ANTIBODIES TO TAU AND USES THEREOF
The present invention is in the field of medicine. Particularly, the present
invention relates to antibodies to tau, compositions comprising such tau antibodies, and
methods of using such tau antibodies for the treatment of neurodegenerative diseases
including Alzheimer's Disease (AD), Progressive Supranuclear Palsy (PSP), and Pick's
Disease (PD).
Tau is an axonal microtubule binding protein that promotes microtubule assembly
and stability. AD and PSP are neurodegenerative diseases pathologically characterized
by aberrant tau aggregation. More specifically, in AD and PSP, hyperphosphorylated tau
is believed to promote insoluble tau fibril aggregation leading to microtubule
destabilization, and neuronal toxicity. Cell culture and murine model studies have shown
tau aggregates spread across neuronal synapse junctions and sequester monomeric (native
or non-aggregated) tau, inducing tau aggregate formation. Neuroanatomical progression
of tau aggregation and accumulation in neurodegenerative diseases such as AD and PSP
suggests that tau fibril aggregation propagates along neuronal networks, ultimately
resulting in destabilization of microtubules and ultimately localized impaired neuronal
function.
The density and neuroanatomical localization of tau aggregation correlates
strongly with AD and PSP neurologic symptoms and disease progression. For example,
in AD, tau forms intraneuronal neurofibrillary tangles (NFTs), which tend to develop in
sequence from transentorhinal, to limbic, to neocortical regions, and which correlate with
severity of dementia and extent of neuronal loss. In PSP, tau aggregation is seen in
neurons, astrocytes, and oligodendrocytes within subcortical and cortical regions, and the
density of aggregated tau has been shown to correlate with the severity of neuronal loss.
Antibodies to tau are known. For example, U.S. Patent No. 8,926,974, and
International Publication Nos. WO2011/026031, WO2012/049570, and WO2013/050567
disclose antibodies to tau and uses of tau antibodies for the treatment of
neurodegenerative diseases such as AD. However, to date no antibody targeting tau has
been approved for therapeutic use and there are currently no approved disease modifying
therapies for AD or PSP. Thus, there remains a need for alternative tau antibodies. In
particular, there remains a need for alternative tau antibodies which specifically bind tau
aggregates and which reduce the propagation of tau aggregate formation, NFT formation
and neuronal loss. Such tau antibodies preferably also possess good physical-chemical
properties to facilitate development, manufacturing, and/or formulation.
The present invention provides a monoclonal antibody that binds human tau and
which comprises a light chain variable region (LCVR) and a heavy chain variable region
(HCVR), wherein the LCVR comprises complementarity determining regions (CDRs)
LCDR1, LCDR2 and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2 and
HCDR3. According to particular embodiments of the present invention the amino acid
sequence of LCDR1 is given by SEQ ID NO.3, the amino acid sequence of LCDR2 is
given by SEQ ID NO.4, the amino acid sequence of LCDR3 is given by SEQ ID NO.5,
the amino acid sequence of HCDR1 is given by SEQ ID NO.6, the amino acid sequence
of HCDR2 is given by SEQ ID NO.7, and the amino acid sequence of HCDR3 is given
by SEQ ID NO.8. In an embodiment, the present invention provides a monoclonal
antibody that binds human tau, comprising a LCVR and a HCVR, wherein the amino acid
sequence of the LCVR is given by SEQ ID NO.9 and the amino acid sequence of the
HCVR is given by SEQ ID NO. 10. In a further embodiment, the present invention
provides a monoclonal antibody that binds human tau, comprising a light chain (LC) and
a heavy chain (HC), wherein the amino acid sequence of the LC is given by SEQ ID
NO.l and the amino acid sequence of the HC is given by SEQ ID NO.2.
