U.S. patent application number 15/612695 was filed with the patent office on 2018-02-08 for antibodies to tau.
The applicant listed for this patent is Washington University. Invention is credited to Marc Diamond, Brandon Holmes, David Holtzman, Hong Jiang, Najla Kfoury.
Application Number | 20180037641 15/612695 |
Document ID | / |
Family ID | 49882495 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180037641 |
Kind Code |
A1 |
Diamond; Marc ; et
al. |
February 8, 2018 |
ANTIBODIES TO TAU
Abstract
This invention relates to antibodies to tau and methods of use
thereof.
Inventors: |
Diamond; Marc; (St. Louis,
MO) ; Jiang; Hong; (St. Louis, MO) ; Holtzman;
David; (St. Louis, MO) ; Kfoury; Najla; (St.
Louis, MO) ; Holmes; Brandon; (St. Louis,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Washington University |
St. Louis |
MO |
US |
|
|
Family ID: |
49882495 |
Appl. No.: |
15/612695 |
Filed: |
June 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14412309 |
Dec 31, 2014 |
9834596 |
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PCT/US2013/049333 |
Jul 3, 2013 |
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15612695 |
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61667515 |
Jul 3, 2012 |
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61694989 |
Aug 30, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/28 20180101;
C07K 2317/76 20130101; A61P 25/00 20180101; C07K 2317/565 20130101;
C07K 2317/34 20130101; C07K 2317/92 20130101; A61P 43/00 20180101;
A61P 25/18 20180101; G01N 33/6896 20130101; A61K 2039/505 20130101;
G01N 2800/2821 20130101; C07K 16/18 20130101; A61P 25/14
20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; G01N 33/68 20060101 G01N033/68 |
Goverment Interests
GOVERNMENT SUPPORT
[0001] This invention was made with government support under
1R01NS071835 awarded by National Institute of Neurological
Disorders and Stroke. The government has certain rights in the
invention.
Claims
1. An isolated monoclonal anti-tau antibody, wherein the antibody
recognizes an epitope within an amino acid sequence selected from
the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID
NO: 8.
2. The isolated monoclonal anti-tau antibody of claim 1, wherein
the antibody comprises an amino acid sequence selected from the
group consisting of SEQ ID NO: 14 and SEQ ID NO: 15.
3. The isolated monoclonal anti-tau antibody of claim 1, wherein
the antibody is encoded by a nucleic acid sequence comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 12 and SEQ ID NO: 13.
4. An isolated monoclonal anti-tau antibody, wherein the antibody
comprises a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 18 with zero to two amino acid substitutions.
5. An isolated monoclonal anti-tau antibody, wherein the antibody
comprises a heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 21 with zero to two amino acid substitutions.
6. An isolated monoclonal anti-tau antibody of any of the preceding
claims, wherein the antibody is selected from the group consisting
of a single-chain antibody, an antibody fragment, a chimeric
antibody, or a humanized antibody.
7. An isolated monoclonal anti-tau antibody of claim 1, wherein the
antibody is specifically able to block tau seeding activity in a
cellular tau aggregation assay.
8. A method for reducing the spread of tau aggregation in the brain
of a subject, the method comprising administering a
pharmacologically effective amount of an isolated monoclonal
anti-tau antibody to the subject, wherein the isolated monoclonal
anti-tau antibody is an isolated monoclonal anti-tau antibody
according to claim 1.
9. The method of claim 8, wherein the method further comprises
improving in a subject at least one symptom associated with tau
aggregation.
10. The method of claim 9, wherein the at least one symptom
associated with tau aggregation is selected from the group
consisting of tau pathology, impaired cognitive function, altered
behavior, abnormal language function, emotional dysregulation,
seizures, impaired nervous system structure or function, and an
increased risk of development of Alzheimer's disease.
11. The method of claim 8, wherein the administration comprises an
effective systemic route of administration.
12. The method of claim 8, wherein the administration comprises an
effective local route of administration, including directly within
the central nervous system.
13. An immunoassay comprising at least two isolated monoclonal
anti-tau antibody according to claim 1.
14. The immunoassay of claim 13, wherein the immunoassay comprises
at least two captures antibodies and a detection antibody, and
wherein each capture antibody is an isolated monoclonal anti-tau
antibody that recognizes a tau epitope distinct from the other.
15. The immunoassay of claim 14, wherein a first capture antibody
is an isolated monoclonal anti-tau antibody that recognizes an
epitope within SEQ ID NO: 7, a second capture antibody is an
isolated monoclonal anti-tau antibody that recognizes an epitope
within SEQ ID NO: 8, and a detection antibody is an isolated
monoclonal anti-tau antibody that recognizes an epitope within SEQ
ID NO: 8.
16. A method of measuring the amount of tau aggregate in a sample
of a biological fluid obtained from a subject, the method
comprising measuring the amount of tau aggregate using an
immunoassay of claim 15.
17. The method of claim 16, wherein the tau aggregate is
immunoprecipitated from the sample using an isolated monoclonal
anti-tau antibody and then the amount of immunoprecipitated tau
aggregate is measured.
Description
FIELD OF THE INVENTION
[0002] This invention relates to antibodies to tau and methods of
use thereof.
BACKGROUND OF THE INVENTION
[0003] Aggregation of the microtubule associated protein tau is
associated with several neurodegenerative disorders, including
Alzheimer's disease (AD) and frontotemporal dementia. In AD,
pathological tau aggregation spreads progressively throughout the
brain, possibly along existing neural networks. AD is the most
common cause of dementia and is an increasing public health
problem. It is currently estimated to afflict 5 million people in
the United States, with an expected increase to 13 million by the
year 2050. Alzheimer's Disease leads to loss of memory, cognitive
function, and ultimately loss of independence. It takes a heavy
personal and financial toll on the patient and the family. Because
of the severity and increasing prevalence of the AD and other
neurodegenerative diseases associated with aggregation of tau in
the population, it is urgent that better treatments and detection
methods be developed.
REFERENCE TO COLOR FIGURES
[0004] The application file contains at least one photograph
executed in color. Copies of this patent application publication
with color photographs will be provided by the Office upon request
and payment of the necessary fee.
BRIEF DESCRIPTION OF THE FIGURES
[0005] FIGS. 1A and 1B depict the amino acid sequence for
N-terminal (FIG. 1A) and C-terminal (FIG. 1B) human tau (htau).
[0006] FIGS. 2A and 2B depict graphs showing the KD for HJ8.1
towards Human Tau (FIG. 2A) and Mouse Tau (FIG. 2B).
[0007] FIGS. 3A and 3B depict graphs showing the KD for HJ8.2
towards Human Tau (FIG. 3A) and Mouse Tau (FIG. 3B).
[0008] FIGS. 4A and 4B depict graphs showing the KD for HJ8.3
towards Human Tau (FIG. 4A) and Mouse Tau (FIG. 4B).
[0009] FIGS. 5A and 5B depict graphs showing the KD for HJ8.4
towards Human Tau (FIG. 5A) and Mouse Tau (FIG. 5B).
[0010] FIGS. 6A and 6B depict graphs showing the KD for HJ8.5
towards Human Tau (FIG. 6A) and Mouse Tau (FIG. 6B).
[0011] FIGS. 7A and 7B depict graphs showing the KD for HJ8.7
towards Human Tau (FIG. 7A) and Mouse Tau (FIG. 7B).
[0012] FIGS. 8A and 8B depict graphs showing the KD for HJ8.8
towards Human Tau (FIG. 8A) and Mouse Tau (FIG. 8B).
[0013] FIGS. 9A and 9B depict graphs showing the KD for HJ9.1
towards Human Tau (FIG. 9A) and Mouse Tau (FIG. 9B).
[0014] FIGS. 10A and 10B depict graphs showing the KD for HJ9.2
towards Human Tau (FIG. 10A) and Mouse Tau (FIG. 10B).
[0015] FIGS. 11A and 11B depict graphs showing the KD for HJ9.3
towards Human Tau (FIG. 11A) and Mouse Tau (FIG. 11B).
[0016] FIGS. 12A and 12B depict graphs showing the KD for HJ9.4
towards Human Tau (FIG. 12A) and Mouse Tau (FIG. 12B).
[0017] FIGS. 13A and 13B depict graphs showing the KD for HJ9.5
towards Human Tau (FIG. 13A) and Mouse Tau (FIG. 13B).
[0018] FIGS. 14A, 14B and 14C depict immunoblots showing the
presence of full-length tau in ISF of wild-type and P301 S tg mice.
(FIG. 14A) Hippocampal lysates from Tau KO (KO), wild-type (WT),
and P301 S tg (P301 S tg) mice were analyzed by immunoblot with the
anti-tau antibody BT-2 or anti-actin antibody. Thirteen micrograms
of protein were loaded per well. Four bands corresponding to
endogenous murine tau and one band corresponding to human tau are
indicated as white circles and a black circle, respectively. There
is also a 39 kDa band representing a form of human tau in the P301
S tg hippocampal lysate. This may represent a tau degradation
product. ISF tau from wild-type (WT) and P301 S tg (P301 S tg) mice
was immunoprecipitated by anti-tau monoclonal antibodies HJ9.3
(FIG. 14B) or HJ8.1 (FIG. 14C) and analyzed by immunoblot. The
bands were visualized by biotinylated BT-2 antibody. The gray and
black arrows indicate endogenous murine tau and human tau,
respectively.
[0019] FIGS. 15A, 15B, 15C and 15D. FIG. 15A illustrates a
schematic representation of the different mutant tau constructs
used in this study, and (FIGS. 15B-15D) depict images showing Tau
RD proteins form fibrillar aggregates in transfected HEK293 cells.
(FIG. 15A) Depending on the experimental design, each form of
mutant tau was either fused at the carboxyl terminus to cyan or
yellow fluorescent protein (CFP or YFP), or to a hemagglutinin (HA)
tag. (FIG. 15B) Atomic force microscopy (AFM) performed on
SDS-insoluble material from HEK293 cells transiently transfected
with the various forms of RD reveals that RD(.DELTA.K)-HA and
RD(LM)-HA produced obvious fibrillar species. No fibrils were
detected in the aggregation-resistant RD(PP)-HA. (n=2), Scale bars,
1 .mu.m. (FIG. 15C) HEK293 cells transiently transfected with the
various forms of RD-YFP and YFP alone were stained with X-34, an
amyloid-specific dye. Inclusions formed by RD(wt)-YFP,
RD(.DELTA.K)-YFP and RD(LM)-YFP, visualized by confocal microscopy,
also stained positive for X-34. No X-34 positive cells were
detected upon expression of YFP alone or RD(PP)-YFP. Arrows
indicate inclusions stained with X-34. (n=3) (FIG. 15D)
Non-Transfected cells (NT) and various forms of RD-YFP/CFP were
transfected into HEK293 cells, followed by Triton/SDS extraction
and Western blotting using an antibody against the RD region. Both
monomer and higher order molecular weight species were detected.
(S=Soluble protein and P=Pellet insoluble protein). This was
repeated three times with identical results.
[0020] FIGS. 16A, 16B and 16C show Tau RD aggregates in HEK293
cells are detected by FRET. To quantitate intracellular RD protein
aggregation by fluorescence resonance energy transfer (FRET),
various RD mutants (wt, .DELTA.K, PP, LM) fused to YFP and CFP were
co-transfected into HEK293 cells. (FIG. 16A). HEK293 cells
co-transfected with RD(LM)-CFP/YFP were imaged and intracellular
aggregate formation was quantified using FRET acceptor
photobleaching microscopy. Donor signal before (Pre) and after
(Post) acceptor photobleaching confirmed that RD(LM)-CFP/YFP
inclusions produced a mean FRET efficiency of 18.2%.+-.0.058 SD
(n=6). The upper and lower panels depict the acceptor and donor
channels, respectively, before and after photobleaching. The top
right image is a representative heat map of the calculated FRET
efficiency. The scale bar of the histogram depicts the calculated
FRET efficiency on a pixel-by-pixel basis. The FRET efficiency of
Tau RD aggregate was -34% in this cell. (FIG. 16B). Using a FPR,
relative FRET from various constructs was determined. No
significant FRET from RD(PP)-CFP/YFP was observed. However,
RD(.DELTA.K)-CFP/YFP and RD(LM)-CFP/YFP each produced a strong FRET
signal (n=3). (FIG. 16C). HEK293 cells expressing
RD(.DELTA.K)-CFP/YFP were exposed to various concentrations of
RD(wt)-HA fibrils (monomer equivalents of 0.01, 0.03, 0.1 and 0.3
.mu.M) for 9 h. Extracellular RD(wt)-HA fibrils dose-dependently
induced aggregation of RD(.DELTA.K)-CFP/YFP (n=3). (* indicates a
p-value <0.05, ** indicates a p-value <0.001, error bars
represent the SEM).
[0021] FIGS. 17A, 17B and 17C depict images and graphs showing
Tau-RD aggregates transfer between cells and induce further
aggregation. (FIG. 17A). HEK293 cells transfected with
RD(.DELTA.K)-YFP were co-cultured for 48 h with an equivalent
number of cells expressing RD(LM)-HA. Cells were fixed with 4%
paraformaldehyde and immunofluorescence/X-34 staining was
performed. Multiple cells showed colocalization of RD(LM)-HA and
RD(.DELTA.K)-YFP within inclusions. These inclusions also stained
positive for X-34, indicating beta sheet structure (solid arrows).
In addition, some RD(LM)-HA inclusions stained positive for X-34
but did not colocalize with RD(.DELTA.K)-YFP inclusions (open
arrow). (FIG. 17B). Two populations of cells, one expressing
RD(.DELTA.K)-CFP/YFP, and the other expressing RD(LM)-HA, were
co-cultured for 48 h. RD(PP)-HA or non-transfected cells, NT, were
used as controls. FRET was increased by co-culture with RD(LM)-HA,
but not with RD(PP)-HA, or mock-transfected cells (n=3). (FIG.
17C). To test for cell death induced by tau aggregates as a
mechanism of tau release, HEK293 cells were transfected for 48 h
with RD-HA (PP, .DELTA.K, or LM), or were mock-transfected.
Mock-transfected cells were treated with varying concentrations of
staurosporine (1, 2, 4, 20 .mu.M) for 30 minutes at 37.degree. C.
to induce cell death. Cells were then exposed to 5 .mu.g/ml of
propidium iodide and fluorescence was determined via plate reader.
No evidence for cell death in the various transfected populations
was observed. (** indicates a p-value <0.001, error bars
represent the SEM).
[0022] FIGS. 18A, 18B and 18C depict images and graphs showing RD
aggregates propagate misfolding between cells. HEK293 cells were
co-transfected with various RD-CFP and RD-HA constructs. 15 h
later, these cells were co-cultured with cells expressing
RD(.DELTA.K)-YFP or RD(PP)-YFP for 48 h (FIG. 18A) FRET microscopy
was performed to determine whether co-aggregation occurred via
direct protein contact. CFP signal was measured before and after
photobleaching of YFP. RD(LM)-CFP and RD(LM)-YFP aggregates had a
mean FRET efficiency of 14.2%.+-.0.053 SD (n=11) indicative of
RD(LM)-CFP and RD(LM)-YFP in direct contact. The upper and lower
panels depict the acceptor and donor channels, respectively, before
(Pre) and after (Post) photobleaching. A representative heat map of
the calculated FRET efficiency is shown at top right. The histogram
depicts the calculated FRET efficiency on a pixel-by-pixel basis.
The FRET efficiency of Tau RD aggregate was -25% in this cell.
Negative values are derived from unpaired CFP. (FIG. 18B) A FRET
signal was observed when cells expressing RD(.DELTA.K)-CFP/RD-HA
were co-cultured with cells expressing RD(.DELTA.K)-YFP. This
signal increased when aggregation of RD(.DELTA.K)-CFP was induced
by co-expression of aggregation-prone forms of tau, either
.DELTA.K, or LM mutants. No significant signal was noted when
either RD-CFP or RD-YFP contained the PP mutation that blocks
.beta.-sheet formation (n=3). (FIG. 18C) To test for amplification
of misfolding, populations of cells expressing CFP alone or
RD(LM)-CFP were preexposed for 48 h to cells expressing RD-HA with
either PP, .DELTA.K, or LM mutations to promote misfolding to
varying degrees. These co-cultured populations were then split and
co-cultured for 48 h with cells expressing RD(.DELTA.K)-YFP to
determine the degree of aggregation reported by cell-cell transfer
and FRET. Prior exposure of RD(LM)-HA cells to the RD(.DELTA.K)-CFP
cell population increased FRET signal by 2.6 fold vs. prior
exposure to RD(PP)-HA. Interposition of cells expressing pure CFP
in the second population of cells completely blocked the effect of
prior exposure to aggregation-prone RD-HA mutants (n=3). (*
indicates a p-value <0.05, ** indicates a p-value <0.001,
error bars represent the SEM).
[0023] FIGS. 19A, 19B, 19C, 19D, 19E, 19F, 19G and 1911 depict
graphs and an immunoblot showing propagation of tau aggregates
through the extracellular medium. (FIG. 19A) HEK293 cells
transfected with RD(LM)-HA were co-cultured for 48 h with an
equivalent number of RD(.DELTA.K)-CFP/YFP cells prior to FRET
analysis. Increasing the volume of cell culture medium reduced the
efficiency of trans-cellular movement of aggregates. (FIG. 19B)
Transfer of conditioned medium from cells expressing RD(LM)-HA to
cells expressing RD(.DELTA.K)-CFP/YFP was sufficient to induce
aggregation by 60%. (FIG. 19C) HJ9.3 antibody added to the media
reduced FRET, consistent with interference with propagation of
aggregation. (FIG. 19D) Non-specific IgG had no effect on
propagation. (FIG. 19E) HJ9.3 had no effect on intracellular
aggregation of RD(.DELTA.K)-CFP/YFP co-expressed within the same
cell. (FIG. 19F) HJ9.3 blocked the effect of RD(LM)-HA to induce
RD(.DELTA.K)-YFP in co-cultured cells, as determined by detergent
fractionation and western blot. (T=Total protein, S=Soluble protein
and P=Pellet insoluble protein, (FIG. 19G) Quantitative analysis of
three independent Western blots revealed a -60% decrease in the
pellet fraction, relative to the total fraction, after exposure to
HJ9.3. (FIG. 1911) Cells expressing RD(LM)-YFP and mCherry were
co-cultured and analyzed by flow cytometry. HJ9.3 decreased the
percentage of dual positive cells from 2.07% to 1.31%. Cells mixed
just prior to cytometry were a background control (* indicates a
p-value <0.05, ** indicates a p-value <0.001, error bars
represent the SEM).
[0024] FIG. 20 depicts images of HEK293 cells transfected with
RD(.DELTA.K)-YFP (top panels) or mock-transfected (lower panels).
HJ9.3 was added to the culture medium for the 48 h period. At the
end of the experiment, the cells were fixed, permeabilized, and
stained with an anti-mouse secondary antibody (labeled with Alexa
546). Confocal microscopy was used to analyze the localization of
HJ9.3/tau complexes. The top panels show that many complexes are
identified when RD.DELTA.(K)-YFP is expressed, but none in its
absence (lower panels). Orthogonal analyses (right panel)
demonstrate that most complexes are present at the cell surface,
although occasional intracellular complexes were observed.
[0025] FIGS. 21A, 21B, 21C, and 21D depict images and a graph
showing Tau fibrils mediate cell-cell propagation. (FIG. 21A)
Conditioned media was collected from transfected cell populations
co-cultured for 0 h or 48 h with HJ9.3 or control IgG antibody
(1:1000), followed by immunoprecipitation and Western blot. HJ9.3
specifically captured tau RD species from the cell media, while IgG
did not. Higher-order aggregated species were present upon
expression of RD(.DELTA.K)-YFP or RD(LM)-YFP but not RD(PP)-YFP.
(FIG. 21B) Quantitative analyses of three independent Western blots
showed a .about.10-fold increase in the tau after 48 h incubation.
(FIG. 21C) Cells were exposed to HJ9.3 for various times. (FIG.
21D) Purified antibody/antigen complexes from media exposed for 48
h to HJ9.3 were deposited on AFM chips for imaging. Obvious
fibrillar species in the media of cells expressing RD(.DELTA.K)-HA
and RD(LM)-HA were observed, while RD(PP)-HA produced only
amorphous aggregates. Scale bar, 1 .mu.m.
[0026] FIGS. 22A, 22B, and 22C depict a schematic and graphs
showing HJ8.5 and HJ9.4 activity against recombinant human tau.
(FIG. 22A) depicts a schematic illustrating a co-culture of RD
(LM)-CFP and RD(.DELTA.K280)-YFP cells in presence and absence of
different monoclonal full length tau antibodies. (FIG. 22B) depicts
a graph showing HJ8.5, HJ9.3 and HJ9.4 were able to block tau
propagation. (FIG. 22C) depicts a graph showing HJ8.5, HJ9.3 and
HJ9.4 were able to detect RD-tau fibrils in an ELISA assay.
[0027] FIGS. 23A, 23B, and 23C depict a schematic illustrating the
experimental plan for (FIG. 23A) intracerebroventricular injection
and (FIG. 23B) implantation of an osmotic pump in the lateral
ventricle of each mouse. (FIG. 23C) shows an image verifying the
placement of the cannula by cresyl violet staining.
[0028] FIGS. 24A, and 24B depict images of the anti-tau antibodies
after 6 weeks infusion in P301 S tg mice by (FIG. 24A) Coomassie
blue staining and (FIG. 24B) immunoblotting against recombinant
longest human tau isoform hTau40 using antibodies taken from the
pump before and after 6 weeks infusion.
[0029] FIG. 25 depicts a graph showing lack of interference of
infused tau antibodies in HJ8.7-BT2B ELISA for total tau. Indicated
concentrations of antibodies were pre-incubated with recombinant
human tau protein before applying to ELISA.
[0030] FIG. 26 depicts images of coronal sections of piriform
cortex of treated 9 month old P301 S tg mice treated with
vehicle/PBS (top panels) or different anti-tau monoclonal
antibodies (HJ8.5, HJ9.3 as labeled in bottom panels). Sections
were stained with biotinylated AT8 antibody, which recognizes an
abnormally phosphorylated form of tau.
[0031] FIGS. 27A, 27B, 27C, and 27D depict graphs showing the
percent of area covered by AT8 staining of neurofibrillary tangles
in the (FIG. 27A) hippocampus CA2 and CA3, (FIG. 27B) amygdala,
(FIG. 27C) piriform cortex, and (FIG. 27D) entorhinal cortex.
[0032] FIG. 28 graphs showing HJ9.3 antibody detection of tau
fibrils and RD-tau monomer by ELISA. Different concentrations of
RD-wt tau monomers and fibrils were coated on ELISA plate. HJ9.3
was used as the primary antibody. For the detection anti-mouse HRP
linked antibody was used.
[0033] FIG. 29 depicts a schematic illustrating trans-cellular
propagation of tau aggregation occurring via transfer of fibrils
within the cell medium. Protein aggregate in a donor cell escapes
the cell, enters a recipient cell, and directly contacts natively
folded protein to amplify the misfolded state. This cell-cell
movement is mediated by fibrils that are released directly into the
medium. These fibrils can be trapped within the extracellular space
by an anti-tau antibody (HJ9.3) that interferes with cell-cell
propagation.
[0034] FIGS. 30A, 30B, 30C, 30D, 30E, 30F and 30G. Characterization
of anti-tau antibodies by surface plasmon resonance (SPR) and
Immunoblotting. The figure depicts SPR sensorgrams showing the
binding of each anti-tau antibody towards immobilized recombinant
human tau (longest isoforms hTau40, 441 aa) and immobilized mouse
tau (longest isoforms mTau40, 432 aa). Each antibody was run with
various concentrations (0.11, 0.23, 0.46, 0.90, 1 0.8, 3.7, 7.5
.mu.g/ml) and plots are shown in the corresponding color. (FIG.
30A) SPR sensorgrams of HJ9.3 antibody binding to immobilized human
tau and immobilized mouse tau (FIG. 30B). (FIG. 30C) SPR
sensorgrams of HJ9.4 antibody binding to immobilized human tau and
immobilized mouse tau (FIG. 30D). SPR sensorgrams of HJ8.5 antibody
binding to immobilized (FIG. 30E) human and (FIG. 30F) mouse tau.
(FIG. 30G) RAB soluble fractions of 3 month old tau knockout (KO),
3 month old wild-type (WT), 3 month old P301 S (3 mo) and 9 month
old P301 S (9 mo) mice were analyzed by immunoblot by using the
indicated anti-tau antibodies.
[0035] FIGS. 31A, 31B, and 31C. SPR sensorgram of the interaction
between anti-tau antibodies towards immobilized human tau fibrils.
SPR sensorgrams of HJ9.3 (FIG. 31A), HJ9.4 (FIG. 31B) and HJ8.5
(FIG. 31C) anti tau antibodies run with various concentrations
towards immobilized human tau fibrils.
[0036] FIG. 32. Characterization of anti-tau antibodies in
different assays. Immunostaining of brain sections from 3 month old
tau knockout (KO), 3 month old wild type (WT), 3 month old P301 S
(3 mo), 12 month old P301 S (12 mo) mice from the region of the
piriform cortex and from the frontal cortex of Alzheimer's disease
(AD) tissue were stained with biotinylated HJ8.5 antibody. Insert
in 12 month old P301 S micrograph shows cell body staining in
addition to diffuse neuropil staining. Black arrow indicates the
area magnified. Insert in human AD brain cortex micrograph shows
the staining of neurofibrillary tangles (NFT) in higher
magnification. Black arrow indicates the area magnified. Scale bar
is 250 .mu.m in panel with tau KO, same magnification images. Scale
bar 50 .mu.m in inserts of P301 S 12 mo and AD.
