, see,
sag, intematienai appiieations WSW/$66955 and WOG2I’0S36948A2, This vector is
ered te delete the CH2 demain and provide a synthetie vector exeressing a n
deleted igfil cetrstant region.
in n embodiments, an‘tibedies, er antigen—binding fragments, variants, er
derivatives thereof dfthe present inventien are minibedies. Mirrihedies can be made using
methods bed in the art, see, e.g., US patent 5,837,821 or international application
WO 94/09817.
In one embodiment, an antibody, or antigen—binding fragment, variant, or
derivative thereof of the inveetion comprises an globulin heavy chain having
deletion or substitution of a few or even a single amino acid as long as it permits
association between the monomeric subunits. For example, the mutation of a single amino
acid in selected areas of the CH2 domain can be enough to substantially reduce Fc
binding and thereby increase tau localization. Similarly, it can be desirable to simply
delete that part of one or more constant region domains that control the effector on
(e. g. complement binding) to be modulated. Such partial deletions of the constant regions
can improve selected characteristics of the antibody (serum half-life) while leaving other
desirable functions associated with the subject constant region domain intact. Moreover,
as alluded to above, the constant regions of the disclosed antibodies can be synthetic
through the mutation or substitution of one or more amino acids that enhances the profile
of the resulting construct. In this respect, the ty provided by a conserved binding site
(e. g. Fc binding) can be disrupted while substantially ining the configuration and
immunogenic profile of the modified antibody. Yet other embodiments comprise the
addition of one enhance desirable
or more amino acids to the constant region to
characteristics such as effector function or provide for more cytotoxin or carbohydrate
attachment. In such embodiments it can be desirable to insert or replicate specific
domains.
sequences derived from ed constant region
The present invention also es dies that comprise, consist essentially
of, varéants (including derivatives) of antibody molecules (e. g., the VH of, or consist
thereof
regions and/or VL regions) bed herein, which antibodies or fragments
immunospecifically bind to tau. Standard techniques known to those of skill in the art can
be used to introduce mutations in the nucleotide sequence encoding an antibody,
including, but reot limited to, site-directed mutagenesis and PCR-mediated mutagenesis
which result in amino acid substitutions. In one ment, the variants (including
derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid
substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions,
less than 10
less than 20 amino acid substitutions, less than 15 amino acid substitutions,
amino acid substitutions, less than 5 amino acid tutions, less than 4 amino acid
substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions
relative to the reference VH region, VH—CDRI, VH-CDR2, VH-CDR3, VL region, VL-
in which
CDRl, VL-CDRZ, or VL-CDR3. A "conservative amino acid tution" is one
with a
the amino acid residue is replaced with an amino acid residue having a side chain
similar charge. Families of amino acid residues having side chains with r charges
have been defined in the art. These es include amino acids with basic side chains
(e. g, lysine, arginine, histidine), acidic side chains (e. g., aspartic acid, glutamic acid),
uncharged polar side chains (e. g., e, asparagine, glutamine, serine, threonine,
ne, cysteine), nonpolar side chains (e. g., alanine, , leucine, cine, proline,
alanine, methionine, tryptophan), beta-branched side chains ( e. g., threonine,
valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan,
of the
histidine). Alternatively, mutations can be introduced randomly along all or part
coding sequence, such as by saturation mutagenesis, and the resultant mutants can
screened for biological activity to identify mutants that retain activity (e.g., the ability to
bind tau).
[0i83] For example, it is possible to introduce mutations only in framework regions or
only in CETER regions of an antibody molecule. Introduced mutations can be
silent or
neutral missense mutations, to bind
e. g., have no, or little, effect on an antibody’s ability
W0 00600 PCT/USZOl3/076952
antigen, indeed some such mutations do not alter the amino acid sequence ever.
These types Sf mutations can be useful to optimize codon usage, or improve a
hybridenia’s antibody production. optimized ceding regiens mending antibodies
0f the invention are sed eisewhere herein. newnentrai
present Alternatively,
missense mutations can alter an antibody’s ability to bind antigen. The location of most
silent and neutral missense mutations is likely to be in the franeework regions, while the
location of most non-neutral missense mutations is likely to be in CDR, though this is not
and test mutant
an absolute requirement. One of skill in the art would be able to design
molecules with desired properties such as no alteration in antigen-binding activity or
alteration in binding activity (e.g., improvements in antigen-binding activity or change in
antibody specificity). ing mutagenesis, the encoded protein can routinely be
expressed and the functional and/or biological activity of the encoded protein, (e. g.,
ability to immunospecifically bind at least one epitope of tau) can be determined using
techniques described herein or by routinely modifying techniques known in the art.
[0E84] Tau binding agents, for example, but not limited to, tau binding antibodies of the
present invention can be characterized using vivo in models of
any in or Vitro
neurodegenerative thies. A skilled artisan y understands that a tau binding
(e.g., an antibody) of the invention can be characterized in a mouse model for agent
neurodegenerative thies. for example, but not limited to, any one of the following
2O three different animal models for thies can be used to characterize and validate the
tau antibodies (and molecules with the binding cities thereof) of the present
invention.
1. Transgenic TauP301L mice (line183): expressing human Tau40 with P301L
mutation under the murine Thy1.2 promoter (Generation of these transgenic animals is
described in Gotz et al., J. Biol. Chem. 276 (2001), 529-534 and in international
application , the disclosure content of which is incorporated herein by
reference)
2. JNPL3 mice expressing the shortest 4R human tau isoform with P301L
mutation under the murine PrP promoter (available from Taconic, , NY, USA).
[0187] 3. P3OISTau (line P819) mice expressing human tau with P3018 mutation under
the murine PrP promoter able from the n tory, Bar Harbor, Maine,
LLSnA)
' 61 '
A skilled artisan understands that an mental model of neurodegenerative
tauopathies can be used in a preventative setting or it can be used in a therapeutic setting.
In a preventative setting, the dosing of animals starts prior to the onset of the
neurodegenerative tauopathies or symptoms thereof. In preventative settings, a tau
g agent (e.g., antibody) of the invention is evaluated for its ability to prevent,
reduce or delay the onset of neurodegenerative tauopathies or symptoms thereof, In a
therapeutic setting, the dosing of animals start after the onset of neurodegenerative
tauopathies or a symptom thereof. In a eutic setting, a tau g agent (e.g.,
antibody) of the invention is evaluated for its y to treat, reduce or alleviate the
neurodegenerative tauopathies or a symptom thereof. Symptoms of the neurodegenerative
tauopathies include, but are not limited to, accumulation of pathological tau deposits,
neurofibrillary tangles (NFT), hyperphosphorylated tau polypeptide, insoluble tau
fractions in the s, brain, spinal cord, cerebrospinal fluid or serum of the
experimental object. A skilled artisan understands that a positive preventative or
therapeutic outcome in any animal model of neurodegenerative tauopathies indicates that
the particular tau binding agent (e.g., antibody) can be used for tative or
therapeutic purposes in a subject other than the experimental model organism, for
example, it can be used to treat neurodegenerative tauopathies in a human subject in need
thereof
[0189] In one embodiment, a tau binding agent (e.g., an antibody) of the invention can be
administered to a tauopathy mouse model and corresponding control Wild type mice. The
antibody stered ean be a minimized dy eftlie present inventien er a human—
murine chimera {if an antibedy of the present inventien. 'l‘lie tan binding agent. (e.g., an
dy) can be stered by any means linemr in the art, fer example, by
intraperiteneal, intracraniai, intramuscular, eneus, subcutaneous, anal, and aeresel
administration. Experimental animals ean be given 011e, twe, three, liter, live or more
doses of tire tau binding agent (e.g., an antibedy) er a. centre} eempesitien, such as PBS.
in one embediment, experimental animals Wiil be administered Gm: or twe doses of a tau
g agent (e.g., an antibody). See, for example, Example 9. In another embodiment,
the animals are chronically dosed with the tau binding agent (e.g., an antibody) over
several weeks or months. See, for example, Example 10. A skilled n can readily
design a dosing regimen that fits the experimental purpose, for example, dosing regimen
for acute studies, dosing regimen for chronic studies, dosing regimen for toxicity studies,
dosing n for preventative or therapeutic studies. The ce of tlee tau binding
animals, for
agent (e.g., dy) in a particular tissue compartment of the mental
example, but not limited to, serum, blood, cerebrospinal fluid, brain tissue, can be
established using well know methods of the art. See, for example, Example 9 and 10. In
one embodiment, a tau binding agent (e.g., antibody) of the invention is capable to
the tau
penetrate the blood brain barrier. A skilled artisan understands that by adjusting
binding agent (e.g., antibody) dose and the dosing frequency, a desired tau binding agent
effect
(e.g., antibody) concentration can be maintained in the experimental animals. Any
of a tan binding agent (e.g., antibody) of the present invention in the tauopathy models
or distribution of tau
can be assessed by comparing the level, biochemical teristics
in the treated and l animals. In one example, the neurofibrillary tangles (NFT) are
examined using the silver impregnation technique of Gallyas or by immunostaining with
monoclonal mouse antibody AT100 and AT180, which recognize pathologically
phosphorylated tau in NFT. The number or frequency of Gallyas-positive neurons and/or
ATIOO, AT180 labeled neurons in the brain and spinal cord in antibody d mice
control animals can be ined to evaluate the effect of antibody treatment. In one
embodiment, an antibody of the present invention is capable of reducing the level,
in an animal
amount or tration of neurofibrillary tangles in the brain or spinal cord
model. The antibody can reduce the level, amount or concentration of neurofibrillary
tangles by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In another
embodiment, an antibody of the present invention is capable of reducing the number or
frequency of Gallyas—positive neurons in the brain or spinal cord in an animal model,
example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In a further
embodiment, an antibody of the present invention is capable of reducing the number or
in an
frequency of AT100 or AT180 antibody positive neurons in the brain or spinal cord
animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or
more. The effect of an antibody of the t invention can also be ed by
examining the distribution and biochemical properties of tau following dy
stration. In one embodiment, an antibody of the present invention is capable of
reducing the amount or concentration of tau protein in the brain or spinal cord of an
animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or
more. In another embodiment, an antibody of the present invention is e ofreducing
the amount or concentration of insoluble tau protein in the brain or spinal cord of an
WO 00600 ' 63 ' 2013/076952
animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or
more. ble tau fraction can be prepared as described, for example, in Example 10 or
in t M, Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992). The
amount of tau protein in a biological sample can be ined by any method known to
one of skill, for example, as described in Example 10. In a further embodiment, an
antibody of the present invention can reduce the amount or concentration of
hyperphosphorylated tau protein in the brain or spinal cord in an animal model, for
example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more.
Hyperphosphorylated tau can be detected using antibodies specific for pathologically
hquoerphosphorylaterl forms ot‘tau, sueh as ATlilll or A3380. An. antibody ofthe present
invention can also alter, for example, reduce or inerease, tau concentration in the hiood,
serum or cerebrospinal fluid or an animal model, for example, by at least ahout 5%, lil%,
%, 30%, 50%, 70%, 90% or more. in one embodiment, the % reduction or increase is
relative compared to the level, ntunltter, fiequenoy, amount or tration that existed
before treatment, or to the level, number, frequency, amount or concentration that exist in
an untreated/control treated subj eet.
In one embodiment, an antibody of the t invention can prevent or delay the
onset of at least one symptom of a neurodegenerative tauopathy in a subject. In one
ment, an antibody of the present invention can reduce or eliminate at least one
symptom of a neurodegenerative tauopathy in a subject. The symptom can be the
formation of pathological tau deposits, hyperphosphorylated tau ts, ble tau
deposits, neurofibrillary fibers, brillary fibers, pre—tangle phosphor tau aggregates,
intraneuronal neurofibrillary tangles or extraneuronal neurofibrillary tangles in the brain
or spinal cord of a subject. See, e.g., Augustinack et a1, Acta athol 103:26-35
(2002). The symptom can also be the presence, or elevated concentration or amount, of
tau in the serum, blood, urine or cerebrospinal fluid, wherein elevated concentration
amount is compared to a healthy subject. The symptom can be a neurological symptom,
for example, altered conditioned taste aversion, altered contextual fear conditioning,
memory impairment, loss of motor function. In one embodiment, memory impairment is
assessed using a two-trial Y-maze task. In a specific ment, the two-trial Y-maze
task is performed ntially as described in Example 10. In one embodiment, the at
least one symptom is reduced by at least about 5%, 10%, 15%, 20%, 30%, 50%, 70%, or
90%. In another embodiment, the two-trial Y-maze task ratio is significantly higher in an
antibody treated t than in a control subject. In a specific embodiment, the two-trial
Y-maze task ratio is increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90%. In another embodiment, the two-trial Y-maze task ratio is at least
about two times, three times, four times, five times, ten times, or twenty times higher. The
present invention also provides a method of preventing or delaying the onset of at least
one m of a neurodegenerative thy in a subject in need thereof, comprising
administeréng a therapeutically effective amount of a tau antibody described herein. The
present invention further provides a method of reducing or ating least one symptom
of a neurodegenerative tauopathy in a subject in need thereof, comprising administeréng a
therapeutically effective amount of a tau antibody described herein. In one embodiment,
the subject is an experimental organism, such as, but not limited to, transgenic mouse. In
one embodiment, the subject is a human.
IiI. Polynucleotides Encoding Antibodies
[019i] A polynucleotide encoding an antibody, or antigen-binding fragment, t, or
derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide,
which can be unmodified RNA or DNA or modified RNA or DNA. For example, a
cleotide encoding an antibody, or antigen-binding fragment, variant, or derivative
thereof can be composed of single- and double—stranded DNA, DNA that is a mixture of
single- and —stranded regions, single- and double-stranded RNA, and RNA that is
mixture of single— and double-stranded regions, hybrid molecules comprising DNA and
RNA that can be single-stranded or, more typically, double-stranded or a mixture of
single- and double-stranded s. In addition, a cleotide encoding an antibody,
or antigen-binding fragment, variant, or derivative thereof can be composed of triple-
stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide
encoding an antibody, or antigen-binding fragment, variant, or tive thereof can also
contain one or more modified bases or DNA or RNA nes modified for stability or
for other reasons. "Modified" bases include, for example, ated bases and unusual
bases such as inosine. A variety of modifications can be made to DNA and RNA; thus,
"polynucleotide" es chemically, enzymatically, or metabolically modified forms.
[0192] An isolated polynucleotide ng a non-natural variant of a polypeptide
derived from an immunoglobulin (e.g., an immunoglobulin heavy chain n or light
chain portion) can be created by introducing one or more tide substitutions,
W0 2014/100600 ' 65 '
additions or ons into the nucleotide sequence of the globulin such that one
or more amino acid substitutions, additions or deletions are introduced into the encoded
protein. Mutations can be introduced by standard techniques, such as site-directed
mutagenesis and PCR—mediated mutagenesis. In one embodiment, conservative amino
acid substitutions are made at one or more non-essential amino acid residues.
As is well known, RNA can be isolated from the original B cells, hybridorna cells
or from other transformed cells by standard techniques, such as guanidinium
isothiocyanate extraction and precipitation followed by centrifugation or chromatography.
Where desirable, mRNA can be isolated from total RNA by standard techniques such as
chromatography on oligo dT ose. Suitable techniques are ar in the art. In one
embodiment, cDNAs that encode the light and the heavy chains of the antibody can be
made, either simultaneously or separately, using reverse transcriptase and DNA
polymerase in accordance with well-known methods. FCR can be initiated by consensus
constant region primers or by more specific s based on the hed heavy and
light chain DNA and amino acid sequences. As discussed above, PCR also can be used to
isolate DNA clones encoding the antibody light and heavy chains. In this case the
libraries can be screened by sus primers or larger homologous , such as
human constant region probes.
DNA, typically d DNA, can be isolated from the cells using techniques
known in the art, restriction mapped and sequenced in accordance with standard, well
known techniques set forth in detail, e. g., in the foregoing references relating to
recombinant DNA techniques. Of , the DNA can be synthetic according to the
present invention at any point during the isolation process or uent analysis.
In one embodiment, the present ion provides an isolated polynucleotide
comprising, ting essentially of, or consisting of a nucleic acid encoding an
immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the
heavy chain variable region or at least two of the VH-CDRS of the heavy chain variable
region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to reference
heavy chain VH—CDRI, VH-CDR2, or VH-CDR3 amino acid sequences from the
antibodies disclosed herein. Alternatively, the VH-CDEI, VH-CDRZ, or VH-CDR3
regions of the VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% cal to
reference heavy chain VH-CDRl, VH-CDRZ, and VH-CDR3 amino acid sequences from
the antibodies disclosed herein. Thus, according to this embodiment a heavy chain
variable region of the invention has VH-CDRI, VH-CDRZ, or VH-CDR3 polypeptide
shown in Fig. 7. In one embodiment, the
sequences related to the polypeptide sequences
amino acid sequence of the reference VH CDRI is SEQ ID NO: 79, 85, 91, 97, 103, 109,
115, 121, 127, 133, 139, 145, 151, 157, or 163; the amino acid sequence ofthe reference
VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152,
NO: 81,
158, or 164; and the amino acid sequence of the reference VH CDR3 is SEQ ID
87, 93, 99,105,111,117,123,129,135,141,147,153,159, or 165.
In one embodiment, the present invention provides an ed polynucleotide
comprising, ting essentially of, or consisting of a nucleic acid encoding an
immunoglobulin heavy chain variable region (VH), in which the VH-CDRl, VH-CDR2
and VH-CDR3 regions have polypeptéde sequences which are identical to the VH-CDRI,
VH-CDRZ and VH-CDR3 groups shown in Fig. 7, except for one, two, three, four, five,
six, seven, eight, nine, or ten amino acid substitutions in any one VH-CDR. In certain
embodiments the amino acid substitutions are conservative. In one embodiment, the
amino acid sequence of the VH CDRI is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121,
is SEQ
127, 133, 139, 145, 151, 157, or 163; the amino acid sequence of the VH CDR2
ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and
amino acid sequence of the VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123,
129,135,141,147,153,159, or 165.
[0197] In another embodiment, the present invention provides an isolated polynucleotide
comprising, consisting essentially of, or consisting of a nucleic acid ng an
immunoglobulin light chain variable region (VL), where at least one of the S of
variable
the light chain variable region or at least two of the s of the light chain
region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nce
dies
light chain VL-CDRI, VL-CDRZ, 0r VL-CDR3 amino acid sequences from the
disclosed herein. Alternatively, the VL-CDRl, Z, or VL-CDR3 regions of the VL
99% identical to reference light
are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
chain I, VL—CDRZ, and VL-CDR3 amino acid sequences from the antibodies
sed herein. Thus, according to this embodiment a light chain variable region of
ion has VL-CDRl, VL-CDRZ, or VL—CDR3 polypeptide sequences related to the
polypeptide sequences shown in Fig. 7. In one embodiment, the amino acid sequence of
the reference VL CDRl is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136,
CDR2 is
142, 148, 154, 160, 166, or 224; the amino acid sequence of the reference VL
SEQ ID NO: 83, 89, 95,101,107, 113, 119, 125, 131, 137, 143, 149, 155, 161, or 167;
and the amino acid sequence of the reference VL CDR3 is SEQ ID NO: 84, 90, 96, 102,
4,120,126,132,138,144,150,156, 162, or 168.