The present invention provides a monoclonal antibody that binds human tau. In
an embodiment, the present invention provides a monoclonal antibody that binds a
conformational epitope of human tau. In a particular embodiment, the conformational
epitope of human tau includes amino acid residues 7-9 and 312-322 of human tau,
wherein the amino acid sequence of the human tau is given by SEQ ID NO. 13.
The present invention further provides pharmaceutical compositions comprising a
monoclonal antibody of the present invention and one or more pharmaceutically
acceptable carriers, diluents or excipients. Further, the present invention provides a
method of treating AD, PSP, or PD comprising administering to a patient in need thereof
a pharmaceutical composition of the present invention.
In addition, the present invention provides a method of treating neurodegenerative
diseases. More particularly, the present invention provides a method of treating AD, PSP,
or PD comprising administering to a patient in need thereof an effective amount of a
monoclonal antibody of the present invention.
The present invention also provides the monoclonal antibody of the present
invention for use in therapy. More particularly, the present invention also provides the
monoclonal antibody of the present invention for use in treatment of AD, PSP, or PD.
In an embodiment, the present invention provides the use of the monoclonal
antibody of the present invention in the manufacture of a medicament for the treatment of
AD, PSP, or PD.
The present invention also relates to nucleic acid molecules and expression
vectors encoding the monoclonal antibody of the present invention. In an embodiment,
the present invention provides a DNA molecule comprising a polynucleotide sequence
encoding a polypeptide having the amino acid sequence of SEQ ID NO. 1. In an
embodiment, the present invention provides a DNA molecule comprising a
polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ
ID NO.2. In a further embodiment, the present invention provides a DNA molecule
comprising a polynucleotide sequence encoding a polypeptide having the amino acid
sequence of SEQ ID NO.l, and comprising a polynucleotide sequence encoding a
polypeptide having the amino acid sequence of SEQ ID NO.2. In a particular
embodiment the polynucleotide sequence encoding a polypeptide having the amino acid
sequence of SEQ ID NO.l is given by SEQ ID NO.l 1 and the polynucleotide sequence
encoding a polypeptide having the amino acid sequence of SEQ ID NO.2 is given by SEQ
ID NO. 12.
Further, the present invention provides a monoclonal antibody prepared according
to a process, wherein said process comprises cultivating a host cell comprising a
polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ
ID NO.l and a polynucleotide sequence encoding a polypeptide having the amino acid
sequence of SEQ ID NO.2, under conditions such that the monoclonal antibody is
expressed, and recovering from said host cell a monoclonal antibody comprising a LC
and a HC, wherein the amino acid sequence of the LC is given by SEQ ID NO.l and the
amino acid sequence of the HC is given by SEQ ID NO.2.
As used herein, an "antibody" is an immunoglobulin molecule comprising 2 HCs
and 2 LCs interconnected by disulfide bonds. The amino terminal portion of each LC and
HC includes a variable region of about 100-120 amino acids primarily responsible for
antigen recognition via the CDRs contained therein. The CDRs are interspersed with
regions that are more conserved, termed framework regions ("FR"). Each LCVR and
HCVR is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxyterminus
in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3
CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3," and the 3 CDRs of
the HC are referred to as "HCDR1, HCDR2, and HCDR3." The CDRs contain most of
the residues which form specific interactions with the antigen. The functional ability of
an antibody to bind a particular antigen is largely influenced by the six CDRs.
Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the
antibodies of the present invention is based on the well-known Kabat numbering
convention (Kabat, et al., Ann. NYAcad. Sci. 190:382-93 (1971); Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and
Human Services, NIH Publication No. 91-3242 (1991)), and North numbering convention
(North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of
Molecular Biology, 406:228-256 (2011)).