[0037] FIGS. 33A, 33B, and 33C. Tau-antibodies block the uptake and
seeding activity of P301 S tau aggregates as detected by a FRET
assay. HEK293 cells expressing RD(.DELTA.K280)-CFP/YFP were exposed
to 2.5 .mu.g of total protein of 1 xTBS brain lysates for 24 h.
(FIG. 33A) Brain lysates collected from 12 mo old P301 S mice
induced much greater seeding activity (n=5) as compared to lysates
from knockout (KO) mice (n=7), wild type (WT) mice (n=6) or young
3-mo old P301 S mice (n=2) (****p<0.0001). (FIG. 33B) HEK293
cells were co-transfected with RD (.DELTA.K280)-CFP and RD
(.DELTA.K280)-YFP. 18 hrs later, pre-incubated P301 S brain lysates
with or without incubation of anti-tau antibodies (HJ8.5, HJ9.3 and
HJ9.4) or control antibody (HJ3.4, anti A.beta. antibody) were
added to cells. All the tau antibodies incubated with P301 S brain
lysates significantly blocked seeding activity. Statistical
significance was determined by one-way ANOVA followed by Dunnett's
post hoc test for multiple comparisons by using GraphPad Prism 5.0
software (***p>0.001). (FIG. 33C) Titration of these antibodies
with various concentrations (0.125 .mu.g/ml, 0.25 .mu.g/ml, 0.5
.mu.g/ml, 1 .mu.g/ml and 2 .mu.g/ml) was performed with a fixed
amount of P301 S brain lysates. 24 hrs later, FRET analysis was
performed. Out of all tau-antibodies we used, HJ8.5 was the most
potent in blocking the uptake and seeding activity of P301 S brain
lysates. Statistical significance was determined by two-way ANOVA
followed by Bonferroni post hoc test for multiple comparisons. (**
p<0.0001, * p<0.01, Values represent mean.+-.SEM).
[0038] FIG. 34. No detected cellular uptake of tau antibodies bound
to P301 S Tau aggregates. P301 S brain lysates were added to HEK293
cells for 3 hrs. For detection of tau, all 3 different anti-tau or
control (HJ3.4, A.beta. antibody) antibodies were used followed by
Alexa-fluor546 anti-mouse IgG staining. In addition, P301 S brain
lysates were pre-incubated with and without 3 different anti-tau
antibodies and HJ3.4 antibody, then added to HEK293 cells, fixed
and permeabilized. Alexa-fluor546 anti-mouse IgG were used to
identify the internalized antibodies.
4',6'-diamidino-2-phenylindole (DAPI; shown in blue) was used for
nuclear stain.
[0039] FIGS. 35A and 35B. Experimental outline of ICV infusion of
antibodies and efficacy of antibody by different treatment method.
(FIG. 35A) Experimental plan for infusion of antibodies or vehicle
(PBS) by intracerebroventricular injection into the left lateral
ventricle of the brain. (FIG. 35B) Representative cresyl violet
staining of the coronally sectioned brain region to verify the
surgically implanted probe placement into the left lateral
ventricle. In this study, we included the mice which had correct
probe placements into the left lateral ventricle.
[0040] FIGS. 36A, 36B, 36C, 36D, and 36E. Anti-tau antibodies
strongly decreased AT8 staining in P301 S mouse brain.
Representative coronal sections of PBS (FIG. 36A), HJ3.4 antibody
(FIG. 36B), HJ8.5 antibody (FIG. 36C), HJ9.3 antibody (FIG. 36D)
and HJ9.4 antibody (FIG. 36E) treated 9 month old P301 S mice
stained with biotinylated AT8 antibody in regions including the
piriform cortex and amygdala. Scale bar is 250 .mu.m. inserts in A
to E show the higher magnification of biotinylated AT8 antibody
staining of phosphorylated tau, scale bar is 50 .mu.m.
[0041] FIGS. 37A, 37B, 37C, and 37D. Certain anti-tau antibodies
strongly decrease AT8 staining in P301 S mouse brain. Percent of
the area covered by biotinylated AT8 staining of abnormally
phosphorylated tau in piriform cortex (FIG. 37A), entorhinal cortex
(FIG. 37B), amygdala (FIG. 37C) and hippocampus CA1 region (FIG.
37D) in mice treated with the anti-tau antibodies HJ8.5 (N=13),
HJ9.3 (N=15), HJ9.4 (N=13), the anti-A.beta. antibody, HJ3.4 (N=8),
or PBS (N=16) in 9 month old P301 S mice. There was reduced AT8
staining in several different brain regions in the anti-tau
antibody treated mice compared to PBS or HJ3.4 antibody treated
mice. HJ8.5 had the largest effects. ** p<0.01, * p<0.05,
values represent mean.+-.SEM.
[0042] FIGS. 38A, 38B, 38C, 38D, 38E, 38F, 38G and 3811.
Quantification of biotinylated AT8 antibody staining in male and
female P301 S mice. Percent of area covered by biotinylated AT8
staining of abnormally phosphorylated tau in male (FIG. 38A) and
female P301 S mice (FIG. 38B) in piriform cortex (FIG. 38A and FIG.
38E), entorhinal cortex (FIG. 38B and FIG. 38F), amygdala (FIG. 38C
and FIG. 38G) and hippocampal CA1 regions (FIG. 38D and FIG. 3811)
in anti-tau antibody (HJ8.5, HJ9.3 and HJ9.4), control antibody
(HJ3.4) plus PBS treated mice.
[0043] FIGS. 39A and 39B. Some anti-tau antibodies strongly
decrease ThioS staining of neurofibrillary tangles in P301 S mouse
brain. (FIG. 39A) Representative images of ThioS staining of
neurofibrillary tangles in the piriform cortex of 9 month old P301
S mice treated for 3 months with PBS, HJ3.4, HJ8.5, HJ9.3 and HJ9.4
antibodies. ThioS staining of neurofibrillary tangles was reduced
in HJ8.5 antibody treated mice compared to the PBS or HJ3.4
antibody treated mice. Scale bar represents 100 .mu.m. (FIG. 39B)
Semi quantitative assessment of ThioS staining by scoring from 1
(no staining) to 5 (maximum staining) in all anti-tau antibody and
control treated mice. HJ8.5 antibody treated mice had significantly
less ThioS staining compared to PBS or HJ3.4 antibody treated mice.
*p<0.05, **p<0.01.
[0044] FIGS. 40A, 40B, and 40C. Correlations between phospho-tau
staining, and activated microglial staining. (FIG. 40A)
Biotinylated AT8 staining of phospho-tau in HJ8.5 (N=6), HJ9.3
(N=6) and PBS treated 9 month old P301 S mice (N=6 per each group)
showed strong correlation with PHF1 staining, another phospho-tau
antibody. (FIG. 40B) Strong correlation was observed between CD68
staining of activated microglia and biotinylated AT8 staining of
phospho-tau in all groups (N=6 per each group) (FIG. 40C)
Immunoblotting of representative 70% FA fraction samples (N=4) were
analyzed with polyclonal mouse anti-tau antibodies (Abeam).
[0045] FIGS. 41A, 41B, 41C. 41D, and 41E. CD68 staining of
activated microglia. Mice were assessed for microglial activation
in P301 S mice. Representative images of CD68 staining of activated
microglia in the piriform cortex of 9 month old P301 S mice treated
with PBS (FIG. 41A), HJ3.4 antibody (FIG. 41B), HJ8.5 antibody
(FIG. 41C), HJ9.3 antibody (FIG. 41D) and HJ9.4 antibody (FIG.
41E).
[0046] FIGS. 42A, 42B, 42C. 42D, 42E, and 42F. Insoluble tau levels
are reduced by antibodies HJ8.5 and HJ9.3 in P301 S mice. The
cortex of all the treated mice [PBS (N=16), HJ3.4 antibody (N=8)
HJ8.5 (N=13), HJ9.3 (N=15), HJ9.4 (N=13)] were sequentially
extracted by RAB (FIG. 42A), RIPA (FIG. 42B) and 70% FA (FIG. 42C)
and their tau levels were quantified by ELISA. There were no
statistical differences in soluble tau levels in RAB and RIPA
fractions between the groups. However, there was a significant
decrease of insoluble tau levels in 70% FA fractions in the HJ8.5
and HJ9.3 anti-tau antibodies treated mice compared to the PBS or
HJ3.4 antibody treated groups. Insoluble tau levels in the HJ9.4
antibody treated mice were not different from the control groups
(**p<0.01). Levels of human tau (FIG. 42D), mouse tau (FIG. 42E)
and phospho tau at Ser202 and Thr205 (FIG. 42F) levels were
assessed in 70% FA fractions by specific anti-human, anti-mouse, or
anti-phospho tau antibodies by ELISA (n=6 mice per treatment
group). There was a decrease in human tau levels in all groups of
anti-tau antibody treated mice and no change in mouse tau levels.
In 70% FA fractions, we also found that phospho tau at Ser202 and
Thr205 as detected by AT8 reactivity was reduced in anti-tau
antibody treated mice compared to controls, similar to total human
tau.
[0047] FIGS. 43A, 43B, 43C. 43D, and 43E. Anti-tau antibody treated
P301 S mice have decreased tau seeding activity in cortical
extracts as detected by FRET assay. (FIG. 43A) Tau seeding activity
was measured with RAB soluble fractions of all PBS (N=16), HJ3.4
(N=8), HJ8.5 (N=13), HJ9.3 (N=15), and HJ9.4 (N=13) treated mice on
HEK293 cells by FRET assay. HEK293 cells were co-transfected with
RD (.DELTA.K280)-CFP and RD (.DELTA.K280)-YFP. 18 hrs later, RAB
soluble fractions were added to cells. Seeding activity was
significantly reduced in HJ8.5, and HJ9.3 antibody treated mice
compared to the PBS or HJ3.4 antibody treated mice. RAB soluble
fractions from HJ9.4 antibody treated mice did not have decreased
seeding activity compared to the PBS or HJ3.4 antibody RAB soluble
fractions (***p<0.001, Values represent mean.+-.SEM). (FIG. 43B)
RAB soluble fractions were immunoprecipitated from tau knockout,
PBS, or anti-tau antibody treated mice. Elution of any seeding
activity from the antibody/bead complexes was measured by FRET
assay. There was significantly less seeding activity observed in
HJ8.5 and HJ9.3 antibody treated mice versus PBS-treated mice
(****p<0.0001, values represent mean.+-.SEM). (FIG. 43C) 70% FA
fractions of 9 month old P301 S brain cortex region of all treated
groups analyzed by ELISA showed a strong correlation with FRET
analysis performed with the RAB soluble fractions. (FIG. 43D)
Comparison between tau levels (X-axis) and seeding activity
(Y-axis) present in RAB soluble fractions of 9 month old P301 S
brain cortex of all treated mice assessed. There was no significant
correlation between these 2 measures. (FIG. 43E) Tau species in the
RAB soluble fractions of 3 month old knockout (KO), 3 month old
wild type (WT), 3 month old P301 S, and 9 month old PBS-treated
P301 S mice were separated on SDD-AGE, followed by western
blotting. Polyclonal mouse anti-tau antibody was used for detecting
tau species. High molecular weight tau species present in the RAB
soluble fraction in both 3 month old P301 S mice and larger amounts
present in 9 month old P301 S mice.
[0048] FIGS. 44A, 44B, 44C. 44D, 44E, and 44F. Groups did not
differ significantly in terms of locomotor activity, sensorimotor
function or on the auditory cue component of the conditioned fear
test. The results of rmANOVAs failed to reveal significant main or
interaction effects involving Treatment for total ambulations in
the holeboard test (FIG. 44A), for the ledge test (FIG. 44B) or any
other of the sensorimotor measures (not shown), or on the
accelerating rotarod (FIG. 44C). Data from the altered context
baseline on day 3 of conditioned fear testing yielded a significant
effect of Treatment (*p=0.027) and subsequent comparisons showed
that a large portion of this effect was due to significant
differences between the HJ9.4 mice and the PBS+HJ3.4 control group
(p=0.0007). (FIG. 44D). However, no significant main or interaction
effects of Treatment were found following an rmANOVA on the
auditory cue data (min 3-10) suggesting that the freezing levels
were not significantly different among the groups during this time
(FIG. 44E). To assess whether activity levels may have had an
effect on freezing during the contextual fear test on day 2, we
computed Pearson's correlation coefficient (r) between total
ambulations measured during the holeboard test versus % time spent
freezing during the contextual fear test and found that they were
not significantly correlated (p=0.39) (FIG. 44F).
[0049] FIGS. 45A, 45B, and 45C. Contextual fear conditioning
deficits in P301 S tau transgenic mice are rescued by HJ8.5 and
HJ9.4 antibody treatments. (FIG. 45A) On day 1 of conditioned fear
testing, no differences were observed among groups in freezing
levels during either the 2-min baseline condition or the tone/shock
(T/S) training as indicated by the lack of a significant main or
interaction effects involving Treatment following rmANOVAs on these
data. (FIG. 45B) In contrast, a significant effect of Treatment
(*p=0.019) and a significant Treatment by Minutes interaction
(**p=0.0001) were observed following an rmANOVA on freezing levels
during the contextual fear testing on day 2. Only the HJ9.4 group
showed significant habituation from minute 1 versus minute 8,
(#p=0.002). (FIG. 45C) Subsequent planned comparisons showed that
freezing in the HJ8.5 and HJ9.4 tau antibody groups was
significantly increased relative to the PBS+HJ3.4 control group
when averaged across the 8-min session (**p=0.006 and *p=0.022,
respectively). However, further analyses of the data showed that
the largest differences between the HJ9.4 group and the PBS+HJ3.4
controls occurred during minute 2 (.dagger.p=0.004), while the
largest differences between the HJ8.5 treated mice and the control
group were found during minutes 4-7 (.dagger..dagger.p<0.004) as
depicted in "B".
[0050] FIG. 46 depicts a graph showing a sandwich Tau ELISA assay
can be used to discriminate between plasma samples that are
positive for seeding activity and plasma samples that are negative
for seeding activity. Seeding activity was determined as described
in Kfoury et al 2012 J Biol Chem 287(23). Amount of tau aggregate
is reported as relative fold-change induction over signal from
plasma collected from healthy young humans (i.e. background signal
of the assay).
[0051] FIGS. 47A, 47B, 47C, 47D, 47E and 47F depict graphs showing
the effect of anti-tau antibodies of the invention on a tau
cellular propagation assay. In each graph, the first bar represents
medium without added antibody, representing baseline efficiency of
propagation. (FIG. 47A) HJ8.1 and HJ8.2; (FIG. 47B) HJ8.3 and
HJ8.4; (FIG. 47C) HJ8.5 and HJ8.7; (FIG. 47D) HJ8.8 and HJ9.1;
(FIG. 47E) HJ9.2 and HJ9.3; (FIG. 47F) HJ9.4 and HJ9.5.
[0052] FIG. 48 depicts a graph showing the effect on tau
propagation of individual anti-tau antibodies or equimolar mixtures
of anti-tau antibodies in a cell-based assay.
[0053] FIGS. 49A and 49B depict in (FIG. 49A) a graph showing HJ9.3
antibody has no effect on intracellular tau aggregation when
RD(.DELTA.K)-CFP/YFP are co-expressed within the same cell, and in
(FIG. 49B) a graph showing that nonspecific IgG has no effect on
trans-cellular propagation of tau aggregation.
[0054] FIG. 50 depicts a graph showing HJ9.3 inhibits tau aggregate
uptake, as measured by flow cytometry. Cells were exposed to
recombinant RD fibrils that were chemically labeled with a
fluorescent dye. After trypsinization and dispersion, the cells
were counted using a flow cytometer. HJ9.3 dose-dependently reduces
the number of fluorescently labeled cells, indicating inhibition of
aggregate uptake.
DETAILED DESCRIPTION
[0055] The common minimal connection between Alzheimer's Disease
and all the tauopathies is the aggregation state of tau. Under all
these diseased conditions, monomeric tau is known to be converted
into polymeric ordered fibrils. Neurofibrillary tangles (NFTs),
which are comprised of fibrillar tau aggregates, are a
neuropathological hallmark of tauopathies. Applicants have
discovered that spreading of tau pathology in the brain may be
caused by a form of tau aggregate released from a "donor" cell
entering a second "recipient" cell, and inducing further misfolding
and aggregation of tau in the recipient cell via direct
protein-protein contact. The specific form of tau aggregate which
facilitates this cell-to-cell spread of tau aggregates is referred
to as "tau seeds" and the activity may be referred to herein as
"seeding activity", since this form of tau aggregate seeds or
nucleates tau aggregation in the cell it enters (i.e. the
"recipient cell").
[0056] Tau can exist in both a monomeric form and in different
aggregated forms. As used herein, the term "tau aggregate" refers
to a molecular complex that comprises two or more tau monomers.
Without wishing to be bound by theory, a tau aggregate may comprise
a nearly unlimited number of monomers bound together. For example,
a tau aggregate may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tau
monomers. Alternatively, a tau aggregate may comprise 20, 30, 40,
50, 60, 70, 80, 90, 100 or more tau monomers. A tau aggregate may
also comprise 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
5000 or more tau monomers. The terms "fibrillar tau aggregate" and
"tau fibril" refer to forms of tau aggregates, and these terms are
used interchangeably herein. A fibrillar tau aggregate is a
polymeric, ordered fiber comprising tau. Tau fibrils are generally
not soluble, but shorter assemblies, or oligomers, can be soluble.
Tau aggregate also refers to soluble tau oligomers and
protofibrils, which may act as intermediates during tau
aggregation. Also included in the definition of tau aggregate is
the term "tau seed", which refers to a tau aggregate that is
capable of nucleating or "seeding" intracellular tau aggregation
when internalized by a cell, or when exposed to monomeric tau in
vitro. Tau seeding activity may be assessed in a cellular tau
aggregation assay as described herein.
[0057] In addition, applicants have discovered antibodies that
specifically bind to tau and methods of use thereof. In an aspect,
the present invention provides antibodies that specifically bind
tau. In another aspect, the present invention provides means for
effectively slowing and/or reducing cell-to-cell propagation of tau
aggregation. Antibodies of the invention may slow and/or reduce the
propagation of tau aggregation by promoting the disaggregation of
protein fibrils, blockading the conversion of monomeric tau into
aggregated tau in the cell, promoting intracellular degradation of
tau aggregates, preventing entry of the tau aggregates into
neighboring cells, or a combination thereof. In another aspect, the
present invention provides means to detect tau aggregate in a
sample of biological fluid obtained from a subject. In another
aspect, the present invention provides means to measure the amount
of tau aggregate in a sample of biological fluid obtained from a
subject. In another aspect, the present invention provides means to
classify a subject based on the amount of tau aggregate measured in
a sample of biological fluid obtained from a subject. Classifying a
subject based on the amount of tau aggregate measured in a sample
of biological fluid obtained from the subject may be used to
identify subjects that will develop a symptom and/or disease
associated with tau aggregation in the subject's lifetime.
[0058] The present invention encompasses the discovery that
anti-tau antibodies may slow the propagation of fibrillar tau
aggregates by binding extracellular tau released from cells,
thereby preventing entry of the tau aggregates into neighboring
cells and slowing spread of tau aggregation. In an aspect, the
present invention provides means for preventing entry of a tau
aggregate into a cell. In another aspect, the present invention
provides means for reducing intracellular tau aggregation. In
another aspect, the present invention provides means for decreasing
tau seeding activity. Antibodies of the invention useful in
preventing entry of the tau aggregates into neighboring cells
include those which bind an epitope within tau.
I. Antibodies that Bind to Tau
[0059] In humans, there are six isoforms of tau that are generated
by alternative splicing of exons 2, 3, and 10. The isoforms ranging
from 352 to 441 amino acids. Exons 2 and 3 encode 29-amino acid
inserts each in the N-terminus (called N, and hence, tau isoforms
may be 2N (both inserts), 1 N (exon 2 only), or ON (neither). All
tau isoforms have three repeats of the microtubule binding domain.
Inclusion of exon 10 at the C-terminus leads to inclusion of a
fourth microtubule binding domain encoded by exon 10. Hence, tau
isoforms may be comprised of four repeats of the microtubule
binding domain (exon 10 included) or three repeats of the
microtubule binding domain (exon 10 excluded). Anti-tau antibodies
of the invention may include antibodies that bind any of the
isoforms of tau. In an exemplary embodiment, anti-tau antibodies of
the invention may include antibodies that bind to an isoform of tau
that comprises exon 10.
[0060] As noted above, tau can be found in soluble and insoluble
compartments, in monomeric and aggregated forms, in ordered or
disordered structures, intracellularly and extracellularly, and may
be complexed with other proteins or molecules. Anti-tau antibodies
of the invention may include antibodies that bind to one or more
forms of tau as described. In some embodiments, an anti-tau
antibody binds a tau monomer. In other embodiments, an anti-tau
antibody binds a tau aggregate. In still other embodiments, an
anti-tau antibody binds a tau fibril. In different embodiments, an
anti-tau antibody binds a tau monomer and a tau aggregate. In
alternative embodiments, an anti-tau antibody binds to a tau
aggregate and a tau fibril. In different embodiments, an anti-tau
antibody binds to a tau fibril and a tau monomer.
[0061] Anti-tau antibodies useful herein also include all
antibodies that specifically bind tau aggregates present in a
biological sample. Anti-tau antibodies useful herein also include
all antibodies that reduce cell-to-cell propagation of tau
aggregation. In other words, useful antibodies slow and/or decrease
the amount of tau that enters recipient cells, compared to the
amount that would enter a recipient cell in the absence of an
antibody of the invention. Hence, useful antibodies decrease the
amount of tau aggregation that occurs in the recipient cells.
[0062] In an aspect, antibodies useful herein include those
antibodies which have been isolated, characterized, purified, are
functional and have been recovered (obtained) for use in a
functional therapeutic composition which is administered to a
living subject. In another aspect, antibodies useful herein include
those antibodies which have been isolated, characterized, purified,
are functional and have been recovered (obtained) for use in an
assay to detect tau aggregates in a biological sample obtained from
a living subject and predict the development of symptoms associated
with tau aggregation over the lifetime of the subject. In another
aspect, antibodies useful herein include those antibodies which
have been isolated, characterized, purified, are functional and
have been recovered (obtained) or for use in an assay to detect tau
aggregates in a biological sample obtained from a living subject
and classify the subject as having an increased risk of developing
symptoms associated with tau aggregation over the subject's
lifetime. In another aspect, antibodies useful herein include those
antibodies which have been isolated, characterized, purified, are
functional and have been recovered (obtained) for use and are
listed in Table A, as well as variants thereof (e.g. humanized
forms, chimeric forms, and immunological fragments).
TABLE-US-00001 TABLE A Antibodies of the invention Antibody Name
Tau epitope HJ8.1.1 DRKDQGGYTMHQD (SEQ ID NO: 1) HJ8.1.2 TDHGAE
(SEQ ID NO: 10) HJ8.2 PRHLSNV (SEQ ID NO: 3) HJ8.3 PRHLSNV (SEQ ID
NO: 3) HJ8.4 KTDHGA (SEQ ID NO: 11) HJ8.5 DRKDQGGYTMHQD (SEQ ID NO:
1) HJ8.7 AAGHV (SEQ ID NO: 5) HJ8.8 EPRQ (SEQ ID NO: 4) HJ9.1
TDHGAEIVYKSPVVSG (SEQ ID NO: 6) HJ9.2 EFEVMED (SEQ ID NO: 7) HJ9.3
GGKVQIINKK (SEQ ID NO: 8) HJ9.4 EFEVMED (SEQ ID NO: 7) HJ9.5
EFEVMED (SEQ ID NO: 7)
[0063] The term "antibody" includes the term "monoclonal antibody".
"Monoclonal antibody" refers to an antibody that is derived from a
single copy or clone, including e.g., any eukaryotic, prokaryotic,
or phage clone. "Monoclonal antibody" is not limited to antibodies
produced through hybridoma technology. Monoclonal antibodies can be
produced using e.g., hybridoma techniques well known in the art, as
well as recombinant technologies, phage display technologies,
synthetic technologies or combinations of such technologies and
other technologies readily known in the art. Furthermore, the
monoclonal antibody may be labeled with a detectable label,
immobilized on a solid phase and/or conjugated with a heterologous
compound (e.g., an enzyme or toxin) according to methods known in
the art.
[0064] Further by "antibody" is meant a functional monoclonal
antibody, or an immunologically effective fragment thereof such as
an Fab, Fab', or F(ab')2 fragment thereof. In some contexts herein,
fragments will be mentioned specifically for emphasis;
nevertheless, it will be understood that regardless of whether
fragments are specified, the term "antibody" includes such
fragments as well as single-chain forms. As long as the protein
retains the ability specifically to bind its intended target, it is
included within the term "antibody." Also included within the
definition "antibody" for example are single chain forms, generally
designated Fv regions, of antibodies with this specificity.
Preferably, but not necessarily, the antibodies useful in the
discovery are produced recombinantly, as manipulation of the
typically murine or other non-human antibodies with the appropriate
specificity is required in order to convert them to humanized form.
Antibodies may or may not be glycosylated. Antibodies are properly
cross-linked via disulfide bonds, as is known.
[0065] The basic antibody unit of an antibody useful herein
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light` (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal
portion of each chain includes a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The carboxy-terminal portion of each chain defines a
constant region primarily responsible for effector function.