In another embodiment, the present invention provides an isolated polynucleotide
comprising, consisting essentially of, or consisting of a nucleic acid ng an
immunoglobulin light chain variable region (V1) in which the VL-CDRl, VL-CDRZ and
VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRI, VL-
CDR2 and VL—CDR3 groups shown in Fig. 7, except for one, two, three, four, five, six,
seven, eight, nine, or ten amino acid tutions in any one VL-CDR. In certain
ments the amino acid substitutions are conservative. in one embodiment, the
amino acid sequence of the VL CDRl is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124,
130, 136, 142, 148, 154, 160, 166, or 224; the amino acid sequence of the VL CDR2 is
SEQ ID NO: 83,89, 95, 101,107,113, 119, 125,131,137, 143, 149,155,161, or 167;
and the amino acid sequence of the VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114,
120,126,132,138,144,150,156,162, or 168.
In another embodiment, the present invention provides an isolated polynucleotide
comprising, ting ially of, or consisting of a nucleic acid encoding an
immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDRZ,
and VH—CDR3 regions have polypeptide sequences which are identical to the VH-CDRl,
VH—CDR2, and VH-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid
sequence ofthe VH CDRl is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, 127, 133,
139, 145, 151, 157, or 163; the amino acid sequence of the VH CDR2 is SEQ ID NO: 80,
86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and the amino acid
sequence ofthe VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135,
141,147,153,159, or 165.
In r embodiment, the t invention provides an isolated polynucleotide
comprising, consisting essentially of, or consisting of a nucleic acid ng an
immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDFi2, and
VL—CDR3 regions have polypeptide sequences which are identical to the VL-CDRI, VL-
CDR2, and VL-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid
sequence ofthe VL CDRl is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136,
142, 148, 154, 160, 166, or 224; the amino acid sequence of the VL CDR2 is SEQ ID
NO: 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, or 167; and the
WO 00600
amino acid sequence ofthe VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126,
132, 138,144, 150,156, 162, or 168.
As known in the art, "sequence identity" n two polypeptides or two
polynucleotides is determined by comparing the amino acid or nucleic acid sequence of
to polypeptide or
one polypeptide or polynucleotide the sequence of a second
polynucleotide. When sed , whether any ular polypeptide is at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to
another polypeptide can be determined using methods and computer programs/software
known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, n, WI 53711). BES’i‘FIT uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489,
to find the best segment of homology between two sequences. When using BESTFIT or
determine whether a particular sequence is, for
any other sequence alignment program to
example, 95% identical to a reference sequence ing to the present invention, the
parameters are set, of course, such that the percentage of identity is calculated over
filll length of the reference polypeptide sequence and that gaps in homology of up to 5%
of the total number of amino acids in the reference ce are allowed.
In one embodiment of the present invention, the polynucleotide comprises,
2O consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of
the VH or VL region of an anti—tau antibody as depicted in Table 4. In this respect, the
that the cleotides encoding at least
person skilled in the art will readily appreciate
the variable domain of the light and/0r heavy chain can encode the variable domain of
both immunoglobulin chains or only one.
Table 4: Nucleotide sequences ofthe VH and VL region of tau
specific antibodies. BG — before ning
may eotiéigéxélifiéfié‘e“;SRWE‘EVETM;
and variableulight (VL) chains
BG vH SEQ. ID. NO:169
‘ *
.17C1 vH SEQ. ID. NO:170
= [siémdf'iii‘l‘ifiou71
NI-105”,6‘C5 iBG-VH §SEQ.ID.N0:172
W0 2014/100600 ' 69 ' PCT/USZOl3/076952
Antibody Ilm‘m‘EnNucleotidesequences of varlable heavy (VH)
and variable light (VL) chams
NI-105.6L9
NI-105.40E8
$11111111
NI-105.48E5 I‘N111112
“‘Z‘Nez1z13
NI-105.6E3 ‘ 1111:1111
NI-105.22E1
‘: . \111117
. 111111111
1111221
1121;;113?31170191 1.111..“
'N1—1115111137 ...._.m“““
SEQ ED “543192
E...u.....1___......._....m“‘“
NI-105.14E2
i VH SEQ111N13: 191
.39E2
VL SEQ111N11 1111;
“""VH .111IN111117
NI-105.19C6 V11‘1W
{EE‘VH131111 1111 N11: 1119
NI-105.9C4 ESEQ 113.
In one embodiment, the present invention provides an isolated polynucleotide
comprising, consisting essentially of, or consisting of a nucleic acid encoding an
globulin heavy chain le region at least 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% or 95% identical to reference heavy chain VH. In one embodiment,
ID NO:
amino acid sequence of the reference heavy chain variable region comprises SEQ
44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, or 220.
In one embodiment, the present invention provides an isolated polynucleotide
comprising, consisting essentially of, or consisting of a nucleic acid encoding an
immunoglobulin light chain variable region at least 80%, 85%, 90%, 95%, 96%, 97%,
the amino
98%, or 99% or 95% identical to reference light chain VL. in one embodiment,
NO: 46, 49,
acid sequence of the reference light chain variable region comprises SEQ ID
51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or 222.
of the
The present invention also includes fragments of the polynucleotides
invention, as bed elsewhere. Additionally polynucleotides which encode fusion
polynucleotides, Fab fragments, and other derivatives, as described herein, are also
contemplated by the invention.
known in
The polynucleotides can be produced or manufactured by any method
the art. For example, if the nucleotide sequence of the antibody is known, a
polynucleotide encoding the antibody can be assembled from chemically synthesized
17 (1994), 242,
oligonucleotides, e. g., as described in Kutmeier et al., BioTechniques
which, briefly, involves the synthesis of overlapping ucleotides ning portions
of the sequence ng the antibody, annealing and ligating of those oligonucleotides,
and then amplification of tEe ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody, or n-binding
from a suitable
fragment, variant, or derivative f can be generated from nucleic acid
is not ble,
source. If a clone containing a nucleic acid encoding a particular antibody
the dy
but the sequence of the antibody molecule is known, a nucleic acid encoding
le source (e.g., an antibody cDNA
can be chemically synthesized or ed from a
library, or a cDNA library ted from, or nucleic acid, preferably polyAJr RNA,
isolated from, any tissue or cells expressing the tau-specific antibody, such as hybridoma
cells selected to express an antibody) by PCR cation using synthetic s
hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide
probe specific for the particular gene sequence to identify, e. g., a cDNA clone from a
cDNA y that encodes tlee antibody. Amplified nucleic acids ted by PCR can
then be cloned into replicable cloning vectors using any d well known in the art.
Once the nucleotide sequence and ponding amino acid sequence of the
or derivative thereof is determined, its dy, or antigen-binding fragment, t,
i3“; nucleotide sequence can be manipulated using methods well known in the art for the
manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for e, the techniques described in Sambrook et al.,
Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY. (1990) and Ausubel et al., eds, Current Protocols in Molecular
herein
Biology, John Wiley & Sons, NY , which are both incorporated by reference
in their entireties), to generate antibodies having a different amino acid sequence,
example to create amino acid substitutions, deletions, and/or insertions.
IV. Expression of Antibody Polypeptides
Following lation of the isolated genetic material to provide antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the invention, the
cleotides encoding the antibodies are typically inserted in an expression vector for
introduction into host cells that can be used to produce the desired quantity of antibody.
Recombinant expression of an antibody, or fragment, derivative or analog f, e.g., a
herein.
heavy or light chain of an antibody which binds to a target molecule is described
of an
Once a polynucleotide encoding an antibody molecule or a heavy or light chain
antibody, or portion thereof (preferably containing the heavy or light chain variable
domain), of the invention has been obtained, the vector for the production of the antibody
molecule can be produced by recombinant DNA logy using techniques well known
in the art. Thus, methods for preparing a protein by expressing a cleotide
herein. s
containing an dy encoding nucleotide sequence are described
which are well known to those skilled in the art can be used to construct expression
vectors containing antibody coding sequences and appropriate transcriptional and
translational control signals. These methods include, for example, in vitro recombinant
DNA techniques, synthetic ques, and in vivo genetic recombination. The invention,
thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody
molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain
variable domain, operably linked to a promoter. Such vectors can include the nucleotide
PCT/USZOl3/076952
sequence encoding the constant region of the antibody molecule (see, e. g., international
applications WO 07 and WO 89/01036; and US patent no. 5,122,464) and the
variable domain of the antibody can be cloned into such a vector for expression of the
entire heavy or light chain.
[0210] The term "vector" or "expression vector" is used herein to mean vectors used in
accordance with the present invention as a vehicle for introducing into and expressing a
desired gene in a host cell. As known to those skilled in the art, such vectors can easily be
ed from the group consisting of plasmids, phages, Viruses and retroviruses. In
general, vectors compatible with the instant invention will comprise a selection marker,
appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter
and/or replicate in eukaryotic or prokaryotic cells. For the purposes of this invention,
us expression vector systems can be employed. For example, one class of vector
utilizes DNA elements which are derived from animal viruses such as bovine papilloma
virus, polyoma virus, adenovirus, vaccinia Virus, baculovirus, retroviruses (RSV, MMTV
or MOMLV) or SV40 virus. Others e the use of polycistronic systems with internal
ribosome binding sites. Additionally, cells which have integrated the DNA into their
chromosomes can be ed by introducing one or more s which allow selection
of ected host cells. The marker can provide for prototrophy to an auxotrophic host,
biocide resistance (e. g., antibiotics) or resistance to heavy metals such as copper. The
selectable marker gene can either be directly linked to the DNA sequences to be
expressed, or uced into the same cell by co-transformation. onal elements can
also be needed for l synthesis of mRNA. These elements can include signal
and termination
sequences, splice signals, as well as transcriptional promoters, enhancers,
signals.
[0211] In particular embodiments the cloned variable region genes are inserted into an
expression vector along with the heavy and light chain nt region genes (e.g., human
heavy and light chain constant region genes) as discussed above. In one embodiment, this
is effected using a proprietary sion vector of Biogen IDEC, Inc., referred to as
NEOSPLA, disclosed in US patent no. 6,159,730. This vector contains the
galovirus promoter/enhancer, the mouse beta globin major promoter, the SV40
origin of replication, the bovine growth hormone polyadenylation sequence, neomycin
phosphotransferase exon 1 and exon 2, the dihydrofclate ase gene and leader
level expression of antibodies
sequence. This vector has been found to result in very high
' 73 '
transfection in CHO upon incorporation of variable and constant region genes, cells,
followed by selection in G418 containing medium and methotrexate amplification. Of
course, any sion vector which is capable of eliciting expression in eukaryotic cells
can be used in the present invention. Examples of le s include, but are not
limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEFl/His, piND/GS, MVZ,
pSV40/Zeo2, pTRACER—HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from
Invitrogen, San Diego, CA), and plasmid pCI able from Promega, Madison, WI). In
general, screening large numbers of transformed cells for those which express suitably
high levels if immunoglobulin heavy and light chains is routine experimentation which
1e can be carréed out, for example, by robotic systems. Vector s are also taught in US
patent nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its
entirety herein. This system provides for high expression levels, e.g., > 30 pg/cell/day.
Other exemplary vector systems are disclosed e.g., in US patent no. 6,413,777.
In other embodiments the antibodies, or antigen-binding fragments, variants, or
derivatives thereof of the invention can be expressed using polycistronic constructs such
as those disclosed in US patent application publication no. 2003-0157641 Al and
incorporated herein in its entirety. In these expression systems, multiple gene products of
interest such as heavy and light chains of antibodies can be produced from a single
stronic construct. These systems advantageously use an internal ribosome entry site
(IRES) to provide vely high levels of antibodies. ible IRES sequences are
disclosed in US patent no. 6,193,980 which is also incorporated herein. Those skilled in
the art will appreciate that such expression systems can be used to effectively produce the
full range of antibodies disclosed in the instant application.
More generally, once the vector or DNA sequence encoding a monomeric t
of the antibody has been prepared, the expression vector can be introduced into an
appropriate host cell. uction of the plasmid into the host cell can be accomplished
by various techniques well known to those of skill in the art. These include, but are not
limited to, transfection including lipotransfection using, e. g., Fugene® or lipofectamine,
protoplast , calcium phosphate precipitation, cell fusion with enveloped DNA,
njection, and infection with intact virus. Typically, plasmid introduction into the
host is via standard calcium phosphate co—precipitation method. The host cells harboring
the sion construct are grown under conditions appropréate to the production of the
light chains and heavy chains,, and assayed for heavy and/0r light chain protein synthesis.
- 74 _
Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), or fluorescence-activated cell sorter is (FACS),
immunohistochemistry and the like.
The expression vector is transferred to a host cell by tional techniques and
the transfected cells are then cultured by conventional techniques to produce an antibody
for use in the methods described herein. Thus, the invention includes host cells containing
or a heavy or light chain f,
a cleotide encoding an antibody of the invention,
operably linked to a heterologous promoter. In particular embodiments for the expression
of double-chained antibodies, vectors encoding both the heavy and light chains can be c0-
expressed in the host cell for sion of the entire immunoglobulin molecule, as
detailed below.
[0215} The host cell can be co—transfected with two expression vectors of the invention,
the first vector
vector encoding a heavy chain derived polypeptide and the second
encoding a light chain derived polypeptide. The two vectors can contain cal
able markers which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single vector. can be used which encodes both heavy and light chain
polypeptides. In such situations, the light chain is advantageously placed before the heavy
chain to avoid an excess of toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52;
, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy
and liglet chains can comprise cDNA or genomic DNA.
As used herein, "host cells" refers to cells which harbor vectors constructed using
recombinant DNA techniques and ng at least one heterologous gene. In
descriptions of processes for ion of antibodies from recombinant hosts, the terms
"cell" and "cell culture" are used interchangeably to denote the source of antibody unless
it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells"
culture containing both the
can mean either from spun down whole cells, or from the cell
medium and the ded cells.
A variety of host-expression vector systems can be utilized to express antibody
molecules for use in the methods described herein. Such host-expression systems
represent vehicles by which the coding sequences of interest can be produced and
subsequently purified, but also represent cells which can, when transformed or transfected
with the appropriate nucleotide coding sequences, s an antibody le of the
invention in situ. These include but are not limited to microorganisms such as bacteria
W0 2014/100600
(e. g., E. coli, B. subtilis) transformed with inant bacteriophage DNA, plasmid
DNA or cosmid DNA sion vectors containing antibody coding sequences; yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors
containing antibody coding sequences; insect cell systems infected with recombinant
virus sion vectors (e. g., baculovirus) containing antibody coding sequences; plant
cell systems infected with recombinant virus expression vectors 1:e. g., cauliflower mosaic
virus, CaMV; tobacco mosaic Virus, TMV) or transformed with recombinant d
expression vectors (e. g., Ti plasmid) containing antibody coding sequences; or
mammalian cell systems (e.g, COS, CHO, NSC, BLK, 293, 3T3 cells) harboring
recombinant expression constructs containing promoters d from the genome of
mammalian cells (e. g., metallothionein promoter) or from mammalian viruses tag, the
adenovirus late promoter; the vaccinia virus 7.5K promoter). In one embodiment,
bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially
for the expression of whole recombinant dy le, are used for the expression
of a recombinant antibody molecule. For e, mammalian cells such as Chinese
Hamster Ovary (CHO) cells, in conjunction with a vector such as the major intermediate
early gene promoter element from human cytomegalovirus is an effective expression
system for antibodies; see, e. g., Foecking et al., Gene 45 (1986), 101; Cockett et al.,
Bio/Technology 8 (1990), 2.
[E218] Tile host cell line used for protein expression is often of mammalian origin; those
skilled in the art are credited with ability to determine ular host cell lines which are
best suited for the desired gene product to be sed therein. Exemplary host cell lines
include, but are not limited to, CHO (Chinese Hamster , DG44 and DUXBll
(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI
(monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK
(baby r kidney), MDCK, W13 8, R1610 (Chinese hamster fibroblast) BALBC/3T3
(mouse ast), HAK er kidney line), SP2/O (mouse myeloma), P3x63-
Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human
lymphocyte) and 293 (human kidney). In a specific embodiment, host cell lines are CHO
or 293 cells. Host cell lines are typically available from commercial services, the
American Tissue Culture Collection or from published literature.
In addition, a host cell strain can be chosen which modulates the expression of the
ed sequences, or modifies and processes the gene product in the c fashion
_ 76 _
desired. Such modifications (e.g., ylation) and processing (e. g., ge) of
protein products can be important for the function of the n. ent host cells have
characteristic and specific mechanisms for the post-translational processing and
modification of proteins and gene products. Appropriate cell lines or host s can be
chosen to ensure the correct modification and processing ofthe foreign protein expressed.
To this end, otic host cells which possess the cellular machinery for proper
processing of the primary transcript, glycosylation, and phosphorylation of the gene
product can be used.
For erm, high—yield production of recombinant proteins, stable expression is
preferred. For example, cell lines which stably express the antibody molecule can be
ered. Rather than using sion s which contain viral origins of
replication, host cells can be transformed with DNA controlled by appropriate expression
control elements (e. g., promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a able marker. ing the introduction of the
foreign DNA, engineered cells can be d to grow for 1-2 days in an enriched media,
and then are swétched to a selective media. The selectable marker in tlee recombinant
plasmid confers resistance to the selection and allows cells to stably integrate the plasmid
into their chromosomes and grow to form foci which in turn can be cloned and expanded
into cell lines. This method can advantageously be used to engineer cell lines which
stably express the antibody molecule.
{0221} A number of selection systems can be used, including but not limited to the herpes
simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine—
guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA
48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980),
817) genes can be ed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite
resistance can be used as the basis of selection for the following genes: dhfr, which
confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357;
O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance
to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072);
Clinical
resistance to the aminoglycoside G—418 iel et
neo, which confers al.,
Pharmacy 12 (1993), 488—505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev,
926-
Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993),
932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH
- 77 —
(1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al.,
Gene 30 (1984), 147. Methods commonly known in the art of recombinant DNA
technology which can be used are descrébed in l et al. (eds), Current Protocols in
Molecular Biology, John Wiley & Sons, NY ; Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and
13, oli et al. (eds), t Protocols in Human Genetics, John Wiley & Sons, NY
(1994); re-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by
reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector
amplification, for a review, see Bebbington and Hentschel, The use of vectors based on
in DNA
gene amplification for the expression of cloned genes in mammalian cells
cloning, Academic Press, New York, Vol. 3. (1987). When a marker in the vector system
expressing antibody is able, increase in the level of inhibitor present in culture of
host cell will increase the number of copies of the marker gene. Since the amplified
region is ated with the antibody gene, production of the antibody will also increase;
see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.