LCs are classified as kappa or lambda, which are each characterized by a
particular constant region as known in the art. The monoclonal antibodies of the present
invention include kappa LCs. HCs are classified as gamma, mu, alpha, delta, or epsilon,
and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. The
monoclonal antibodies of the present invention include IgG HCs. IgG antibodies can be
further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4. In a particular
embodiment, the monoclonal antibodies of the present invention are IgG4. The carboxyterminal
portion of each HC defines a constant region primarily responsible for effector
function. In a particular embodiment, the monoclonal antibodies of the present invention
have one or more modifications in the constant region of each HC that reduces effector
function. In a more particular embodiment, the monoclonal antibodies of the present
invention are IgG4 and have modifications in the constant region of both HCs that reduce
effector function including the amino acid alanine at both residues 230 and 231 (residue
numbering based on the exemplified HC of SEQ ID NO.2). In an even more particular
embodiment, the monoclonal antibodies of the present invention are IgG4 and have
modifications in the constant region of both HCs that reduce effector function including
the amino acid alanine at both residues 230 and 231 and have further modifications in the
constant region of both HCs promoting stability including the amino acid proline at
residue 224 and the deletion of the amino acid lysine at residue 443 (residue numbering
based on the exemplified HC of SEQ ID NO.2).
The antibodies of the present invention are monoclonal antibodies ("mAbs"). The
mAbs for the present invention are complete mAbs containing 2 HCs and 2 LCs. As
referred to herein, mAbs are antibodies derived from a single copy or clone including, for
example, any eukaryotic, prokaryotic or phage clone, and not the method by which it is
produced. Monoclonal antibodies can be produced, for example, by hybridoma
technologies, recombinant technologies, phage display technologies, synthetic
technologies, e.g., CDR-grafting, or combinations of such or other technologies known in
the art.
Methods of producing and purifying antibodies are well known in the art and can
be found, for example, in Harlow and Lane (1988), Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring harbor, N.Y., chapters 5-8 and 15,
ISBN 0-87969-314-2. For example, mice can be immunized with human tau paired
helical filaments ("PHF") from brain tissue of patients characterized as having AD (Jicha
et al., J. Neurosci. Res., 15:48(2), 128-132 (April, 1997)), and the resulting antibodies can
be recovered, purified, and the amino acid sequences determined using conventional
methods well known in the art. The monoclonal antibodies of the present invention are
engineered to contain one or more human framework regions surrounding CDRs derived
from a non-human antibody. Human framework germline sequences can be obtained
from ImMunoGeneTics (INGT) via their website, http://imgt.cines.fr, or from The
Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic
Press, 2001, ISBN 01244 1351. According to particular embodiments of the present
invention, particular germline HC framework and LC framework regions for use in
monoclonal antibodies of the present invention include 5-51 and A27, respectively.
In particular embodiments of the present invention, the antibody, or the nucleic
acid encoding same, is provided in isolated form. As used herein, the term "isolated"
refers to a protein, peptide, or nucleic acid which is free or substantially free from other
macromolecular species found in a cellular environment.
The monoclonal antibodies of the present invention may be prepared and purified
using known methods. For example, cDNA sequences encoding a HC (for example the
amino acid sequence given by SEQ ID NO.2) and a LC (for example, the amino acid
sequence given by SEQ ID NO.l) may be cloned and engineered into a GS (glutamine
synthetase) expression vector. The engineered immunoglobulin expression vector may
then be stably transfected into CHO cells. As one of skill in the art will appreciate,
mammalian expression of antibodies will result in glycosylation, typically at highly
conserved N-glycosylation sites in the Fc region. Stable clones may be verified for
expression of an antibody specifically binding to tau aggregates. Positive clones may be
expanded into serum-free culture medium for antibody production in bioreactors. Media,
into which an antibody has been secreted, may be purified by conventional techniques.
For example, the medium may be conveniently applied to a Protein A or G Sepharose FF
column that has been equilibrated with a compatible buffer, such as phosphate buffered
saline. The column is washed to remove nonspecific binding components. The bound
antibody is eluted, for example, by pH gradient and antibody fractions are detected, such
as by SDS-PAGE, and then pooled. The antibody may be concentrated and/or sterile
filtered using common techniques. Soluble aggregate and multimers may be effectively
removed by common techniques, including size exclusion, hydrophobic interaction, ion
exchange, or hydroxyapatite chromatography. The product may be immediately frozen,
for example at -70°C, or may be lyophilized.