[0066] Anti-tau antibodies useful herein include those which are
isolated, characterized, purified, function and have been recovered
(obtained) from a process for their preparation and thus available
for use herein in a useful form in a therapeutically, medicinally,
or diagnostically sufficient amount.
[0067] Light chains are classified as gamma, mu, alpha, and lambda.
Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
and define the antibody's isotype as IgO, IgM, IgA, IgD and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids.
[0068] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, an intact antibody has two binding
sites. The chains exhibit the same general structure of relatively
conserved framework regions (FR) joined by three hypervariable
regions, also called complementarily determining regions
(hereinafter referred to as "CDRs.") The CDRs from the two chains
are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4 respectively. The assignment of amino acids to each domain
is in accordance with known conventions (See, Kabat "Sequences of
Proteins of Immunological Interest" National Institutes of Health,
Bethesda, Md., 1987 and 1991; Chothia, et al, J. Mol. Bio. (1987)
196:901-917; Chothia, et al., Nature (1989) 342:878-883).
[0069] In an aspect, monoclonal anti-tau antibodies are generated
with appropriate specificity by standard techniques of immunization
of mammals, forming hybridomas from the antibody-producing cells of
said mammals or otherwise immortalizing them, and culturing the
hybridomas or immortalized cells to assess them for the appropriate
specificity. In the present case, such antibodies could be
generated by immunizing a human, rabbit, rat or mouse, for example,
with a peptide representing an epitope encompassing a region of the
tau protein coding sequence or an appropriate subregion thereof.
Materials for recombinant manipulation can be obtained by
retrieving the nucleotide sequences encoding the desired antibody
from the hybridoma or other cell that produces it. These nucleotide
sequences can then be manipulated and isolated, characterized,
purified and, recovered to provide them in humanized form, for use
herein if desired.
[0070] As used herein "humanized antibody" includes an anti-tau
antibody that is composed partially or fully of amino acid
sequences derived from a human antibody germline by altering the
sequence of an antibody having non-human complementarity
determining regions ("CDR"). The simplest such alteration may
consist simply of substituting the constant region of a human
antibody for the murine constant region, thus resulting in a
human/murine chimera which may have sufficiently low immunogenicity
to be acceptable for pharmaceutical use. Preferably, however, the
variable region of the antibody and even the CDR is also humanized
by techniques that are by now well known in the art. The framework
regions of the variable regions are substituted by the
corresponding human framework regions leaving the non-human CDR
substantially intact, or even replacing the CDR with sequences
derived from a human genome. CDRs may also be randomly mutated such
that binding activity and affinity for tau is maintained or
enhanced in the context of fully human germline framework regions
or framework regions that are substantially human. Substantially
human frameworks have at least 90%, 95%, or 99% sequence identity
with a known human framework sequence. Fully useful human
antibodies may also be produced in genetically modified mice whose
immune systems have been altered to correspond to human immune
systems. As mentioned above, it is sufficient for use in the
methods of this discovery, to employ an immunologically specific
fragment of the antibody, including fragments representing single
chain forms.
[0071] Further, as used herein the term "humanized antibody" refers
to an anti-tau antibody comprising a human framework, at least one
CDR from a nonhuman antibody, and in which any constant region
present is substantially identical to a human immunoglobulin
constant region, i.e., at least about 85-90%, preferably at least
95% identical. Hence, all parts of a humanized antibody, except
possibly the CDRs, are substantially identical to corresponding
pairs of one or more native human immunoglobulin sequences.
[0072] If desired, the design of humanized immunoglobulins may be
carried out as follows. When an amino acid falls under the
following category, the framework amino acid of a human
immunoglobulin to be used (acceptor immunoglobulin) is replaced by
a framework amino acid from a CDR-providing nonhuman immunoglobulin
(donor immunoglobulin): (a) the amino acid in the human framework
region of the acceptor immunoglobulin is unusual for human
immunoglobulin at that position, whereas the corresponding amino
acid in the donor immunoglobulin is typical for human
immunoglobulin at that position; (b) the position of the amino acid
is immediately adjacent to one of the CDRs; or (c) any side chain
atom of a framework amino acid is within about 5-6 angstroms
(center-to-center) of any atom of a CDR amino acid in a three
dimensional immunoglobulin model (Queen, et al., op. cit., and Co,
ct al, Proc. Natl. Acad. Sci. USA (1991) 88:2869). When each of the
amino acids in the human framework region of the acceptor
immunoglobulin and a corresponding amino acid in the donor
immunoglobulin is unusual for human immunoglobulin at that
position, such an amino acid is replaced by an amino acid typical
for human immunoglobulin at that position.
[0073] In all instances, an antibody of the invention specifically
binds tau. In exemplary embodiments, an antibody of the invention
specifically binds human tau. The phrase "specifically binds"
herein means antibodies bind to the protein with an affinity
constant or Affinity of interaction (KD) in the range of 0.1 pM to
10 nM, with a preferred range being 0.1 pM to 1 nM. The sequence of
tau from a variety of species is known in the art, and methods of
determining whether an antibody binds to tau are known in the art.
For instance, see the Examples.
[0074] The antibodies of the present invention may also be used as
fusion proteins known as single chain variable fragments (scFv).
These scFvs are comprised of the heavy and light chain variable
regions connected by a linker. In most instances, but not all, the
linker may be a peptide. A linker peptide is preferably from about
10 to 25 amino acids in length. Preferably, a linker peptide is
rich in glycine, as well as serine or theronine. ScFvs can be used
to facilitate phage display or can be used for flow cytometry,
immunohistochemistry, or as targeting domains. Methods of making
and using scFvs are known in the art.
[0075] In a preferred embodiment, the scFvs of the present
invention are conjugated to a human constant domain. In some
embodiments, the heavy constant domain is derived from an IgG
domain, such as lgG1, lgG2, lgG3, or lgG4. In other embodiments,
the heavy chain constant domain may be derived from IgA, IgM, or
IgE.
[0076] An isolated antibody of the present invention that binds to
tau preferably recognizes one of several epitopes. In one
embodiment, the isolated antibody of the present invention that
binds to tau recognizes an epitope listed in Table A. In another
embodiment, the isolated antibody of the present invention that
binds to tau recognizes an epitope within the amino acid sequences
of SEQ ID NO: 1 (DRKDQGGYTMHQD). Preferably, the isolated antibody
recognizes an epitope within at least three contiguous amino acids
of SEQ ID NO: 1, including within at least 6 contiguous amino acids
of SEQ ID NO: 1, within at least 7 contiguous amino acids of SEQ ID
NO: 1, within at least 8 contiguous amino acids of SEQ ID NO: 1,
within at least 9 contiguous amino acids of SEQ ID NO: 1, within at
least 10 contiguous amino acids of SEQ ID NO: 1, within at least 11
contiguous amino acids of SEQ ID NO: 1, within at least 12
contiguous amino acids of SEQ ID NO: 1, and within at least 13
contiguous amino acids of SEQ ID NO: 1. In an exemplary embodiment,
an isolated antibody of the present invention that recognizes an
epitope within SEQ ID NO: 1 is the antibody HJ8.5. In another
exemplary embodiment, an isolated antibody of the present invention
that recognizes an epitope within SEQ ID NO: 1 is the antibody
HJ8.1.1.
[0077] In another embodiment, the isolated antibody of the present
invention that binds to tau recognizes an epitope within the amino
acid sequence of SEQ ID NO: 2 (KTDHGAE). Preferably, the isolated
antibody recognizes an epitope within at least three contiguous
amino acids of SEQ ID NO: 2, including within at least 4 contiguous
amino acids of SEQ ID NO: 2 within at least 5 contiguous amino
acids of SEQ ID NO: 2 within at least 6 contiguous amino acids of
SEQ ID NO: 2, and within at least 7 contiguous amino acids of SEQ
ID NO: 2. In an exemplary embodiment, an isolated antibody of the
present invention that recognizes an epitope within SEQ ID NO: 2 is
the antibody HJ8.1.2. In another exemplary embodiment, an isolated
antibody of the present invention that recognizes an epitope within
SEQ ID NO: 2 is the antibody HJ8.4.
[0078] In another embodiment, the isolated antibody of the present
invention that binds to tau recognizes an epitope within the amino
acid sequence of SEQ ID NO: 3 (PRHLSNV). Preferably, the isolated
antibody recognizes an epitope within at least three contiguous
amino acids of SEQ ID NO: 3, including within at least 4 contiguous
amino acids of SEQ ID NO: 3, within at least 5 contiguous amino
acids of SEQ ID NO: 3, within at least 6 contiguous amino acids of
SEQ ID NO: 3, and within at least 7 contiguous amino acids of SEQ
ID NO: 3. In an exemplary embodiment, an isolated antibody of the
present invention that recognizes an epitope within SEQ ID NO: 3 is
the antibody HJ8.2. In another exemplary embodiment, an isolated
antibody of the present invention that recognizes an epitope within
SEQ ID NO: 3 is the antibody HJ8.3.
[0079] In still another embodiment, the isolated antibody of the
present invention that binds to tau recognizes an epitope within
the amino acid sequences of SEQ ID NO: 4 (EPRQ). Preferably, the
isolated antibody recognizes an epitope within at least three
contiguous amino acids of SEQ ID NO: 4, including within at least 4
contiguous amino acids of SEQ ID NO: 4. In an exemplary embodiment,
an isolated antibody of the present invention that recognizes an
epitope within SEQ ID NO: 4 is the antibody HJ8.8.
[0080] In yet a further embodiment, the isolated antibody of the
present invention that binds to tau recognizes an epitope within
the amino acid sequence of SEQ ID NO: 5 (AAGHV). Preferably, the
isolated antibody recognizes an epitope within at least three
contiguous amino acids of SEQ ID NO: 5, including within at least 4
contiguous amino acids of SEQ ID NO: 5, and within at least 5
contiguous amino acids of SEQ ID NO: 5. In an exemplary embodiment,
an isolated antibody of the present invention that recognizes an
epitope within SEQ ID NO: 5 is the antibody HJ8.7.
[0081] In an additional embodiment, the isolated antibody of the
present invention that binds to tau recognizes an epitope within
the amino acid sequence of SEQ ID NO: 6 (TDHGAEIVYKSPVVSG).
Preferably, the isolated antibody recognizes an epitope within at
least five contiguous amino acids of SEQ ID NO: 6, including within
at least 6 contiguous amino acids of SEQ ID NO: 6, within at least
7 contiguous amino acids of SEQ ID NO: 6, within at least 8
contiguous amino acids of SEQ ID NO: 6, within at least 9
contiguous amino acids of SEQ ID NO: 5, within at least 9
contiguous amino acids of SEQ ID NO: 6, within at least 10
contiguous amino acids of SEQ ID NO: 6, within at least 11
contiguous amino acids of SEQ ID NO: 6, within at least 12
contiguous amino acids of SEQ ID NO: 6, within at least 13
contiguous amino acids of SEQ ID NO: 6, within at least 14
contiguous amino acids of SEQ ID NO: 6, within at least 15
contiguous amino acids of SEQ ID NO: 6, and within at least 16
contiguous amino acids of SEQ ID NO: 6. In an exemplary embodiment,
an isolated antibody of the present invention that recognizes an
epitope within SEQ ID NO: 6 is the antibody HJ9.1.
[0082] In another embodiment, the isolated antibody of the present
invention that binds to tau recognizes an epitope within the amino
acid sequence of SEQ ID NO: 7 (EFEVMED). Preferably, the isolated
antibody recognizes an epitope within at least three contiguous
amino acids of SEQ ID NO: 7, including within at least 4 contiguous
amino acids of SEQ ID NO: 6, within at least 5 contiguous amino
acids of SEQ ID NO: 7, within at least 6 contiguous amino acids of
SEQ ID NO: 7, and within at least 7 contiguous amino acids of SEQ
ID NO: 7. In an exemplary embodiment, an isolated antibody of the
present invention that recognizes an epitope within SEQ ID NO: 7 is
the antibody HJ9.2. In an exemplary embodiment, an isolated
antibody of the present invention that recognizes an epitope within
SEQ ID NO: 7 is the antibody HJ9.4. In an exemplary embodiment, an
isolated antibody of the present invention that recognizes an
epitope within SEQ ID NO: 7 is the antibody HJ9.5.
[0083] In yet another embodiment, the isolated antibody of the
present invention that binds to tau recognizes an epitope within
the amino acid sequence of SEQ ID NO: 8 (GGKVQIINKK). Preferably,
the isolated antibody recognizes an epitope within at least three
contiguous amino acids of SEQ ID NO: 8, including within at least 4
contiguous amino acids of SEQ ID NO: 8, within at least 5
contiguous amino acids of SEQ ID NO: 8, within at least 6
contiguous amino acids of SEQ ID NO: 8, within at least 7
contiguous amino acids of SEQ ID NO: 8, within at least 8
contiguous amino acids of SEQ ID NO: 8, within at least 9
contiguous amino acids of SEQ ID NO: 8, and within at least 10
contiguous amino acids of SEQ ID NO: 8. In an exemplary embodiment,
an isolated antibody of the present invention that recognizes an
epitope within SEQ ID NO: 8 is the antibody HJ9.3.
[0084] A preferred antibody is a humanized form of mouse antibody
derived from a hybridoma designated HJ8.5. As used herein, the term
"derived from" means that the "derived" antibody comprises at least
one CDR region from the antibody produced hybridoma HJ8.5. Stated
another way, the "derived antibody" comprises at least one CDR
region comprised of the amino acid sequence selected from the group
consisting of SEQ ID NO: 16, 17, 18, 19, 20 and 21.
[0085] In one embodiment, an antibody of the invention may be
derived from the hybridoma HJ8.5, and may be encoded by a nucleic
acid sequence comprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%
identity to the light chain variable region of SEQ ID NO:12, or may
be encoded by a nucleic acid sequence comprising 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99% identity to the heavy chain variable
region of SEQ ID NO:13. In another embodiment, an antibody of the
invention may be derived from the hybridoma HJ8.5, and may comprise
an amino acid sequence with 90, 91, 92, 93, 94, 95, 96, 97, 98, or
99% identity to the light chain variable region of SEQ ID NO:14, or
may comprise an amino acid sequence with 90, 91, 92, 93, 94, 95,
96, 97, 98, or 99% identity to the heavy chain variable region of
SEQ ID NO:15. In each of the above embodiments, the antibody may be
humanized.
[0086] In an exemplary embodiment of an antibody of the invention
that binds to tau, the antibody comprises the light chain nucleic
acid sequence of SEQ ID NO:12 and the heavy chain nucleic acid
sequence of SEQ ID NO:13 [i.e. the monoclonal antibody referred to
herein as HJ8.5]. In another exemplary embodiment of an antibody of
the invention that binds to tau, the antibody comprises the light
chain amino acid sequence of SEQ ID NO:14 and the heavy chain amino
acid sequence of SEQ ID NO:15 [i.e. the monoclonal antibody
referred to herein as HJ8.5].
[0087] In one embodiment, an antibody of the invention may comprise
a light chain CDR1, such as antibody 1 of Table B. In another
embodiment, an antibody of the invention may comprise a light chain
CDR2, such as antibody 4 of Table B. In yet another embodiment, an
antibody of the invention may comprise a light chain CDR3, such as
antibody 6 of Table B. In an alternative embodiment, an antibody of
the invention may comprise a combination of two or three light
chain CDRs, such as the antibodies 2, 3, and 5 of Table B.
[0088] Similarly, in one embodiment, an antibody of the invention
may comprise a heavy chain CDR1, such as antibody 7 of Table B. In
another embodiment, an antibody of the invention may comprise a
heavy chain CDR2, such as antibody 10 of Table B. In yet another
embodiment, an antibody of the invention may comprise a heavy chain
CDR3, such as antibody 12 of Table B. In an alternative embodiment,
an antibody of the invention may comprise a combination of two or
three heavy chain CDRs, such as the antibodies 8, 9, 11 of Table
B.
[0089] Alternatively, an antibody of the invention may comprise one
or more light chain CDRs and one or more heavy chain CDRs, such as
the antibodies 13-48 of Table B.
TABLE-US-00002 TABLE B Anti- Light Chain Heavy Chain body CDR1 CDR2
CDR3 CDR1 CDR2 CDR3 1 SEQ ID NO: 16 2 SEQ ID SEQ ID NO: 16 NO: 17 3
SEQ ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 18 4 SEQ ID NO: 17 5 SEQ ID
SEQ ID NO: 17 NO: 18 6 SEQ ID NO: 18 7 SEQ ID NO: 19 8 SEQ ID SEQ
ID NO: 19 NO: 20 9 SEQ ID SEQ ID SEQ ID NO: 19 NO: 20 NO: 21 10 SEQ
ID NO: 20 11 SEQ ID SEQ ID NO: 20 NO: 21 12 SEQ ID NO: 21 13 SEQ ID
SEQ ID NO: 16 NO: 19 14 SEQ ID SEQ ID SEQ ID NO: 16 NO: 19 NO: 20
15 SEQ ID SEQ ID SEQ ID SEQ ID NO: 16 NO: 19 NO: 20 NO: 21 16 SEQ
ID SEQ ID NO: 16 NO: 20 16 SEQ ID SEQ ID SEQ ID NO: 16 NO: 20 NO:
21 17 SEQ ID SEQ ID NO: 16 NO: 21 19 SEQ ID SEQ ID SEQ ID NO: 16
NO: 17 NO: 19 20 SEQ ID SEQ ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 19
NO: 20 21 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 19
NO: 20 NO: 21 22 SEQ ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 20 23 SEQ
ID SEQ ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 20 NO: 21 24 SEQ ID SEQ
ID SEQ ID NO: 16 NO: 17 NO: 21 25 SEQ ID SEQ ID SEQ ID SEQ ID NO:
16 NO: 17 NO: 18 NO: 19 26 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO:
16 NO: 17 NO: 18 NO: 19 NO: 20 27 SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID NO: 16 NO: 17 NO: 18 NO: 19 NO: 20 NO: 21 28 SEQ ID SEQ
ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 18 NO: 20 29 SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 18 NO: 20 NO: 21 30 SEQ ID SEQ
ID SEQ ID SEQ ID NO: 16 NO: 17 NO: 18 NO: 21 31 SEQ ID SEQ ID NO:
17 NO: 19 32 SEQ ID SEQ ID SEQ ID NO: 17 NO: 19 NO: 20 33 SEQ ID
SEQ ID SEQ ID SEQ ID NO: 17 NO: 19 NO: 20 NO: 21 34 SEQ ID SEQ ID
NO: 17 NO: 20 35 SEQ ID SEQ ID SEQ ID NO: 17 NO: 20 NO: 21 36 SEQ
ID SEQ ID NO: 17 NO: 21 37 SEQ ID SEQ ID SEQ ID NO: 17 NO: 18 NO:
19 38 SEQ ID SEQ ID SEQ ID SEQ ID NO: 17 NO: 18 NO: 19 NO: 20 39
SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 17 NO: 18 NO: 19 NO: 20 NO:
21 40 SEQ ID SEQ ID SEQ ID NO: 17 NO: 18 NO: 20 41 SEQ ID SEQ ID
SEQ ID SEQ ID NO: 17 NO: 18 NO: 20 NO: 21 42 SEQ ID SEQ ID SEQ ID
NO: 17 NO: 18 NO: 21 43 SEQ ID SEQ ID NO: 18 NO: 19 44 SEQ ID SEQ
ID SEQ ID NO: 18 NO: 19 NO: 20 45 SEQ ID SEQ ID SEQ ID SEQ ID NO:
18 NO: 19 NO: 20 NO: 21 46 SEQ ID SEQ ID NO: 18 NO: 20 47 SEQ ID
SEQ ID SEQ ID NO: 18 NO: 20 NO: 21 48 SEQ ID SEQ ID NO: 18 NO:
21
[0090] In various embodiments, an antibody of the invention is
humanized. For instance, in one embodiment, a humanized antibody of
the invention may comprise a light chain variable region comprising
a CDR1 of amino acid sequence SEQ ID NO: 16 with zero to two amino
acid substitutions, a CDR2 of amino acid sequence SEQ ID NO: 17
with zero to two amino acid substitutions, and a CDR3 of amino acid
sequence SEQ ID NO: 18 with zero to two amino acid substitutions,
or may comprise a heavy chain variable region comprising a CDR1 of
amino acid sequence SEQ ID NO: 19 with zero to two amino acid
substitutions, a CDR2 of amino acid sequence SEQ ID NO: 20 with
zero to two amino acid substitutions, and a CDR3 of amino acid
sequence SEQ ID NO: 21 with zero to two amino acid substitutions.
In a preferred embodiment, a humanized antibody of the invention
may comprise a light chain variable region comprising a CDR1 of
amino acid sequence SEQ ID NO: 16 with zero to two amino acid
substitutions, a CDR2 of amino acid sequence SEQ ID NO: 17 with
zero to two amino acid substitutions, a CDR3 of amino acid SEQ ID
NO: 18 with zero to two amino acid substitutions, a heavy chain
variable region comprising a CDR1 of amino acid sequence SEQ ID NO:
19 with zero to two amino acid substitutions, a CDR2 of amino acid
sequence SEQ ID NO: 20 with zero to two amino acid substitutions,
and a CDR3 of amino acid sequence SEQ ID NO: 21 with zero to two
amino acid substitutions. In an exemplary embodiment, a humanized
antibody of the invention may comprise a light chain variable
region comprising a CDR1 of amino acid sequence SEQ ID NO: 16, a
CDR2 of amino acid sequence SEQ ID NO: 17, a CDR3 of amino acid
sequence SEQ ID NO: 18, a heavy chain variable region comprising a
CDR1 of amino acid sequence SEQ ID NO: 19, a CDR2 of amino acid
sequence SEQ ID NO: 20, and a CDR3 of amino acid sequence SEQ ID
NO: 21. The invention also encompasses the corresponding nucleic
acid sequences of SEQ ID NO: 16, 17, 18, 19, 20, and 21, which can
readily be determined by one of skill in the art, and may be
incorporated into a vector or other large DNA molecule, such as a
chromosome, in order to express an antibody of the invention.
II. Method of Use
[0091] In an aspect, the present invention provides antibodies for
use in a functional therapeutic composition which is administered
to a living subject. In another aspect, the present invention
provides antibodies for use in an immunoassay to detect tau
aggregates in a sample of biological fluid obtained from a living
subject. In another aspect, the present invention provides
antibodies for use in an immunoassay to measure the amount of tau
aggregate in a sample of biological fluid obtained from a living
subject. The amount of tau aggregate in a sample of biological
fluid obtained from a subject can be used to classify a subject as
having high or low amounts of tau aggregate, and may be further
used to predict the risk of developing symptoms and/or disease
associated with tau aggregation over the lifetime of the
subject.
[0092] Suitable subjects include, but are not limited to, a human,
a livestock animal, a companion animal, a lab animal, and a
zoological animal. In one embodiment, the subject may be a rodent,
e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the
subject may be a livestock animal. Non-limiting examples of
suitable livestock animals may include pigs, cows, horses, goats,
sheep, llamas and alpacas. In yet another embodiment, the subject
may be a companion animal. Non-limiting examples of companion
animals may include pets such as dogs, cats, rabbits, and birds. In
yet another embodiment, the subject may be a zoological animal. As
used herein, a "zoological animal" refers to an animal that may be
found in a zoo. Such animals may include non-human primates, large
cats, wolves, and bears. In preferred embodiments, the animal is a
laboratory animal. Non-limiting examples of a laboratory animal may
include rodents, canines, felines, and non-human primates. In
certain embodiments, the animal is a rodent. Non-limiting examples
of rodents may include mice, rats, guinea pigs, etc. In embodiments
where the animal is a mouse, the mouse may be a C57BL/6 mouse, a
Balb/c mouse, a 129sv, or any other laboratory strain. In an
exemplary embodiment, the subject is a C57BL/6J mouse. In a
preferred embodiment, the subject is human.
A. Method of Treatment
[0093] In an aspect, the present invention comprises a method of
reducing the spread of tau aggregation in the brain of a subject.
In another aspect the present invention comprises a method for
reducing intracellular aggregation of tau induced by tau seeds. In
each aspect, the method comprises administering a pharmacologically
effective amount of anti-tau antibody to a subject. Suitable
Antibodies are described above in Section I. In a preferred
embodiment, an antibody is selected from the group consisting of an
antibody from Table 1 and an antibody from Table 2, including a
humanized antibody, a chimeric antibody or an immunological
fragment thereof.
[0094] A subject may or may not be having a symptom associated with
tau aggregation prior to administration of a pharmacologically
effective amount of anti-tau antibody. Stated another way, a
subject may or may not be experiencing a symptom associated with
tau aggregation. A skilled artisan will appreciate that
pathological tau aggregation likely commences prior to diagnosis or
the onset of symptoms associated with tau aggregation. In some
embodiments, a subject is having a symptom associated with tau
aggregation. In other embodiments, a subject is not having a
symptom associated with tau aggregation. In still other
embodiments, a subject has detectable tau pathology but is not
having any other symptom associated with tau aggregation. Reducing
the spread of tau aggregation in the brain of a subject may reduce
the development and/or progression of symptoms associated with the
pathological aggregation of tau.