In vitro production allows scale—up to give large amounts of the desired
polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions
in an airlift reactor
are known in the art and include homogeneous suspension culture, e.g.
e. g. in hollow
or in a uous stirrer reactor, or immobilized or entrapped cell culture,
fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or
desired, the solutions of polypeptides can be purified by the customary chromatography
methods, for e gel filtration, ion-exchange chromatography, chromatography over
DEAE—cellulose or (immuno-)affinity tography, e. g., after preferential
biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC
chromatography step described herein.
Genes encoding antibodies, or antigen-binding fragments, variants, or tives
thereof of the invention can also be expressed in non-mammalian cells such as bacteria or
insect or yeast or plant cells. Bacteria which readily take up nucleic acids include
members of the bacteriaceae, such as strains of Escherichia coli or Salmonella;
aceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus
influenzae. It will further be appreciated that, when sed in bacteria, the
heterologous polypeptides typically become part of inclusion bodies. The heterologous
W0 2014/100600 PCT/USZOl3/076952
polypeptides must be isolated, purified and then led into functional molecules.
Where tetravalent forms of antibodies are desired, the subunits will then self-assemble
into tetravalent antibodies; see, 8.g. , international ation W002/096948.
In bacterial systems, a number of expression vectors can be ageously
selected depending upon the use intended for the antibody molecule being expressed. For
example, when a large quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, s which direct the
expression of high levels of fusion protein ts that are readily purified can be
desirable. Such vectors include, but are not limited, to the E. coli expression vector
pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody coding sequence
can be ligated individually into the vector in frame with the lacZ coding region so that a
fusion protein is produced; pIN vectors e & Inouye, Nucleic Acids Res. 13 (1985),
3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 509); and the like.
pGEX s can also be used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins are soluble and can
easily be d from lysed cells by adsorption and binding to a matrix of glutathione-
agarose beads followed by elution in the presence of free glutathione. The pGEX vectors
are designed to include thrombin or factor Xa protease cleavage sites so that the cloned
target gene product can be released from the GST moiety.
[0226] In addition to prokaryotes, otic microbes can also be used. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic
microorganisms although a number of other s are commonly available, e.g., Pichia
pastoris. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb
et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,
Gene 10 (1980), 157) is commonly used. This plasmid already ns the TRPl gene
which provides a selection marker for a mutant strain of yeast lacking the ability to grow
in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12).
The ce of the trpl lesion as a teristic of the yeast host cell genome then
provides an effective environment for detecting transformation by growth in the absence
of tryptophan.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is typically used as a vector to express foreign genes. The virus grows in Spodoptera
fiugz‘perda cells. The antibody coding sequence can be cloned individually into nor:—
_ 79 _
essential regions (for example the polyhedrin gene) of the Virus and placed under control
of an AcNPV er (for example the polyhedrin promoter).
Once an antibody le of the invention has been recombinantly expressed,
the whole antibodies, their dimers, individual. light and heavy chains, or other
immunoglobulin forms of the present invention, can be purified according to standard
procedures of the art, including for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, e. g. um sulfate
precipitation, or by any other standard technique for the purification of proteins; see, e.g.,
Scopes, "Protein Purification", Springer Verlag, NY. (1982). Alternatively, another
method for increasing the affinity of antibodies of the ion is disclosed in US patent
publication 2002-0123057 A1.
V. Fusion Proteins and Conjugates
In certain embodiments, the antibody polypeptide comprises an amino acid
associated with an dy. Exemplary
sequence or one or more moieties not normally
modifications are described in more detail below. For e, a single—chain Fv
antibody fragment of the invention can comprise a flexible linker sequence, or can be
modified to add a functional moiety (e. g., PEG, a drug, a toxin, or a label such as a
fluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and the like)
[0230] An antibody polypeptide of the invention can comprise, consist essentially of, or
consist of a fiision n. Fusion proteins are chimeric molecules which comprise, for
example, an immunoglobulin tau-binding domain with at least one target binding site, and
at least one heterologous n, i.e., a portion with which it is not naturally linked in
nature. The amino acid sequences can normally exist in separate proteins that are brought
together in the fusion polypeptide or they can normally exist in the same protein but are
placed in a new arrangement in the fusion ptide. Fusion proteins can be created, for
e, by chemical synthesis, or by ng and translating a polynucleotide in which
the peptide regions are encoded in the desired relationship.
The term ologous" as applied to a polynucleotide or a ptide, means
that the polynucleotide or polypeptide is derived from a ct entity from that of the
rest of the entity to which it is being compared. For ce, as used herein, a
"heterologous polypeptide" to be fused to an antibody, or an antigen-binding fragment,
W0 2014/100600 PCT/USZOl3/076952
variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same
species, or an immunoglobulin or munoglobulin polypeptide of a different s.
As discussed in more detail elsewhere , antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention can r be recombinantly
fused to a heterologous polypeptide at the N— or C-terminus or chemically conjugated
(including covalent and non—covalent conjugations) to ptides or other
compositions. For example, antibodies can be recombinantly fused or conjugated to
les useful as labels in detection assays and effector molecules such as logous
polypeptides, drugs, radionuclides, or toxins; see, e. g., international applications
WO92/08495; WO91/14438; WO89/12624; US patent no. 995; and European
patent application EP 0 396 387.
Antibodies, or antigen-binding fragments, variants, or derivatives thereof of the
invention can be composed of amino acids joined to each other by peptide bonds or
modified peptide bonds, 1'. e. and can contain amino acids other than the
, peptide isosteres,
20 gene-encoded amino acids. Antibodies can be d by natural processes, such as
anslational processing, or by chemical modification techniques which are well
known in the art. Sue}: modifications are well described in basic texts and in more
detailed monographs, as well as in a voluminous research literature. ations can
occur anywhere in the antibody, including the peptide backbone, the amino acid side-
chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be
appreciated that the same type of ation can be present in the same or varying
degrees at several sites in a given antibody. Also, a given antibody can contain many
types of modifications. Antibodies can be branched, for example, as a result of
ubiquitination, and they can be cyclic, with or t branching. Cyclic, branched, and
branched cyclic dies can result from posttranslation natural processes or can be
made by synthetic methods. Modifications include acetylation, acylation, ADP-
ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme
moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment
of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking,
ation, disulfide bond formation, demethylation, formation of covalent cross-links,
formation of cysteine, ion of pyroglutamate, forrnylation, gamma-carboxylation,
glycosylation, GPI anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, pegylation, lytic processing, phosphorylation,
_ 81 _
prenylation, racemization, selenoylation, ion, transfer-RNA mediated on of
amino acids to ns such as arginylation, and ubiquitination; see, e. g., Proteins -
Structure And Molecular ties, T. E. Creighton, W. H. Freeman and Company, New
York 2nd Ed., (1993); Positranslational Covalent Modification 0f Proteins, B. C.
Johnson, Ed., Academic Press, New York, Meth.
pgs. 1-12 (1983); Seifter et al.,
Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).
The present ion also provides for fusion proteins comprising an antibody, or
antigen-binding fragment, variant, or derivative f, and a heterologous polypeptide.
In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or
consists of, a polypeptide having the amino acid sequence of any one or more of the VH
regions of an dy of the invention or the amino acid sequence of any one or more of
the VL regions of an antibody of the invention or fragments or variants thereof, and a
heterologous polypeptide sequence. In another embodiment, a fusion protein for use in
the diagnostic and ent methods disclosed herein comprises, consists ially of,
of any one, two, three of the
or consists of a polypeptide having the amino acid sequence
VH-CDRs of an antibody, or nts, variants, or derivatives thereof, or the amino acid
of an dy, or fragments, variants, or
sequence of any one, two, three of the VL-CDRS
derivatives thereof, and a heterologous polypeptide sequence. In one embodiment, the
fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of
or t thereof, and a
an antibody of the present ion, or fragment, derivative,
logous polypeptide binds to tau. In
sequence, which fusion protein specifically
another embodiment, a fusion protein comprises a polypeptide having the amino acid
of the invention and the amino acid
sequence of at least one VH region of an antibody
of at least one VL region of an antibody of the invention or
sequence fragments,
derivatives or variants thereof, and a heterologous polypeptide sequence. In one
embodiment, the VH and VL regions of the fusion protein correspond to a single source
antibody (or scFv or Fab fragment) which specifically binds tau. In yet another
ment, a fusion n for use in the diagnostic and treatment methods disclosed
herein comprises a polypeptide having the amino acid sequence of any one, two, three or
of any one, two, three
more of the VH CDRs of an antibody and the amino acid sequence
of the VL CDRs of an antibody, or fragments or variants thereof, and a
or more
heterologous polypeptide sequence. In one embodiment, two, three, four, five, six, or
scFv or
more of the VH-CDR(s) or VL—CDR(S) correspond to single source antibody (or
WO 00600 - 82 —
Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are
also encompassed by the ion.
Exemplary fusion proteins reported in the literature include fusions of the T cell
receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4
(Capon et al., Nature 337 (1989), 525-531; cker et al., Nature 339 (1989), 68-70;
Zettmeissl et 611., DNA Cell Biol. USA 9 (1990), 347-353; and Bym er al., Nature 344
(1990), 667-670); L-selectin g receptor) (Watson et al., J. Cell. Biol. 110 (1990),
2221-2229; and Watson et al., Nature 349 (1991), 164—167); CD44 (Aruffo et al., Cell 61
(1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (l99l),721-730);
CTLA-4 (Lisley et al., J. Exp. Med. 174 (1991), 561—569); CD22 (Stamenkovic et al.,
Cell 66 (1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA
88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886; and
Peppel et al., J. Exp. Med. 174 (1991), 489 (1991); and IgE or a (Ridgway
and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).
[0236] As discussed elsewhere herein, antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention can be fused to heterologous polypeptides to
increase the in Vivo half-life of the polypeptides or for use in immunoassays using
methods known in the art. For example, in one embodiment, PEG can be conjugated to
the antibodies of the invention to increase their half-life in vivo; see, e. g., Leong et al.,
Cytokine 16 , 9; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et (1].,
m. Soc. Transactions 30 (2002), 512.
er, antibodies, or antigen—binding fragments, variants, or derivatives
thereof of the invention can be fused to marker sequences, such as a peptide to facilitate
their purification or detection. In particular embodiments, the marker amino acid
sequence is a hexa-histidine peptide (HIS), such as the tag provided in a pQE vector
N, Inc., 9259 Eton Avenue, orth, Calif, 91311), among others, many of
which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci.
USA 86 (1989), 821-824, for instance, hexa—histidine provides for ient ation
of the fusion protein. Other peptide tags useful for purification include, but are not limited
to, the "HA" tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al. , Cell 37 (1984), 767) and the "flag" tag.
Fusion proteins can be prepared using methods that are well known in the art; see
for example US patent nos. 5,116,964 and 5,225,538. The precise site at which the fusion
W0 2014/100600 PCT/U82013/076952
is made can be selected empirically to optimize the secretion or binding characteristics of
the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for
expression.
Antibodies of the present invention can be used in non—conjugated form or can be
conjugated to at least one of a variety of molecules, e. g., to improve the therapeutic
properties of the le, to facilitate target detection, or for imaging or therapy of the
patient. Antibodies, or n-binding fragments, variants, or derivatives thereof of the
ion can be labeled or conjugated either before or after purification, when
purification is med. In particular, antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the ion can be conjugated to therapeutic agents, prodrugs,
peptides, proteins, enzymes, Viruses, lipids, biological response modifiers, pharmaceutical
agents, or PEG.
ates that are immunotoxins including conventional antibodies have been
widely described in the art. The toxins can be coupled to the antibodies by conventional
coupling techniques or immunotoxins containing protein toxin ns can be produced
as fusion proteins. The antibodies of the present invention can be used in a corresponding
way to obtain such immunotoxins. Illustrative of such immunotoxins are those described
by Byers, Seminars Cell. Biol. 2 , 59-70 and by Fanger, Immunol. Today 12
(1991), 51-54.
[0241] Those skilled in the art will iate that ates can also be led
using a variety of techniques depending on the selected agent to be conjugated. For
example, conjugates with biotin are prepared e. g. by ng a tau binding polypeptide
with an ted ester of biotin such as the biotin N-hydroxysuccinimide ester. rly,
conjugates with a fluorescent marker can be prepared in the presence of a coupling agent,
e. g. those listed herein, or by reaction with an isothiocyanate, or fluorescein—
isothiocyanate. Conjugates of the antibodies, or antigen-binding fragments, variants or
tives thereof of the invention are prepared in an analogous manner.
The present invention further encompasses antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or
therapeutic agent. The antibodies can be used diagnostically to, for example, demonstrate
presence of a neurological disease, to indicate the risk of getting a neurological disease, to
r the development or progression of a neurological e, i. e. tauopathic disease
as part of a clinical testing procedure to, e,g., determine the efficacy of a given treatment
_ 84 _
and/or prevention regimen. Detection can be facilitated by coupling the antibody, or
antigen—binding fragment, t, or derivative thereof to a detectable substance.
Examples of detectable substances e various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent materials, radioactive
materials, positron emitting metals using various positron emission tomographies, and
nonradioactive paramagnetic metal ions; see, e. g., US patent no. 4,741,900 for metal ions
which can be ated to antibodies for use as stics according to the present
invention. Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, B-galactosidase, or acetylcholinesterase; examples of suitable prosthetic
complexes include streptavidin/biotin and
group avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin; and examples of le ctive
material include 125I, 131i, 111In or 99Tc.
An antibody, or antigen-binding fragment, variant, or derivative f also can
be ably labeled by coupling it to a chemiluminescent compound. The presence of
the chemiluminescent-tagged antibody is then determined by detecting the presence of
luminescence that arises during the course of a chemical reaction. Examples of
particularly useful chemiluminescent labeling compounds are luminol, inol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
One of the ways in which an antibody, or antigen—binding fragment, variant, or
tive thereof can be detectably labeled is by linking the same to an enzyme and
using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme
Linked Immunosorbent Assay )" Microbiological Associates Quarterly
ation, sville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin.
. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E.
(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); lshikawa, E. et al.,
(eds.), Enzyme assay, Kgaku Shoin, Tokyo (1981). The enzyme, which is 1Eound
to the antibody will react with an appropriate substrate, preferably a chromogenic
substrate, in such a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, etric or by visual means. Enzymes which can be
used to detectably label the antibody e, but are not limited to, malate
_ 85 _
dehydrogenase, staphylococcal nuclease, delta-S-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-
galactosidase, ribonuclease, , catalase, glucosephosphate dehydrogenase,
glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished
by colorimetric s which employ a genic substrate for the enzyme.
Detection can also be accomplished by visual comparison of the extent of enzymatic
reaction of a substrate in comparison with similarly prepared standards.
Detection can also be accomplished using any of a variety of other immunoassays.
For example, by radioactively labeling the antibody, or antigen—binding fragment, variant,
or derivative f, it is possible to detect the antibody through the use of a
radioimmunoassay (RIA) (see, for example, Weintraub, 13., ples of
Radioimmunoassays, Seventh Training Course on Radioligand Assay ques, The
Endocrine Society, (March, 1986)), which is incorporated by reference herein). The
radioactive isotope can be detected by means including, but not limited to, a gamma
counter, a scintillation r, or autoradiography.
An antibody, or antigen-binding nt, variant, or derivative thereof can also
be detectably labeled using fluorescence emitting metals such as 152Eu, or others of
lanthanide series. These metals can be attached to the antibody using such metal
chelating groups as diethylenetriaminepentacetic acid (DTPA) or
nediaminetetraacetic acid (EDTA).
Techniques for ating various es to an antibody, or antigen-binding
nt, variant, or derivative thereof are well known, see, e. g., Arnon et al.,
"Monoclonal Antibodies For targeting Of Drugs In Cancer Therapy“, in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds), pp. 243-56 (Alan R.
Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug ry", in Controlled Drug
Delivery (2nd Ed.), Robinson et al. (eds), Marcel Dekker, Inc., pp. 623-53 (1987);
Thorpe, "Antibody Carriers Of xic Agents In Cancer Therapy: A Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds),
And Future Prospective Of The eutic Use
pp. 475-506 (1985); "Analysis, Results,
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer
Detection And Therapy, Baldwin et al. (eds), Academic Press pp. 303-16 (1985), and
PCT/USZOl3/076952
Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin
Conjugates", Immunol. Rev. 62 (1982), 119—158.
As ned, in certain embodiments, a moiety that enhances the stability or
efficacy of a binding molecule, e.g., a binding polypeptide, e.g, an antibody or
immunospecific nt thereof can be conjugated. For example, in one embodiment,
PEG can be conjugated to the binding molecules of the ion to increase their half-
life in viva. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 (2002),
531; or Weir et 0]., Biochem. Soc. Transactions 30 (2002), 512.
VI. Compositions and Methods of Use
[0249] The present invention s to compositions comprising the aforementioned tau
binding molecule, e.g, antibody or antigen-binding fragment thereof of the present
invention or derivative or variant f, or the polynucleotide, vector or cell of the
invention. The composition of the present invention can further comprise a
pharmaceutically able carrier. Furthermore, the ceutical composition of the
present invention can se further agents such as interleukins or interferons
depending on the intended use of the pharmaceutical composition. For use in the
treatment of a tauopathic disease, e.g. , of the Alzheimer’s disease the additional agent can
be selected from the group consisting of small organic molecules, anti-tau antibodies, and
combinations thereof. Hence, in a particular embodiment the present invention relates to
the use of the tau binding molecule, e. g., antibody or antigen-binding fragment f of
the present invention or of a binding molecule having substantially the same binding
specificities of any one thereof, the polynucleotide, the vector or the cell of the present
invention for the preparation of a pharmaceutical or stic composition for
prophylactic and therapeutic treatment of a tauopathic e, monitoring the progression
of a tauopathic disease or a se to a tauopathic disease treatment in a subject or for
determining a subj ect's risk for developing a tauopathic disease.