The monoclonal antibodies of the present invention can be used in the treatment
of patients. More particularly the antibodies of the present invention are expected to treat
a class of neurodegenerative disorders, termed tauopathies, which includes AD, PSP, and
PD. Although monoclonal antibodies of the present invention are expected to be useful in
the treatment of AD, PSP, and PD, such antibodies may also be useful in the treatment of
other tauopathies, including chronic traumatic encephalopathy. As used interchangeably
herein, "treatment" and/or "treating" and/or "treat" are intended to refer to all processes
wherein there may be a slowing, interrupting, arresting, controlling, stopping, or reversing
of the progression of the disorders described herein, but does not necessarily indicate a
total elimination of all disorder symptoms. Treatment includes administration of an
antibody of the present invention for treatment of a disease or condition in a human that
would benefit from a reduction in the propagation of at least one of tau aggregate
formation, NFT formation and neuronal loss, and includes: (a) inhibiting further
progression of the disease, i.e., arresting its development; and (b) relieving the disease,
i.e., causing regression of the disease or disorder or alleviating symptoms or
complications thereof.
As used interchangeably herein, the term "patient," "subject," and "individual,"
refers to a human. In certain embodiments, the patient is further characterized with a
disease, disorder, or condition (e.g., a neurodegenerative disorder) that would benefit
from a reduction in the propagation of at least one of tau aggregate formation, NFT
formation, and neuronal loss. In another embodiment, the patient is further characterized
as being at risk of developing a neurodegenerative disorder, disease, or condition that
would benefit from a reduction in the propagation of at least one of tau aggregate
formation, NFT formation, and neuronal loss.
As used herein, the term "bind (or binds)" tau refers to an interaction of an
antibody with an epitope of human tau aggregate. More preferably, the epitope is a
conformational epitope of human tau. In a particular embodiment, the term "bind (or
binds)" tau refers to an interaction with a conformational epitope including amino acid
residues 7-9 and 312-322 of human tau aggregate (residue numbering based on the
exemplified human tau of SEQ ID NO. 13). It should be understood that there are known
variations of human tau protein, for example resulting from splice variants. Such known
variations, however, possess the conformational epitope including amino acid residues 7-
9 and 312-322 of SEQ ID NO. 13. Known variants, however, may result in altered
residue numbering for amino acid residues 7-9 and 312-322 of SEQ ID NO. 13. Although
the residue numbering may be altered in some variants, the amino acids comprising the
epitope remain the same. The term "epitope" as used herein refers to discrete, threedimensional
sites of an antigen that are recognized by the monoclonal antibodies of the
present invention.
A monoclonal antibody of the present invention can be incorporated into a
pharmaceutical composition which can be prepared by methods well known in the art and
comprise a monoclonal antibody of the present invention and one or more
pharmaceutically acceptable carrier(s) and/or diluent(s) (e.g., Remington. The Science
and Practice of Pharmacy, 22nd Edition, Loyd V., Ed., Pharmaceutical Press, 2012, which
provides a compendium of formulation techniques as are generally known to
practitioners). Suitable carriers for pharmaceutical compositions include any material
which, when combined with the monoclonal antibody of the present invention, retains the
molecule's activity and is non-reactive with the patient's immune system.