[0095] Preventing propagation of fibrillar tau aggregates may treat
pathologies associated with generation and spread of tau
aggregates. As used herein, the terms "treating" or "treatment"
include prevention, attenuation, reversal, or improvement in at
least one symptom or sign of symptoms associated with tau
aggregation. One definition of symptoms associated with tau
aggregation refers to any symptom caused by the formation of tau
aggregates being composed of, in part, tau fibrils. Exemplary
disorders that have symptoms associated with tau aggregation
include, but are not limited to, progressive supranuclear palsy,
dementia pugilistica (chronic traumatic encephalopathy),
frontotemporal dementia and parkinsonism linked to chromosome 17,
Lytico-Bodig disease (Parkinson-dementia complex of Guam),
tangle-predominant dementia, ganglioglioma and gangliocytoma,
meningioangiomatosis, subacute sclerosing panencephalitis, lead
encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease,
lipofuscinosis, Pick's disease, corticobasal degeneration,
argyrophilic grain disease (AGD), Frontotemporal lobar
degeneration, Alzheimer's Disease, and frontotemporal dementia.
Methods for diagnosing these disorders are known in the art.
[0096] Exemplary symptoms associated with tau aggregation may
include impaired cognitive function, altered behavior, emotional
dysregulation, seizures, and impaired nervous system structure or
function. Impaired cognitive function includes but is not limited
to difficulties with memory, attention, concentration, language,
abstract thought, creativity, executive function, planning, and
organization. Altered behavior includes but is not limited to
physical or verbal aggression, impulsivity, decreased inhibition,
apathy, decreased initiation, changes in personality, abuse of
alcohol, tobacco or drugs, and other addiction-related behaviors.
Emotional dysregulation includes but is not limited to depression,
anxiety, mania, irritability, and emotional incontinence. Seizures
include but are not limited to generalized tonic-clonic seizures,
complex partial seizures, and non-epileptic, psychogenic seizures.
Impaired nervous system structure or function includes but is not
limited to hydrocephalus, Parkinsonism, sleep disorders, psychosis,
impairment of balance and coordination. This includes motor
impairments such as monoparesis, hemiparesis, tetraparesis, ataxia,
ballismus and tremor. This also includes sensory loss or
dysfunction including olfactory, tactile, gustatory, visual and
auditory sensation. Furthermore, this includes autonomic nervous
system impairments such as bowel and bladder dysfunction, sexual
dysfunction, blood pressure and temperature dysregulation. Finally,
this includes hormonal impairments attributable to dysfunction of
the hypothalamus and pituitary gland such as deficiencies and
dysregulation of growth hormone, thyroid stimulating hormone,
lutenizing hormone, follicle stimulating hormone, gonadotropin
releasing hormone, prolactin, and numerous other hormones and
modulators. Methods for detecting and evaluating symptoms
associated with tau aggregation are known in the art.
[0097] In some embodiments, a symptom associated with tau
aggregation refers to dementia. Dementia is not itself a specific
disease, but is an overall term that describes a wide range of
symptoms associated with a decline in memory or other thinking
skills severe enough to reduce a person's ability to perform
everyday activities. Dementia is also a shared clinical feature of
many diseases associated with tau aggregation. A skilled
practitioner will be familiar with the numerous methods available
to diagnose the severity of dementia. For example, several
cognitive tests and screening questionnaires for dementia are known
in the art, all with varying degrees of sensitivity and
specificity. Non-limiting examples include the mini mental state
examination (MMSE), the abbreviated mental test may score (AMTS),
the modified mini mental state exam (3MS), the cognitive abilities
screening instrument (CASI), the Trail-making test, the clock
drawing test, the Informant Questionnaire on cognitive decline in
the elderly, the General practitioner assessment of cognition, the
Clinical Dementia Rating (CDR), Eight-item informant interview to
differentiate aging and dementia (AD8).
[0098] In some embodiments, the severity of the symptoms of
dementia are quantified using the CDR. Using the CDR, a score of 0
indicates no symptoms, a score of 0.5 indicates very mild symptoms,
a score of 1 indicates mild symptoms, a score of 2 indicates
moderate symptoms and a score of 3 indicates severe symptoms. Thus,
any increase in a CDR score for a subject indicates a worsening in
cognition and an increase in dementia. Moreover, change in CDR from
0 to greater than 0, indicates the development or onset of
dementia.
[0099] In some embodiments, a symptom associated with tau
aggregation refers to tau pathology. The term "tau pathology"
refers to the pathological aggregation of tau. In some embodiments,
tau pathology refers to neurofibrially tangles. In other
embodiments, tau pathology refers to hyperphosphorylated tau. In
still other embodiments, tau pathology refers to a high level of
tau aggregates detectable in blood, plasma, serum, CSF, or ISF,
anywhere from 1.2 to approximately 40-fold higher than that
detected in individuals without disease. Methods for detecting
pathological aggregation of tau are in known in the art and further
detailed in the Examples.
[0100] In an exemplary embodiment, a method of reducing the spread
of tau aggregation in the brain of a subject comprises
administering a pharmacologically effective amount of anti-tau
antibody to the subject, wherein the antibody is selected from the
group consisting of an isolated antibody comprising a light chain
variable region comprising a CDR1 of amino acid sequence SEQ ID NO:
16 with zero to two amino acid substitutions, an isolated antibody
comprising a light chain variable region comprising a CDR2 of amino
acid sequence SEQ ID NO: 17 with zero to two amino acid
substitutions, an isolated antibody comprising a light chain
variable region comprising a CDR3 of amino acid sequence SEQ ID NO:
18 with zero to two amino acid substitutions, an isolated antibody
comprising a heavy chain variable region comprising a CDR1 of amino
acid sequence SEQ ID NO: 19 with zero to two amino acid
substitutions, an isolated antibody comprising a heavy chain
variable region comprising a CDR2 of amino acid sequence SEQ ID NO:
20 with zero to two amino acid substitutions, and an isolated
antibody comprising a heavy chain variable region comprising a CDR3
of amino acid sequence SEQ ID NO: 21 with zero to two amino acid
substitutions.
[0101] In another exemplary embodiment, a method of reducing the
spread of tau aggregation in the brain of a subject comprises
administering a pharmacologically effective amount of anti-tau
antibody to the subject, wherein the antibody specifically binds
tau and recognizes an epitope comprising SEQ ID NO: 1
(DRKDQGGYTMHQD).
[0102] In another exemplary embodiment, a method of reducing the
spread of tau aggregation in the brain of a subject comprises
administering a pharmacologically effective amount of anti-tau
antibody to the subject, wherein the antibody specifically binds
tau and recognizes an epitope consisting of SEQ ID NO: 1
(DRKDQGGYTMHQD).
[0103] The antibodies in a pharmacologically effective amount
preferred in pharmaceutical grade, including immunologically
reactive fragments, may be administered to a subject.
Administration is performed using standard effective techniques,
include peripherally (i.e. not by administration into the central
nervous system) or locally to the central nervous system.
Peripheral administration includes but is not limited to
intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal,
intramuscular, intranasal, buccal, sublingual, or suppository
administration. Local administration, including directly into the
central nervous system (CNS) includes, but is not limited to, via a
lumbar, intraventricular or intraparenchymal catheter or using a
surgically implanted controlled release formulation.
[0104] Pharmaceutical compositions for effective administration are
deliberately designed to be appropriate for the selected mode of
administration, and pharmaceutically acceptable excipients such as
compatible dispersing agents, buffers, surfactants, preservatives,
solubilizing agents, isotonicity agents, stabilizing agents and the
like are used as appropriate. Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest
edition, incorporated herein by reference in its entirety, provides
a compendium of formulation techniques as are generally known to
practitioners. It may be particularly useful to alter the
solubility characteristics of the antibodies useful in this
discovery, making them more lipophilic, for example, by
encapsulating them in liposomes or by blocking polar groups.
[0105] Effective peripheral systemic delivery by intravenous or
intraperitoneal or subcutaneous injection is a preferred method of
administration to a living patient. Suitable vehicles for such
injections are straightforward. In addition, however,
administration may also be effected through the mucosal membranes
by means of nasal aerosols or suppositories. Suitable formulations
for such modes of administration are well known and typically
include surfactants that facilitate cross-membrane transfer. Such
surfactants are often derived from steroids or are cationic lipids,
such as N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethyl ammonium
chloride (DOTMA) or various compounds such as cholesterol
hemisuccinate, phosphatidyl glycerols and the like.
[0106] The concentration of humanized antibody in formulations to
be administered is an effective amount and ranges from as low as
about 0.1% by weight to as much as about 15 or about 20% by weight
and will be selected primarily based on fluid volumes, viscosities,
and so forth, in accordance with the particular mode of
administration selected if desired. A typical composition for
injection to a living patient could be made up to contain from 1-5
mL sterile buffered water of phosphate buffered saline and about
1-5000 mg of any one of or a combination of the humanized antibody
of the present discovery. The formulation could be sterile filtered
after making the formulation, or otherwise made microbiologically
acceptable. A typical composition for intravenous infusion could
have volumes between 1-250 mL of fluid, such as sterile Ringer's
solution, and 1-100 mg per ml, or more in anti-tau antibody
concentration. Therapeutic agents of the discovery can be frozen or
lyophilized for storage and reconstituted in a suitable sterile
carrier prior to use. Lyophilization and reconstitution may lead to
varying degrees of antibody activity loss (e.g. with conventional
immune globulins, IgM antibodies tend to have greater activity loss
than IgG antibodies). Dosages administered are effective dosages
and may have to be adjusted to compensate. The pH of the
formulations that are generally of pharmaceutical grade quality
will be selected to balance antibody stability (chemical and
physical) and comfort to the patient when administered. Generally,
a pH between 4 and 8 is tolerated. Doses will vary from individual
to individual based on size, weight, and other physio-biological
characteristics of the individual receiving the successful
administration.
[0107] As used herein, the term "effective amount" means an amount
of a substance such as a compound that leads to measurable and
beneficial effects for the patient administered the substance,
i.e., significant efficacy. The effective amount or dose of
compound administered according to this discovery will be
determined by the circumstances surrounding the case, including the
compound administered, the route of administration, the status of
the symptoms being treated and similar patient and administration
situation considerations among other considerations. In an aspect,
a typical dose contains from about 0.01 mg/kg to about 100 mg/kg of
an anti-tau antibody described herein. Doses can range from about
0.05 mg/kg to about 100 mg/kg, more preferably from about 0.1 mg/kg
to about 50 mg/kg, or from 0.5 mg/kg to about 50 mg/kg. The
frequency of dosing may be daily or once, twice, three times or
more per week or per month, as needed as to effectively treat the
symptoms. Alternatively, the frequency of dosing may be at least
once every three months, as needed as to effectively treat the
symptoms. For example, dosing may be about every 5 weeks, about
every 6 weeks, about every 7 weeks, about every 8 weeks, about
every 9 weeks, about every 10 weeks, about every 11 weeks, or about
every 12 weeks.
[0108] The timing of administration of the treatment relative to
the disease itself and duration of treatment will be determined by
the circumstances surrounding the case. Treatment could begin after
diagnosis of a disease associated with tau aggregation.
Alternatively, treatment could begin after clinical confirmation of
a symptom associated with tau aggregation. Further still, treatment
could begin after detection of tau pathology. Treatment could begin
immediately in a hospital or clinic, or at a later time after
discharge from the hospital or after being seen in an outpatient
clinic. Duration of treatment could range from a single dose
administered on a one-time basis to a life-long course of
therapeutic treatments.
[0109] Although the foregoing methods appear the most convenient
and most appropriate and effective for administration of proteins
such as humanized antibodies, by suitable adaptation, other
effective techniques for administration, such as intraventricular
administration, transdermal administration and oral administration
may be employed provided proper formulation is utilized herein.
[0110] In addition, it may be desirable to employ controlled
release formulations using biodegradable films and matrices, or
osmotic mini-pumps, or delivery systems based on dextran beads,
alginate, or collagen.
[0111] Typical dosage levels can be determined and optimized using
standard clinical techniques and will be dependent on the mode of
administration.
B. Method of Detecting Tau Aggregates in Biological Fluid
[0112] In an aspect, the invention provides means to detect tau
aggregate in a sample of biological fluid obtained from a subject.
In another aspect, the invention provides means to measure the
amount of tau aggregate in a sample of biological fluid obtained
from a subject. The method generally comprises (i) obtaining a
sample of a biological fluid from a subject, and (ii) measuring the
amount of tau aggregate in the sample using an antibody that
specifically binds tau. Suitable antibodies are described above in
Section I. Suitable subjects are described above.
[0113] As used herein, the term "biological fluid" refers to a
fluid obtained from a subject. Any biological fluid comprising a
tau aggregate is suitable. Non-limiting examples include blood,
plasma, serum, urine, CSF and ISF. The fluid may be used "as is",
the cellular components may be isolated from the fluid, or a
protein fraction may be isolated from the fluid using standard
techniques.
[0114] As will be appreciated by a skilled artisan, the method of
collecting a sample of biological fluid can and will vary depending
upon the nature of the biological fluid and the type of analysis to
be performed. Any of a variety of methods generally known in the
art may be utilized to collect a sample of biological fluid.
Generally speaking, the method preferably maintains the integrity
of the sample such that tau aggregate can be accurately detected
and the amount measured according to the invention.
[0115] Once a sample is obtained, it is processed in vitro in order
to detect and measure the amount of tau aggregate using an anti-tau
antibody. In some embodiments, the concentration of tau aggregate
in the sample is increased prior to detection and measurement. In
some embodiments, tau aggregate is immunoprecipitated from a sample
prior to detection and measurement using at least one isolated
anti-tau antibody. In other embodiments, tau aggregate is
immunoprecipitated from a sample prior to detection and measurement
using at least two isolated anti-tau antibodies. In embodiments
where at least two antibodies are used to immunoprecipitate tau
aggregates, preferably a first antibody binds a first epitope of
tau and a second antibody binds a second, non-overlapping epitope
of tau. The use of two antibodies that bind two distinct epitopes
of tau may be more efficient at capturing all possible tau
aggregate conformers. Non-limiting examples of suitable Antibody
pairs for immunoprecipitation are listed in Table C. In a preferred
embodiment, tau aggregate is immunoprecipitated from a sample prior
to detection and measurement using at least two isolated anti-tau
antibodies, wherein at least a first antibody recognizes an epitope
within SEQ ID NO: 1 and at least a second antibody recognizes an
epitope within SEQ ID NO: 8. A skilled artisan will be able to
determine with routine experimentation whether or not tau aggregate
in a sample needs to be concentrated or immunoprecipitated prior to
detection and measurement, and will be able to do so using methods
known in the art.
TABLE-US-00003 TABLE C Second Antibody HJ8.1.1 HJ8.1.2 HJ8.2 HJ8.3
HJ8.4 HJ8.5 HJ8.7 HJ8.8 HJ9.1 HJ9.2 HJ9.3 HJ9.4 HJ9.5 First HJ8.1.1
X X X X X X X X X X X Antibody HJ8.1.2 X X X X X X X X X X X HJ8.2
X X X X X X X X X X X HJ8.3 X X X X X X X X X X X HJ8.4 X X X X X X
X X X X X HJ8.5 X X X X X X X X X X X HJ8.7 X X X X X X X X X X X X
HJ8.8 X X X X X X X X X X X X HJ9.1 X X X X X X X X X X X X HJ9.2 X
X X X X X X X X X HJ9.3 X X X X X X X X X X X X X HJ9.4 X X X X X X
X X X X HJ9.5 X X X X X X X X X X
[0116] Methods for detecting and measuring an amount of protein
using an antibody are well known in the art. All suitable methods
for detecting and measuring an amount of protein using an antibody
known to one of skill in the art are contemplated within the scope
of the invention. Non-limiting examples include an ELISA, a
sandwich immunoassay, a radioimmunoassay, an immunoblot or Western
blot, flow cytometry, immunohistochemistry, and an array.
[0117] In general, an antibody-based method of detecting and
measuring an amount of tau aggregate comprises contacting some or
all of the sample comprising tau aggregate with an anti-tau
antibody under conditions effective to allow for formation of a
complex between the antibody and the tau aggregate. Typically, the
entire sample is not needed, allowing one skilled in the art to
repeatedly detect and measure the amount of tau aggregate in the
sample over time. The method may occur in solution, or the antibody
or tau aggregate may be immobilized on a solid surface.
Non-limiting examples of suitable surfaces include microtitre
plates, test tubes, beads, resins, and other polymers. Attachment
to the substrate may occur in a wide variety of ways, as will be
appreciated by those in the art. For example, the substrate and the
antibody may be derivatized with chemical functional groups for
subsequent attachment of the two. For example, the substrate may be
derivatized with a chemical functional group including, but not
limited to, amino groups, carboxyl groups, oxo groups or thiol
groups. Using these functional groups, the antibody may be attached
directly using the functional groups or indirectly using linkers.
An anti-tau antibody may also be attached to the substrate
non-covalently. For example, a biotinylated anti-tau antibody may
be prepared, which may bind to surfaces covalently coated with
streptavidin, resulting in attachment. Alternatively, an antibody
may be synthesized on the surface using techniques such as
photopolymerization and photolithography.
[0118] Contacting the sample with an antibody under effective
conditions for a period of time sufficient to allow formation of a
complex generally involves adding the anti-tau antibody composition
to the sample (or to the immunopreicipitated or concentrated tau
aggregate) and incubating the mixture for a period of time long
enough for the anti-tau antibody to bind to any antigen present.
After this time, the complex may be washed and then the complex is
detected and the amount measured by any method well known in the
art. Methods of detecting and measuring an amount of an
antibody-polypeptide complex are generally based on the detection
of a label or marker. The term "label", as used herein, refers to
any substance attached to an antibody, or other substrate material,
in which the substance is detectable by a detection method.
Non-limiting examples of suitable labels include luminescent
molecules, chemiluminescent molecules, fluorochromes, fluorescent
quenching agents, colored molecules, radioisotopes, scintillants,
biotin, avidin, stretpavidin, protein A, protein G, antibodies or
fragments thereof, polyhistidine, Ni.sup.2+, Flag tags, myc tags,
heavy metals, and enzymes (including alkaline phosphatase,
peroxidase, and luciferase). Methods of detecting and measuring an
amount of an antibody-polypeptide complex based on the detection of
a label or marker are well known in the art.
[0119] In a preferred embodiment, a method for measuring the amount
of tau aggregate in a sample is an immunoassay comprising two
captures antibodies and a detection antibody, wherein each capture
antibody is an isolated anti-tau antibody that recognizes a tau
epitope distinct from the other, and the detection antibody is an
isolated anti-tau antibody attached to a label. The detection
antibody may be the same antibody as one of the two capture
antibodies or, alternatively, the detection antibody may recognize
a tau epitope not recognized by either capture antibody. Typically,
the first capture antibody and the second capture antibody are used
in an amount from about 10:1 to about 1:10, from about 5:1 to about
1:5, from about 3:1 to about 1:3, or from about 2:1 to about 1:2.
In some embodiments, the first capture antibody and the second
capture antibody are used at about equivalent concentrations.
Non-limiting examples of suitable pairs of capture antibodies
include the antibodies disclosed in Table D and Table E.
Non-limiting examples of suitable detection antibodies include the
antibodies listed in Table A, as well as antibodies that
specifically bind tau and recognize an epitope within an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1-11. In
an exemplary embodiment, a first capture antibody is an isolated
antibody that specifically binds tau and recognizes an epitope
within SEQ ID NO: 7, a second capture antibody is an isolated
antibody that specifically binds tau and recognizes an epitope
within SEQ ID NO: 8, and a detection antibody is an isolated
antibody that specifically binds tau and recognizes an epitope
within SEQ ID NO: 8.
TABLE-US-00004 TABLE D First and Second Capture Antibodies Second
Capture Antibody HJ8.1.1 HJ8.1.2 HJ8.2 HJ8.3 HJ8.4 HJ8.5 HJ8.7
HJ8.8 HJ9.1 HJ9.2 HJ9.3 HJ9.4 HJ9.5 First HJ8.1.1 X X X X X X X X X
X X Capture HJ8.1.2 X X X X X X X X X X X Antibody HJ8.2 X X X X X
X X X X X X HJ8.3 X X X X X X X X X X X HJ8.4 X X X X X X X X X X X
HJ8.5 X X X X X X X X X X X HJ8.7 X X X X X X X X X X X X HJ8.8 X X
X X X X X X X X X X HJ9.1 X X X X X X X X X X X X HJ9.2 X X X X X X
X X X X HJ9.3 X X X X X X X X X X X X X HJ9.4 X X X X X X X X X X
HJ9.5 X X X X X X X X X X
TABLE-US-00005 TABLE E First and Second Capture Antibodies: each
antibody specifically binds tau and recognizes an epitope within
the amino acid sequence indicated by the SEQ ID NO shown. Second
Capture Antibody SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 NO: 7 NO: 8 First SEQ ID
X X X X X X X Capture NO: 1 Antibody SEQ ID X X X X X X X NO: 2 SEQ
ID X X X X X X X NO: 3 SEQ ID X X X X X X X NO: 4 SEQ ID X X X X X
X X NO: 5 SEQ ID X X X X X X X NO: 6 SEQ ID X X X X X X X NO: 7 SEQ
ID X X X X X X X NO: 8
[0120] In another aspect, the invention provides means to classify
a subject based on the amount of tau aggregate measured in a sample
of biological fluid obtained from the subject. The method generally
comprises (i) obtaining a sample of a biological fluid from a
subject and measuring the amount of tau aggregate in the sample
using an antibody that specifically binds tau, (ii) comparing the
amount of tau aggregate in the sample to a reference value, and
(iii) classifying the subject as having a high or low amount of tau
aggregate based on the amount of tau aggregate measured in the
sample. Methods for obtaining a sample of a biological fluid from a
subject and measuring the amount of tau aggregate in the sample
using an antibody that specifically binds tau are detailed above
and further described in the Examples.
[0121] Any suitable reference value known in the art may be used.
For example, a suitable reference value may be the amount of tau
aggregate in a sample of biological fluid obtained from a subject,
or group of subjects, of the same species that has no clinically
detectable symptom of tau aggregation. In another example, a
suitable reference value may be the amount of tau aggregate in a
biological fluid sample obtained from a subject, or group of
subjects, of the same species that has no detectable tau pathology.
In another example, a suitable reference value may be the amount of
tau aggregate in a biological fluid sample obtained from a subject,
or group of subjects, of the same species that has a Clinical
Dementia Rating score of zero (CDR=0). In another example, a
suitable reference value may be the background signal of the assay
as determined by methods known in the art. In another example, a
suitable reference value may be a measurement of the amount of tau
aggregate in a reference sample obtained from the same subject. The
reference sample comprises the same type of biological fluid as the
test sample, and may be obtained from a subject when the subject
had no clinically detectable symptom of tau aggregation. A skilled
artisan will appreciate that it is not always possible or desirable
to obtain a reference sample from a subject when the subject is
otherwise healthy. For example, when monitoring the effectiveness
of a therapy, a reference sample may be a sample obtained from a
subject before therapy began. In such an example, a subject may
have tau pathology but may not have other symptoms of tau
aggregation (e.g. dementia, declined cognition, etc.) or the
subject may have tau pathology and one or more other symptom of tau
aggregation. In an additional example, a suitable reference sample
may be a biological fluid from an individual or group of
individuals that has been shown not to have tau aggregates.
[0122] According to the invention, a subject may be classified
based on the amount of tau aggregate measured in the sample.
Classifying a subject based on the amount of tau aggregate measured
in a sample of biological fluid obtained from the subject may be
used to identify subjects that will develop a disease and/or
symptom associated with tau aggregation in the subject's lifetime.
Generally speaking, a subject may be classified as having a high or
low amount of tau aggregate compared to a reference value, wherein
a high amount of tau aggregate is an amount above the reference
value and a low amount is an amount equal to or below the reference
value. In preferred embodiments, to classify a subject as having a
high amount of tau aggregate, the amount of tau aggregate in the
sample of biological fluid compared to the reference value is
increased at least 2-fold. For example, the amount of tau aggregate
in the sample compared to the reference value is increased at least
2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at
least 20-fold, at least 25-fold, at least 30-fold, at least
35-fold, at least 40-fold, at least 45-fold, or at least 50-fold.
When the amount of tau aggregate in the sample of biological fluid
obtained from a subject is increased at least 2-fold compared to a
reference value, and the reference value is a sample of the same
type of biological fluid obtained from one or more disease free
individuals with no detectable symptom of tau aggregation (or a
sample equivalent thereto), the subject is more likely to develop a
disease and/or symptom associated with tau aggregation in the
subject's lifetime.
Definitions
[0123] As used herein, "antibody" refers to an immunoglobulin
derived molecule that specifically recognizes tau. An antibody of
the invention may be a full length antibody (IgM, IgG, IgA, IgE) or
may be an antibody fragment (Fab, F(ab')2, scFv). An antibody may
be chimeric or may be humanized.
[0124] As used herein, "CDR" means "complementary determining
region." CDRs may also be referred to as hypervariable regions.
[0125] As used herein, "light chain" is the small polypeptide
subunit of the antibody. A typical antibody comprises two light
chains and two heavy chains.
[0126] As used herein, the "heavy chain" is the large polypeptide
subunit of the antibody. The heavy chain of an antibody contain a
series of immunoglobulin domains, with at least one variable domain
and at least one constant domain.
[0127] "Humanized", as used herein, refers to the process where
monoclonal antibodies are produced using recombinant DNA to create
constructs capable of expression in human cell culture. Any known
techniques for producing these constructs will work for purposes of
the present invention.
[0128] As used herein, "single chain variable fragments" or "scFv"
or "scFvs", refer to fusion proteins of the variable regions of the
heavy and light chains of immunoglobulins connected via a linker.
In some embodiment, the linker is a peptide of about 10 to 25 amino
acids.