[0250} Hence, in one ment the present invention relates to a method of treating a
neurological disorder characterized by abnormal accumulation and/or tion of tau in
the brain and the central nervous , respectively, which method ses
administering to a subject in need thereof a therapeutically effective amount of any one of
the afore-described tau binding molecules, antibodies, polynucleotides, vectors or cells of
the instant invention. The term "neurological disorder" includes but is not limited to
thic diseases such as Alzheimer’s disease, amyotrophic lateral
sclerosis/parkinsonism—dementia complex, argyrophilic grain ia, British type
amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-
Jakob disease, ia pugilistica, diffuse brillary tangles with calcification,
Down’s syndrome, frontotemporal dementia, frontotemporal ia with parkinsonisrn
linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-
Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system
atrophy, ic dystrophy, Niemann-Pick disease type C, non-Guamanian motor
neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic
parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical s,
progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle only
dementia, multi-infarct dementia and ischemic stroke. Unless stated otherwise, the terms
neurodegenerative, neurological or neuropsychiatric are used interchangeably herein.
A particular advantage of the therapeutic approach of the present invention lies in
the fact that the antibodies of the present ion are derived from B cells or B memory
cells from healthy human subjects with no signs of a tauopathic e and thus are, with
a certain probability, capable of preventing a clinically st tauopathic disease, or
diminishing the risk of the occurrence of the clinically manifest disease, or of delaying
the onset or progression of the clinically manifest disease. Typically, the antibodies of the
present invention also have y successfully gone through somatic maturation, i.e. the
optimization with respect to ivity and iveness in the high affinity g to
the target tau molecule by means of somatic variation of the variable regions of the
antibody.
The knowledge that such cells in vivo, 2. g. in a human, have not been activated by
means of related or other physiological proteins or cell structures in the sense of an
autoimmunological or allergic reaction is also of great l importance since this
es a corisiderably increased chance of successfully living through the clinical test
phases. So to speak, efficiency, acceptability and tolerability have already been
demonstrated before the preclinical and clinical development of the prophylactic or
therapeutic antibody in at least one human subject. It can thus be expected that the human
antistau antibodies of the present invention, both its target structure-specific efficiency as
therapeutic agent and its decreased probability of side effects significantly increase its
clinical probability of success.
W0 2014/100600 - 88 — PCT/USZOl3/076952
The present invention also provides a pharmaceutical and diagnostic, respectively,
pack or kit comprising one or more containers filled with one or more of the above
described ients, tag, anti~tau dy, binding fragment, derivative or varé‘ant
thereof, cleotide, vector or cell of the present inven‘ticu. Asscciated with such
centainertfis} can be a nutice in the term prescrébed by a gctrerttinentai agency regifiating
the manufacture, use or sale of pharmaceuticais er binicgicai preducts, which natice
reflects apprcvai by the agency ci‘manutacture, use at sate for human administration, in
additicn or alternatively the kit comprises reagents and/or instructions fer use in
appropriate stic assays. The composition, e. g. kit of the present invention is of
course particularly suitable for the risk assessment, diagnosis, prevention and treatment of
a disorder which is accompanied with the presence of tau, and in ular applicable for
the treatment of Alzheimer’s disease (AD), amyotrophic l sclerosis/parkinsonism—
ia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type d
angiopatliy, al amyloid angiopathy, corticobasal degeneration (CBD), Creutzfeldt-
Jakob disease (CJD), dementia pugilistica, e neurofibrillary tangles with
calcification, Down’s syndrome, frontotereporal ia, frontotemporal dementia with
sonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration,
Gerstmann-Straussler-Scheinker disease, vorden-Spatz disease, inclusion body
myositis, multiple system y, myotonic dystrophy, Niemann—Pick disease type C
(NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease
(PiD), postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy,
progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing
panencephalitis, tangle only dementia, multi-infarct dementia and ischemic stroke.
The pharmaceutical compositions of the present invention can be formulated
according to methods well known in the art; see for example Remington: The e and
Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0
306472. Examples of suitable ceutical carriers are well known in the art and
include phosphate buffered saline solutions, water, emulsions, such as oil/water
emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising
such carriers can be formulated by well known conventional methods. These
pharmaceutical compositions can be administered to the subject at a suitable dose.
Administration of the suitable compositions can be effected by different ways, e.g., by
intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or
_ 89 _
intradermal administration or spinal or brain ry. Aerosol formulations such as nasal
solutions of the active agent with
spray formulations include puréfled aqueous or other
preservative agents and isotonic agents. Such formulations are adjusted to a pH and
isotonic state ible with the nasal mucous membranes. Formulations for rectal or
vaginal ad-ministration can be presented as a itory with a suitable carrier.
Furthermore, s the present invention includes the now standard (though
fortunately infrequent) procedure of drilling a small hole in the skull to administer a drug
of the t invention, in one aspect, the binding molecule, especially antibody or
antibody based drug of the present invention can cross the blood-brain barrier, which
allows for intravenous or oral administration.
The dosage regimen will be determined by the attending ian and clinical
factors. As is well known in the medical arts, dosages for any one patient depends upon
surface area, age, the particular compound
many factors, including the patient's size, body
to be administered, sex, time and route of administration, l , and other drugs
being stered concurrently. A typical dose can be, for example, in the range of
0.001 to 1000 pg (or of nucleic acid for expression or for inhibition of expression in this
range); however, doses below or above this exemplary range are envisioned, especially
considering the aforementioned factors. Generally, the dosage can range, e.g, from about
0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e. g., 0.02 mg/kg, 0.25 mg/kg,
0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example
dosages can be 1 mg/kg body weight or 10 mg/kg bedy weight or Within the range of l-
mg/kg, or at least 1 mg/kg. Doses intermediate in the above ranges are also intended to
be within the scope of the invention. Subjects can be administered such doses daily, on
alternative days, weekly or according to any other schedule ined by empirical
analysis. An exemplary treatment s administration in multiple dosages over a
prolonged period, for example, of at least six months. Additional exemplary treatment
regimes entail administration once per every two weeks or once a month or once every 3
to 6 . Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on
consecutive days, 30 mg/kg on ate days or 60 mg/kg weekly. In some methods, two
or more monoclonal antibodies with different binding specificities are administered
simultaneously, in which case the dosage of each antibody administered falls Within the
ranges indicated. Progress can be monitored by periodic assessment. Preparations for
parenteral administration include e s or non—aqueous solutions, suspensions,
W0 2014/100600
and emulsions. Examples of non-aqueous solvents are propylene , polyethylene
glycol, vegetable oils such as olive oil, and able organic esters such as ethyl .
Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions,
including saline and buffered media. Parenteral vehicles include sodium chloride solutiore,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such
as those based on Ringer's dextrose), and the like. Preservatives and other additives can
also be present such as, for example, antimicrobials, anti-oxidants, chelating , and
inert gases and the like. Furthermore, the pharmaceutical composition of the invention
can comprise further agents such as dopamine or psychopharmacologic drugs, depending
on the intended use of the pharmaceutical ition.
Furthermore, in a particular embodiment of the present invention the
pharmaceutical composition can be formulated as a vaccine, for example, if the
ceutical composition of the invention ses an au antibody or binding
nt, derivative or variant thereof for passive immunization. As mentioned in the
background section, phosphor-tau species have been reported ellularly in plasma
and CSF (Aluise et al., Biochim. Biophys. Acta. 1782 (2008), 549—558) andstudies in
transgenic mouse lines using active vaccination with phosphorylated tau peptides
revealed reduced brain levels of tau aggregates in the brain and slowed progression of
behavior impairments (Sigurdsson, J. mers Dis. 15 (2008), 157-168; Boimel et 0].,
Exp Neurol. 224 (2010), 472-485). Accordingly, it is t to expect that passive
immunization with human anti-tau antibodies and equivalent tau binding molecules of the
present ion would help to circumvent several adverse effects of active
immunization therapy concepts as already discussed in the background section.
Therefore, tlee present anti-tau antibodies and their equivalents of the present invention
will be particularly useful as a vaccine for the tion or amelioration of tauopathic
diseases such as AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17, NP-C, PiD, PSP or
other thies as ned before.
{0258] In one embodiment, it can be beneficial to use recombinant bispecific or
multispecific constructs of the antibody of the present invention. For a nce see
Fischer and Léger, Pathobiology 74 (2007), 3-14. Such bispecific molecule might be
designed to target tau with one binding arm and another pathologic entity such as AB or
alphamsynuclein or a different pathological conformation of tau with a second binding
- 91 _
arm. Alternatively the second binding arm can be designed to target a n present
the blood—brain-barrier to facilitate antibody penetration into the brain.
In one embodiment, it can be beneficial to use recombinant Fab (rFab) and single
chain fragments (scFvs) of the antibody of the present invention, which might more
readily penetrate a cell membrane. For example, Robert et 611., Protein Eng. Des. Sci.
(2008) Oct 16; 81741-0134, published online ahead, describe the use of chimeric
recombinant Fab (rFab) and single chain nts (scFvs) of monoclonal dy WO-
2 which recognizes an epitope in the N-terminal region of AB. The engineered fragments
fibréls
were able to (i) t amyloid fibrillization, (ii) disaggregate preformed ABl—42
and (iii) inhibit ABl-42 oligomer-mediated neurotoxicity in vitro as ntly as the
whole IgG le. The perceived advantages of using small Fab and scFV engineered
antibody formats which lack the effector function include more efficient passage across
the blood-brain barrier and minimizing the risk of triggering atory side reactions.
rmore, besides scFV and -domain dies retain the binding specificity of
full-length antibodies, they can be sed as single genes and intracellularly in
mammalian cells as intrabodies, with the potential for alteration of the folding,
interactions, modifications, or subcellular localization of their targets; see for review, e.g,
Miller and Messer, Molecular Therapy 12 (2005), 394—401.
In a different approach Muller et 411., Expert Opin. Biol. Ther. (2005), 237-241,
describe a technology platform, so-called 'SuperAntibody Technology', which is said to
enable antibodies to be shuttled into living cells without g them. Such cell-
penetrating antibodies windows. The
open new diagnostic and therapeutic term
'TransMabs' has been coined for these antibodies.
In a r embodiment, co-administration or sequential administration of other
dies useful for treating a tauopathic disease can be desirable. In one embodiment,
the additional antibody is comprised in the pharmaceutical composition of the present
invention. Examples of antibodies which can be used to treat a subject include, but are not
limited to, antibodies targeting beta—amyloid, alpha—synuclein, TDP—43 and SOD-l.
In a further embodiment, co-administration or sequential administration of other
neuroprotective agents useful for treating a thic disease can be desirable. In one
embodiment, the additional agent is comprised in the pharmaceutical composition of the
present invention. es of neuroprotective agents which can be used to treat a
subject include, but are not limited to, an acetylcholinesterase inhibitor, a glutamatergic
- 92 —
receptor antagonist, kinase inhibitors, HDAC inhibitors, anti—inflammatory ,
divalproex sodium, or any combination thereof. Examples of other neuroprotective agents
that can be used concomitant with pharmaceutical composition of the present invention
are described in the art; see, e. g. international application W02007/011907. In one
embodiment, the additional agent is ne or a dopamine receptor agonist.
A therapeutically effective dose or amount refers to that amount of the active
ingredient sufficient to rate the symptoms or condition. Therapeutic efficacy and
toxicity of such compounds can be ined by standard pharmaceutical procedures in
cell cultures or experimental s, e. g., ED50 (the dose therapeutically effective in
50% of the tion) and LDso (the dose lethal to 50% of the tion). The dose
ratio between therapeutic and toxic effects is the therapeutic index, and it can be
sed as the ratio, LDso/EDSO. In one embodiment, the therapeutic agent in the
composition is present in an amount sufficient to e or preserve normal behavior
and/or cognitive properties in case of AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-l7,
NP-C, PiT), PSP or other tauopathic diseases as mentioned before.
From the foregoing, it is evident that the present invention encompasses any use
of a tau binding molecule comprising at least one CDR of the above described antibody,
in particular for diagnosing and/or treatment of a tauopathic disease as mentioned above,
particularly Alzheimer’s disease. In one embodiment, said binding molecule is an
antibody of the present invention or an immunoglobulin chain thereof. In addition, the
present invention relates to anti-idiotypic antibodies of any one of the mentioned
antibodies described hereinbefore. These are antibodies or other binding molecules which
bind to the unique antigenic peptide sequence located on an antibody's variable region
near the antigen-binding site and are useful, e. g., for the detection of anti-tau antibodies in
sample of a subject.
In another ment the present invention relates to a diagnostic ition
comprising any one of the above described tau g molecules, antibodies, antigen-
binding nts, cleotides, vectors or cells of the invention and optionally
suitable means for detection such as reagents conventionally used in immuno or nucleic
3O acid based diagnostic s. The antibodies of the invention are, for example, suited
for use in immunoassays in which they can be utilized in liquid phase or bound to a solid
phase carrier. Examples of immunoassays which can utilize the antibody of the invention
are itive and non-competitive immunoassays in either a direct or indirect format.
W0 00600 '
PCT/USZOl3/076952
Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich
(immunometric assay), flow cytometry and the Western blot assay. The antigens and
antibodies of the ion can be bound to many different carriers and used to isolate
cells cally bound thereto. Examples of well known carriers include glass,
polystyrene, polyvinyl chloride, opylene, polyethylene, polycarbonate, dextran,
nylon, amyloses, l and modified celluloses, polyacrylamides, es, and
magnetite. The nature of the carrier can be either e or insoluble for the es of
the invention. There are many different labels and methods of labeling known to those of
ordinary skill in the art. Examples of the types of labels which can be used in the present
invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds,
chemiluminescent compounds, and inescent compounds; see also the embodiments
discussed hereinabove.
By a further embodiment, the tau binding molecules, in particular antibodies of
the present invention can also be used in a method for the diagnosis of a disorder in an
individual by obtaining a body fluid sample from the tested individual which can be a
blood , a lymph sample or any other body fluid sample and contacting the body
fluid sample with an antibody of the instant invention under conditions enabling the
formation of antibody-antigen xes. The level of such complexes is then
determined by methods known in the art, a level significantly higher than that formed in a
control sample indicating the disease in the tested individual. In the same manner, the
specific antigen bound by the antibodies of the invention can also be used. Thus, the
present invention relates to an in vitro immunoassay comprising the binding molecule,
e. g., antibody or antigen-binding fragment thereof of the invention.
In this context, the present invention also s to means specifically designed
for this purpose. For example, an antibody-based array can be used, which is for example
loaded with antibodies or equivalent antigen-binding les of the present invention
which specifically recognize tau. Design of microarray imniunoassays is summarized in
Kusnezow et al., Mol. Cell mics 5 , 1681-1696. Accordingly, the present
invention also relates to microarrays loaded with tau binding molecules identified in
accordance with the present invention.
In one embodiment, the present invention relates to a method of diagnosing a
tauopathic disease in a subject, the method comprising determining the presence of tau
and/or pathologically modified and/or aggregated tauin a sample from the subject to be
_ 94 _
diagnosed with at least one antibody of the present invention, an tau binding fragment
thereof or an nding molecule having substantially the same binding specificities of
modified and/or aggregated tau is
any one thereof, wherein the presence of pathologically
indicative of a egenerative tauopathy and an increase of the level of the
pathologically modified and/or aggregated tau in comparison to the level of the
physiological tau forms is indicative for progression of a neurodegenerative tauopathy in
said subject.
The subject to be diagnosed can be asymptomatic or nical for the disease. In
one embodiment, the control subject has a tauopathic disease, for example, AD, ALS—
PDC, AGD, CBD, CJD, FTD, FTDP—17, NP-C, PiD, PSP or other tauopathies as
mentioned before, wherein a rity between the level of pathologically modified
and/or aggregated tau and the reference standard indicates that the subject to be diagnosed
has a tauopathic disease. Alternatively, or in on as a second control the l
subject does not have a tauopathic disease, wherein a difference between the level tau
and/or of pathologically modified and/or aggregated tau and the reference standard
indicates that the subject to be diagnosed has a tauopathic disease. In one embodiment,
the subject to be diagnosed and the control subject(s) are age-matched. The sample to be
analyzed can be any body fluid suspected to contain pathologically modified and/or
aggregated tau, for example a blood, CSF, or urine sample.
[0270] The level tau and/or of ogically modified and/or ated tau can be
assessed by any suitable method known in the art comprising, e. g., analyzing tau by one
or more ques chosen from Western blot, imrnunoprecipitation, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell
sorting , two—dimensional gel electrophoresis, mass spectroscopy (MS), -
assisted laser desorption/ionization-time of flight-MS (MALDI-TOF), surface—enhanced
laser desorption ionization—time of flight (SELDI-TOF), high mance liquid
chromatography (HPLC), fast n liquid chromatography (FPLC), multidimensional
liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), and laser
densitometry. In one embodiment, said in vivo imaging of tau comprises positron
emission tomography (PET), single photon emission tomography (SPECT), near infrared
(NIR) optical imaging or magnetic resonance imaging (MRI).
Methods of sing a tauopathic disease such as mer's disease,
monitoréng a thic disease progression, and monitoring a tauopathic disease
_ 95 -
treatment using antibodies and related means which can be adapted in accordance with
the present ion are also described in ational applications WO93/08302,
WO94/13795, WO95/17429, W096/04309, WO2002/0-62851 and WO2004/Ol6655.
Similarly, antibody based detection methods for tau are described in international
application W02005/080986, the disclosure content of all being incorporated herein by
nce. Those s can be applied as described but with a tau c antibody,
binding fragment, derivative or variant ofthe present invention.
[0272} In a r aspect the present invention also relates to es having an epitope
of tau specifically recognized by any antibody of the present invention. In one
embodiment, such peptide comprises, consists of or consists essentially of an amino acid
sequence selected from the group consisting of: residues 125-131, 397—441, 226-244,
217—227, 37-55, 387-406, 421—427, 427-439, 1-158, 197-207, 57-67, 355-441, 313-319,
309-319, 221-231 of SEQ ID NO:6, and any combination thereof, and a modified
amino acids are
sequence thereof in which one, two, three, four, five, six, seven or more
substituted, deleted and/or added, wherein the peptide is recognized by any antibody of
the present invention.
{@273} In one embodiment of this invention such a peptide can be used for diagnosing a
neurodegenerative tauopathy in a subject, sing a step of determining the ce
of an antibody that binds to a peptide in a biological sample of said subject, and being
used for sis of a tauopathy in said subject by measuring the levels of antibodies
which ize the above descrtébed peptide of the present invention and comparing the
measurements to the levels which are found in healthy subjects of comparable age and
gender. An elevated level of measured antibodies specific for said peptide of the t
invention would be indicative for diagnosing a thy in said t. The peptide of
the present invention can be formulated in an array, a kit and composition, respectively,
as described hereinbefore.