A pharmaceutical composition comprising a monoclonal antibody of the present
invention can be administered to a patient at risk for, or exhibiting, diseases or disorders
as described herein by parental routes (e.g., subcutaneous, intravenous, intraperitoneal,
intramuscular, or transdermal). A pharmaceutical composition of the present invention
contains an "effective" or "therapeutically effective" amount, as used interchangeably
herein, of a monoclonal antibody of the present invention. An effective amount refers to
an amount necessary (at dosages and for periods of time and for the means of
administration) to achieve the desired therapeutic result. An effective amount of the
monoclonal antibody may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the monoclonal antibody to elicit a desired
response in the individual. An effective amount is also one in which any toxic or
detrimental effects of the monoclonal antibody of the present invention are outweighed by
the therapeutically beneficial effects.
Engineered Tau Antibody
Significant problems associated with chemical and physical stability were
encountered when constructing a monoclonal tau antibody of the present invention.
Problems encountered included low binding affinity, immunogenicity, aggregation, HC
dimerization, as well as variable region deamidation, oxidation, isomerization and
misfolding.
For example, murine IgGl antibody MC-1 ("MC-l")(Albert Einstein College of
Medicine, Jicha et al., 1997), which recognizes a conformational epitope of tau protein at
amino acid residues 7-9 and 312-322 (residue numbering based on exemplified human tau
protein having the amino acid sequence of SEQ ID NO. 13), was initially humanized by
engineering the three MC-1 murine HC CDRs into multiple human HC framework
germline genes and the three MC-1 murine LC CDRs into multiple human LC framework
germline genes. Humanized constructs of MC-1 utilized 96 different combinations of
heavy- and light-chain frameworks, representing each of the twelve HC framework
germline families (specific human HC frameworks: 1-24, 1-46, 1-69, 2-05, 3-15, 3-23, 3-
53, 3-72, 4-04, 4-39, 5-51, and 6-01) and each of the eight LC germline families (specific
human LC frameworks: A-26, A-27, B-2, B-3, L-2, L-12, Oil, and 0-2). The respective
framework germline genes were cloned into heavy and light chain human IgG4
expression vectors and transfected into HEK293 cells for expression and analysis of
binding by ELISA. Although multiple framework pairs demonstrated some level of
binding to human tau in ELISA, resulting antibody constructs displayed a myriad of
issues including poor binding affinity, aggregation, HC dimerization, and chemical
stability issues such as deamidation, oxidation, and isomerization in the variable regions.
Modifications were therefore engineered to develop tau antibodies possessing
improved binding affinity, eliminated or reduced HC dimerization, reduced
immunogenicity, and improved chemical and physical stability. Amino acid
modifications (relative to MC-1, Jicha et al., 1997) were engineered in HCDR2 and
HCDR3, and LCDR1, LCDR2, and LCDR3. The modified murine antibody was
humanized by engineering the three HC CDRs into multiple human HC framework
germline genes and the three LC CDRs into multiple human LC framework germline
genes essentially as described above. Further, extensive protein stability studies were
performed and the engineered monoclonal antibodies were screened for expression and
thermostability properties as well as binding affinity properties. A monoclonal antibody
containing seven CDR mutations (amino acid position is based on linear amino acid
residue numbering of an exemplified antibody of the present invention reflected in Table
1: HCDR2 at N61E and E62K; HCDR3 at P103V and Y105D; LCDR1 at G34Q;
LCDR2 at S57D; and LCDR3 at H98L) was identified as improving the binding affinity,
chemical and physical stability, and immunogenicity for monoclonal antibodies of the
present invention (relative to MC-1, Jicha et al., 1997). None of the above modifications
were identified in characterizations of MC-1 or the humanized MC-1 antibody constructs.
An exemplified engineered tau monoclonal antibody of the present invention is
presented in Table 1. The exemplified engineered tau monoclonal antibody includes
human HC framework 5-51 and human LC framework A27. The relationship of the
various regions of the exemplified engineered tau monoclonal antibody is as follows
(numbering of amino acids applies linear numbering; assignment of amino acids to
variable domains is based on the International Immunogenetics Information System®
available at www.imgt.org; assignment of amino acids to CDR domains is based on the
well-known North numbering convention, with the exception of HCDR2 which is based
on the well-known Kabat numbering convention):
Table 1: Amino acid regions of an exemplified engineered tau monoclonal antibody of
the present invention.