EXAMPLES
[0129] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Introduction to Examples 1-8
[0130] Aggregation of the microtubule associated protein tau in
neurons and glia is associated with over 20 neurodegenerative
disorders including Alzheimer disease (AD), progressive
supranuclear palsy, and frontotemporal dementia. Recent evidence
from human studies suggests that tau pathology does not distribute
randomly through the brain, but instead is linked to existing
networks of neuronal connectivity. The fibrillar tau pathology of
AD progresses along known anatomical connections, although the
mechanisms by which networks degenerate are unknown. Importantly,
recent pathological studies suggest that protein aggregates can
move from one cell to another in human and mouse brain. Moreover,
fibrillar forms of recombinant, human disease-associated proteins
such as tau, SOD-1, .alpha.-synuclein and polygutamines are readily
taken up from the extracellular space to trigger intracellular
misfolding. These phenomena are reminiscent of prion propagation,
for which exosomes and tunneling nanotubes have been proposed to
mediate trans-cellular spread. It is an open question as to whether
tau aggregates might spread protein misfolding from cell to cell
via direct cell-cell contact or through extracellular space.
Furthermore, it has not yet been determined whether pathological
tau species can mediate true trans-cellular propagation of
aggregation, whereby an aggregate is released from a "donor" cell,
enters a second "recipient" cell, and induces further misfolding
via direct protein-protein contact, as opposed to more indirect
mechanisms. Here it is tested whether tau fibrils are released
directly into the extracellular space and can propagate aggregation
by this mechanism.
Example 1. Anti-Tau Antibodies
[0131] Two series of anti-tau antibodies were created using
standard techniques: the HJ8 series (mouse monoclonal antibodies
against recombinant human tau), and the HJ9 series (mouse
monoclonal antibodies against recombinant mouse tau) (Table 1).
Binding epitopes have been mapped for many of the antibodies (Table
A).
TABLE-US-00006 TABLE 1 HJ8 series and HJ9 series against human and
mouse tau Antibody Isotype Application HJ 8.1 IgG2b IgG1 IP WB
IHC(h&m) HJ 8.2 IgG2b IP WB IHC(h&m) HJ 8.3 IgG2b IP WB
IHC(h&m) HJ 8.4 IgG1 IP WB IHC(h&m) HJ 8.5 IgG2b IP WB
IHC(h) ELISA for coating staining 3mon old mice HJ 8.7 IgG2b IP WB
IHC(h&m) HJ8.7B for ELISA detact staining HJ 8.8 IgG2b IP WB
IHC(h&m) staining HJ 9.1 IgG2b IP WB IHC ELISA HJ 9.2 unknown
IP WB IHC ELISA for coating staining HJ 9.3 IgG2b IP WB IHC ELISA
for coating HJ 9.4 IgG2b IP WB IHC HJ 9.5 IgG2b IP WB IHC IP =
Immunoprecipitation; WB = Western] Blot; ELISA = Enzyme-linked
immosorbent Assay, IHC = immunohistochemistry; h = human; m =
mouse
[0132] To characterize the binding affinity of the HJ8 and HJ9
series antibodies to mouse tau and human tau, Biacore's SPR
technology was used. Biacore sensor chip CM-5 (Carboxymethylated
dextran matrix) was activated by using EDC
(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimid) and NHS
(N-hydroxysuccinimide) in 1:1 ratio. 2). Then ligand, either mouse
tau or human Tau, were immobilized (20 ug/ml, in 10 mM sodium
acetate pH 3.5) on Biacore CM-5 sensor chip at a flow rate of 5
.mu.I/min. The remaining unbound area on the Biacore CM-5 sensor
chip was deactivated by passage of 1 M ethanolamine pH 8.5.
[0133] Following preparation of the sensor chip surface, analytes
(e.g. antibody) were injected with different concentrations (0.78
nM-400 nM) in filtered, degassed 0.01 M Hepes buffer, 0.15 M NaCl,
0.005% surfactant P20, pH 7.4 at a flow rate of 10 .mu.I/min. All
the samples were run in duplicates. After each cycle/run with
single antibody concentration, the surface of the chip was
regenerated by using 10 mM glycine pH 1.7, to remove the bound
antibody/analyte, leaving the monomer/fibrils/ligand attached to
the surface.
[0134] From the SPR sensorgram (FIGS. 2A-13B), the rate of
association or On rate (K.sub.a), the rate of dissociation or Off
rate (K.sub.d) and the affinity constant or Affinity of interaction
(KD, where KD=K.sub.d/K.sub.a) were obtained (Tables 2 and 3).
TABLE-US-00007 TABLE 2 Binding data of HJ8 series and HJ9 series to
mouse tau Antibody K.sub.a (1/MS) K.sub.d (1/s) K.sub.D (M) HJ 8.1
3.17 .times. 10.sup.4 1.83 .times. 10.sup.-8 0.578 pM HJ 8.2 2.25
.times. 10.sup.5 3.45 .times. 10.sup.-7 1.57 pM HJ 8.3 1.46 .times.
10.sup.5 7.05 .times. 10.sup.-8 0.48 pM HJ 8.4 2.78 .times.
10.sup.5 1.03 .times. 10.sup.-7 0.37 pM HJ 8.5 No binding detected
HJ 8.7 7.03 .times. 10.sup.5 2.41 .times. 10.sup.-8 0.34 pM HJ 8.8
1.92 .times. 10.sup.5 1.78 .times. 10.sup.-4 0.926 nM HJ 9.1 3.52
.times. 10.sup.5 7.61 .times. 10.sup.-9 0.02 pM HJ 9.2 2.65 .times.
10.sup.5 1.08 .times. 10.sup.-4 0.4 nM HJ 9.3 8.61 .times. 10.sup.4
9.16 .times. 10.sup.-6 0.1 nM HJ 9.4 2.28 .times. 10.sup.5 5.1
.times. 10.sup.-7 2.24 pM HJ 9.5 3.4 .times. 10.sup.5 5.37 .times.
10.sup.-7 1.58 pM
TABLE-US-00008 TABLE 3 Binding data of of HJ8 series and HJ9 series
to human tau Antibody K.sub.a (1/MS) K.sub.d (1/s) K.sub.D (M) HJ
8.1 2.43 .times. 10.sup.4 3.19 .times. 10.sup.-8 1.32 pM HJ 8.2
1.98 .times. 10.sup.5 8.95 .times. 10.sup.-7 4.51 pM HJ 8.3 1.44
.times. 10.sup.5 1.93 .times. 10.sup.-3 0.07 pM HJ 8.4 2.46 .times.
10.sup.5 3 .times. 10.sup.-8 0.122 pM HJ 8.5 1.3 .times. 10.sup.5
4.34 .times. 10.sup.-8 0.336 pM HJ 8.7 6.8 .times. 10.sup.4 2.33
.times. 10.sup.-8 0.34 pM HJ 8.8 1.6 .times. 10.sup.5 9.57 .times.
10.sup.-7 5.95 pM HJ 9.1 3.1 .times. 10.sup.5 1.84 .times.
10.sup.-3 0.5 pM HJ 9.2 6.13 .times. 10.sup.4 1.15 .times.
10.sup.-3 24.6 nM HJ 9.3 7.55 .times. 10.sup.4 7.51 .times.
10.sup.-6 99 pM HJ 9.4 1.53 .times. 10.sup.5 1.07 .times. 10.sup.-3
6.9 nM HJ 9.5 5.14 .times. 10.sup.5 1.97 .times. 10.sup.-3 3.82
nM
Example 2. Full Length Tau is Present in ISF
[0135] Tau was immunoprecipitated from ISF samples of both
wild-type mice and P301 S human tau transgenic mice (P301 S tg
mice, details in Methods) using tau antibodies recognizing both
mouse and human tau. Two anti-tau monoclonal antibodies that worked
well in immunoprecipitation assays were used, as the amount of
monomeric tau in ISF is relatively low. Following
immunoprecipitation, tau was analyzed by immunoblot. Endogenous
murine tau isoforms migrate at 48-62 kDa. In wild-type brain
lysate, tau appeared in four separate bands on SDS-PAGE (FIG. 14A).
The most abundant species in wild-type mice migrated at 48 kDa. In
P301 S tg mice brain, in addition to the four endogenous murine tau
bands, overexpressed human 1 N4R tau was observed as an intense
band migrating at 55 kDa as well as a 39 kDa band, which may
represent a tau degradation product.
[0136] In contrast to total brain lysates, upon immunoprecipitation
a single tau band was detected with antibody HJ9.3 recognizing the
microtubule binding region (MTBR) of tau in ISF from wild-type mice
(FIG. 14B). This band corresponded to the largest isoform 2N4R
observed in mouse brain lysate. In ISF of P301 S tg mice, a
human-specific tau band was co-precipitated with the aforementioned
mouse tau band and was slightly lower in molecular weight (FIG.
14B). These two bands were also precipitated by another mouse
monoclonal antibody raised against tau HJ8.1 (FIG. 14C). These data
suggested that the major species in ISF that is assessed by ELISA
is likely full-length monomeric tau.
Example 3. Tau RD Proteins Form Fibrillar Aggregates in Transfected
HEK293 Cells
[0137] The tau gene encodes six protein isoforms, and multiple
mutations cause dominantly inherited neurodegenerative disease.
Depending on splicing, the tau protein has either three or four
repeat regions that constitute the aggregation-prone core of the
protein, which is termed the repeat domain (RD). Expression of the
tau RD causes pathology in transgenic mice, and there is evidence
for truncation of full-length tau to form fragments that comprise
fibrils in patients. This construct was used rather than
full-length tau because it reliably forms fibrils in cultured
cells. Various mutations known to increase tau aggregation were
engineered into a four-repeat RD protein: .DELTA.K280 (termed
.DELTA.K), P301 L, and V337M. The P301 L and V337M mutants were
combined in one protein (termed LM) to create a mutant form of RD
with strongly increased aggregation potential, similar to what has
been described previously. This "nonphysiologic" mutant facilitates
assays of transfer events and trans-cellular propagation of
misfolding that depend on efficient formation of intracellular
aggregates, and complements similar, but less robust aggregation
phenotypes of the "physiologic" .DELTA.K mutant. Also engineered
were two proline substitutions into the .DELTA.K mutant, I227P and
1308P (termed PP), which inhibit .beta.-sheet formation and
fibrillization, although they do not block formation of amorphous
aggregates. Each form of mutant tau was fused either at the
carboxyl terminus to cyan or yellow fluorescent protein (CFP or
YFP), or to an HA tag. Constructs are diagrammed in FIG. 15A.
[0138] To evaluate the characteristics of tau RD intracellular
aggregates, the various forms of RD were transiently transfected
into HEK293 cells. Atomic force microscopy (AFM) was used to
evaluate SDS-insoluble material. RD(.DELTA.K)-HA and RD(LM)-HA
produced evident fibrillar species (FIG. 15B). RD(.DELTA.K)-HA and
RD(LM)-HA aggregates within cells also stained positive for X-34, a
thioflavin derivative that labels beta sheet fibrils and emits in
the blue spectrum (FIG. 15C). Additionally, detergent fractionation
was used to test whether the inclusions visible by light microscopy
had a biochemical correlate. In SDS insoluble pellets (1% Triton
X-100 in 1.times.PBS with protease inhibitors for isolation of
soluble pellet followed by SDS/RIPA extraction of insoluble
pellets), monomer and higher molecular weight species consistent
with oligomers were detected (FIG. 15D).
[0139] The applicants previously used fluorescence resonance energy
transfer (FRET) to quantitate intracellular huntingtin protein
aggregation. To test whether this method could be used to track tau
RD aggregation, the various RD mutants (wt, .DELTA.K, PP, LM) were
fused to yellow fluorescent protein (YFP:FRET acceptor) and cyan
fluorescent protein (CFP:FRET donor). These constructs were
co-transfected into HEK293 cells (denoted as RD-CFP/RD-YFP), and
intracellular aggregate formation was quantified using FRET
acceptor photobleaching confocal microscopy and spectral emission
FRET using a fluorescence plate reader (FPR). For confocal
microscopy, cells co-expressing RD(LM)-CFP/RD(LM) YFP were imaged
and donor signal was measured before and after partial and complete
acceptor photobleaching. The increase in donor signal after
photobleaching resulted in a mean FRET efficiency of 18.2%.+-.0.058
(n=6, data are .+-.standard deviation) confirming inter-molecular
interactions between the FRET-paired RD species (FIG. 16A). To
measure RD-CFP/YFP aggregation by spectral FRET with a FPR,
established methods were used. This was based on co-transfection of
RD-YFP and RD-CFP in a 3:1 ratio, to maximize donor quenching
within the limits of signal detection. Significant FRET from
RD(PP)-CFP/YFP was not observed. However, RD(.DELTA.K)-CFP/YFP and
RD(LM)-CFP/YFP each produced a strong FRET signal (FIG. 16B),
corroborating the microscopy findings.
[0140] It has been previously observed that a variety of cells will
take up recombinant tau fibrils from the extracellular media. This
triggers intracellular fibrillization of natively folded, full
length tau protein fused to YFP. To confirm this phenomenon, FRET
was used to monitor aggregation of RD(.DELTA.K)-CFP/YFP induced by
various amounts of recombinant RD fibrils. HEK293 cells were
co-transfected with RD(.DELTA.K)-CFP/YFP and cultured for 15 h.
Various concentrations of RD-HA fibrils (monomer equivalents of
0.01, 0.03, 0.1 and 0.3 .mu.M) were then added to the media for 9
h. Fibrils were then removed by changing the media, and the cells
were allowed to recover for 4 h before being fixed and analyzed
using FRET. A dose dependent increase in the FRET signal induced by
recombinant fibrils relative to untreated RD(.DELTA.K)-CFP/YFP
cells was observed (FIG. 16C). In summary, a correlation between
microscopic, molecular, biochemical, and biophysical measures of
tau RD aggregation and fibril formation within cells was observed.
Within certain limits, especially with controls for protein
expression levels, the plate reader-based FRET assay provides a
facile measure of this process.
Example 4. Trans-Cellular Induction of RD Aggregation
[0141] The applicants have previously determined that tau
inclusions from one cell will transfer to naive cells in
co-culture. However it has not yet been demonstrated that these
transferred aggregates can induce further aggregation in the
recipient cells, nor whether induction of aggregation is based on
direct protein-protein interaction. First tested was whether
RD(LM)-HA aggregates derived from one donor cell population would
form inclusions with RD(.DELTA.K)-YFP in a different recipient
population upon co-culture. One group of cells was transfected with
aggregation-prone RD(LM)-HA, and a separate group transfected with
RD(.DELTA.K)-YFP. The next day, the cell populations were re-plated
together and co-cultured for 48 h. After fixation, they were
immunostained using an HA antibody, and counterstained with X-34.
Many cells were observed with RD(LM)-HA and RD(.DELTA.K)-YFP
co-localized in inclusions (FIG. 17A). Frequently these inclusions
also stained positive for X-34, indicating beta sheet structure.
These studies were extended by using the FRET assay to monitor
aggregation of RD(.DELTA.K)-CFP/YFP induced by co-culture with
cells expressing RD(LM)-HA. In this case, two populations of cells
were co-cultured. The donor population expressed RD(LM)-HA and the
recipient population expressed RD(.DELTA.K)-CFP/YFP. The
.beta.-sheet-resistant form of tau RD(PP)-HA or mock transfected
cells were used as negative controls. After 48 h FRET was measured
from the cell monolayers. A strong increase in FRET induced by
co-culture with RD(LM)-HA versus RD(PP)-HA or mock transfected
cells was observed (FIG. 17B). A small increase in FRET signal was
observed following co-culture of RD(LM)-HA cells with
RD(WT)-CFP/YFP recipient cells (data not shown). These results
suggested movement of one aggregation-prone tau species from one
cell to another to trigger co-localization in a beta-sheet rich
inclusion. Aggregate release could potentially occur after cell
death, however, no evidence for this was observed using propidium
iodide staining of the various transfected populations (FIG.
17C).
Example 5. Propagation of Misfolding by Direct Protein Contact
[0142] While strongly suggestive, these results could not formally
address whether co-aggregation occurred via direct protein contact,
with intermolecular association between tau RD derived from donor
contacting the corresponding protein in recipient cells. FRET was
used to address this question. First, RD(LM)-CFP was co-expressed
within a donor cell population, and RD(LM)-YFP in a second
recipient population. FRET from the cell monolayers was measured
after 48 with both confocal microscopy and the FPR. Using confocal
microscopy, CFP signal was measured before and after photobleaching
of YFP. A mean FRET efficiency of -14.2% was recorded, indicating
that inclusions contained RD(LM)-CFP and RD(LM)-YFP in direct
contact (FIG. 18A). Relative FRET signals were then compared via
FPR, using different forms of unlabeled RD to induce aggregation of
RD-CFP. First, RD(.DELTA.K)-CFP and RD(LM)-HA were co-expressed
within a donor cell population, and RD(.DELTA.K)-YFP in a second
recipient cell population. RD(LM)-HA serves as an enhancer of both
RD(.DELTA.K)-CFP aggregation and movement, prompting its subsequent
transfer into the RD(.DELTA.K)-YFP recipient cells. This led to a
small but reproducible FRET signal increase in the co-cultured
cells. This signal disappeared when either the CFP- or YFP-tagged
RD constructs contained the PP mutation that blocks .beta.-sheet
formation (FIG. 18B), indicating that both members of the pair must
have the capacity to form a beta sheet structure. Taken together
with the prior experiments, these results suggested that
propagation of misfolding by direct contact occurs, i.e. an
aggregate from one cell exits to contact and trigger misfolding of
natively folded protein in a second cell. This data implied that
amplification of misfolding might also occur in serial cell
co-cultures. It was predicted that pre-exposure of a "donor" cell
population to aggregation seeds would increase final aggregation
detected in a recipient cell population. This was tested by
successively culturing three populations of cells. The first
population expressed various forms of non-fluorescent RD-HA to form
aggregation "seeds." The second group expressed CFP or
RD(.DELTA.K)-CFP, to be either non-permissive (CFP) or permissive
RD(.DELTA.K)-CFP) for aggregate maintenance. These two groups were
co-cultured for 48 h to allow amplification of misfolding. Next,
50% of the combined first and second groups were then co-cultured
for 48 h with a third group of cells expressing RD(.DELTA.K)-YFP.
This third recipient group served as a "reporter" to indicate the
degree of RD(.DELTA.K)-CFP intracellular aggregation and
propagation. Prior exposure of RD(LM)-HA to the RD(.DELTA.K)-CFP
population increased final FRET by 2.6 fold vs. cells that had not
been pre-exposed to aggregation-prone tau. As expected,
interposition of cells expressing pure CFP in the second population
of cells completely blocked the effect of prior exposure to tau RD
"seeds" (FIG. 18C). Taken together these data indicate an
amplification of tau aggregation within serially cultured cell
populations.
Example 6. Cell-Cell Propagation Mediated by Release of Aggregates
into the Extracellular Space
[0143] The mechanism by which protein aggregates move between cells
is unknown. For example, some have postulated prion protein
propagation via tunneling nanotubes, while others have suggested
exosomes. Since antibodies against tau protein have previously been
reported to reduce pathology in vivo, it was hypothesized that tau
aggregates might be released directly into the extracellular space.
Whereas trans-cellular movement based on cell-cell contact should
be independent of the volume of extracellular media, it was
predicted that transcellular movement of tau might be sensitive to
extracellular volume, as has been described for SOD1. To start, the
effect of co-culture in the setting of various volumes of media was
first tested. It was observed that increasing the cell culture
medium volume reduced the efficiency of transcellular movement of
aggregates (FIG. 19A). Further, transfer of conditioned medium from
cells expressing RD(LM)-HA was sufficient to induce aggregation in
cells expressing RD-CFP/YFP (FIG. 19B). These results were
consistent with the movement of tau between cells through the
extracellular space, but could not determine whether the protein
was encapsulated in an endosome.
[0144] It was reasoned that access to encapsulated tau would be
blocked by the lipid membrane, whereas free tau would be accessible
to an antibody. Thus, it was tested whether a mouse monoclonal
antibody (HJ9.3) that can immunprecipitate tau would block
transcellular propagation. A modification of the cellular model of
tau RD propagation described above was used, in which RD(LM)-HA and
RD(.DELTA.K)-CFP were co-expressed within one cell population, and
co-cultured for 48 h with cells that express RD(.DELTA.K)-YFP,
prior to analysis by FRET. HJ9.3 versus pooled mouse IgG was tested
for the 48 h co-culture period. A dose dependent reduction in
trans-cellular propagation with HJ9.3 was observed, while
non-specific IgG had no effect (FIGS. 19C and 19D). Importantly,
HJ9.3 had no effect on intracellular aggregation of
RD(.DELTA.K)-CFP and RD(.DELTA.K)-YFP when the two proteins were
co-expressed within the same cell (FIG. 19E), indicating the
antibody was not directly inhibiting intracellular aggregation. The
role of free tau was further tested in transcellular propagation by
evaluating induction of tau misfolding using biochemistry. The
induction of aggregation by detergent fractionation and Western
blot was confirmed, which revealed an increase in RD(.DELTA.K)-YFP
in the insoluble fraction induced by co-culture with RD(LM)-HA.
HJ9.3 blocked the effect of RD(LM)-HA to induce insolubility of
RD-YFP in co-cultured cells (FIGS. 19F and 19G).
[0145] The effectiveness of antibody addition suggested that free
tau was directly transferring between cells, but left uncertain the
mechanism of antibody inhibition. It was hypothesized that HJ9.3
was blocking uptake of tau fibrils into cells. To test this idea
flow cytometry was used to monitor the effect of the antibody on
transcellular movement of aggregates. The applicants have
previously established a cytometry paradigm whereby one population
of cells is labeled with mCherry, and the second contains tau-YFP
fusions. After co-culture, it is possible to monitor trans-cellular
movement based on the relative percentage of dual-positive
(YFP/mCherry) cells. A population of HEK293 cells was transfected
with tau RD(LM)-YFP, and a second population was transduced with
lentivirus expressing mCherry. After washing and resuspending the
two populations, the cells were then co-cultured for 48 h in the
presence or absence of 10-fold dilutions of HJ9.3 in the medium.
Cells were harvested and the relative number of dual positive cells
measured using flow cytometry. Negative controls consisted of the
same cell populations mixed prior to sorting. Each data point
consisted of biological triplicates. Co-cultured cells had
significantly more RD(LM)-YFP/mCherry dual positive cells (2.07%)
compared to 0.142% of premixed cells (background). HJ9.3 decreased
the percentage of dual positive cells from 2.07% to 1.31% (FIG.
19H). This parallels the effect of this antibody on transcellular
propagation of aggregation as measured by FRET. The difference in
the potency of this antibody in blocking propagation as measured by
FRET and flow cytometry is most probably due to the differences
between the two techniques used to measure this event.
[0146] To further monitor the effect of the HJ9.3 antibody on
trans-cellular movement of aggregates, direct immunofluorescence
was used in an attempt to define where the HJ9.3/antibody complexes
deposited. RD(.DELTA.K)-YFP cells or non-transfected cells were
cultured in the presence of HJ9.3 for 48 hrs. Cells were fixed with
4% PFA, permeabilized with 0.25% TritonX-100 and then exposed to
goat anti-mouse Alexa 546 labeled secondary antibody. A very small
number of HJ9.3/tau complexes were present inside cells. However,
most complexes were found outside of the cells, mainly bound to the
cell membrane. This antibody decoration was not present in
nontransfected cells indicating that the signal is specific to the
HJ9.3/tau complexes (FIG. 20). Thus HJ9.3 blocks tau aggregate
uptake, trapping aggregates outside the cell.
Example 7. Tau Fibrils Mediate Cell-Cell Propagation
[0147] The activity of HJ9.3 in the propagation assay created an
opportunity to define the tau species responsible. HJ9.3 was used
to extract tau from the cell media. HJ9.3 or control IgG was added
to the media of cells expressing a variety of RD constructs (wt,
PP, .DELTA.K, LM). Antibodies were added either at the beginning or
the end of the 48 h culture period. Media were harvested for
affinity purification of antibody/antigen complexes using
protein-G-agarose beads. The complexes were washed, and then boiled
in SDS loading buffer for analysis by Western blot. HJ9.3
specifically captured tau RD species from the cell media, while IgG
had no appreciable effect (FIG. 21A). A .about.10-fold increase in
the tau protein present in the media was observed when HJ9.3 was
present throughout the culture period, as opposed to addition at
the end of this period (FIG. 21B). Higher-order molecular weight
species were also noted in the media of RD(.DELTA.K)-HA and
RD(LM)-HA transfected cells, consistent with RD aggregates.
RD(PP)-HA tau had the least protein present in the medium, and no
higher-order species were observed on Western blot. A time course
(Oh, 3 h, 6 h, 9 h, 12 h, 24 h and 48 h) of the previously
described experiment showed a time-dependent increase in the levels
of tau in the media, implying that HJ9.3 incubation was indeed
increasing the steady-state level of tau protein present in the
conditioned medium (FIG. 21C). Taken together, these data indicated
that HJ9.3 blocks cell-to-cell propagation by interference with
aggregate uptake into cells, and is consistent with a steady state
flux of tau aggregates in and out of cells.
[0148] The precise nature of the tau species that mediate
trans-cellular propagation is not known. Thus, HJ9.3 was used to
trap these species for imaging via AFM. HEK293 cells that were
transfected with the various tau mutants were cultured in the
presence of HJ9.3. After 48 the antibody/antigen complexes were
purified with protein-G agarose beads. The complexes were then
eluted from the beads in high salt buffer, and deposited on AFM
chips for imaging. Evident fibrillar species were detected in the
media of cells expressing RD(.DELTA.K)-HA and RD(LM)-HA, while
RD(PP)-HA produced only amorphous aggregates, (FIG. 21D), and
mock-transfected cells produced no signal (data not shown). These
findings are consistent with free tau fibrils mediating
trans-cellular propagation of tau aggregation by their release into
the extracellular space.