[E274] These and other embodiments are disclosed and encompassed by the description
and es of the present invention. Further literature concerning any one of the
materials, methods, uses and compounds to be employed in accordance with the t
invention can be retrieved from public libraries and databases, using for example
onic devices. For example the public database "Medline" can be utilized, which is
hosted by the National Center for Biotechnology Information and/or the National Library
of Medicine at the National Institutes of Health. Further databases and web addresses,
- 96 _
such as those of the European Bioinformatics Institute (EBI), which is part of the
an Molecular Biology Laboratory (EMBL) are known to the person skilled in the
art and can also be obtained using internet search s. An overview of patent
information in biotechnology and a survey of relevant sources of patent information
useful for retrospective ing and for current awareness is given in Berks, TIBTECH
12 (1994), 352-364.
The above disclosure generally describes the present invention. Unless otherwise
stated, a term as used herein is given the definition as provided in the Oxford Dictionary
of Biochemistry and lar Biology, Oxford University Press, 1997, revised 2000 and
ted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of
this specification. Full bibliographic citations can be found at the end of the specification
immediately preceding the claims. The contents of all cited references (including
literature references, issued patents, published patent ations as cited throughout this
application and manufacturer's specifications, ctions, etc.) are hereby expressly
incorporated by reference; however, there is no admission that any nt cited is
indeed prior art as to the present invention.
A more complete understanding can be obtained by reference to the following
specific es which are provided herein for purposes of illustration only and are not
intended to limit the scope of the invention.
EXAMPLES
The examples which follow further illustrate the invention, but should not be
construed to limit the scope of the invention in any way. The experiments in the following
Examples are illustrated and bed with respect to antibodies NI-105.4E4, NI-
105.24B2 and Nl-105.4A3 as cloned, i.e. the framework 1 (FRI) Ig-Variable regions
without being adjusted to the germ line (GL) sequences of human le heavy and
light chains; see Figure 1.
Material and methods
Detailed descriptions of conventional methods, such as those employed herein can
be found in the cited literature; see also "The Merck Manual of Diagnosis and y"
Seventeenth Ed. edited by Beers and Berkow (Merck & Co., Inc. 2003) and US. Patent
_ 97 _
Application Publication No. 2012/0087861, the content of which is orated herein by
reference in its entirety.
The practice of the present ion will employ, unless ise indicated,
conventional ques of cell biology, cell culture, molecular biology, transgenic
biology, microbiology, recombinant DNA, and immunology, which are within the skill of
the art. For further elaboration of general ques useful in the practice of this
invention, the practitioner can refer to standard textbooks and reviews in cell biology and
tissue culture; see also the references cited in the es. General methods in molecular
and cellular biochemistry can be found in such standard textbooks as Molecular Cloning:
A Laboratory Manual, 3rd Ed. ook et al., Harbor Laboratory Press 2001); Short
Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999);
DNA Cloning, Volumes I and II (Glover ed., 1985); Oligoreucleotide Synthesis (Gait ed.,
1984); Nucleic Acid Hybridization (Harnes and Higgins eds. 1984); ription And
Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and
Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos,
eds.); Current Protocols in lar Biology and Short Protocols in Molecular Biology,
3rd Edition (Ausubel et al., eds.); and Recombinant DNA Methodology (Wu, ed.,
Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu
et al., eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical
Guide To lar Cloning (1984); the treatise, Methods In Enzymology (Academic
Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and
Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology,
Volumes I-IV (Weir and ell, eds., 1986). Protein Methods (Bollag et al., John
Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. eds., Academic
Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology
Methods Manual (Leflcovits ed., Academic Press 1997); and Cell and Tissue Culture:
Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).
ts, cloning vectors and kits for genetic manipulation ed to in this disclosure
are available from commercial vendors such as BioRad, Stratagene, rogen, Sigma-
Aldrich, and ClonTech. General techniques in cell culture and media collection are
outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8
(1997), 148); free Media (Kitano, hnology 17 (1991), 73); Large Scale
_ 98 -
Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture
of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251); ting
ation from cDNA , Herzel et al. , CHAOS 11 , 98-107.
“dagger...._r..__.r_._».__._.“nmmmm-----------------------------------------
[0280] Unless indicated otherwise below, identification of tau-specific B cells and
molecular g of anti-tau antibodies displaying specificity of interest as well as their
recombinant expression and functional characterization has been or can be generally
med as described in the Examples and Supplementary Methods section of
international application published as W02008/081008, the
disclosure content of which is incorporated herein by reference in its entirety. See also
U.S. Patent Application Publication No. 2012/0087861, the content of which is incorporated
herein by reference in its entirety. A new method for identification of ecific B cells
and molecular cloning of tau antibodies displaying specificity of interest as well as their
recombinant expression and functional characterization is ed within this
application. As described above in one embodiment of the present invention cultures of
single or oligoclonal B-cells are cultured and the supernatant of the culture, which
contains antibodies produced by said s is ed for presence and affinity of new
anti-tau antibodies therein. The screening process comprises the steps of a sensitive tissue
amyloid plaque immunoreactivity (TAPIR) assay such as described in international
application , the sure content of which is incorporated herein by
reference, and shown in Figure 3; screen on brain extracts for binding to PHFTau as
descrébed in Example 2; screening for binding of a peptide derived from tau of the amino
acid sequence represented by SEQ ID NO:6 with phosphate groups on amino acids Ser-
202 and Ser—205; on amino acid Thr-231; and/or on amino acids Ser-396 and Ser-404 of
said ce as analogously described in Example 3 with non-phosphorylated peptides
due to the epitope confirmation ments for antibody NI-105.4E4; a screen for
binding of full-length tau of the amino acid sequence represented by SEQ ID N016 and
isolating the antibody for which binding is detected or the cell producing said antibody as
described in international patent WO2008/081008 and as bed in Example 1.
W0 2014/100600 PCT/USZOl3/076952
Euéikraruigianflssa
Recombireant human Tau40 was purchased from rPeptide (Bogart, GA, USA).
PHFTau was extracted from AD brain.
Isolation of paired helical ts containing pathologically phosphorylated tau
filaments (PHFTau) was performed following the method by Goedert et a1. (Goedert et
al., Neuron 8 (1992), 159-168) with modifications. One gram of AD brain tissue was cut
into 5mm pieces with all visible blood vessels removed. The tissue was washed with 40
ml ice cold washing on (lOOmM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1
mM NaF) for three times followed by homogenization with 20 ml lysis buffer (lOmM
Tris pH 7.4, 0.8M NaCl, 1mM EGTA, 1 x protease inhibitor cocktail, 1 mM Na3VO4,
lmM NaF, lmM AEBSF, 10% sucrose). The homogenate was centrifuged at 4°C at
’000xg for 20 min. Supernatant was collected with addition of N-lauroyl sarcosinate
(Sigma, Switzerland) to 1% (w/V). After two hours incubation at 37°C with shaking, the
supernatant was then centrifuged at 4°C at lOO’OOOxg for one hour. The pellet was
collected and re-suspended in PBS. After ng out possible inating
globulins with protein A magnetic beads, the PHFTau suspension was stored at -
80°C before use. A control extract from healthy control human brain tissue was prepared
accordingly.
Lhamnrbnanfihndsanegnna
ELISA:
96 well half area microplates (Corning) were coated with recombinant Tau protein
(rPeptide, , USA) at a standard concentration of l 11ng in ate ELISA
coating buffer (pH 9.6) overnight at 4°C. For PHFTau screening, 96 well Immobilizer
Microplates (Nunc, k) were coated with PHFTau extracted from human AD brain
at 1:100 dilutions in carbonate ELISA coating buffer (pH9.6) overnight at 4°C. Plates
were washed in PBS-T pH 7.6 and non-specific binding sites were blocked for 2 hrs at
RT with PBS-T ning 2% BSA (Sigma, Buchs, Switzerland). B cell conditioned
medium was transferred from memory B cell culture plates to ELISA plates and
incubated for one hour at RT. ELISA plates were washed in PBS-T and then incubated
with horse radish peroxidase (HRP)-conjugated donkey anti-human IgG (Fcy fragment
specific) polyclonal antibodies (Jackson ImmunoResearch, Newmarket, UK). After
W0 2014/100600 — 10G — ZOl3/076952
washing with PBS-T, binding of human antibodies was determined by measurement of
HRP activity in a standard colorimetric assay.
MULTI-ARRAY® micro rlate in‘r
{02843 Standard 96 well 10-Spot MULTI-SPOT plates (Meso Scale Discovery, USA)
were coated with 30 ug/ml rTau (rPeptide), PHFTau brain extract and healthy control
brain t in PBS. Non-specific g sites were d for 1 hr at RT with PBS—T
containing 3% BSA followed by incubation with B cell conditioned medium for 1 hr at
RT. Plates were washed in PBS-T and then incubated with SULFO-Tag conjugated anti-
human polyclonal antibody (Meso Scale Discovery, USA). After washing with PBS-T,
bound of antibody was detected by electrochemiluminescence measurement using a
SECTOR Imager 6000 (Meso Scale Discovery, USA).
hBflganarghnunsrnlauannhgdka
Samples containing memory B cells were obtained from healthy human subjects.
Living B cells of selected memory B cell cultures are harvested and mRNA is prepared.
Immunoglobulin heavy and light chain sequences are then obtained using a nested PCR
approach.
A combination of primers representing all sequence families of the human
immunoglobulin germline oire are used for the amplifications of leader peptides, V-
segments and J-segments. The first round amplification is performed using leader
peptide-specific primers in 5’-end and constant region-specific primers in 3’-end (Smith
et al., Nat Protoc. 4 (2009), 372-384). For heavy chains and kappa light chains, the
second round amplification is performed using V-segment-specific primers at the 5’-end
ar‘2d J-segment—specific primers at the 3’end. For lambda light , the second round
amplification is performed using V-segment-specific primers at the 5’-end and a C-
region-specific primer at the 3’end (Marks er al., Mol. Biol. 222 (1991), 581-597; de
Haard et al., J. Biol. Chem. 26 , 18218-18230).
Identification of the antibody clone with the desired specificity is performed by re-
screening on ELISA upon recombinant expression of te antibodies. Recombinant
expreSSion of complete human IgG1 antibodies or chimeric IgG2a antibodies is achieved
upon insertion of the le heavy and light chain sequences "in the correct reading
frame" into expression s that ment the variable region sequence with a
sequence encoding a leader peptide at the 5’-end and at the 3’-end with a sequence
encoding the appropriate constant (s). To that end the primers ned restriction
sites designed to tate cloning of the variable heavy and light chain sequences into
antibody expression vectors. Heavy chain immunoglobulins are sed by inserting
the immunoglobulin heavy chain RT-PCR product in frame into a heavy chain expression
vector g a signal peptide and the nt domains of human immunoglobulin
gamma 1 or mouse immunoglobulin gamma 2a. Kappa light chain immunoglobulins are
expressed by inserting the kappa light chain RT—PCR-product in frame into a light chain
expression vector providing a signal peptide and the constant domain of human kappa
light chain immunoglobulin Lambda light chain immunoglobulins are expressed by
ing the lambda light chain RT—PCR—product in frame into a lambda light chain
expression vector providing a signal e and the constant domain of human or mouse
lambda light chain immunoglobulin.
Functional recombinant monoclonal antibodies are obtained upon co-transfection
into HEK293 or CHO cells (or any other appropriate recipient cell line of human or
mouse origin) of an Ig- heavy—chain expression vector and a kappa or lambda Ig—light—
chain expression vector. Recombinant human monoclonal antibody is subsequently
purified from the ioned medium using a standard Protein A column purification.
Recombinant human monoclonal antibody can produced in unlimited quantities using
either transiently or stably transfected cells. Cell lined producing recombinant human
monoclonal antibody can be established either by using the Ig-expression vectors directly
or by re—cloning of Ig-Variable regions into different expression vectors. Derivatives such
as F(ab), F(ab)2 and scFv can also be generated from these Ig-variable regions.
Antibodies
{mag} Mouse monoclonal uman tau antibody Tau12 (Covance, California, USA.)
and mouse monoclonal tau antibody AT180 (Thermo Scientific, USA.) were used
according to manufacturer’s protocol. Recombinant human tau antibodies NI-105.4E4,
Nl-105.24BZ and NI-105.4A3 are described in U.S. Patent Application Publication No.
2012/0087861, the content of which is orated herein by reference in its ty.
Recombinant human tau antibodies NI-105.17C1, NI-105.17C1(N31Q), NI-105.6C5, NI-
105.29G10, NI—105.6L9, NI-105.40E8, NI-105.48E5, NI-105.6E3, .22El, NI-
105.26Bl2, NI-105.12E12, NI—105.60E7, Nl—105.14E2, NI-105.39E2, NI-105.19C6, and
_ 102 ..
NI-105.9C4 are antibodies of this invention. They were expressed in HEK293 or CHO
cells, purified from conditioned media and were directly used in subsequent applications
unless otherwise stated.
Direct ELISA
[0290] 96 well microplates (Costar, Corning, USA) were coated with recombinant Tau
protein (hTau40, rPeptide, Bogart, USA) diluted to a concentration of l ug/ml in
carbonate ELISA coating buffer (SOmM, pH9.6) at 4°C overnight. Non-specific g
sites were blocked for 2 hr at RT wéth PBS ning 2% BSA , Buchs,
Switzerland) and 0.5% Tween20. Binding of human antibodies of the present invention
was determined using HRP conjugated goat anti-human IgG Fey (Jackson
immunoResearch, Newmarket, UK), followed by measurement of HRP activity in a
standard colorimetric assay. ECso values were estimated by a non-linear regression using
GraphPad Prism software (San Diego, USA).
WWestemBlottiinasztgtndnstaiuins
[0291] PHFTau and recombinant hTau40 were resolved by gradient SDS-PAGE
(NuPAGE 4-12%; Invitrogen, Basel, Switzerland) followed by electroblotting on
nitrocellulose membranes. After blocking the non-specific binding with 2% BSA at room
temperature for one hour, blots were ted overnight with primary human anti-tau
antibodies or Tau12 (mouse monoclonal antibody, Covance, California, USA),
followed by a HRP—conjugated goat anti-human IgGFcy (for human primary antibodies)
or a HRP-conjugated goat anti-mouse IgG secondary antibody.
Blots were developed using ECL and ImageQuant 350 ion (GE Healthcare,
Otelfingen, Switzerland).
PHFTau extraction from AD brain,
[0293] ion of paired l filaments containing pathologically phosphorylated tau
filaments (PHFTau) was performed following the method by Goedert et a]. (Goedert et
al., Neuron 8 (1992), 8) with ations. One gram of AD brain tissue was cut
into 5mm pieces with all visible blood vessels removed. The tissue was washed with 40
ml ice cold g solution (IOOmM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1
mM NaF) for three times followed by homogenization with 20 ml lysis buffer (IOmM
W0 2014/100600 PCT/USZOl3/076952
Tris pH 7.4, 0.8M NaCl, lmM EGTA, 1 x protease inhibitor cocktail, 1 mM Na3VO4,
lmM NaF, lmM AEBSF, 10% sucrose). The nate was centrifuged at 4°C at
’000xg for 20 min. Supernatant was collected with addition of N-lauroyl sarcosinate
(Sigma, Switzerland) to 1% (w/V). After two hours incubation at 37°C wth shaking, the
supernatant was then centrifuged at 4°C at 0xg for one hour. The pellet was
collected and resuspended in PBS. After clearing out possible contaminating
globulins with proteir: A magnetic beads, the PHFTau suspension was stored at —
80°C before use. A control extract from y control human brain tissue was ed
accordingly.
.Iahnentidjs synthesis,““““““““““““““ -—.a ——-—--
A peptide corresponding to amino acids 333-346 of hTau40
(333GGGQVEVKSEKLDF346) which includes the e of NI—105.4E4 identified by
Pepspot g (amino acids 337-343) was synthesized by Schafer—N (Copenhagen,
Denmark). An onal cysteine was added to the C-terminus to allow for covalent
g to Immobilizer Microplates (Nunc, Denmark). A second peptide corresponding to
amino acids 226-239 of human tau (226VAVVRpTPPKSPSSA239), the cognate e of
the commercially available mouse monoclonal tau antibody AT180 (Therrno Scientific,
USA) was synthesized accordingly and used as control.
Transgenic mice
[0295] Three different animal models for tauopathies are used to validate the tau
antibodies (and molecules with the binding specificities thereof) of the present invention.
{£32953 1. Transgenic TauP301L mice (linel83): expressing human Tau40 with P301L
mutation under the murine Thy1.2 promoter (Generation of these transgenic animals is
described in Gotz et al., J. Biol. Chem. 276 (2001), 529-534 and in international
application , the disclosure content of which is incorporated herein by
reference)
2. JNPL3 mice expressing the shortest 4R human tau isoform with P301L
mutation under the murine PrP promoter (available from Taconic, Hudson, NY, USA).
3. P3OISTau (line P819) mice expressing human tau with P3018 mutation under
the murine PrP er (available from the Jackson Laboratory, Bar Harbor, Maine,
U.S.A).
_ 104 _
Tauopathies mouse models and corresponding wild type mice are kept under
standard housing conditions on a reversed 12h212h light/dark cycle with free access to
food and water. The treatment groups are balanced for age and gender.
Example 1
Validation of target and binding specificity of human tau-antibodies
To validate tau as a recognized target of isolated antibodies direct ELISA assays
were performed as described above. For the exemplary recombinant human .4A3
antibody 96 well microplates (Costar, Corning, USA) were coated with recombinant
human tau (hTau40, rPeptide, Bogart, USA) diluted to a tration of 3 ug/ml or with
BSA in carbonate ELISA coating buffer (pH 9.6) and binding efficiency of the antibody
was tested. The ary NI-105.4A3 antibody specifically bound to human tau by
ELISA. No binding was ed to BSA .
For a determination of the half maximal effective concentration (EC50) of the
exemplary antibodies NI-105.4E4 andNI-105.24B2 additional direct ELISA ments
with varying antibody concentrations were performed. 96 well microplates (Costar,
Corning, USA) were coated with recombinant human tau (hTau40, rPeptide, Bogart,
USA) diluted to a concentration of 1 ng/ml (for the assay with NI-105.4E4Antibody), or
of 3 pg/ml (for the assay with Nl-105.24B2 Antibody) in carbonate ELISA coating buffer
and binding ncy of the antibody was tested. The EC50 values were estimated by a
2O non-linear regression using GraphPad Prism re. Recombinant human-derived
antibody NI-105.4E4 bound to hTau40 with high y in the low nanomolar range at
2.4 nM EC50 . NI—105.24B2 bound to hTau40 with high y in the low nanomolar
range at 6.6 nM EC50.