The following Examples and assays demonstrate that the monoclonal antibodies
of the present invention are useful for treating neurodegenerative disorders associated
with propagation of tau aggregates such as AD, PSP, or PD. It should be understood
however, that the following Examples are set forth by way of illustration and not
limitation, and that various modifications may be made by one of ordinary skill in the art.
Examples
Expression of Engineered Tau Antibody
Engineered tau monoclonal antibodies of the present invention can be expressed
and purified essentially as follows. A glutamine synthetase (GS) expression vector
containing the DNA sequence of SEQ ID NO.l 1 (encoding LC amino acid sequence of
SEQ ID NO.l) and the DNA sequence of SEQ ID NO. 12 (encoding HC amino acid
sequence of SEQ ID NO.2) is used to transfect a Chinese hamster ovary cell line (CHO)
by electroporation. The expression vector encodes an SV Early (Simian Virus 40E)
promoter and the gene for GS. Expression of GS allows for the biochemical synthesis of
glutamine, an amino acid required by the CHO cells. Post-transfection, cells undergo
bulk selection with 50mM L-methionine sulfoximine (MSX). The inhibition of GS by
MSX is utilized to increase the stringency of selection. Cells with integration of the
expression vector cDNA into transcriptionally active regions of the host cell genome can
be selected against CHO wild type cells, which express an endogenous level of GS.
Transfected pools are plated at low density to allow for close-to-clonal outgrowth of
stable expressing cells. The masterwells are screened for antibody expression and then
scaled up in serum-free, suspension cultures to be used for production. Clarified medium,
into which the antibody has been secreted, is applied to a Protein A affinity column that
has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH
7.4). The column is washed with 1M NaCl to remove nonspecific binding components.
The bound tau monoclonal antibody is eluted, for example, with sodium citrate at pH
(approx.) 3.5 and fractions are neutralized with 1M Tris buffer. Tau monoclonal antibody
fractions are detected, such as by SDS-PAGE or analytical size-exclusion, and then are
pooled. Soluble aggregate and multimers may be effectively removed by common
techniques, including size exclusion, hydrophobic interaction, ion exchange, or
hydroxyapatite chromatography. The tau monoclonal antibody of the present invention is
concentrated and/or sterile filtered using common techniques. The purity of the tau
monoclonal antibody after these chromatography steps is greater than 95%. The tau
monoclonal antibody of the present invention may be immediately frozen at -70°C or
stored at 4°C for several months.
Binding Kinetics and Affinity
Surface Plasmon Resonance (SPR) assay, measured with a BIACORE 2000
instrument (primed with HBS-EP+ running buffer (GE Healthcare, 10 mM Hepes pH7.4
+ 150 mM NaCl + 3 mM EDTA + 0.05% surfactant P20) at 25°C), is used to measure
binding of exemplified tau monoclonal antibody of Example 1 to both human monomeric
(e.g., native or non-aggregate) tau and human tau aggregates (both having the amino acid
sequence as set forth in SEQ ID NO: 13). Binding of humanized MC-1 antibody construct
(having the framework combination: 5-51 heavy-chain, A27 light-chain) to human
monomeric tau and human tau aggregate is measured in the same manner.
Except as noted, all reagents and materials are from BIACORE® AB (Upsala,
Sweden). A CM5 chip containing immobilized protein A (generated using standard
NHS-EDC amine coupling) on all four flow cells (FC) is used to employ a capture
methodology. Antibody samples are prepared at O^g/mL by dilution into running
buffer. Monomeric tau and fibril tau are prepared to concentrations of 2000, 1000, 500,
250, 125, 62.5, 31.25, 15.63, 7.82, 3.91, 1.95, and 0 (blank) nM by dilution into running
buffer. Each analysis cycle consists of: (1) capturing antibody samples on separate flow
cells (FC2, FC3, and FC4); (2) injection of 250mI (300 sec) of either monomeric tau or
tau fibril aggregate over respective FC at a rate of 50 mI h h; (3) return to buffer flow for
20 mins. to monitor dissociation phase; (4) regeneration of chip surfaces with 25 mI_, (30
sec) injection of glycine, pH1.5; (5) equilibration of chip surfaces with a 50 mI_, (60 sec)
injection ofHBS-EP+.