Example 8. Effect of Anti-Tau Antibodies on Tau Pathology In
Vivo
[0149] The activity of two additional antibodies against full
length, recombinant human tau were tested in the propagation assay.
RD(LM)-CFP and RD(.DELTA.K)-YFP cells were co-cultured for 48 hrs
in the presence and absence of different monoclonal antibodies that
target different tau epitopes (HJ8.5, HJ9.3 and HJ9.4, FIG. 22A).
HJ3.4 antibody against A.beta. peptide was used as a negative
control. All three anti-tau antibodies blocked the trans-cellular
propagation of pro-aggregation mutants of RD-tau between cells
(FIG. 22B). The negative control, HJ3.4, did not block
trans-cellular propagation. HJ8.5, HJ9.3 and HJ9.4 also detected
RD-tau fibrils by ELISA (FIG. 22C).
[0150] To block the propagation of tau aggregates from cell to cell
in vivo, a passive vaccination approach was used with antibodies
targeting different epitopes on tau. Anti-tau antibodies, HJ8.5 and
HJ9.3, or vehicle were each infused into the lateral ventricle of 6
month old, P301 S tg mice by intracerebroventricular injection
using Alzet osmotic pumps (2006 model, FIG. 23A). Brain cannula
attached to an Alzet pump assembly were surgically implanted into
the left lateral ventricle of each mouse at the position 0.4 mm
anteroposterior to bregma, 1.0 mm lateral to midline and 2.5 mm
dorsoventral (FIG. 23B). After treatment, placement of the cannula
was verified by cresyl violet staining (FIG. 23C). The Alzet
osmotic pumped was replaced after 6 weeks, and the experiment
concluded on day 84.
[0151] To confirm that the experimental design did not result in
antibody degradation and/or inactivity, antibodies were collected
from the Alzet pump after 6 weeks of infusion into mouse brain and
loaded onto an SDS-PAGE gel. The gel was first stained by Coomassie
blue dye (FIG. 24A) and then analyzed by western blotting using
antibodies taken from the pump before and after the 6 week infusion
(FIG. 24B). All the antibodies were stable and active after 6 weeks
in the Alzet pump at physiological temperature in vivo. It was
further confirmed that spiking of recombinant human tau protein
with different infusion antibodies did not interfere with
HJ8.7-BT2B ELISA assay for measuring total tau (FIG. 25).
[0152] To determine whether antibody treatment reduced pathological
tau staining, tau staining was assessed in tissue sections of the
9-month old, P301 S tg mice treated with Vehicle/PBS or the
anti-tau monoclonal antibodies. Coronal sections of the piriform
cortex were stained with biotinylated AT8 antibody, which
recognizes an abnormally phosphorylated form of tau. Quantitative
analyses of preliminary immunohistochemistry data showed that
abnormally phosphorylated tau load was remarkably reduced after
infusion of HJ8.5 and HJ9.3 in mouse brain (FIG. 26 and FIGS.
27A-27D). Biochemical analysis of these effects are underway. If
successful, passive immunization against tau propagation and
pathology could become a therapeutic approach to treat Alzheimer's
Disease, fronto-termporal dementia or other tauopathies.
Discussion for Examples 3-7
[0153] It has been previously proposed that prion-like mechanisms
involving templated conformational change and trans-cellular
propagation of aggregation could explain the relentless progression
of tauopathies and other neurodegenerative diseases. This would
consist of the release of a protein aggregate from a donor cell,
entry into a recipient cell, and direct contact with natively
folded protein to amplify the misfolded state. However, mechanistic
evidence to support this model of tauopathy has been incomplete,
and trans-cellular propagation of tau misfolding in this manner has
not previously been demonstrated. Examples 3-7 now describe
transcellular propagation of tau aggregation in cultured cells via
secreted tau aggregates, and propose a likely mechanism. First
documented was spontaneous formation of RD tau fibrils in
transfected cells using X-34 staining and AFM of extracted
material. Then observed was the coincidence of tau derived from two
separate cells in intracellular inclusions using confocal
microscopy. This was associated with increased detergent
insolubility of tau RD(.DELTA.K)-YFP upon co-culture with cells
expressing an aggregation-prone form of the protein, RD(LM)-HA.
Also documented was this increase in aggregation using FRET between
RD(LM)-CFP/YFP that were co-expressed within the same cells. This
was detected by acceptor photobleaching (microscopy), and spectral
methods (FPR). Next used was FRET between RD(LM)-CFP and RD(LM)-YFP
expressed in separate cell populations to document that propagation
occurred by direct protein contact. This method was then extended
to document amplification of tau protein misfolding within the cell
populations in successive culture conditions. Transcellular
propagation of tau aggregation is mediated by fibrils that are
released directly into the extracellular space, because transfer is
sensitive to extracellular volume, conditioned medium can increase
intracellular aggregation, and an anti-tau antibody (HJ9.3)
interfered with cellcell propagation, and trapped extracellular tau
fibrils. Using a variety of techniques, the applicants have thus
documented the trans-cellular aggregate propagation via templated
conformational change and propose a simple model to explain these
phenomena (FIG. 29).
[0154] Trans-cellular propagation--Although spontaneous movement of
aggregated tau between cells has been previously described, it was
unknown whether tau protein aggregates could propagate a misfolded
state between cells by direct contact of the proteins, as opposed
to indirect effects on the cell. Cell culture studies of
.alpha.-synuclein have also suggested propagation, but it is
unclear what is the nature of the species (e.g. aggregates vs.
dimers vs. monomer) derived from donor cells and those formed in
recipient cells. Likewise, SOD1 aggregates can transfer between
cells via the medium to induce further aggregation, but the precise
nature of the responsible protein conformers, and whether direct
protein-protein contact occurs is unclear. Injection of purified
A.beta.42 and tau fibrils into transgenic mouse brain induces
aggregation of endogenous tau, with nearby development of tau
fibrils, but it is difficult to rule out seeding by injected
protein. Work from the Applicants' lab, and subsequently from
others has documented movement of tau aggregates and induction of
aggregation by recombinant protein from the outside to the inside
of the cell. But no prior study of the tau protein has demonstrated
bona fide propagation: aggregate movement from one cell to another,
direct contact with the native protein, conversion of the protein
in the recipient cell to a fibrillar state, and amplification of
the misfolded species.
[0155] This work demonstrated these phenomena in several ways.
First, it was found that co-culture of an aggregation-prone form of
tau RD(LM)-HA with cells expressing RD(.DELTA.K)-YFP leads to
co-localization in .beta.-sheet positive inclusions. Next, it was
observed that co-culture of cells expressing RD(LM)-HA with another
population expressing both RD(.DELTA.K)-CFP and RD(.DELTA.K)-YFP
led to an increase of FRET signal, suggesting that movement of
RD(LM)-HA into cells expressing the FRET pair was inducing their
aggregation. To demonstrate direct contact and coaggregation of tau
aggregates moving between cells, RD(.DELTA.K)-CFP and
RD(.DELTA.K)-YFP were expressed in separate populations. This led
to a FRET signal derived from trans-cellular movement and
co-aggregation that disappeared if either one of the constructs
contained a double proline mutation to block .beta.-sheet
formation. Induction of full-length tau-YFP aggregation by transfer
of RD-CFP aggregates was also observed, but the efficiency is
reduced (data not shown). Finally, the efficiency of FRET induced
by trans-cellular movement of protein aggregates increased
significantly by preliminary co-culture of RD(LM)-HA expressing
cells with those expressing RD(.DELTA.K)-CFP, demonstrating that an
aggregated state can be amplified within a population of cells.
[0156] Antibody modulation of tau aggregate propagation--Antibodies
against A.beta. peptide, which is predominantly extracellular, can
prevent A.beta. aggregation in the brain and remove existing
aggregates. While there are potential side effects, such antibodies
hold promise as treatments. However, the success of vaccination in
mouse models of tauopathy and synucleinopathy has been puzzling in
light of the fact that the target proteins are predominantly
intracellular. It was observed that HJ9.3, a mouse monoclonal
antibody against tau-RD, inhibited the trans-cellular propagation
of tau aggregation. However, this antibody had no effect on
intracellular aggregation of tau. Chronic exposure of the cell
medium to this antibody strongly increased the steady state tau
levels in the media. This was corroborated by flow cytometry
studies which indicated that HJ9.3 blocks transfer of aggregates
from one cell to another. Finally, HJ9.3/tau complexes trapped at
the cell surface were observed. The effect of this antibody
suggested strongly that tau fibrils are released into the
extracellular space, and are not propagating misfolding primarily
via cell-cell transfer in exosomes or tunneling nanotubes, as has
been proposed for prions. Further, aggregates present outside the
cell, if not trapped by HJ9.3, are likely taken up again into
cells. Multiple modes of inhibition are conceivable for therapeutic
antibodies, including disaggregation of protein fibrils, blockade
of conversion within cells, and promotion of intracellular
degradation. Our results with HJ9.3 are most consistent with
interference with cell uptake as one mechanism that could be used
to block tauopathy, and suggest new ways to consider development
and optimization of therapeutic antibodies for neurodegenerative
diseases.
[0157] Trans-cellular propagation via fibrillar tau--The
effectiveness of HJ9.3 in blocking propagation of tau aggregation
allowed use of this antibody to trap the responsible species.
Immuno-affinity purification of tau from conditioned medium
revealed fibrillar tau. No tau fibrils in medium from control cells
were observed, or from those expressing the 0-sheet-resistant
RD(PP)-HA, which produced amorphous aggregates. RD(.DELTA.K)-HA and
RD(LM)-HA expression each caused fibril secretion into the
extracellular space. It has been unclear how protein aggregation in
one cell might influence the aggregation in a neighboring cell, and
it was formally possible that cytokines, exosomes, or direct
connections between cells might facilitate this process. These
possibilities cannot be completely excluded. However, these results
are most consistent with free fibrillar species as mediators of
propagation through the extracellular space. This work suggests
answers to several important questions about the mechanisms by
which protein aggregates propagate from one cell to another in
culture, and thus how they might do so in vivo. In conjunction with
the methods described here to monitor trans-cellular propagation,
it may be possible to target this process with pharmacological and
biological agents for more effective treatment of tauopathies and
other neurodegenerative diseases.
Methods for Examples 1-8
Antibodies
[0158] The longest mouse recombinant tau isoform mTau40 (432 aa)
and the longest human tau isoform hTau40 (441 aa) were produced in
the laboratory of Eva Mandelkow and used as standards in the tau
ELISA. The mouse monoclonal antibody Tau-5, which recognizes both
human and mouse tau (epitope at residues 218-225), was from the
laboratory of L. Binder (LoPresti et al., 1995; Porzig et al.,
2007). Monoclonal antibodies HJ8.1 and HJ9.3 are mouse monoclonal
antibodies raised by immunizing against human tau and mouse tau,
respectively, in tau knock-out mice (The Jackson Laboratory). Both
antibodies recognize mouse and human tau on Western blots, by
immunoprecipitation, and in ELISA assays. HJ9.3 recognizes the
microtubule binding region (MTBR) of tau. Mouse monoclonal antibody
BT-2, which also recognizes human and mouse tau (epitope at
residues 194-198), was obtained from Pierce. Rabbit polyclonal
antibody directed against Tau (ab64193, epitope located in the
repeat domain region) was purchased from Abeam, Cambridge, Mass.
Mouse monoclonal antibody directed against hemagglutinin HA (HA.11
Clone 16B12) was purchased from Covance, Emeryville, Calif. Rabbit
polyclonal GFP antibody (sc-8334) was purchased from Santa Cruz
Biotechnology.
Plasmids
[0159] Sequences encoding the four repeat domain (RD) of the
microtubule associated protein tau were used for protein
expression. In addition to the wild-type form, various tau mutants
were created: .DELTA.K280 .DELTA.(K); P301 L/V337M (LM);
.DELTA.K280/I227P/I308P (PP). These sequences were either subcloned
into pcDNA3.1 (Invitrogen) with a C-terminal hemagglutinin (HA)
tag, or into pEYFP-N1 or pECFP-N1 (Clontech) to create Cterminal
fluorescent protein fusions.
Animals
[0160] P301 S tg mice (line PS19), which overexpress P301 S human
T34 isoform tau (1 N4R), have been generated and characterized
previously and are on a B6C3 background. P301 S tg mice were
obtained from the Jackson Laboratory. Tau knock-out mice were
obtained from The Jackson Laboratory. Age and genetic background
matched nontransgenic mice littermates were used as wildtype mice.
In all experiments, both male and female were used in this
study.
Immunoprecipitation and Immunoblot Analysis
[0161] Immunoprecipitation and immunoblot analysis. Hippocampal
microdialysis samples were collected at 1.0 l/min for 15 h from
P301 S tau transgenic mouse and wild-type mice. ISF was
immunoprecipitated by Dynabeads (Invitrogen) coated with HJ8.1 or
HJ9.3 tau antibody according to the manufacturer's instructions.
Precipitated fractions were loaded on a reducing 4-12% Bis-Tris
mini-gel (Invitrogen) and transferred to nitrocellulose membrane.
Biotinylated BT-2 antibody (Pierce) and Poly-HRP-conjugated
streptavidin (Thermo Scientific) were used to eliminate the
interference of precipitated antibodies. HEK293 cells were cultured
in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10%
fetal bovine serum, 100 .mu.g/mL penicillin and 100 .mu.g/mL
streptomycin. Cultures were maintained in a humidified atmosphere
of 5% CO2 at 37.degree. C. For transient transfections, cells
plated in Optimem medium were transfected using Lipofectamine/Plus
reagent and 600 ng of appropriate DNA constructs (Invitrogen,
Carlsbad, Calif., USA) according to manufacturer's recommendations,
and harvested 24 h or 48 h later for further analyses.
Detergent Fractionation and Western Blot Analyses
[0162] HEK293 cells were plated at 400,000 cells/well in a 12-well
plate. The following day cells were transfected with 600 ng of
plasmid. After 48 h, cells were harvested with 0.05% trypsin for 3
minutes at 37.degree. C., pelleted briefly at 7000.times.g and
lysed in 1000 of 1% Triton in PBS containing protease inhibitors.
Soluble cytosolic proteins were then collected by centrifugation at
14,000.times.g for 10 minutes. Insoluble proteins were obtained by
resuspending the pellet in RIPA/SDS buffer and centrifugation at
20,000.times.g for 15 minutes following benzonase nuclease
digestion of nucleic acids. For co-culture experiments, equal
numbers of cells transfected with RD(LM)-HA and RD(.DELTA.K)-YFP
were co-cultured together for 48 h before harvesting and Western
blotting. Equivalent amounts of HEK293 cell protein extract from
each fraction were analyzed using 4%-20% polyacrylamide gels
(Biorad); antibody directed against tau RD (which recognizes an
epitope in the RD region) at a 1:2000 dilution (ab64193, Abeam,
Cambridge, Mass.) and/or antibody directed against GFP at 1:1000
dilution (sc-8334, Santa Cruz Biotechnology, Inc.). A
chemiluminescence-based peroxidase-conjugated secondary antibody
reaction was performed and detected by X-ray film. Quantification
was performed using Image J analysis software.
Co-Culture Experiments: Measuring RD-CFP/YFP Co-Aggregation by
FRET
[0163] HEK293 cells were plated at 300,000 cells/well in a 12-well
plate. The following day, cells were transfected with 600 ng of
plasmid as described above. Co-transfected cells received a
combination of 150 ng of RD-CFP constructs and 450 ng of RD-YFP
constructs. 15 h later, cells were harvested with 0.05% trypsin for
3 minutes at 37.degree. C., and a fraction of cells were re-plated
in a 96-well plate in quadruplicate, or on ibidi .mu.-slides (ibidi
GmbH, Germany) for imaging by microscopy. Cells were then cultured
an additional 48 h before fixation with 4% paraformaldehyde and
analysis.
Co-Culture Experiments: Measuring Induction of RD-YFP Aggregation
by RD-HA
[0164] HEK293 cells were transfected with either RD(.DELTA.K)-YFP
or RD(LM)-HA in 12-well plates. After 15 h the cells were replated
together onto ibidi .mu.-slides and cocultured an additional 48 h.
They were then fixed and stained with anti-HA antibody and X-34 for
analysis by microscopy.
Co-Culture Experiments: Propagation Assays in Co-Culture
[0165] Two populations of HEK293 cells in a 12-well plate were
co-transfected with 300 ng RD(LM)-HA and 300 ng RD(.DELTA.K)-CFP
together, or with RD.DELTA.(K)-YFP. After 15 h, equal percentages
of the two populations were co-cultured for 48 h in a 96-well plate
format. Cells were then fixed with 4% paraformaldehyde and FRET
analysis was performed using the Fluorescent Plate Reader (FPR).
For FRET microscopy analysis, two populations of HEK293 cells in a
12-well plate were transfected with 600 ng RD(LM)-CFP or with
RD(LM)-YFP. After 15 h, equal percentages of the two populations
were co-cultured for 48 h on ibi i .mu.-slides. Cells were then
fixed with 4% paraformaldehyde and FRET acceptor photobleaching was
conducted.
Co-Culture Experiments: Amplification of Tau Aggregation in Serial
Culture
[0166] HEK293 cells were transfected in a 12-well plate with 600 ng
of various forms of nonfluorescent RD-HA and cultured for 24 h. A
second group of cells were transfected with CFP or
RD(.DELTA.K)-CFP. Equal percentages of the first and second
populations were then co-cultured for 48 h. At this point, 50% of
this population was plated with a population of cells transfected
with RD(.DELTA.K)-YFP in a 96-well plate for 48 h. Cells were then
fixed with 4% paraformaldehyde for FRET analyses using the FPR.
Media Transfer and Conditioned Media Experiments
[0167] HEK293 cells were transfected in a 12-well plate with either
600 ng of RD(LM)-HA or co-transfected with a combination of 150 ng
of RD(.DELTA.K)-CFP construct and 450 ng of RD(.DELTA.K)-YFP
construct. 15 h later, cells were harvested with 0.05% trypsin for
3 minutes at 37.degree. C. An equivalent number of cells expressing
RD(.DELTA.K)-YFP/CFP and RD(LM)-HA were co-cultured for 48 h in
varying amounts of cell culture medium. Cells were then fixed with
4% paraformaldehyde and FRET analysis was performed. For the
conditioned media experiments, 15 h after transfection, media from
RD(LM)-HA cells containing transfection complexes was replaced with
fresh media. Cells expressing RD(.DELTA.K)-YFP/CFP were harvested
with 0.05% trypsin for 3 minutes at 37.degree. C. and replated in
96-well plate. 24 h later, conditioned media from cells transfected
with RD(LM)-HA was collected and added to cells expressing
RD(.DELTA.K)-YFP/CFP. 48 h later cells were fixed with 4%
paraformaldehyde and FRET analysis was performed.
Fluorescence Resonance Energy Transfer (FRET) Assays: FRET
Measurements by Microscopy with Photobleaching
[0168] HEK293 cells transfected for cotransfection and co-culture
experiments as described earlier were prepared for FRET acceptor
photobleaching microscopy. All images were obtained using a
C-Apochromat 40.times.1.2 NA lens (Carl Zeiss Advanced Imaging
Microscopy, 07740 Jena, Germany 100.times.(CFP). Digital images
were acquired using a Zeiss LSM510 Meta NLO Multiphoton/Confocal
laser scanning microscope system on the Zeiss Axiovert 200M.
Channels used for imaging were as follows: the donor CFP was
stimulated using a 458 nm argon laser and fluorescence collected
with a 480-520 nm bandpass filter; the acceptor YFP was stimulated
using a 514 nm argon laser and fluorescence collected with a
long-pass 560 nm filter. To create an image in which the intensity
reflected an estimate of FRET efficiency, the value of the initial
CFP image was subtracted from the final CFP image obtained after
photobleaching on a pixel-by-pixel basis, and this difference was
multiplied by 100 and divided by the final CFP image intensity:
100.times.(CFP.sub.final-CFP.sub.initial)/CFP.sub.final. Proper
adjustments were made for partial acceptor photobleaching. Image
arithmetic and grayscaleto-color image conversion were done using
NIH ImageJ 1 0.44 software.
FRET Assays: Fluorescence Plate Reader
[0169] Spectral FRET measurements (FRET/donor) were obtained using
a TecanM1000 fluorescence plate reader according to methods
previously described. When donor and acceptor are not fused to the
same protein, spectral FRET measurements depend on careful control
for the relative amount of donor and acceptor proteins expressed
within the cell. All values on the plate reader were first
background subtracted against mock-transfected cells. The YFP
signal in each well (Smpl485ex/528em FRET) was used to estimate
RD-YFP expression levels, and it was likewise assumed that under
experimental conditions that RD-CFP/YFP do not vary independently.
This helps eliminate the possibility that changes in apparent FRET
are due simply to variations in RD expression levels. Relative
contribution of acceptor activation (528 nm) by donor excitation
signal (435 nm) to the overall FRET measurement was corrected by
determining the "crossover activation" fraction for acceptor, X,
where X=RD-YFP signal measured at 435ex/528em divided by the signal
measured at 485ex/528em. This "crossover activation" is essentially
constant across different expression levels of RD-YFP encountered
in the experiments. The "measured" FRET value in each sample is
recorded at 435ex/528em, the "donor" value (CFP) is recorded at
435ex/485em. The "actual" FRET/donor value for each well is then
reflected as:
FRET.sub.actual=(SMPl.sub.435
ex/528em-X*(SMPl.sub.435ex/528em))/SMPl.sub.435ex/528em
[0170] This method of measuring protein aggregation by FRET has
reliably allowed detection of subtle changes in response to
pharmacologic as well as genetic manipulations of androgen receptor
and huntingtin protein aggregation that were corroborated by visual
and biochemical analyses. Since the relative amount of spectral
FRET measured depends on the ratio of acceptor:donor, a constant
ratio of 3:1 was used when RD-CFP and RD-YFP are co-expressed
within the same cell. This provides close to maximal FRET
efficiency while allowing for acceptable signal:noise in the
measurement of donor signal.
Atomic Force Microscopy (AFM)
[0171] RIPA-insoluble proteins were extracted from transfected
HEK293 cells and incubated on mica chips (Ted Pella, Inc) for 10
minutes. Samples were then rinsed twice with 100 .mu.I ddH2O and
left at RT to dry. The following day, atomic force microscopy was
performed using a MFP-3D atomic force microscope (Asylum
Research).
Immunofluorescence and Confocal Microscopy
[0172] HEK293 cells transfected for co-culture experiments as
described earlier were prepared for immunofluorescence and X-34
staining. After fixation in 4% paraformaldehyde for 15 min at RT,
cells were washed twice in PBS at room temperature (RT) for 5 min,
and permeabilized in 0.25% Triton X-100 in PBS at RT for 10
minutes. Cells were blocked with a blocking solution containing 1%
normal goat serum, 20 mg/ml BSA, 0.25% Triton X-100 in PBS for 3 h
at RT. Primary mouse monoclonal antibody against HA (Covance,
Emeryville, Calif.) was diluted 1:2000 in blocking solution and
applied to cells overnight at 4.degree. C. Cells were then washed
with PBS containing 0.1% Triton X-100 3 times for 5 minutes each
and incubated with anti-mouse Alexa546-conjugated secondary
antibody (Invitrogen) diluted at 1:400 in blocking solution. Cells
were then washed with PBS containing 0.1% Triton X-100 3 times for
5 min each, and exposed to 1 .mu.M X-34 prepared in a solution of
40% ethanol, 60% PBS, and 20 mM NaOH for 10 min at RT. Cells were
then washed 3 times for 2 min each in 40% EtOH, 60% PBS and rinsed
twice in 1.times.PBS for 5 min each. Images were captured using
confocal microscopy (405 Confocal Microscope-Zeiss). For the
characterization of the mechanism of HJ9.3 antibody blockade of
propagation, HEK293 cells were transfected with RD(.DELTA.K)-YFP or
mock transfected. Following culture of RD(.DELTA.K)-YFP cells or
mock-transfected cells in the presence of HJ9.3 for 48 hrs, cells
were fixed with 4% PFA, permeabilized with 0.25% TritonX-100 and
then exposed to goat anti-mouse Alexa 546 labeled secondary
antibody. Images were captured using confocal microscopy (Confocal
Microscope-Zeiss).
Propidium Iodide (PI) Cell Death Assay
[0173] HEK293 cells were plated at 75,000 cells/well in a 96-well
plate. The following day, cells were transfected in quadruplicate
with 100 ng of various forms of non-fluorescent RD-HA plasmids or
exposed to transfection complexes without DNA. The next day, media
containing transfection complexes were removed, and replaced with
fresh media. Non-transfected cells were treated with varying
concentrations of staurosporine (1, 2, 4, 20 .mu.M) for 30 minutes
at 37.degree. C. as a positive control for cell death.
Staurosporine solution was then removed and all cells were exposed
to 5 .mu.g/ml of propidium iodide for 10 minutes at 37.degree. C.
Propidium iodide solution was then replaced with phenol-free media
and fluorescence was read on the plate reader at 535 nm excitation
and 617 nm emission.