[03021 The half maximal effective concentration (EC50) of the exemplary antibody NI-
105.4A3 was also ined using direct ELISA experiments. ELISA plates were coated
with recombinant human tau (hTau40, lug/m1), PHFTau ( 1:100) and control preparation
(1:100), and incubated with varying antibody concentrations. NI-105.4A3 bound to rTau
with high y in the low nanomolar range at 1.4 nM EC50. NI—105.4A3 binds to
PHFTau with high affinity in the low nanomolar range at 1.2 nM ECso.
W0 2014/100600 - 105 — PCT/USZOl3/076952
Example 2
inant human antibodies g is to recombinant tau and
ogical tau extracted from AD brain
To determine the binding capacity of NI-105.4E4 and Nl-105.24B2 to
pathological tau species extracted from AD brain. SDS—PAGE and n Blot analysis
was performed as described in detail above. Blots were incubated overnight with primary
antibodies NI-105.4E4 (human), NI-105.24B2 (human) or Tau12 (mouse monoclonal
dy, Covance, California, USA), followed by a HRP-conjugated goat anti-human
IgGFcy (for human antibodies) or a HRP—conjugated goat anti-mouse IgG secondary
antibody.
Recombinant antibodies NI-105.4E4 ared NI-105.24B2 recognized recombinant
hTau40 as well as pathologically modified PHFTau extracted from AD brain on Westem
blot. The control antibody Tau12 recognized both tau species as well .
Additionally, as discussed in Example 1 above, the half maximal effective
tration (EC50) of the exemplary antibody NI-105.4A3 was determined in direct
ELISA ments using PHFTau. NI-105.4A3 bound to PHFTau with high affinity in
the low nanomolar range at 1.2 nM EC50 .
Mapping ofthe NT-105.4E4 and NI-105.4A3 binding epitope on hTau40
[0306] A peptide array of 118 peptide sequences covering the full-length hTau4O (amino
acids 1-441) with an overlap of 11 amino acids n two adjacent peptides was
d on a nitrocellulose membrane (JPT Peptide Technologies GmbH, Berlin,
Germany). Immunolabeling of antibodies as well as membrane regeneration were carried
out according to manufacturer’s instructions. To rule out non-specific binding of the
ion antibody, the membrane was first probed by HRP-conjugated goat anti-human
IgG omitting the primary antibody. After regeneration the membrane was probed with
100 nM recombinant NI-105.4E4 antibody. Bound antibody was detected using ECL and
ImageQuant 350 detection (GE Healthcare, Otelfingen, Switzerland).
_ 106 _
Two groups of adjacent peptide spots (peptide 83, 84 and 85; peptide 96 and 97)
were specifically identified by NI105.4E4, when compared to the detection antibody only.
The sequences covered by these two groups of peptides correspond to amino acids 329-
351 and 387-397 of hTau40. These data suggested that Nl-105.4E4 recognized a
discontinuous e sing two linear sequences: one within the R4 microtubule
binding domain and another in the inal domain.
The sequence shared by peptides 83-85 comprises amino acid es 337-343 of
hTau40. The Pepspot (JPT) data suggested that NI-105.4E4 recognized an epitope in
hTau that comprises amino acids 3 of human tau. This region is located within the
ubule g domain of tau and is conserved among all neuronal human tau
isoforms as well as across other species including mouse and rat.
As this domain is bound to microtubules in physiological microtubule—associated
tau, NI—105.4E4 is expected to entially target the pathologically relevant pool of tau
that is detached from the microtubules.
[0310] To determine key residues within the NI-105.4E4 binding peptides, e
scanning was performed to substitute each residue with e one at a time. The alanine
residues in the original sequence (A384 and A390) were substituted to proline and
glycine. Spots 35-50 and 51-68 are the original peptides (spot 35 and spot 51) and their
alanine substituted variants,. Alanine scan suggested V339, E342, D387, E391 and K395
were necessary for NI-105.4E4 binding.
An additional experiment has been performed by testing the binding of NI-
105.4E4 to tau es. Direct ELISA showed that .4E4 specifically recognized a
peptide corresponding to amino acid 333-346 of , which ns the amino acid
residues 337-343 identified by Pepspot mapping. No cross-reactivity of NI-105.4E4 was
observed to the control peptide covering the AT180 epitope. Vice versa, AT180
recognized its cognate epitope containing peptide but failed to bind to the NI—105.4E4
specific peptide. Species-specific secondary antibodies did not bind to any of the
peptides. Together, direct ELISA with coated peptides confirmed that NI-105.4E4
specifically recognized a peptide containing the amino acid residues 337-343 of human
tau identified by Pepspot mapping.
To grossly map the .4A3 binding epitope on hTau40, four tau domain
polypeptides (Tau domain 1, domain 11, domain III and domain IV) were produced. DNA
fragments, synthesized using GeneArt® (Invitrogen), which encode each Tau domain
with 6xHis tagged at the N-terminus were cloned into the pRSET-A expression vector
(Invitrogen), were ected into E. Coli BL21 (DE3) (New England Biolabs). The
sions of the His-tagged Tau domains were induced by 0.5mM IPTG for six hours
before bacteria were lysed with lysozyme with sonication. The lysate was boiled for five
minutes before being further purified wéth Ni-NTA Superflow Columns (Qiagen). The
eluted gged Tau domains were coated on ELISA plates or loaded on
polyacrylamide gel for Western Blot. These sequentially overlapping tau domain
polypeptides covered the full lengeh of . Puréfied tau domains were coated on
ELISA plate and the binding of NI—105.4A3 was tested. NI-105.4A3 binds only to tau
domain I and the full length hTau40, indicating the epitope was within the N—terminal
part of the hTau40 (aa1-136). n blot confirmed the specific binding of NI—105.4A3
to tau domain 1. NI—105.4A3 epitope mapping with PepSpot (JPT) technology identified
amino acids Q35-Q49 of the human Tau40. To ine key es within the epitope
for NI-105.4A3 binding, alanine scanning was performed to substitute each residue with
alanine one at a time. The alanine residue in the original sequence (A41) was substituted
With glycine or proline. Alanine scan showed that D40, A41 and K44 are key residues for
NI-105.4A3 binding.
Example 4
Assessment of the binding ofNI-105.4E4 to physiological forms
as well as pathological aggregates of tau AD brain tissues and in
human tau transgenic mice
Neurofibrillary tangles (NFT) composed of hyperphosphorylated tau filaments are
a neuropathological hallmarks of AD. Hyperphosphorylated tau filaments are also the
major ents of dystrophic neurites and neuropil threads, both of which are common
neuropathological features in AD. Overexpression of human tau ning the al
P301L tau on in mice induces NFT formation at six months of age (Gotz et al.,
2001a).
To assess the binding of recombinant human tau antibody to physiological forms
as well as pathological aggregates of tau, immunohistological stainings were med in
AD brain tissues and in TauP301L transgenic mice with the exemplary NI—105.4E4
antibody of this invention.
_ 108 _
Mice were perfused with 20 m1 100 mM /6 mM EGTA (pH7.4) at room
temperature under deep anesthesia. Brains were taken out and immersed in 4% PFA in
PBS (pH 7.4) at 4°C overnight for fixation ed by embedding in n. For
human tissue, paraffin blocks of brain tissues from AD and healthy control subjects were
used. DAB staining was cariéed out following standard protocols. As positive control,
mouse monoclonal antibody Tau-12 (Covance, California, USA.) was used. HRP-
conjugated detection antibodies without primary antibodies were also included.
Recombinant human antibody NI-105.4E4 identified numerous NFTs and
neuropil threads in AD brain (Figure 2A), which were absent in healthy control brain
(Figure 2B). Secondary antibody alone did not give s in both AD (Figure 2C) and
control brain (Figure 2D). In P301L tau enic mouse brain, Nl—105.4E4 b0und
strongly to the pathological tau resembling NFT (Figure 2E, F and H), neuropil threads
(Figure 2E and G) and dystrophic es (Figure 2E and H). In addition, Nl-105.4E4
also identified tau aggregates at pre-tangle stage e 21). In the brain of transgenic
mice overexpressing both human P301L tau and human APP with Swedish and Arctic
ons, NI-105.4E4 bound cally to dystrophic neurites surrounding beta-
amyloid plaques (Figure 2]).
Example 5
In vivo tests of the antibodies of the present ion
[0317] As already bed above studies in transgenic mouse lines using active
vaccination with phosphorylated tau peptides revealed reduced brain levels of tau
aggregates in the brain and slowed progression of behavior impairments (Sigurdsson, J.
Alzheimers Dis. 15 (2008), 157-168; Boimel et all, Exp. Neurol. 224 , 472-485).
However, active vaccination may not be particularly useable in humans because a
significant fraction of the elderly population is expected to be non-responders t0
vaccination. Furthermore, the potential side effects associated wéth a tau-directed immune
response can be difficult to l. Tau binding molecules of the present invention can
be reasonably expected to achieve similar reductions in brain levels of tau aggregates as
bed anve for the mouse antibodies, e of their similar binding specificities
against pathologically tau species. However, because of the evolutionarély optimization
and affinity maturation within the human immune system antibodies of the present
invention e a valuable therapeutic tool due to being isolated from healthy human
subjects with high probability for excellent safety profile and lack of immunogenicity.
Confirmation of these expected therapeutic effects can be provided by test methods as
described in the above mentioned experiments with mouse antibodies. In particular, the
antibodies to be screened can be applied on diverse possible routes to the animals such as
intraperitoneal antibody injection, intracranial injection, intraventricular brain infusion
and tested for treatment effects. Either of the above mentioned application possibilities
can be also used after prior brain injection of beta-amyloid preparations into the brain of
tau enic mice to evaluate ent effects on beta amyloid-induced tau pathology.
Evaluation of the treatment effects can be performed by hemical methods
comprising quantification of Gallyas positive cells , total human tau staining, brain
burden of phosphorylated tau and/or a biochemical determination of brain soluble and
insoluble tau and phosphor—tau levels upon sequential brain extraction. Further on,
behavior testing of the treated mice can be performed, e. g., conditioned taste aversion or
contextual fear conditioning for a confirmation of the therapeutic effects of the antibodies
of the present invention (Pennanen, Genes Brain Behav. 5 , 369-79, Pennanen
Neurobiol Dis. 15 (2004), 500-9.)
Example 6
Chimerization of antibodies Nl-105.4E4 and NI-105.4A3 with mouse IgG2a
nt domains
[03,19] In order to generate dies with reduced immunogenicity for use in chronic
treatment studies, mouse chimeric versions of antibodies NI—105.4E4 and NI-105.4A3
were generated using recombinant DNA technology. A mouse IgG2a/lambda isotype was
selected for these chimeric dies, in order to generate a le which bound with
high affinity to mouse Fc-gamma receptors, and was therefore capable of inducing an
immune effector response. The amino acid sequences of the ic NI—105.4E4
4") and chimeric NI—105.4A3 ("ch4A3") heavy and light chain constructs are
shown below.
_ 1 10 _
Table 5: Amino acid sequences of chimeric "NI—1 05.4E4 (ch4E4) and chimeric
NI—105.4A3 (ch4A3)
inmhuech4E4 aEVQLVESGGGLVQPGGSLKLSCAASGFNFNISAIHWVRQASGkéiEWVGR‘
‘ iIRSKSHNYATLYAASLKGRFTLSRDDSRNTAYLQMSSLQTEDMAVYYCTVi
heavy chain
: iLSANYDTFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV
lggzg) iKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSi
t ITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPK
iSEQ IDNO: 20 2 ~
iIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYi
iNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP'
iQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP
iVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG
inmnuech4E4 ESYELTQEPE§§V§EEQTARISCFGDTLPKQYTYWYQQKPGQAPVLVIYKD
’ iTERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCLSADNSATWVFGG
i light chain
, EGTKVTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDEYPGVVTVDWK
lmnbmfi iVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEG
‘ EHTVEKSLSRADCS
SEQ ID NO: 21 g
Example 7
l of consensus N—Iinked glycosylation site from ch4E4 heavy chain (mouse
IgG2a)
A consensus N-linked glycosylation site was identified in the CDRI region of the
NI—105.4E4 heavy chain. Upon mammalian (CHO) cell expression, the predicted N-
glycosylation site (Asn-30) was fully occupied by glycan, as trated by mass
spectrometry. In order to eliminate N-glycosylation in this region and reduce product
heterogeneity, Asn-30 of the heavy chain of ch4E4 was d to Gln (Table 4). When
produced and purified from CHO cells, the modified antibody bound to recombinant tau
with ~4—fold higher apparent binding affinity relative to the al, glycosylated
antibody.
Table 6: Amino acid sequences of mature ch4E4(N30Q) heavy chain (mouse
IgG2a). Substituted Gln e is in bold, underlined.
imam EVQLVEsGGGLre‘fiaeg‘m‘é‘emé‘é‘fifi‘igiémwvtars:5&3vaGR
i N30Q) I RSKSHNYATLYAASLKGRFTLSRDDSRNTAYLQMSSEQTEDMfiRVYYCTV
i LSANYDTFDYWGQGTLVTVS SAKTTAPSVYPLAPVCGDTTGS SVTLGCLV
i heavy chain KGYFPEPVTLTWNSGSLS SGVHTFPAVLQS DLYTLSSSVTVT SSTWPSQS
ti... ITCNVAHPASSTKVDKKIEPRGPTIKPCPPQECPAPNLLGGPSVFI FP Pia;
]' (mouse IgGié)M§f"iKD\/LMISLSPtvrevaDVSEDDPDVQISWFVNqu‘v‘fiT'AQTQTHREDY
NSTLRVVSALP-EQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP
SEQID N022 gQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP.
" l:VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG
Example 8
Production of aglycosylated chimeric NI-105.4E4(N30Q) (ch4E4(N3OQ)
mIgGl Agly)
A mouse chimeric aglycosylated variant of germlined NI-105.4E4 was produced
("ch4E4(N3OQ) IgGl-Agly") in order to evaluate the relationship between dy
effector function and activity. For the heavy chain (SEQ ID 214), the variable domain of
NI-105.4E4(N30Q) (SEQ ID NO: 43) was fused to a mouse IgGl heavy chain constant
region containing an Asn to Gln mutation to eliminate the consensus Fc glycosylation
site. The heavy chain variable region contained the N30Q change in order to eliminate the
sus osylation site in CDRl (Example 7). The light chain was the ch4E4
lambda light chain described above (SEQ ID 21).
Example 9
Acute brain penetration study ofhuman 4E4 and 4A3
Human NI-105.4E4 and NI—105.4A3 germlined antibodies were produced by
transient transfection of CHO cells and d by y purification. The endotoxin
levels were controlled and were all bellow 1 EU/rng. TauP301L mice were
intraperitoneally injected with 30 mg/kg NI—105.4E4 (n=7), 4A3 (n=7) antibody or equal
volume of PBS (n=7) at day 1 and day 4. At day 5, mice were perfused under anesthesia
with PBS containing 1 Unit/m1 heparin. Blood, brain and spinal cord were collected for
es. Right hemisphere of the brain was frozen at -80°C, left hemisphere of the brain
and the spinal cord were post fixed in 10% neutralized formalin at 4°C for two days
before being ed in paraffin block and sectioned. Plasma was stored at —80°C in
aliquots.
Brain protein extraction: frozen right hemisphere was weighed and homogenized
in 5 volumes (5 mL/g of wet tissue) of a solution containing 50 mM NaCl, 0.2%
_ 112 -
diethylamine, protease inhibitors (Roche stics GmbH) and phosphatase inhibitor
(Roche Diagnostics GmbH). Samples were then erred to polycarbonate tubes and
added another 5 volume of homogenization solution, and kept on ice for 30 min. Soluble
fraction was then collected after centrifugation at 100,000 g, 4°C for 30 min. This soluble
fraction was used in human IgG assay. The pellet was re—suspended in 3 volumes ofPBS
with protease and phosphatase inhibitor. After centrifugation at 16,000 g, 4°C for 30min,
supernatants and pellets were stored separately at -80°C for further insoluble tau
tion. Pellets further extracted with modified sarcosyl extraction (Goedert M,
Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992)).
[0324] Human IgG-specific sandwich ELISA: 2 ug/ml of goat anti-human IgG Fab
(Jackson) in 50 mM carbonate ELISA coating buffer (pH9.6) was used as capture
antibody. Half-area l microtiter plates was coated with 30 ill/well with capture
antibody at 4°C overnight. The plate was then washed 4 times with PBS containing 0.1%
Tween 20 before incubating with 50 l PBS containing 2% BSA at room
temperature for one hour. Soluble fractions of brain extracts, plasma samples and
antibody standard (4A3) were diluted in PBS containing 2% BSA and 0.1% Tween 20. 30
ul of the diluted s were added into each well and incubated at room temperature
for one hour. The plate was then washed with 200 ul/well PBS containing 0.1% Tween
for four times before incubated with HRP—conjugated donkey anti-human Fcy
(Jackson, diluted at 1:10,000 in PBS ning 2% BSA and 0.1% Tween 20) at room
temperature for one hour. The plate was then washed with 200 ul/well PBS containing
0.1% Tween 20 for four times before adding 20 til/well TMB (1:20 in 10 mM citrate
solution pH=4.l). The on was then stopped by adding 10 u] 1M H2SO4to each well.
Antibody rd curve was obtained from serial dilutions of NI-105.4A3. Antibody
concentrations in plasma and brain s were calculated according to the standards.
Brain human IgG level was then converted to ug antibody/gram fresh brain tissue
(assuming 1g/10 ml) as indicated in Figure 6.
High levels of human IgG were detected in the plasma of all NI-105.4E4 and NI—
105.4A3 d mice. In contaast, no human IgG was detected in the plasma of PBS
d mice (Figure 5). Significant amount of human IgG was detected in brain
homogenates of4E4 and 4A3 treated mice (Figure 6).
- 113 —
Example 10
Chronic study with chimeric Nl-105.4E4 and Nl—105.4A3
Chimeric NI-105.4E4 and NI-105.4A3 containing the varéable domains of the
original human antibody and the constant regions of mouse IgGZa can be ed by
transient transfection of CHO cells and purified by affinity ation. The endotoxin
levels in each batch of the antibodies wll be controlled and kept below 1 Eng. Gender
balanced TauP301L mice at age of 7.5-8 months wéll be intraperitoneally injected with 10
mg/kg, 3 mg/kg of antibody solution, or equal volume of PBS control. Each treatment
gafoup will have 20-25 mice. The ent will be d out once a week for 26 weeks.
Alternatively, the treatment will be carried out twice a week for 13 weeks. Body weight
will be monitored every two weeks. Mice wéll be perfused under anesthesia at the end of
the treatment period. Brain, spinal cord and blood will be ted. Half brain and spinal
cord can be post-fixed in 10% formalin for three days before being embedded in paraffin
block. 4-6 mm thick ns cut from these tissue blocks can be used for
immunohistochemistry studies. The other half brain will be weighted and deep frozen at -
80°C for mical analyses.