Data of binding to tau aggregate is processed using standard double-referencing
and fit to a 1:1 binding model using Biacore 2000 Evaluation software, version 4.1, to
determine the association rate (ko , M Y units), dissociation rate (ko , s units), and Rmax
(RU units). The equilibrium dissociation constant (K ) was calculated from the
relationship K = k0f ko , and is in molar units. Data of binding to monomeric tau cannot
be determined accurately by SPR as described above due to rapid on- and off-rates.
Therefore, for binding to monomeric tau is obtained by using a steady state binding fit
model from plotting the concentration of antigen versus the response unit. Resulting
binding data is provided in Table 2.
Table 2 : SPR binding data to both human monomeric and aggregate tau.
results are considered relative as the results are not normalized for influence of
avidity.
The results provided in Table 2 demonstrate tau monoclonal antibody of Example
1 does not possess measureable binding to monomeric tau such that an affinity value can
be accurately determined by Biacore analysis (due to rapid on- and off-rates).
Conversely, the results provided in Table 2 demonstrate tau monoclonal antibody of
Example 1 possesses improved affinity to tau aggregate compared to humanized MC-1
antibody construct.
Enzyme-Linked Immunosorbant Assay (ELISA) is used to determine relative
binding affinity of the exemplified tau monoclonal antibody of Example 1 to aggregate
tau fibrils from AD brain homogenates. AD brain homogenates are prepared from
approx. 80g of cortex from brain of AD patients. Briefly, buffer (TBS/lmM PMSF/1X
Complete® protease inhibitor cocktail (Roche, p/n. 11 697 498 001) and phosphatase
inhibitor (ThermoFischer, p/n. 78428)) is added to the AD brain tissue at about lOml/lg
(tissue). Tissue is homogenized using a handheld Kinematica Polytron at speed 6-7.
Tissue is then further homogenized using Parr Bomb (Parr Instrument, p/n. 4653) at 1500
psi of nitrogen for 30 mins. Homogenate is spun at 28,000g (J14 Beckman rotor) for 30
min at 4 C. Supernatant is collected, pooled and run over a 4 cm high guard column of
Sepharose 400 Superflow to remove larger debris, then run over 25 ml MCl-Affigel 10
column at a flow rate of 50 - 60 ml per hour, in order to purify MC1-binding tau fibrils.
To maximize the recovery of purification, supernatants are recycled through MC-1
column over 18-20 hours at 4°C. Guard column is removed and MCI column is washed
with TBS with at least 40 column volumes. Bound tau aggregates are then eluted with 2
column volumes of 3M KSCN, collecting in approx. 1ml fractions. Protein concentration
in each eluted fraction is checked by microtiter plate Bradford assay. Fractions
containing positive protein levels are pooled, concentrated to about 2ml using Centricon
(Millipore Ultracel-30K) at 4°C, and dialyzed using a Slide-A-Lyzer cassette (10K
MWCO 3-12ml, Pierce) overnight against 1 liter TBS. The concentration of tau within
the tau fibrils purified from AD brain homogenate is measured by sandwich ELISA using
DA-9 capture antibody and CP27 detection antibody.