Immune Precipitation
[0174] Transfected cell populations were co-cultured either alone
or in the presence of mouse monoclonal antibody HJ9.3 (1:1000 which
is equivalent to 2.5 ng/.mu.I of antibody) or pooled mouse IgG
antibody for 3 h, 6 h, 9 h, 12 h, 24 h or 48 h. Conditioned media
were collected and protein-G-agarose beads (100 .mu.I of 50% slurry
beads from Pierce) were added to the media and incubated overnight
at 4.degree. C. with rotation. 18 h later, 500 .mu.I of binding
buffer (Pierce) was added to samples and centrifuged at
2000.times.g for 3 minutes. Supernatant was discarded, and this
wash step was repeated three times. Proteins bound to beads were
then eluted using a high salt elution buffer (50 .mu.I) with
incubation at room temperature for 5 minutes. Samples were then
centrifuged at 2000.times.g for three minutes and supernatant
collected. This elution step was repeated once for a total of 100p
eluate. Another sample of conditioned media not initially exposed
to HJ9.3 or IgG was incubated with the HJ9.3 (1:1000) or IgG
antibodies overnight at 4.degree. C. with rotation, followed by the
same immunoprecipitation protocol as described above. Samples from
all conditions were analyzed on 4-20% polyacrylamide gels (BioRad)
and detected with rabbit polyclonal antibody directed against tau
RD at 1:2000 dilution in 5% dry milk in TBS/Tween (ab64193, Abeam,
Cambridge, Mass.). A chemiluminescence-based peroxidaseconjugated
secondary antibody reaction was performed and detected by X-ray
film.
Flow Cytometry
[0175] HEK293 cells were plated in a 10-cm plate at -80%
confluency. Cells were then transfected with 24 .mu.g of RD(LM)-YFP
construct or transduced with mCherry lentivirus. The following day,
cells were harvested by treating with 0.05% trypsin for 3 minutes
at 37.degree. C., pelleted and resuspended in fresh media. The two
cell populations were co-cultured either alone or in the presence
of mouse monoclonal antibody HJ9.3 directed against Tau-RD at
1:1000 or 1:10,000 dilutions for 48 h (1:1000 is equivalent to 2.5
ng/.mu.I of antibody). After this time, cells were harvested and
resuspended in Hanks balanced medium containing 1% FBS and 1 mM of
EDTA. Cells premixed just prior to cytometry were used as negative
controls. Cells were counted using the MoFlo high speed cell sorter
(Beckman Coulter) and the percentage of dual positive cells was
analyzed for each of the conditions. Each condition had three
biological replicates, with 50,000 cells analyzed in each
experimental condition.
Intracerebroventricular (ICV) Injection of Anti-Tau Monoclonal
Antibodies
[0176] P301 S tau transgenic mice which express P301 S human T34
isoform (1 N4R) were used in this study. At 6 months age these mice
develop tau pathology. Therefore, antibodies were infused into the
left lateral ventricle by cerebroventricular injection at 6 months
of age and these infusions were carried for 12 weeks. After
treatment, mice brains were processed for immunohistochemistry and
biochemical analysis by ELISA and immunoblotting.
[0177] Intracerebroventricular injections were performed by using
Alzet osmotic pumps, 2006 model. Brain cannula attached to an Alzet
pump assembly were surgically implanted into the left lateral
ventricle of each mouse at the position 0.4 mm anteroposterior to
bregma, 1.0 mm lateral to midline and 2.5 mm dorsoventral. After
treatment, placement of the cannula was verified by cresyl violet
staining.
Introduction for Examples 9-15
[0178] Tau is a microtubule-associated protein that forms
intracellular aggregates in several neurodegenerative diseases
collectively termed tauopathies. These include Alzheimer's disease
(AD), progressive supranculear palsy (PSP), corticobasal
degeneration (CBD), and frontotemporal dementia (FTD). Tau is a
highly soluble and natively unfolded protein which binds and
promotes the assembly of microtubules. In tauopathies, tau
accumulates in hyperphosphorylated neurofibrillary tangles (NFTs)
that are visualized within dystrophic neurites and cell bodies upon
appropriate staining. The amount of tau pathology correlates with
progressive neuronal dysfunction and synaptic loss, and functional
decline in humans and transgenic mouse models.
[0179] In human tauopathies, pathology progresses from one brain
region to another in disease-specific patterns, although the
underlying mechanism is not yet clear. The prion hypothesis holds
that tau aggregates escape cells of origin to enter adjacent cells,
where they seed further tau aggregation and propagate pathology.
The inventors have previously observed that recombinant tau fibrils
will induce aggregation of full-length intracellular tau in
cultured cells, and that aggregated forms of tau transfer between
cells (Frost et al., 2009; Nat Rev Neurosci 11, 155-159). Further,
the inventors found that intracellular tau fibrils are released
free into the media, where they propagate aggregation by direct
interaction with native tau in recipient cells. An anti-tau
antibody (HJ9.3) blocks this process by preventing tau aggregate
uptake into recipient cells (Kfoury et al., 2012; J Biol Chem 287,
19440-19451). In addition to similar experiments with recombinant
tau, it has been shown that paired helical filaments from AD brain
induce cytoplasmic tau aggregation. Injection of brain extract from
human P301 S tau transgenic mice into the brains of mice expressing
wild-type human tau induces assembly of wild-type human tau into
filaments and spreading of pathology. Similar effects occurred
after injection of recombinant full-length or truncated tau
fibrils, which caused rapid induction of NFT-like inclusions that
propagated from injected sites to connected brain regions in a
time-dependent manner. Finally, selective tau expression in the
entorhinal cortex caused late pathology in the axonal terminal
zones in cells in the dentate gyrus and hippocampus, consistent
with trans-synaptic movement of aggregates. A growing body of work
thus supports the idea that tau aggregates transfer between cells,
and might be targeted with therapeutic antibodies.
[0180] In mouse models that mimic aspects of AD and Parkinson's
disease (PD), passive immunization using antibodies against A.beta.
and alpha synuclein can reduce A.beta. and alpha-synuclein
deposition in brain, and improve behavioral deficits. Active
immunization in tauopathy mouse models using tau phospho peptides
reduced tau pathology and in some studies improved behavior
deficits. However, in one study active immunization of C57BL/6 wild
type mice with full length recombinant tau induced tau pathology
and neurologic deficits. In two passive vaccination studies, there
was reduced tau pathology and improved motor function when the
antibody was given prior to the onset of pathology. While several
of the tau immunization studies appear to have some beneficial
effects, the maximal expected efficacy of anti-tau antibodies
administered after the onset of pathology, the optimal tau species
to target, and the mechanism of the therapeutic effect have
remained unknown.
Example 9. Characterization of Anti-Tau Antibodies
[0181] The inventors have previously observed that tau aggregates,
but not monomer, are up taken by cultured cells, and that
internalized tau aggregates trigger intracellular tau aggregation
in recipient cells (Frost et al., 2009; Nat Rev Neurosci 11,
155-159; Kfoury et al., 2012; J Biol Chem 287, 19440-19451). The
HJ8 series of 8 mouse monoclonal antibodies (raised against
full-length human tau) and HJ9 series of 5 antibodies (raised
against full-length mouse tau) were characterized in an adapted
cellular biosensor system previously described in Kfoury et al.
(2012; J Biol Chem 287, 19440-19451) that measures cellular tau
aggregation induced by the addition of brain lysates containing tau
aggregates. The antibodies had variable effects in blocking
seeding, despite the fact that all antibodies efficiently bind tau
monomer and stain neurofibrillary tangles. Three antibodies were
selected with different potencies in blocking seeding for the
studies presented herein.
[0182] Prior to testing in vivo, the binding affinities and
epitopes of the antibodies, which are all lgG2b isotype, were
determined. Human and mouse tau was immobilized on a sensor chip
CMS for surface plasmon resonance (SPR) (FIGS. 30A-30G). The HJ9.3
antibody, raised against mouse tau, recognizes both human (FIG.
30A) and mouse (FIG. 30B) tau with the same binding constant
(K.sub.D=K.sub.d/K.sub.a=100 pM) (FIG. 30G). The association
(K.sub.a) and dissociation (K.sub.d) was calculated by using
BIAevaluation software (Biacore AB) selecting Fit kinetics
simultaneous K.sub.d/K.sub.a (Global fitting) with 1:1 (Langmuir)
interaction model. The K.sub.a and K.sub.d of HJ9.3 towards human
(Ka=7.5.times.104 Ms.sup.-1, K.sub.d=7.5.times.10-6 s.sup.-1) and
mouse tau (K.sub.a=8.6.times.104 Ms.sup.-1, K.sub.d=9.1.times.10-6
s.sup.-1) indicate strong binding to both. The epitope of HJ9.3 was
mapped to the repeat domain (RD) region, between amino acids
306-320. HJ9.4, raised against mouse tau, had high affinity K.sub.D
(2.2 pM) towards mouse tau with a high association rate constant
(K.sub.a=2.28.times.105 Ms.sup.-1) and very low dissociation
constant (K.sub.d=5.1.times.10-7 s.sup.-1) (FIG. 30D and Table 4.
However, the same antibody had a much lower affinity (K.sub.D=6.9
nM) toward human tau (FIG. 30C and Table 4) with a similar
association rate constant (K.sub.a=1.5.times.105 Ms.sup.-1) as with
mouse tau but with much faster dissociation (K.sub.d=1
0.07.times.10-3 s.sup.-1). Thus, the HJ9.4 interaction with human
tau is less stable than with mouse tau. The epitope for this
antibody is amino acids 7-13. HJ8.5 was raised against human tau.
It binds to human tau (FIG. 30E) but not to mouse tau (FIG. 30F).
The K.sub.D (0.3 pM) (FIG. 30E and Table 4) and low dissociation
rate (K.sub.d=4.38.times.10.sup.-8 s.sup.-1), indicate that HJ8.5
binds human tau with very high affinity. The epitope of HJ8.5 was
mapped to amino acids 25-30. All 3 anti-tau antibodies strongly
recognized human tau fibrils on SPR (FIG. 31). Because the fibrils
have multiple identical epitopes, the association and dissociation
rates could not be directly calculate.
TABLE-US-00009 TABLE 4 Association rate constant (K.sub.a),
dissociation rate constant (K.sub.d) and binding constant (K.sub.D)
of each antibody towards human and mouse tau. BIAevaluation
software (BiacoreAB) was used to calculate K.sub.a and K.sub.d by
selecting Fit kinetics simultaneous K.sub.a/K.sub.d (Global
fitting) with 1:1 (Langmuir) interaction model. HJ9.3 HJ9.4 HJ8.5
Human K.sub.a (Ms.sup.-1) 7.55 .times. 10.sup.4 1.53 .times.
10.sup.5 1.3 .times. 10.sup.5 K.sub.d (s.sup.-1) 7.51 .times.
10.sup.-6 1.07 .times. 10.sup.-3 4.34 .times. 10.sup.-8 K.sub.D (M)
99 pM 6.9 nM 0.336 pM Mouse K.sub.a (Ms.sup.-1) 8.61 .times.
10.sup.4 2.28 .times. 10.sup.5 -- K.sub.d (s.sup.-1) 9.16 .times.
10.sup.-6 5.1 .times. 10.sup.-7 -- K.sub.D (M) 100 pM 2.24 pM --
Ms.sup.-1 = millisecond, M = molar, s = second
[0183] The antibodies were also assessed by immunoblotting and
immunostaining. On Western blots, all 3 antibodies bound to human
tau (FIG. 30G). HJ9.3 and HJ9.4 bound to mouse tau while HJ8.5 did
not (FIG. 30G). Consistent with our prior findings of the inventors
(Yamada et al., 2011; J Neurosci 31, 131 10-131 17), there appeared
to be less reassembly buffer (RAB) soluble tau in 9 month old
compared to 3 month old P301 S mice. It was also found that HJ8.5
stained human tau in 3 month and 9-12 month old transgenic P301 S
mouse brains. Tau immunoreactivity was present throughout the cell
bodies and processes (FIG. 32). In 9-12 month old P301 S mice with
tau aggregates, HJ8.5 detected tau aggregates in cell bodies (FIG.
32). Other antibodies produced similar results (Table 5). All
antibodies bound to neurofibrillary tangles and neuropil threads in
AD brain (FIG. 32).
TABLE-US-00010 TABLE 5 Relative efficacy of anti-tau antibodies in
different assays. HJ8.5 HJ9.3 HJ9.4 Human Mouse Human Mouse Human
Mouse Method tau tau tau tau tau tau Western blot ++ - +++ +++ ++
++ Immuno- +++ - + + ++ +++ staining Human AD +++ N/A + N/A + N/A
brain NFT's
Example 10. Tau-Antibodies Block the Uptake and Seeding Activity of
P301 S Tau Aggregates
[0184] To evaluate seeding activity present in P301 S brain
lysates, a cellular biosensor system previously described by the
inventors (Kfoury et al., 2012) was adapted. This is based on
expression of the repeat domain of tau (aa 243-375) containing the
AK280 mutation fused either to cyan or yellow fluorescent protein
(RD(.DELTA.K)-CFP/YFP). Uptake of exogenous aggregates into these
cells triggers intracellular aggregation of RD(.DELTA.K)-CFP/YFP
that is detected by fluorescence resonance energy transfer (FRET)
recorded on a fluorescence plate reader. Clarified brain lysates
from 12 month old P301 S mice added to the biosensor cell system
induced strong aggregation of the RD(.DELTA.K)-CFP/YFP reporter,
indicating the presence of tau seeding activity (FIG. 33A). The
seeding activity from 12-mo P301 S brain homogenate mice roughly
corresponds to 50 nM (monomer equivalent) of recombinant full
length fibrils (data not shown).
[0185] There was little to no aggregation induced by lysates from
tau knockout mice, wild-type mice, or 3 month old P301 S mice
lacking tau pathology (FIG. 33A). The anti-tau antibodies (HJ8.5,
HJ9.3 and HJ9.4) were assessed for their ability to block the
uptake, and seeding activity of these lysates. HJ3.4 (mouse
monoclonal anti-A.beta. antibody) was a negative control. The
anti-tau antibodies effectively blocked seeding activity (FIG.
33B). To determine their relative efficacy, the antibodies (0.125,
0.25, 0.5, 1, 2 .mu.g/ml) were titrated against a fixed amount of
P301 S brain lysate (FIG. 33C). The HJ8.5 antibody blocked seeding
activity at concentrations as low as 0.25 .mu.g/ml compared to
controls. At 0.5 .mu.g/ml, both HJ8.5 and HJ9.3 antibody
significantly blocked uptake and seeding activity compared to
control. HJ9.4 was least potent in blocking the uptake and seeding
activity, consistent with its higher affinity for mouse tau. All 3
anti-tau antibodies detected tau aggregates internalized following
uptake by HEK293 cells, as detected by post-hoc cellular
permeabilization and staining. However, when these antibodies were
pre-incubated with and without P301 S brain lysates, none of these
antibodies were detected inside cells upon staining with anti-mouse
secondary antibody (FIG. 34). While other modes of inhibition are
possible, these data are consistent with a mechanism based on
blocking cellular uptake of tau aggregates.
Example 11. Intracerebroventricular Infusion of Anti-Tau
Antibodies
[0186] In the mouse colonies, P301 S mice first develop
intracellular tau pathology beginning at 5 months of age. To test
the efficacy of the 3 antibodies by chronic intracerebroventricular
(ICV) administration, a catheter was surgically implanted into the
left lateral ventricle of each mouse at 6 months of age and
continuously infused anti-tau antibodies for 3 months via Alzet
subcutaneous osmotic mini-pump (FIG. 35A). Anti-A.beta. antibody
HJ3.4 and phosphate buffered saline (PBS) were used as negative
controls. After 6 weeks, each pump was replaced with one filled
with fresh antibody solution or PBS. At the time of brain
dissection, catheter placement in the left lateral ventricle of
each mouse was verified by cresyl violet staining (FIG. 35B). Only
mice with correctly placed catheters were included in the analyses.
To test the stability of the antibodies after 6 weeks in vivo (FIG.
35A), residual pump contents were collected upon removal from the
animals, and the antibodies were assessed using SDS-PAGE and
Coomassie blue staining. Light and heavy chains were intact, with
no fragmentation, and retained tau binding activity on western blot
(data not shown). To estimate the concentration of anti-tau
antibodies in CSF and serum during the infusion, biotinylated HJ8.5
(HJ8.5B) was administered for 48 hours (-7.2 .mu.g/day) (FIG. 35A).
The concentration of free HJ8.5B was 7.3 .mu.g/m I in the CSF and
6.2 .mu.9/ml in the serum, indicating significant clearance of the
antibody from the CNS to the periphery (Table 6). HJ8.5B bound to
human tau was also detected in both CSF and serum, though the
concentration was lower than that of free antibody (Table 6).
TABLE-US-00011 TABLE 6 Levels of biotinylated HJ8.5 antibody that
is free (not bound to tau) and HJ8.5 antibody bound to tau in serum
and cerebrospinal fluid (CSF) 48 hrs after IP or ICV
administration. CSF Conc. As % Treatment CSF Serum Serum Conc.
Conc. Of free HJ8.5B (.mu.g/ml) HJ8.5B injected IP 0.9 .+-. 0.1 552
.+-. 38.6 0.16 .+-. 0.02 (50 mg/kg/48 hrs) HJ8.5B injected ICV 7.3
.+-. 1.6 6.2 .+-. 0.5 95.4 .+-. 19.4 (ca. 14 .mu.g/48 hrs) Conc. Of
HJ8.5B bound to tau (.mu.g/ml) HJ8.5B injected ICV 0.10 .+-. 0.02
0.04 .+-. 0.03 53 .+-. 4.6 (ca. 14 .mu.g/48 hrs)
Example 12. Anti-Tau Antibody Treatment Reduces Abnormally
Phosphorylated Tau
[0187] To determine the extent of tau pathology in P301 S mice
after 3 months of treatment, multiple stains for tau pathology were
carried out. Brain sections were first assessed by immunostaining
with the anti-phospho tau antibody AT8 (FIGS. 36A-36E). AT8 binds
phosphorylated residues Ser202 and Thr205 of both mouse and human
tau (FIGS. 36A-36E). In mice treated with PBS and HJ3.4, AT8
strongly stained neuronal cell bodies and the neuropil in multiple
brain regions, particularly in the piriform cortex, entorhinal
cortex, amygdala, and hippocampus (FIGS. 36A and 36B). HJ8.5
treatment strongly reduced AT8 staining (FIG. 36C), especially in
the neuropil. HJ9.3 and HJ9.4 also decreased AT8 staining but the
effects were slightly less (FIGS. 36D and 36E). Quantitative
analysis of AT8 staining in piriform cortex (FIG. 37A), entorhinal
cortex (FIG. 37B), and amygdala (FIG. 37C) demonstrated a strong
but variable reduction in phospho-tau in all anti-tau antibody
treated mice. HJ8.5 antibody markedly reduced AT8 staining in
piriform cortex, entorhinal cortex, and amygdala. HJ9.3 had
slightly decreased effects compared to HJ8.5, and HJ9.4 had
significant effects in both entorhinal cortex and amygdala but not
in the piriform cortex (FIGS. 37A-37D). The hippocampus exhibited
much more variable AT8 staining vs. other brain regions,
predominantly in cell bodies, and thus was not statistically
different in treatment vs. control groups (FIG. 37D). Because it
has been reported that male P301 S mice have greater tau pathology
than females, the effect of both gender and treatment were also
assessed (FIGS. 38A-38H). In addition to an effect of treatment,
there was significantly more AT8 staining in all brain regions
analyzed in male mice (Table 7). However, the effects of the
antibodies were still highly significant and virtually identical
after adjusting for gender (Table 8). The treatment groups versus
controls in males and females were also compared separately, and
the effects of antibody HJ8.5 remained most significant (FIGS. 38A
and 38B).
TABLE-US-00012 TABLE 7 p Values of Treatment/Gender Entorhinal
Amygdala cortex Hippocampus Piriform cortex Treatment 0.0107 0.0053
0.2917 0.0147 Gender 0.0026 0.0027 0.0244 0.0067 p values
determined by two-way ANOVA considering treatment and gender as
factors. For amygdala, entorhinal cortex, and piriform cortex
regions, treatment and gender are both significant factors with p
values <0.05, but for hippocampus CA1 region, treatment is not a
significant factor with p value = 0.2917 while gender is a
significant factor with p value = 0.0244.
TABLE-US-00013 TABLE 8 Amygdala Entorhinal cortex Hippocampus CA1
Piriform cortex p p p p p p p p value-1 value-2 value-1 value-2
value-1 value-2 value-1 value-2 Control 0.0009 0.0009 0.0022 0.0022
0.0421 0.0526 0.011 0.0113 vs. HJ8.5 Control 0.0956 0.1605 0.0335
0.0576 0.2486 0.3889 0.0566 0.0982 vs. HJ9.3 Control 0.0106 0.0072
0.0077 0.005 0.2427 0.2427 0.1787 0.1569 vs. HJ9.4 p values were
calculated before and after adjustment of gender. p value-1: not
adjusted by gender; p value-2: adjusted by gender. p value-1 was
determined by one-way ANOVA, treatment is the independent variable.
p value-2 was determined by two-way ANOVA, treatment and gender are
independent variables.
Example 13. Correlation of Multiple Staining Modalities
[0188] To test for tau amyloid deposition, thioflavin S (ThioS) was
used to stain brain sections (FIGS. 39A-39B). ThioS staining was
semi-quantitatively assessed using a blinded rater who gave a score
from 1 (no staining) to 5 (maximum staining) in all control and
anti-tau antibody treated mice. By semi-quantitative assessment,
HJ8.5 treatment significantly reduced ThioS staining compared to
PBS and HJ3.4 (FIGS. 39A and 39B). Mice treated with PBS, HJ8.5,
and HJ9.3 (n=6 from each group) were also stained with PHF1
monoclonal antibody, which recognizes tau phospho-residues Ser396
and Ser404. AT8 and PHF1 staining significantly correlated
(r=0.630, p=0.005) (FIG. 40A) showing that 2 anti-phospho tau
antibodies to different tau epitopes give similar results.
[0189] Many neurodegenerative diseases, including tauopathies,
exhibit microglial activation in areas of the brain surrounding
protein aggregation and cell injury. Microglial activation was
assessed in the treatment groups using anti-CD68 antibody (FIGS.
41A-41E). HJ8.5 and HJ9.3 treatment reduced microglial activation
in piriform cortex, entorhinal cortex, and amygdala compared to
controls (FIGS. 41A-41 D). HJ9.4 had a weaker effect in the
piriform cortex compared to HJ8.5 and HJ9.3 (FIGS. 41 C-41 E),
consistent with the AT8 staining results (FIG. 37A). Microglial
activation strongly correlated with AT8 staining in all samples
(r=0.51 1, p=0.0038) (FIG. 40B).
Example 14. Anti-Tau Antibodies Reduce Detergent-Insoluble Tau and
Seeding Activity
[0190] To determine the level of soluble and insoluble tau in the
cortex, sequential biochemical extraction with RAB (aqueous
buffer), radio immunoprecipitation assay (RIPA)(detergent buffer),
and 70% formic acid (FA) were performed to solubilize the final
pellet. Total tau was quantified by ELISA with anti-tau antibody
HJ8.7, which detects both human and mouse tau with the same K.sub.D
(0.34 pM). The possibility that the treatment antibodies would
interfere with the ELISA was excluded by spiking positive control
samples with these antibodies prior to analysis and observing no
interference (data not shown). All mice that were assessed by
pathological analysis in FIGS. 37A-37D were analyzed. Total tau
levels in the RAB (FIG. 42A) or RIPA (FIG. 42B) soluble fractions
were similar among all groups. The detergent-insoluble/70% FA
soluble fractions were analyzed by neutralizing the samples prior
to ELISA and western blot. Every animal studied was analyzed, and
it was found that HJ8.5 and HJ9.3 decreased detergent-insoluble tau
by >50% vs. controls (FIG. 42C). Representative samples (n=4
from each group) illustrate by western blot decreased levels of
insoluble tau in mice treated with HJ8.5 and HJ9.3 (FIG. 40C).
Insoluble tau levels were no different in HJ9.4-treated groups
versus PBS or HJ3.4. Human and mouse tau were also assessed
specifically in the detergent-insoluble/70% FA soluble fractions in
N=6 mice per group in which the mean AT8 staining reflected the
mean values of results in FIGS. 37A-37D. There was significantly
more human tau than mouse tau in the 70% FA soluble fraction, and
the antibodies significantly lowered human but not mouse tau in
this fraction (FIGS. 42D and 42E). In these same samples, levels of
AT8 immunoreactive signal were assessed by ELISA. The AT8 signal
was lower in the antibody treated samples (FIG. 42F), similar to
what was seen for total tau in this fraction.
[0191] It was hypothesized that a reduction of tau aggregation in
brain would correlate with a reduction in seeding activity. Thus,
the cellular biosensor assay was used to test for P301 S brain
seeding activity in the cortical RAB soluble fractions from the
different treatment groups. Prior data by the inventors assessing
ISF tau in P301 S mice suggested the possible presence of
extracellular tau aggregates in equilibrium with both the
biochemically soluble and insoluble pools of tau (Yamada et al.,
2011; J Neurosci 31, 131 10-131 17). First, intracellular
aggregation of RD(.DELTA.K)-CFP/YFP was assessed after treating the
cells with lysates from mice treated with PBS or HJ3.4. Lysates
from these groups strongly induced FRET signal (FIG. 43A). Markedly
less seeding activity was observed in lysates from the cortical
tissue of mice treated with HJ8.5 and HJ9.3 (FIG. 43A). This was
not due to residual antibody in the brain lysates, because
immunoprecipitation of the brain lysates followed by elution of
seeding activity from the antibody/bead complexes produced the same
pattern (FIG. 43B). Thus HJ8.5 and HJ9.3 reduce seeding activity in
the P301 S tau transgenic mouse brain. HJ9.4 did not significantly
reduce seeding activity (FIG. 43A). Seeding activity strongly
correlated with the amount of detergent-insoluble/formic
acid-soluble tau detected by ELISA (Pearson's r=0.529, p=0.0001)
(FIG. 43C), but did not correlate with total tau in RAB fractions
(FIG. 43D). It was hypothesized that seeding activity is due to tau
aggregates present in the RAB soluble fraction. To test for this,
Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE) was
performed followed by Western blot. In addition to tau monomer,
higher molecular weight tau species present in 3 month old P301 S
mice and a larger amount present in 9 month old P301 S mice was
observed (FIG. 43E). A component of these higher molecular weight
species likely constitutes the seeding activity detected in the
FRET assay and may be in equilibrium with the tau present in the
detergent-insoluble/formic acid-soluble fraction.