Drug effects will be evaluated by comparing the level of neurofibrillary tangles
(NFT) and the level of tau with different solubility characteristics in treated and control
samples. NFT can be visualized by Gallyas silver impregnation (F s Acta MOIphol.
Acad. Sci. Hung 19.1 (1971)), or by immunostaining with monoclonal mouse dy
ATIOO and AT180, which recognize pathologically phosphorylated tau in NFT. The
number or frequency of Gallyas-positive neurons and/or ATlOO, AT180 labeled neurons
in the brain and spinal cord in antibody treated mice and control animals can be
determined to evaluate the effect of antibody treatment.
[0328] e and insoluble tau can be extracted following the brain protein extraction
ol described herein. Alternatively, soluble and insoluble tau can be extracted with
modified sarcosyl extraction (Goedert M, Spillantini MG, Cairns NJ Crowther RA.
Neuron 8, 159 (1992)). Briefly, frozen brain is homogenized in 10 volumes (wt/vol) of 10
% sucrose homogenate buffer consisting of 10 mM Tris-HCI (pH 7.4), 0.8 M NaCl, 1
mM EGTA, 1mM Na3VO4, 1 mM NaF, 1mM AEBSF, protease inhibitors (Roche
Diagnostics GmbI-l) and phosphatase inhibitor (Roche Diagnostics GmbH). The
WO 00600 _ 1 14 _
homogenate is spun for 20 min at 20,000g, and the supernatant retained. The pellet is
homogenized in 10 volumes of homogenization buffer and centrifuged for a second time.
The supematants can be pooled together, and N-lauryl—sarkosinate (Sigma) is added to
1% l) final concentration, and incubated at 37°C with 300 rpm rotation for 1.5
hour, followed by centrifugation at 0 g for 1 h. The supernatant is collected as
sarcosyl soluble fraction and the pellet of 1 g brain tissue is re-suspended in 0.2 ml 50
mM TriS’HCI (pH 7’.4) as PHF fraction.
The levels of soluble and insoluble tau will be ed with commercially
available Tau ELISA kits (Invitrogen). In addition, brain protein extracts will be
separated with 4-12% Bis-Tris SDS-PAGE followed immunoblotting with Tau12 (human
tau), AT8 (pS202/pT205), ATIOO (pT212/p8214), AT180 (pT231) and E178 (pS396)
antibodies. uantitative analysis will be performed with measuring the integrated
density of each sample against standards of known quantities of tau.
{0330] Additionally, behavioral tests can be performed as ted in Example 5, above.
For e, improvement of working memory in antibody treated TauP301L mice can
be tested using a two-trial Y-maze task (e.g., Pennanen, Genes Brain Behav. 5 (2006), 369-
79, which is herein incorporated by reference in its entirety). The three arms of the maze
are 22cm long, 5 cm wide and 15 cm deep. Black and white abstractive clues are placed
on a black curtain surrounding the maze. Experiments are ted wéth an ambient
light level of 6 lux during the dark phase. Each experiment comprises a ng session
and an observation session. During the training session, a mouse is assigned to two of the
three arms (the start arm and the second arm), which can be freely explored during 4 min,
with no access to the third arm (the novel arm). The mouse is then removed from the
maze and kept in a holding cage for 1.5-5 min, while the maze is thoroughly cleaned with
7’0‘5’9 ethanoi to remove any Olfactory eiues. The mouse is then put back again in the maze
fer observation with ali three arms accessible for 4 min. The sequence ef entries, the
number of entry to each arm and the time spent in each arm is recorded. From that the
ratio of time spent in the nevei third arm over the average of time spent in the other two
arms (start arm and second arm) is calculated and compared among different treatment
wild type mice. Rodents
groups in tauopathy mouse model and ponding l
typically prefer to investigate a new arm of the maze rather than returning to one that was
previously visited. Effects of the antibodies can be monitored in regard of regaining this
preference by treated tauopathy model mice in comparison to non-discriminative behavior
WO 00600 — 115 -
of untreated mice due to their disorder-related working memory impairment. Therefore, a
ratio close to 1 indicates impaired working memory. A higher ratio indicates better
working . Impaired working memory in TauP301L mice is considered to be due
to tau pathology resulting from the overexpression of human tau. ore a
U2 significantly higher ratio observed in anti-tau antibody treated TauP301L mice than in the
control TauP301L mice will indicate that the anti-tau antibody has therapeutic effect on
tau ogy.
Example 11
Identification ofhuman anti-tau antibodies.
[0331] Recombinant human tau antibodies NI-105.17C1, NI—105.6C5, .29G10,
.6L9, NI-105.4OE8, NI-105.48E5, NI-105.6E3, NI-105.22E1, NI-105.26B12, NI-
105.12E12, NI—105.60E7, .14E2, NI-105.39E2, NI-105.19C6, and NI-105.9C4
were isolated according to the methods described herein. The target and binding
specificity of these human tau-antibodies were validated as described above. A summary
of the findings is provided in Table 5. All antibodies used except NI-105.17C1 were
germlined.
Table 7. In Vitro characterization of human anti-tau antibodies.
Antlbody ECso TauECSO [nM]/ PHFTau Bmdmg reglon Phosphorylation
= ELISA ELISA reamed“
E-NI 1056C5 E0033 E004 N0
E33 E
.NP. WY“
217-227 N0
E:NI 105.48E5 E>500 E132 pS396, pS46
37-55; 387-406
unconfirmed
W .__
.....
Phosphorylation at
ENI—105.60E7 E018 E
197—207 either 198,199 202 orE
: a .
. .
EMMWWWM..“1.1...“...w.“Wm““wfl....................................'1 . ..............______.1.E205 dlsrupts bmdlng
....
: \
ENI-105 14E2 E0.65 57; 67
355-441
313319
‘-.-.».».».».».».».».»...._...A...1.“....n..eee“‘“uW...~e«««““-,.,......
... . .:-. . -.-. .
WEI".1:1::1:...-.......1:3.00 ‘309-319 ENe
E—NI105.9C4 E221-231 1-111deaES23:1 disrupts
*..blnding region
on hTau40 Was Identlfied by combinedapproaches of PepSPOTs tau—
fragments western blot and ELISA, tau—peptide ELESA and Alanine scanning.
**2 whether antibody binding requires phosphorylation at certain amino acids on tau
n was verified by comparing the binding of antibody to rTau, PHFTau, PHFTau
dephosphorylated by calf intestine atase, rTau in vitro phosphorylated by GSK3B
or GSK3B /CDK5/p35, and phosphorylated tau peptides on PepSPOTs and direct ELISA.
Example 12
Chimerization of human dies with mouse IgGZa constant domains.
In order to generate antibodies with reduced genicity for use in chronic
treatment studies, mouse chimeric versions of dies NI-105.17C1 ("ch17C1"), NI-
105.6C5 ("ch6C5"), .40E8 ("ch40E8"), and NI-105.6E3 ("ch6E3") were generated
using recombinant DNA technology. A mouse IgGZa/lambda isotype was ed for
these ic antibodies, in order to generate a molecule which bound with high affinity
to mouse Fc-gamma receptors and was therefore capable of inducing are immune effector
response. The amino acid sequences of ch17C1, ch6C5, ch40E8, ch40E8(R104W), and
ch6E3 heavy and light chain constructs are shown below.
hlvcnmmmbdywq1Dmm
"‘"eh3155‘héWfifiBfiéfigfiéjiWWWIDNO:205
»»»»»»»»»»»
“0116135llght chaln (mouse lambda) ESEQ ID NO266“
“61140138heavychain (mouse IgG2a) “ESEQ: ID NO:207
E”ch40138(R104Wheavy chain (mouse IgG2a ‘sM3Q1D NO:208 1
01'{3‘1A205m;001b—‘I;1—i-(IQ!E: O W .... 5‘1N;r—I-52 A.—1%0‘CL3’2
ch6E3 heavy chain(mouse IgG2a)W SEQ1D NO210
33 llgh’t Chaln(m0useW““““““““““WWWWSEQ1DNo:211
35S‘
WO 00600 - 1 17 — 2013/076952
Example 13
Elimination of CDR glycosylation site in NI-105.17C1 light chain.
A consensus ed glycosylation site was identified in the CDRl region of the
NI-105.17C1 light chain. Upon mammalian (CHO) cell expression, the predicted N glycosylation site (Asn-31) was fully occupied by glycan, as demonstrated by mass
spectrometry. In order to eliminate N-glycosylation in this region and improve product
heterogeneity, Asn-31 of the light chain of ch17C1 was changed to Gln (see sequence
below). When produced and purified from CHO cells, the modified antibody
(ch17C1(N31Q) mi'gGZa) bound to recombinant tau with similar nt binding
affinity relative to the original, glycosylated antibody (see Figure 8A). The NI-
105.17C1(N31Q) light chain variable region comprises the amino acid sequence of SEQ
ID NO:221.
‘ch17C1(N31Q)Welambda) SEQIDN0212
humanN110517C1(N31Q) VL ‘ SEQIDN0221_
e 14
tion of antibodies with reduced effector on.
Antibody variants ning mutations Within the consensus N—glycosylation site
in the heavy chain Fc domain were generated. These variants, designated “Agly”, were
designed to generate anti-tau antibodies with reduced immune effector function. The
amino acid sequences of Agly variants of the tau antibodies are provided below.
ch4A3-mIgG1Aglyheavycha1nSEQ ID N0:213
ch4E4(N30Q)-m1g5ifi§i§ii€§€9Efiaifi"""”“”"‘"EmsiéEiifimfio‘E‘iz14
cthhamSEQlDNOZlS
ch17WychamSEQlDN0216
- l 18 -
Example 15
Comparison of Binding Activity of chl 7C1-mIgG2a and ch17C1-mIgGl-Agly.
The relationship between antibody effector function and activity was assessed for
ClEl7Cl Agly (Figure 8A). The ch17C1 dy comprised the ch17C1 heavy chain
(SEQ ID NO:203), and the ch17C1 light chain (SEQ ID NO:204) The ch17C1(N31Q)
mIgGZa antibody comprised the ch17C1 heavy chain (SEQ ID ), and the
ch17C1(N31Q) light chain (SEQ ID NO:212), which incorporates the N31Q mutation in
CDRl to eliminate the CDR glycosylation site. The ch17C1(N31Q) mIgGl Agly
antibody comprised the ch17C1-mIgG1-Agly heavy chain (SEQ ID ), wherein the
variable domains of 17C1 are fused to a mouse IgGl heavy chain containing an Asn to
Gln mutation at position 294 (Kabat residue 297) to ate the consensus Fe
glycosylation site, and the ch17C1 (N3 1Q) light chain (SEQ ID ). When produced
and purified from CHO cells, ch17C1(N3 1 Q) mIgGl Agly bound to recombinant tau with
similar apparent binding affinity relative to the original, glycosylated antibody (see Figure
8A).
Example 16
Comparison of Valine vs. cine at position 48 ofthe 17C1 light chain.
: In the process of generating the mouse chimeric IgG2a version of germlined
antibody NI—105.17C1, residue 48 of the light chain was also d from valine to
‘ isoleucine. To confirm that this suEstitution did not affect the binding y of NI-
Cl, a mouse chimeric IgG2a version of NI—105.17C1 with valine at position 48
was prepared. When produced and purified from CHO cells, ch17C1(N31Q, I48V)
mIgGZa antibody bound to recombinant tau with similar apparent binding affinity relative
to the ch17C1(N31Q) mIgG2a (see Figure8B). The ch17C1(N31Q) mIgG2a antibody
comprised the ch17C1 heavy chain (SEQ ID NO:203), and the ch17C1(N31Q, I48V)
light chain (SEQ ID NO:217).
(N31Q148WSEQIDNOZI7
lambda)
huTnWVWLSEQIDNozzz
- 1 19 -
e 17
Comparison of Arg vs. Trp at on 104 ofNI-105.40E8.
[173371 Antibody NI-105.4OE8, which is selective for the phosphorylated form of tau
found in human paired helical filaments (PHF), contains an unusual arginine residue at
position 104 of the NI—105.40E8 VH. lly this on within the human
immunoglobulin oire is occupied by a tryptophan e. A form of the NI-
105.40E8 heavy chain, NI-l05.40E8(R104W)-hIgGl, was generated in which residue
Arg104 was replaced with tryptophan. When produced and purified from CHO cells, NI—
105.40E8(R104Wj—hIgG1 antibody bound to humar‘e PHF tau with similar apparent
binding affinity relative to NI—105.40E8-hIgGl (see Figure 9). The light chain of the two
antibodies was identical.
"Ni-1‘65fibfiéfifiifiktwyhlgG1, heavy SEQ 115N0218
Echain
{“hfiihiih“““i§iI-105.40E8 light cha1n(humanSEQIDN5219
lambda)
humanN110540E8(R104W)VH1SEQIDN022OM
Example 18
Human anti-tau antibodies bind to pathologically aggregated tau in AD brain and in
the brain of transgenic mouse model oftauopathy.
Brain tissue samples obtained from Alzheimer's disease and control patients, as
well as from the brain of transgenic mouse of tauopathy and wild-type control were
stained with the germlined human au antibodies provided herein. Representative
images of germlined human .40E8, NI—105.48E5, NI-105.6C5 and NI-105.17C1
anti-tau antibodies binding to pathological tau aggregates in the brain of Alzheimer’s
disease (AD) and in the brain of transgenic mouse of tauopathy (Tg) are shown in Figure
. None of these antibodies bind to normal tan in mentally healthy subject (Ctr) or wild
type mouse brain (Wt). The different patterns among these antibodies reflected their
differences in epitope specificity and binding affinity.
Example 19
Brain penetration of antibodies in TauP301L mice.
[0339] Animals and Antibody treatments: Human NI-105.6C5, NI-105.4GE8 and NI-
105.6E3 anti—tau antibodies were produced by transient ection of CHO cells and
d using standard s. A humanized antibody with no cross-reactivity to
mouse antigens was used as an isotype control (hIgGl). In the first experiment, half of
the injected NI-105.6C5, NI—105.6E3 and hIgGl antibodies were labeled with Cy3 (GE
Healthcare, PA13105) wéth an approximate antibody: Cy3 ratio of 1:3. Cy3-labeling did
not change the antibody g as confirmed by ELISA and immune-staining with
TauP301L brain (data not shown). In the second experiment, unlabeled NI-105.6C5 and
.4OE8 anti-tau antibodies were used.
1L mice between 18-22 months of age received two doses of 30 mg/kg
NI-105.6C5, NI-105.6E3 or human IgG1 isotype control hIgGl via i.p. injection within
seven days. Tissue samples were collected from three mice of each d group at time
points of one day, eight days and 22 days post the second dosing. In the second
experiment, TauP301L mice were ip injected with 30 mg/kg h4OE8 and h6C5 twice
within seven days and tissue samples were collected from those mice one day post the
second injection.
For tissue sample collection, mice were deeply anaesthetized with
ketamine/xylazine before blood was collected through the right atrium. CSF was then
collected by cistema magna re. Brain and spine were subsequently collected
following perfusion for 2 min with ice cold PBS, containing 10 UI/ml heparin and five
minutes with 10% neutralized formalin through the left ventricle. The brain was fixed in
% lized formalin for another 3 h at 4°C, following immersion in 30% sucrose for
48 h. The brain was then frozen in dry ice and subsequently sectioned into 30 pm thick
coronal section series. The section series were stored at -20°C in antifreeze solution
containing 1M e, 37.5% ethylene glycol in 50 mM sodium phosphate buffer pH7.4
_ 121 _
with 0.025% sodium azide before use. The spine was post fixed in 10% neutralized
formalin for two days and embedded in paraffin blocks.
iasazg Immunohistochemistry: Coronal ns of 30 um thickness were probed with
biotinylated donkey anti-human IgG (H+L) by free floating staining. Free-floating
sections were washed in Tris—Triton pH7.4 (SOmM Tris, 150 mM NaCl, 0.05% Triton X-
100), incubated in 1% H202 PBS for 30 min, and incubated with a ng solution
containing 2% normal goat— and horse serum in Tris-Triton with additional 0.2% Triton
X-100 for 1 h at room temperature. The sections were then incubated with biotinylated
donkey anti-human IgG (H+L) (Jackson research Labs, 709149) at 1:200 in
blocking solution for 16 h at 4°C with agitation at 100 rpm to detect neuronal human IgG.
The —bound biotinylated antibody was visualized by peroxidase chromogenic
reaction using the Vectastain Elite ABC kit (Vector Laboratories, PK6100, 1:100). The
tic reaction was stopped with ice cold PBS and the sections were washed in PBS 3
times. The sections were then mounted on glass slides and air dried over night before they
were counterstained with hemalum solution to visualize the nuclei (Carl Roth GmbH +
Co., T8651). After dehydration steps, the slides were covered with coverslips before
being scanned with the s dotSlide 2.1 virtual microscopy system.
Human antibodies were detected in the brains of TauP301L mice, which had
ed either human au antibodies or hlgGl control antibody via i.p. injection, but
not in TauP301L and wild type mice without antibody treatment (Fig. 11). However,
neuronal staining was only observed in the hippocampi of NI-105.6C5, NI-105.6E3 and
NI-105.40E8 anti-tau antibody treated mice, but not in hIgGl treated mice. Neuronal
staining with anti-tau antibodies was readily detectable one day post injection, less
pronounced at eight days post injection, and was undetectable at 22 days post ion
(data not shown). TauP301L mice produce high levels of transgenic human tau in the
hippocampal formation, and the hippocampus is one of the earliest regions which p
neurofibrillary tangles. Thus, peripherally injected anti-tau antibodies not only entered
the brain but also likely entered into neurons which contained high levels of tau.
Example 20
Effects of chronic treatment of TauP301L mice with ch4E4(N30Q) and
chl7C1(N31Q).
{0344] s and Antibody treatments: Chimeric NI-105.4E4(N30Q)
("ch4E4(N30Q)") and chimeric NI-105.17C1(N31Q) ("ch17C1(N31Q)") containing the
variable domains of the human dy and the constant regions of mouse IgG2a were
produced by transient transfection of CHO cells and purified using standard methods.