Purified tau fibrils (50m1) in PBS are coated on wells of 96-well plates (Coastar,
p/n. 3690) at a concentration corresponding to 0.7 mg/ml of total tau. Plates are incubated
overnight at 4°C, then washed three times with 150m1of PBST (PBS containing 0.05%
Tween-20), blocked in IOOmI BB3 (ImmunoChemistry Technology, p/n. 643) at room
temperature for at least 1 hr (usually 2hrs). Following blocking, the blocking buffer is
removed from the wells. Exemplified tau monoclonal antibody of Example 1 and a
humanized MC-1 antibody construct (having the framework combination: 5-51 heavy

WE CLAIM:
1. A monoclonal antibody that binds human tau comprising a light chain variable region
(LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises
complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 and the
HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein the amino acid
sequence of LCDR1 is given by SEQ ID NO: 3, the amino acid sequence of LCDR2
is given by SEQ ID NO: 4, the amino acid sequence of LCDR3 is given by SEQ ID
NO: 5, the amino acid sequence of HCDR1 is given by SEQ ID NO: 6, the amino acid
sequence of HCDR2 is given by SEQ ID NO: 7, and the amino acid sequence of
HCDR3 is given by SEQ ID NO: 8.
2. The monoclonal antibody of Claim 1, comprising a light chain variable region (LCVR)
and a heavy chain variable region (HCVR), wherein the amino acid sequence of the
LCVR is given by SEQ ID NO: 9 and the amino acid sequence of the HCVR is given
by SEQ ID NO: 10.
3. The monoclonal antibody of any of Claims 1-2, comprising a light chain (LC) and a
heavy chain (HC), wherein the amino acid sequence of the LC is given by SEQ ID
NO: 1 and the amino acid sequence of the HC is given by SEQ ID NO: 2.
4. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide
having the amino acid sequence of SEQ ID NO: 1.
5. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide
having the amino acid sequence of SEQ ID NO: 2.
6. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide
having the amino acid sequence of SEQ ID NO: 1, and comprising a polynucleotide
sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
7. A mammalian cell comprising the DNA molecule of Claim 4 and the DNA molecule
of Claim 5, wherein the cell is capable of expressing a monoclonal antibody
comprising a light chain having an amino acid sequence of SEQ ID NO: 1 and a
heavy chain having an amino acid sequence of SEQ ID NO: 2.
8. A mammalian cell comprising the DNA molecule of Claim 6, wherein the cell is
capable of expressing a monoclonal antibody comprising a light chain having an
amino acid sequence of SEQ ID NO: 1 and a heavy chain having an amino acid
sequence of SEQ ID NO: 2.
9. A process for producing a monoclonal antibody comprising a light chain having an
amino acid sequence of SEQ ID NO: 1 and a heavy chain having an amino acid
sequence of SEQ ID NO: 2, the process comprising cultivating the mammalian cell of
any one of Claims 7 and 8 under conditions such that the monoclonal antibody is
expressed, and recovering the expressed monoclonal antibody.
10. A monoclonal antibody produced by the process of Claim 9.
11. A pharmaceutical composition comprising a monoclonal antibody of any one of
Claims 1-3 and one or more pharmaceutically acceptable carriers, diluents or
excipients.
12. A method of treating Alzheimer's disease, Progressive Supranuclear Palsy or Pick's
disease comprising administering to a patient in need thereof an effective amount of a
monoclonal antibody of any one of Claims 1-3.
13. A method of treating Alzheimer's disease, Progressive Supranuclear Palsy or Pick's
disease comprising administering to a patient in need thereof the pharmaceutical
composition of Claim 11.
14. A monoclonal antibody of any one of Claims 1-3 for use in therapy.
15. A monoclonal antibody of any one of Claims 1-3 for use in the treatment of a disease
selected from the group consisting of Alzheimer's disease, Progressive Supranuclear
Palsy and Pick's disease.
16. A monoclonal antibody of any one of Claims 1-3 for use in the manufacturer of a
medicament for the treatment of Alzheimer's disease, Progressive Supranuclear Palsy
or Pick's disease.