Gender balanced TauP301L mice at ages of 7.5-8 months were weekly given 10
mg/kg ch17C1(N31Q) (n=20), 10 mg/kg ch4E4(N30Q) (n=20) or an equal volume of
PBS (nreZO) through intraperitoneal injection. Body weight was monitored every two
weeks. No significant weight loss was observed. Two mice from the PBS group and one
mouse from the chl7C1(N31Q) treated group died prematurely. Mice were anaesthetized
one day after the 25th treatment for tissue collection. Blood was collected through the
orbital sinus. Brain and spine were uently ted following perfusion for 2 min
with ice cold PBS, containing 10 UI/ml heparin, through the left ventricle. The left half
brain was then weighed, rozen in dry ice and stored at -80°C before use. The réght
half of the brain and the spine were post-fixed in lized 10% formalin at 4°C for two
days followed by further e in PBS before being processed to paraffin embedded
brain and spinal cord blocks.
[0346] Brain protein extraction: Brain protein was sequentially extracted based on the
solubility. The left half brain was first homogenized in 10 times w/v of 50 mM NaCl
ning 0.2 % diethylamine, 1X protease inhibitor (Roche Diagnostics GmbH) and 1 X
phosphatase inhibitor (Roche Diagnostics GmbH). After 30 min incubation on ice, the
nate was centrifuged at 100,000 g at 4°C for 30 min. Subsequently, the
supernatant was collected and defined as the e fraction. The remaining pellet was
homogenized in 12 times w/v of 10 % sucrose lysis buffer containing 10 mM Tris 7.4,
0.8M NaCl, lmM EGTA, 1X phosphatase inhibitor,1X protease inhibitor,1 mM Na3VO4,
1 mM NaF and 1 mM AEBSF. After 30 min tion on ice, the homogenate was
centrifuged at 20,500 g at 4°C for 20 min. 95% of the supernatant was carefully collected
for sarcosyl extraction. The pellet was stored at -80°C.N—1aury1—sarcosinate was added to
the supernatant (1% (w/v) final concentration). Following 1 h incubation at 37°C with
W0 2014/100600 PCT/USZOl3/076952
agitation at 220 rpm, the solution was centrifuged at 100,000g at 4°C for one hour. The
supernatant was collected and defined as the sarcosyl soluble on. The pellet was left
to dry at room temperature for 30 min, then dissolved in 50 mM Tris pH 7.4 (20% v/w
initial brain weight) and defined as the PHF ble fraction, which was stored at -80°C
until use.
ELISA measurements Human total tau and phosphorylated tau in three brain
protein ons were fied with commercial ELISA kits (Life Technologies)
following the manufacturer’s protocol. Total human tau, human tau phosphorylated at
Threonine 231 (pT231 tau), human tau phosphorylated at Serine 199 (pSl99 tau) and
human tau orylated at Threonine 181 (pT181) tau were detected. s of the
soluble fraction were standardized to 1 mg/ml based on the total protein content measured
with BCA protein assay (Pierce) with 50 mM Tris pH7.4. Aliquots of the standardized
samples were prepared for ELISA measurements and Western blotting. To e
solubilized PHF insoluble fraction for ELISA, 10 ul PHFTau was ted with 10 pl
8M guanidine hydrochloride at room temperature for one hour followed by addition of
180 pl 50 mM Tris 7.4. ELISA measurements were d out following standard
protocols. The tau levels in each sample measured by ELISA were normalized to initial
brain weight for final is.
End point plasma drug levels were measured using a sandwich ELISA. Briefly, 3
ug/ml rTau (rPeptide) (SEQ ID NO:6) in 100 nM carbonate ELISA coating buffer
(pH9.6) was incubated in Costar half-area ELISA plates at 4°C overnight. The plates
were blocked with 3% BSA in PBS at room temperature for one hour. Plasma samples
were diluted in 3% BSA in PBS containing 0.1% Tween®20 to 1:200, 1:400 and 1:800.
Serial dilutions of ch17C1(N31Q) and ch4E4(N30Q) were used to generate standard
curves. After one hour incubation, the plates were washed 4 times with PBS containing
0.1% Tween®20 followed by a one hour incubation with donkey anti-mouse IgGFcy-
HRP (1:10,000). After washing, the bound antibody was further determined by a rd
colorimetric assay. Standard curves were generated by dal curve fit with GraphPad
Prism 5.
[0349] Western blot: Protein samples of the three fractions were heated at 70°C for 10
min in 4X NuPAGE® LDS sample buffer (Life Technologies) and an equal amount of
total protein from each sample was electrophoresed on a NuPAGE® 4-12% (w/v) gel.
Following semi-dry transfer of protein to PVDF membrane, the membrane was blocked
— 124 —
in 3% BSA containing 0.1% Tween-20 in TBS and subsequently probed with different
anti-tau antibodies at 4°C overnight. Peroxidase—conjugated ary antibodies were
then incubated at room temperature for 1 h following 4 washes with TBST.
Subsequently, the bound antibodies were detected by enhanced chemiluminescence
(ECL) (Pierce). Densitometric analysis of immunoblots was performed with the National
utes of Health Image] program.
Two-trial Y maze: Mice were tested for short-term spatial memory using a two-
trial Y-maze test. The arms of the maze were 35 cm long, 5 cm wide and 10 cm deep.
Abstractive cues were placed on the n surrounding the maze. Experiments were
conducted wéth an ambient light level of 6 lux mice were
. During the re phase,
assigned to two arms (the start arm and one other arm), which can be freely explored
during 4 min, without access to the third arm (new arm), blocked by a door made of the
same material as the maze. Mice were then removed from the maze and kept in the
holding cage for 2 min, when the maze was cleaned with 50% ethanol. During the test
please, mice were placed at the end of the start arm and allowed to freely explore all. three
arms during 4 min. The test phase was recorded with the TSE VideoMot2 software for
Video tracking and analysis of animal or (TSE Systems, Bad Homburg, Germany).
The number of arm entries and time spent in the new arm were recorded. The average of
number of arm entries and time spent in the other two arms that were open during the
training session was calculated. A ratio n the number of arm entries into (or time
spent in) the new arm and the average of the other two arms were calculated. Wild type
control animals which do not have a deficit in spatial working memory will typically have
a ratio n 1.5 and 2 in this test. Pennanen et al., Genes Brain Behav 5(5):369-79
(2006).
[0351] Data analysis: ELISA data were log teansformed to meet the normality
assumption for the two-way analysis of variance. The ence was considered
significant when p<0.05.
ch4E4(N30Q) significantly d soluble human tau in TauP301L mice:
Human tau levels in the DEA-soluble, sarcosyl-soluble, and insoluble fractions of brain
n extracts were quantified by ELISA. The majority of the human tau was found in
the DEA-soluble fraction (data not shown). Total human tau (hTau) was reduced in the
DEA-soluble fraction from ch17Cl(N3lQ) and ch4E4(N30Q) treated mice compared
with that of PBS treated mice (29% reduction on e in ch17Cl(N3lQ) and 37% in
- 125 _
ch4E4(N30Q), Fig. 12A). Reductions were also seen in phosphorylated tau (pT231,
pSl99 and pT181) in the DEA-soluble fraction (Fig. 12B, C and D respectively). We
have previously observed lower human tau expression in female 1L mice than
their male counterparts. Therefore, to accurately analyze the data we used two-way
ANOVA with gender and treatment as the two variables. A gender effect was confirmed
with a p<0.01 in all soluble human tau measurements (total human tau, pSl99, pT181 and
pT231 human tau). There was no interaction between gender and ent (0.49 <p<
0.91 in all e human tau measurements). Importantly, there was a significant
treatment effect in DEA-soluble human tau 5 for hTau, p8199 and pT231, and
p=0.06 for pT181). The treatment effect was inantly driven by ch4E4(N30Q).
When compared with PBS control, ch4E4(N3OQ) significantly reduced hTau (p<0.05),
pSl99 (p<0.01), pT23l (p<0.01) and pTl81 (p<0.05). ELISA measurements using the
sarcosyl-insoluble fraction showed a high variability among animals, and no sigraificant
gender effect was observed. No significant treatment effect was observed in the sarcosyl-
insoluble on. Similarly, no cant treatment effect was observed by ELISA in
the yl-soluble fraction.
n blots using human tau-specific monoclonal antibody Tau12 showed full
length human tau, as a single band at 62 kDa, as the major tau immunoreactive
component in the DEA-soluble fraction. A clear reduction of the full length human tau
was ed in majority of the mice treated with ch4E4(N30Q) and ch17Cl(N31Q). In
the sarcosyl insoluble fractior’a, a 64 kDa band and several other higher molecular weight
bands were observed, as well as smaller molecular weight bands presumably
corresponding to human tau fragments. Densitometric analysis showed a high degree of
variability among individual animals, which prevented any quantitative comparison.
However, there was an overall qualitative ion in all human tau proteins in the
sarcosyl insoluble fraction detected by Tau12 in ch17Cl(N31Q) and ch4E4(N30Q)
treated mice (representative Western blot shown in Fig. 13).
Plasma drug levels: Mice were treated with ch17Cl(N31Q) and ch4E4(N30Q) at
mg/kg weekly through i.p. injection. To assess the drug exposure, plasma samples
were collected 24 h after the last treatment. Plasma drug levels were measured with
ELISA using rTau as capture agent. The average plasma levels of ch17Cl(N31Q) and
N30Q) were 200 ug/ml and 145 ug/ml, respectively, suggesting both antibodies
had good blood exposure (Fig. 14).
- 126 _
Antibody treatment and spatial memory: An earlier study suggested deficits in
spatial reference memory, which is hippocampus—dependent, in TauP301L mice
(Pennanen et at, Genes Brain Behav 5(5):369—79 (2006)). The two-trial Y maze was
reported as a sensitive test to detect deficits in short—term spatial memory in tau transgenic
mice (Troquier et al., Curr Alzheimer Res. 9(4):397—405 (2012)). During the exposure
phase, all groups explored the maze equally, ng a similar amount of time in each
available arm (data not shown). No differences were found comparing distance moved.
During the test phase, PBS treated TauP301L mice made almost equal number of entries
in the new arm as the average of the other two arms explored during the exposure phase,
=1.18), suggesting a poor spatial working memory in PBS treated TauP301L mice.
Both ch17C1(N31Q) and ch4E4(N30Q) treated mice showed a preference for the new
arm ve to the other two arms, and they made more visits to the new arm than the
e of the other two arms (ratio=150 for ch17C1(N31Q) and 1.30 for
ch4E4(N30Q)). The ratio of new arm entey in ch17C1(N31Q) and ch4E4(N30Q) treated
TauP301L mice compared with that ofthe PBS d group is shown in Fig. 15.
The present invention is not to be limited in scope by the specific embodiments
bed which are intended as single rations of individual aspects of the invention,
and any compositions or methods which are flinctionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in the art from the
foregoing description and accompanying drawings. Such ations are intended to
fall within the scope of the appended claims.
Definitions of specific embodiments of the invention as claimed herein .
According to a first embodiment of the invention, there is provided an isolated anti-tau
antibody or tau-binding fragment thereof comprising a heavy chain variable domain (VH) and a
light chain variable domain (VL), n the VH comprises VH complementarity determining
regions : VHCDR1; VHCDR2; and VHCDR3, and the VL comprises VL CDRs:
VLCDR1; VLCDR2; and VLCDR3, wherein: the VHCDR1 comprises the amino acid sequence
as set forth in SEQ ID NO:85, the VHCDR2 ses the amino acid sequence as set forth in
SEQ ID NO:86 and the VHCDR3 comprises the amino acid sequence as set forth in SEQ ID
NO:87; and the VLCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:88, the
VLCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:89 and the VLCDR3
ses the amino acid as sequence as set forth in SEQ ID NO:90.
According to a second embodiment of the invention, there is ed an anti-tau
antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the
amino acid sequence of SEQ ID NO:215 or SEQ ID NO:205 and the light chain comprises the
amino acid sequence of SEQ ID NO:206.
According to a third ment of the invention, there is provided a pharmaceutical
ition comprising the anti-tau antibody or tau-binding fragment of the first or second
embodiment, and a pharmaceutically acceptable carrier.
According to a fourth embodiment of the invention, there is provided a vector or s
comprising an isolated polynucleotide or polynucleotides comprising a nucleotide sequence or
nucleotide sequences encoding the anti-tau antibody or tau-binding fragment of the first or
second embodiment.
According to a fifth embodiment of the ion, there is provided an isolated host cell
comprising the vector or vectors of the fourth embodiment.
According to a sixth embodiment of the invention, there is provided a method of
preparing an anti-tau antibody or the tau-binding nt thereof, the method comprising:
culturing the host cell of the fifth embodiment in a cell culture; and
isolating the anti-tau dy or tau-binding fragment thereof from the cell culture.
According to a seventh embodiment of the present invention, there is provided a use of
the anti-tau antibody or the tau-binding fragment of the first or second embodiment or the
pharmaceutical ition of the third embodiment in the cture of a medicament for
treating a neurodegenerative tauopathy.
According to an eighth ment of the present ion, there is provided a use of
the anti-tau antibody or the tau-binding fragment thereof of the first or second embodiment or
the pharmaceutical composition of the third embodiment in the manufacture of a medicament
for treating abnormal accumulation or deposition of tau in the central nervous system in a
human subject in need thereof.
Claims (23)
1. An isolated anti-tau antibody or tau-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain le domain (VL), wherein the VH comprises VH complementarity determining regions : VHCDR1; VHCDR2; and VHCDR3, and the VL comprises VL CDRs: VLCDR1; VLCDR2; and VLCDR3, wherein: the VHCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:85, the VHCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:86 and the VHCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:87; and the VLCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:88, the VLCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:89 and the VLCDR3 ses the amino acid as sequence as set forth in SEQ ID NO:90.
2. The au dy or tau-binding fragment f of claim 1, wherein VHCDR1 consists of the amino acid sequence as set forth in SEQ ID NO:85, VHCDR2 consists of the amino acid sequence as set forth in SEQ ID NO:86 and VHCDR3 consists of the amino acid sequence as set forth in SEQ ID NO:87; and the VLCDR1 consists of the amino acid sequence as set forth in SEQ ID NO:88, the VLCDR2 consists of the amino acid sequence as set forth in SEQ ID NO:89 and the VLCDR3 consists of the amino acid sequence as set forth in SEQ ID NO:90.
3. The anti-tau antibody or tau-binding fragment thereof of claim 2, wherein: (a) the VH is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:48; (b) the VH is identical to the amino acid sequence set forth in SEQ ID NO:48; (c) the VH is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:47; (d) the VH is identical to the amino acid sequence set forth in SEQ ID NO:47; (e) the VL is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:49; (f) the VL is identical to the amino acid sequence set forth in SEQ ID NO:49; (g) the VH is at least 90% cal to the amino acid sequence set forth in SEQ ID NO:48 and the VL is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:49; (h) the VH is identical to the amino acid sequence set forth in SEQ ID NO:48 and the VL is identical to the amino acid sequence set forth in SEQ ID NO:49; (i) the VH is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:47 and the VL is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:49; or (j) the VH is identical to the amino acid sequence set forth in SEQ ID NO:47 and the VL is identical to the amino acid sequence set forth in SEQ ID NO:49.
4. The au antibody or tau-binding fragment of claim 1, wherein the VH comprises the amino acid sequences as set forth in SEQ ID NO:47 and the VL comprises the amino acid sequence as set forth in SEQ ID NO:49.
5. The anti-tau antibody or tau-binding fragment of claim 1, wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO:48 and the VL comprises the amino acid sequence as set forth in SEQ ID NO:49.
6. The anti-tau antibody or tau-binding fragment of any one of claims 1 to 5, which comprises a human IgG1 heavy chain constant .
7. The anti-tau antibody or tau-binding fragment of any one of claims 1 to 5, which comprises a human lambda light chain constant region.
8. The anti-tau antibody or tau-binding nt of any one of claims 1 to 5, which comprises a human IgG1 heavy chain constant region and a human lambda light chain nt region.
9. The anti-tau antibody or tau binding fragment of claim 1, wherein the antibody is a human dy or a humanized antibody.
10. The anti-tau dy or tau binding fragment of any one of claims 1 to 5, wherein the tau-binding fragment is a single chain Fv fragment (scFv), an F(ab') fragment, an F(ab) fragment or an F(ab')2 fragment.
11. The au antibody or tau binding fragment of claim 1, which is an anti-tau antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid ce of SEQ ID NO:215 or SEQ ID NO:205 and the light chain comprises the amino acid sequence of SEQ ID NO:206.
12. A ceutical composition comprising the anti-tau antibody or tau-binding fragment of any one of claims 1-11, and a pharmaceutically acceptable carrier.
13. A vector or vectors comprising an ed polynucleotide or polynucleotides comprising a nucleotide sequence or nucleotide sequences encoding the anti-tau antibody or tau-binding fragment of any one of claims 1-11.
14. The vector or s of claim 13, wherein the polynucleotide or polynucleotides comprise a sequence of nucleotides ng a VH that comprises the nucleotides as set forth in SEQ ID NO:172 or SEQ ID NO:173 and a sequence of nucleotides encoding a VL that ses the nucleotides as set forth in SEQ ID NO:174.
15. An isolated host cell comprising the vector or vectors of claim 13 or 14.
16. A method of preparing an anti-tau antibody or the nding fragment thereof, the method comprising: culturing the host cell of claim 15 in a cell culture; and isolating the anti-tau dy or tau-binding fragment thereof from the cell culture.
17. The method of claim 16, further comprising formulating the anti-tau antibody or ding nt thereof into a sterile pharmaceutical composition suitable for administration to a human subject.
18. Use of the anti-tau dy or the tau-binding fragment of any one of claims 1-11 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for treating a neurodegenerative tauopathy.
19. The use of claim 18, wherein the neurodegenerative tauopathy is selected from the group consisting of Alzheimer’s e, amyotrophic lateral sclerosis/parkinsonism–dementia complex, argyrophilic grain dementia, British type d athy, cerebral amyloid angiopathy, corticobasal ration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic sonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, ssive supranuclear palsy, subacute sclerosing panencephalitis, Tangle only dementia, multi-infarct dementia and ischemic stroke.
20. The use of claim 18, wherein the neurodegenerative tauopathy is mer’s disease.
21. The use of claim 18, wherein the neurodegenerative tauopathy is frontotemporal dementia with parkinsonism linked to chromosome 17 or frontotemporal lobar ration.
22. The use of claim 18, wherein the neurodegenerative tauopathy is progressive supranuclear palsy.
23. Use of the anti-tau antibody or the tau-binding fragment thereof of any one of claims 1- 11 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for treating al accumulation or deposition of tau in the central nervous system in a human subject in need thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261745410P | 2012-12-21 | 2012-12-21 | |
US61/745,410 | 2012-12-21 | ||
PCT/US2013/076952 WO2014100600A2 (en) | 2012-12-21 | 2013-12-20 | Human anti-tau antibodies |
Publications (2)
Publication Number | Publication Date |
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NZ709976A NZ709976A (en) | 2020-10-30 |
NZ709976B2 true NZ709976B2 (en) | 2021-02-02 |
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