Example 15. Anti-Tau Antibodies Rescue Contextual Fear Deficits
[0192] In studies of P301 S Tau transgenic mice at 9 months of age,
the control and anti-tau antibody treated groups were compared in a
variety of behaviors. The groups did not differ in locomotor
activity, exploration, or measures of sensorimotor function (FIGS.
44A-44F). The ability of the anti-tau antibody treatments to rescue
cognitive deficits in P301 S mice was evaluated by assessing the
performance of the mice on the conditioned fear procedure. On day
1, all four treatment groups of mice exhibited similar levels of
baseline freezing during the first two minutes in the training
chamber. This was confirmed by rmANOVA, which failed to reveal any
significant overall main effects or interactions involving
treatment (FIG. 45A). In addition, all four groups showed similar
levels of freezing during the tone-shock (T/S) conditioned
stimulus-unconditioned stimulus (CS-US) pairings (FIG. 45A). The
general lack of differences in freezing levels between groups
across the three T/S pairings was documented by a non-significant
effect of Treatment and a non-significant Genotype by Minutes
interaction.
[0193] In contrast to the absence of differences among groups
during testing on day 1, there were robust differences in freezing
levels from the contextual fear test (form of associative learning)
conducted on day 2 between two of the anti-tau antibody groups and
the PBS+HJ3.4 control mice (FIG. 45B). Subsequent planned
comparisons indicated that the HJ8.5 mice showed significantly
elevated freezing levels averaged across the 8-minute test session
(FIG. 45C) compared to the PBS+HJ3.4 control group, [F(1, 45)=8.30,
p=0.006], as did to a lesser extent the HJ9.4 mice, [F(1, 45)=5.60,
p=0.022]. Thus, HJ8.5 appeared to have a stronger effect overall in
preserving associative learning.
Discussion for Examples 9-15
[0194] One model for the pathogenesis of the tauopathies holds that
aggregates produced in one cell escape or are released into the
extracellular space to promote aggregation in neighboring or
connected cells. It was observed that selection of therapeutic
antibodies that specifically block tau seeding activity from brain
lysates predicts potent in vivo responses at least as strong if not
stronger than prior reports of active or passive tau vaccination.
Experiments were began with a cellular biosensor assay that is
sensitive to the presence of extracellular tau aggregates. It was
found that brain lysates from P301 S transgenic mice contained
seeding activity that could induce further intracellular
aggregation. After screening a panel of anti-tau antibodies, three
were selected with variable activities in blocking tau seeding
activity. These antibodies were infused ICV over three months into
P301 S tauopathy mice, beginning at a time when pathology had
initiated (6 months). Infusion of the antibodies resulted in
appreciable concentrations of antibody present in both CSF and
serum, consistent with previous reports of efflux of antibodies
from the CNS to the periphery. Treatment with HJ8.5, the most
potent antibody in vitro, profoundly reduced tau pathology,
strongly decreasing it from the neuropil. This effect was detected
with multiple independent stains, biochemical analyses of insoluble
tau, and by analysis of residual tau seeding activity present in
brain lysates. Further, this treatment improved the one behavioral
deficit detected in this model. All antibodies block tau aggregate
uptake into cells, and none is observed within cells in the
presence or absence of extracellular aggregates in the assays. The
efficacy of these antibodies implies a clear role for extracellular
tau in the pathogenesis of neuropathology that was previously
thought to be cell-autonomous. Further, this work extends prior
findings by the inventors, which suggest that aggregate flux may
occur in the setting of intracellular pathology, raising the
possibility of therapies that can assist in aggregate clearance by
targeting extracellular species. Finally, this work has important
implications for the design of therapeutic antibodies, and suggests
that targeting seeding activity in particular may produce the most
effective agents.
[0195] Mechanism-based antibody therapy Several prior active and
passive peripheral immunotherapy approaches against tau have also
reduced tau pathology and improved behavioral deficits, but the
underlying rationale for antibody choice was based either on a
phospho-epitope, reactivity with neurofibrillary tangles, or was
not stated. One tau immunization study, performed by vaccinating
mice with full length tau, induced pathology in wild type mice.
However, subsequent active immunization approaches with phospho-tau
peptides in tau transgenic models reduced tau pathology and showed
behavioral improvement. In a passive immunization study, JNPL3 tau
transgenic mice were administered the PHF1 antibody
intraperitoneally at 2-3 months of age, prior to the onset of
tauopathy. PHF-1 targets a pathological form of abnormally
phosphorylated tau. Treatment reduced tau pathology and improved
behavior. However, while it decreased insoluble phosphorylated tau,
total insoluble tau did not change. In another passive immunization
study, JNPL3 and P301 S mice (at age 2-3 months, prior to the onset
of tauopathy) were peripherally administered the PHF1 or MC1
antibody, which targets an aggregate-associated epitope. Both
treatments improved tau pathology and delayed the onset of motor
dysfunction. In these prior studies, the mechanism of action of the
antibodies was not clear, and none was explicitly tested. Indeed,
some proposed an intracellular mechanism. Moreover, no study
appears to have produced the magnitude of reduction in tau
pathology described in the examples provided herein, with the
caveats that antibodies were infused into the CNS while the other
studies utilized peripheral infusion; and different animal models
were utilized.
[0196] This study was explicitly designed to test a prediction that
extracellular tau seeds are a key component of pathogenesis. The
study began with a selection process to pick antibodies capable of
blocking tau seeding in vitro, purposely testing agents with a
range of predicted activities. All antibodies tested in vivo
effectively block aggregate uptake and seeding, providing a basis
for their observed activity. In addition, correlation of antibody
affinity, epitope, isotype, glycosylation, and ability to bind
phosphorylated forms of tau may be important to assess in future
studies. This is also the first study to report the effects of
direct, intra-CNS infusion of anti-tau antibodies. Despite the fact
that the antibodies utilized each target different tau epitopes and
none targets phospho-tau, 2 of 3 strongly reduced abnormal tau load
both immunohistologically and biochemically, and two significantly
improved memory, one to a greater extent than the other. Effects on
tau pathology also correlated very well with a reduction in
intrinsic seeding activity.
[0197] HJ8.5 and HJ9.3 strongly decreased pathological tau seeds in
vivo. A strong reduction in tau pathology might occur by preventing
induction of tau aggregation in neighboring cells. While HJ9.4 did
not decrease pathology as potently, it did decrease tau pathology
in the amygdala. The variation in effectiveness in different brain
regions among the antibodies may be due to the formation of
region-specific aggregate conformers for which the antibodies have
subtle differences in binding affinity.
[0198] Once extracellular tau aggregates are sequestered by
anti-tau antibodies in vivo, their metabolic fate is not yet clear.
After 3 months of antibody administration, reduced microglial
activation were found, presumably due to less tau-related pathology
and neurodegeneration. However, this could be due to more efficient
clearance of extracellular aggregates, with a reduction in related
microglial activation. Several months of passive immunization with
anti-A.beta. antibodies has also been noted to reduce microgliosis.
The mechanism by which antibody/tau complexes are cleared in vivo,
and the mechanism via which they decrease tau pathology, remains to
be definitively clarified. It has been suggested that immunization
with anti-.alpha.-synuclein antibodies clears .alpha.-synuclein
aggregates by promoting lysosomal degradation. A recent study with
anti-.alpha.-synuclein antibodies showed that the antibodies
targeted .alpha.-synuclein clearance mainly via microglia,
presumably through Fc receptors. Neurons express Fey receptors, and
may be able to internalize IgG complexed with antigen by high
affinity FcyRI receptor. Internalized tau antibodies may also
contact tau in endosomes and eventually induce clearance of
intracellular tau aggregates by the endosomal/autophagy-lysosomal
system. Though the anti-tau antibodies used in the study described
herein can bind extracellular tau assemblies, no evidence of
significant localization within cells was found. That does not,
however, rule out the possibility that cells in vivo take up
antibody/tau complexes to influence tau aggregate clearance as well
as inflammation. For example, it has recently been shown that
antibodies complexed with viruses can bind to the cytosolic IgG
receptor TRIM21, targeting the antibody/virus complex to the
proteasome. In addition, antibodies bound to TRIM21 were shown to
activate immune signaling. While interaction with
antibodies/non-infectious antigen complexes with TRIM21 has not yet
been shown, it may be interesting to determine if such a mechanism
is relevant to the anti-tau antibodies. Interestingly, there is
also evidence in the P301 S model of tauopathy that the innate
immune system is activated prior to the development of significant
tau pathology, and that early immunsuppresion attenuates tau
pathology. It may be possible that antibodies capture tau
aggregates induced by inflammation, reducing subsequent
aggregate-induced inflammation and disease progression.
[0199] Extracellular tau and spreading of tau pathology The work
presented herein implicitly tests the role of extracellular tau in
pathogenesis. It is now abundantly clear that extracellular tau
aggregates can trigger fibril formation of native tau inside cells,
whether their source is recombinant protein or tau extracted from
mammalian cells. A role for free tau aggregates was originally
hypothesized (i.e. not membrane-enclosed) as mediators of
trans-cellular propagation based on our prior work, in which HJ9.3
added to the cell media blocked internalization, and
immunoprecipitated free fibrils (Kfoury et al., 2012; J Biol Chem
287, 19440-19451).
[0200] In animal models, tau aggregates can apparently spread from
one region to another (e.g. entorhinal cortex to neurons downstream
in the dentate gyrus and hippocampus). The inventors have found
that monomeric tau is constantly released in vivo into the brain
interstitial fluid even under non-pathological conditions (Yamada
et al., 2011; J Neurosci 31, 131 10-131 17). The inventors also
found that exogenous aggregates would reduce levels of soluble ISF
tau, suggesting that seeding and/or sequestration phenomena can
occur in this space (Yamada et al., 2011; J Neurosci 31, 131 10-131
17). Taken together, abundant evidence supports the concept that
extracellular tau aggregates form, and can be taken up by adjacent
cells, connected cells, or possibly back into the same cell,
thereby increasing the burden of protein misfolding. This evidence
makes a clear prediction: therapy that captures extracellular
seeding activity should ameliorate disease. The findings described
in the examples presented herein are consistent with this idea.
[0201] The role of tau flux in pathogenesis It would not be
predicted a priori that a mouse model such as P301 S, which drives
mutant tau expression via the prion promoter in virtually all
neurons, should benefit from antibody treatments that block
trans-cellular propagation of aggregation. In theory, pathology
could occur independently in all neurons that express this
aggregation-prone protein. However, prior work by the inventors in
tissue culture suggested a role for flux of tau aggregates, since
HJ9.3 added to the cell media increased the steady state level of
aggregates over time. While the model of aggregate flux requires
further testing, the results presented herein are consistent with
this idea, since antibody treatment profoundly reduced
intracellular tau pathology. It is predicted that antibodies that
block tau uptake may create a "sink" in the extracellular space
that may promote clearance by another mechanism, possibly involving
microglia.
[0202] Therapeutic antibodies and targeting seeding activity The
pharmaceutical industry is devoting increasing efforts to develop
therapeutic antibodies that target aggregation-prone proteins that
accumulate within cells. The principal criteria have been that the
antibodies will bind epitopes known to accumulate in diseased
brain. This approach may or may not lead to antibodies with optimal
activity in vivo. The examples herein supports a new model of
therapeutic antibody development that emphasizes efficacy in
blocking the seeding activity present in the brain. Using this
approach, antibodies with higher apparent efficacy than has
previously been reported were identified. In an extension of the
prion hypothesis, it is further proposed that distinct tau
aggregate "strains" may predominate in patients with different
types of tauopathy, and these may have unique sensitivities to
different antibodies. In any case, the use of sensitive in vitro
assays of antibody efficacy as described here may allow much more
efficient development and optimization of antibody-based
therapies.
[0203] The strong protective effect of the anti-tau antibodies,
particularly with the HJ8.5 antibody, suggests that this type of
approach should be considered as a treatment strategy for human
tauopathies. While chronic administration of antibodies via an ICV
approach may be possible, in future studies, it may be important to
determine the PK/PD response with peripheral administration of
these antibodies when given in both a prevention and treatment
mode. In addition, the tau seeding assay may be useful to monitor
target engagement by the antibodies.
Experimental Procedures for Examples 9-15
[0204] Antibodies HJ9.3 and HJ9.4 mouse monoclonal antibodies were
raised by immunizing tau knockout mice (The Jackson laboratory)
against mouse tau, and HJ8.5 and HJ8.7 monoclonal antibodies were
raised by immunizing tau knockout mice against human tau. HJ9.3,
HJ9.4 and HJ8.7 monoclonal antibodies recognize both mouse and
human tau. However, HJ8.5 monoclonal antibody binds only to human
tau (epitope is at residues 25-30 [NCBI reference sequence:
NP_005901]). HJ9.3 antibody recognizes the RD region of tau
(epitope at residues 306-320). HJ9.4 antibody recognizes the
N-terminal region of tau (epitope is at residues 7-13). As a
control antibody, HJ3.4 mouse monoclonal antibody was used, which
recognizes the N-terminal region of the human A.beta. sequence
(epitope at residues 1-16). HJ8.5, 9.3, and 9.4 monoclonal
antibodies are of the lgG2b isotype. Rabbit polyclonal tau antibody
(ab64193, epitope located at repeat domain region) was purchased
from Abeam. Mouse monoclonal biotinylated BT-2 antibody, recognizes
human and mouse tau (epitope at residues 194-198) and was purchased
from Pierce. Rat anti-mouse monoclonal CD68 antibody was purchased
from AbD SeroTec. Biotinylated AT8 antibody was purchased from
Thermo scientific.
[0205] Surface plasmon resonance Surface plasmon resonance
experiments were performed on BIAcore 2000 surface plasmon
resonance instrument (GE Healthcare-BIAcore). Biacore sensor chip
CM-5 was activated by using EDC
(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and NHS
(N-hydroxysuccinimide) in a 1:1 ratio for 7 minutes. The sensor
chip surface was saturated by immobilizing 5 .mu.g/ml of
recombinant human or mouse tau or human tau fibrils in 10 mM Sodium
acetate pH 3.5 with a flow rate of 5 .mu.l/min. The remaining
unbound area was blocked by 1 M Ethanolamine pH 8.5. For reference,
one flow cell is activated with NHS and EDS, followed by blocking
with 1 M ethanolamine. Then all the anti-tau antibodies were
injected at different concentrations (0.11, 0.23, 0.46, 0.9, 1 0.8,
3.7, 7.5 .mu.g/ml) in filtered, degassed 0.01 M Hepes buffer, 0.15
M NaCl, 0.005% surfactant P20, pH 7.4 at a flow rate of 10
.mu.I/min. All samples were run in duplicate. After each run with a
single antibody concentration, the surface of the chip was totally
regenerated by using 10 mM Glycine pH 1 0.7, to remove the bound
antibody to tau, without disturbing the immobilized tau on the
chip. Data analysis was performs by using BIAevaluation software
(GE healthcare-BIAcore).
[0206] Tau fibrilization 8 .mu.M recombinant full length human tau
was pre-incubated with 2 mM dithiothreitol for 45 min at room
temperature then 10 mM HEPES and 100 mM NaCl and 8 .mu.M heparin
were added for a total volume of 200 .mu.I followed by incubation
for 7 day at 37.degree. C. to form fibrils. After fibril formation,
the remaining monomers of tau in the sample were separated by using
100 kDa Microcon centrifugal filters according to manufacturer's
instructions (Millipore).
[0207] IP and ICV administration of biotinylated HJ8.5 antibody
Mouse monoclonal HJ8.5 antibody was biotinylated according to the
manufacturer's instructions (Sulfo-NHS-LC-Biotin kit, Pierce).
Biotinylated HJ8.5 (HJ8.5B) was administered by interperitoneal
injection (IP) at 50 mg/kg in 5-6 month old P301 S mice (n=3).
After 48 hrs, mice were sacrificed. Serum and CSF was collected and
stored at -80.degree. C. until use. HJ8.5B was also administered by
intracerebroventricular injection (ICV) by surgically implanted
osmotic pumps into the left lateral ventricle of 5-6 month old P301
S mice (n=3). This antibody was continuously infused for 48 hrs.
After 48 hrs, mice were sacrificed. Serum and CSF was collected and
stored at -80.degree. C. until use.
[0208] Inracerebroventricular (ICV) injection procedure ICV
infusions were performed by Alzet osmotic pumps, 2006 model
(Durect). The age of the mice was 6 months at the time of surgery.
Before the surgery, an L-shaped cannula was attached to tubing (3
cm, long), which was then attached to Alzet pumps carrying antibody
or vehicle (phosphate buffer saline--PBS, pH 7.4). This assembly
was pre-incubated in PBS for 60 hrs at 37.degree. C. to activate
the pump prior to placement into the lateral ventricle. The
assembly was surgically implanted with the use of a stereotactic
apparatus (David Kopf Instruments) into the left lateral ventricle
of each mouse at 0.4 mm anteroposteriorly to bregma, 1.0 mm lateral
to midline, and 2.5 mm dorsoventral to the surface of the brain
under isoflurane anesthesia. Alzet osmotic pumps were placed under
the skin by making a subcutaneous pocket with a curved, blunt ended
scissors. Each implanted cannula was secured with dental cement
along with small anchor stainless steel screws. After the cement
dried, the skin was sutured. The antibody (2 mg/ml) or PBS in the
pump was continually infused into the left lateral ventricle of the
brain. These osmotic pumps carry a maximum of 200 .mu.I of volume,
and they pump with a flow rate of 3.6 .mu.I/day resulting in an
infusion of 7.2 .mu.g of antibody per day. In each mouse, osmotic
pumps were changed once after 6 weeks of infusion. The solution
remaining in the Alzet pump was collected after its removal from
each mouse and stored at -80.degree. C. At the age of 9 months, all
mice were sacrificed. All surgically implanted mice were housed
singly.
[0209] Histology After 12 weeks of the treatment, P301 S mice were
anesthetized intraperitoneally with pentobarbital 200 mg/kg),
followed by perfusion with 3 U/ml heparin in cold Dulbecco's PBS.
The brain was removed and cut into two hemispheres. The left side
of the brain was fixed for 24 hrs in 4% paraformaldehyde and
transferred to 30% sucrose in PBS and stored at 4.degree. C. prior
to freezing in powdered dry ice and stored at -80.degree. C. Half
brains were cut coronally into 50 .mu.m sections with a freezing
sliding microtome and all sections were stored in 24 well plates
with cryoprotectant solution (0.2M phosphate buffered saline, 30%
sucrose, 30% ethylene glycol) at -20.degree. C. until use. The
hippocampus and cortex were dissected from the freshly perfused
right hemisphere of each brain for biochemical analysis. All the
dissected tissues were stored at -80.degree. C. until analyzed. The
placement of the cannula into the left lateral ventricle was
verified by mounting brain sections 300 .mu.m apart and stained by
cresyl violet as previously described (Holtzman et al., 1996; Ann
Neurol 39, 1 14-122). The stained tissues were scanned using a
NanoZoomer digital pathology system (Hamamatsu Photonics).
[0210] Cell culture/Seeding Assay: P301S Brain Lvsates and Antibody
Treatment HEK293 cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum, 100
.mu.g/mL penicillin and 100 .mu.g/mL streptomycin. Cultures were
maintained in a humidified atmosphere of 5% CO2 at 37.degree. C.
For transient transfections, HEK293 cells were plated at 250,000
cells/well in a 12-well plate in optimem medium and transfected
using Lipofectamine 2000 reagent and 600 ng of appropriate DNA
constructs (Invitrogen) according to manufacturer's
recommendations. Co-transfected cells received a combination of 150
ng of RD(.DELTA.K280)-CFP constructs and 450 ng of
RD(.DELTA.K280)-YFP constructs. 15 h later, cells were harvested
with 0.05% trypsin for 3 minutes at 37.degree. C. and then
re-plated in a 96-well plate in quadruplicate for 15 hrs. Then,
P301 S brain lysates [prepared in 1.times.TBS with protease (Roche)
and phosphatase inhibitors (Roche)] that were pre-incubated with
all anti-tau monoclonal antibodies (HJ8.5, 9.3 and 9.4) or HJ3.4
antibody (monoclonal anti-A.beta. antibody) were added at various
concentrations (0.125 .mu.g/ml, 0.25 .mu.g/ml, 0.5 .mu.g/ml, 1
.mu.g/ml and 2 .mu.g/ml) for 16 hrs at 4.degree. C. with rotation.
To determine the seeding activity in the P301 S mice treated for 3
months with different antibodies, RAB soluble fractions of all
treated mice were also added to cells at various concentrations.
Cells were cultured an additional 24 h before fixation with 4%
paraformaldehyde, and FRET analysis was performed.
[0211] Immunoprecipitation RAB soluble fractions from PBS or
antibody-treated mice were incubated in the presence of mouse
monoclonal anti-tau antibodies HJ9.3 and HJ8.5 cross-linked to
protein-G-agarose beads (per kit recommendation-Pierce Crosslink
Immunoprecipitation kit) at 4.degree. C. with end-over-end rotation
for 24 hours. In addition, RAB soluble fractions from antibody
treated mice were incubated in the presence of un-conjugated
protein-G-agarose beads at 4.degree. C. with end-over-end rotation
for 24 hours. 18 h later, 500 .mu.I of binding/wash buffer (Pierce)
was added to samples and centrifuged at 2000.times.g for 3 minutes.
Supernatant was discarded, and this wash step was repeated three
times. Proteins bound to beads were then eluted using a low pH
elution buffer (25 .mu.I) with incubation at room temperature for 5
minutes. Samples were then centrifuged at 2000.times.g for three
minutes and supernatant collected. This elution step was repeated
once for a total of 50 .mu.I eluates. Tau-immunoprecipitates (IP)
containing tau aggregates were reapplied to co-transfected
RD(.DELTA.K)-CFP/YFP cells at equivalent amounts to initial brain
lysates experiments for further analysis with the seeding
assay.
[0212] Brain tissue extraction The cortex of each brain was
homogenized in 30 .mu.I/mg of RAB buffer [100 mM MES, 1 mM EDTA,
0.5 mM MgSO4, 750 mM NaCl, 20 mM NaF, 1 mM Na3VO4, supplemented by
protease inhibitor (Roche) and phosphatase inhibitor (Roche)]. In
brief, the samples were centrifuged at 50,000 g for 20 min at
4.degree. C. using an Optima MAX-TL Ultracentrifuge (Beckman). The
supernatants were collected as RAB soluble fractions and pellets
were resuspended in RIPA buffer [150 mM NaCl, 50 mM Tris, 0.5%
deoxycholic acid, 1% Triton X-100, 0.5% SDS-25 mM EDTA, pH 8.0,
supplemented by protease inhibitor (Roche) and phosphatase
inhibitor (Roche)], 30 .mu.I/mg and centrifuged at 50,000 g for 20
min at 4.degree. C. The supernatants were collected as RIPA soluble
fractions. The pellets were further resuspended in 70% formic acid,
10 .mu.I/mg and centrifuged at 50,000 g for 20 min at 4.degree. C.
The supernatants were collected as 70% formic acid fractions. All
fractions were stored in -80.degree. C. until analyzed.
[0213] Electrophoresis and Immune-blotting Gel electrophoresis was
performed under reducing conditions by 4-12% NuPAGE Bis-Tris gels
(Invitrogen) followed by transfer to PVDF membrane by using IBIot
apparatus (Invitrogen). 70% formic acid fractions were neutralized
before loading and subjecting to gel electrophoresis by diluting
1:3 with 1:1 mixture of 10N NaOH and neutralization buffer (1 mol/L
Tris base; 0.5 mol/L NaH.sub.4PO.sub.4). Pre-stained molecular
weight standards "SeeBlue" (Invitrogen) were included in each run.
Membranes were blocked with 5% milk in Tris buffered saline (TBS)
containing 0.1% of Tween 20. Then, membranes were washed 3 times
for 5 minutes each. Rabbit polyclonal tau antibodies (Abeam,
1:2000) were used as primary antibodies for the detection tau in
formic acid fractions. Treated mouse anti-tau antibodies collected
before and after its infusion from osmotic pumps were also used as
primary antibodies. The membranes were subsequently incubated with
Goat anti-rabbit or Goat anti mouse secondary antibody (GE
Healthcare, 1:2000). All the membranes were developed with ECL
prime substrate (GE Healthcare). Bands were visualized with G:Box
Chemiluminescent Imager (Syngene).
[0214] To determine the immunoreactivity of anti-tau antibodies to
tau from brain homogenates, RAB soluble fractions of 9 month old
P301 S and 3 month old P301 S mice, 3 month old wild type mice and
3 month old tau knockout mice samples were separated by SDS-PAGE
followed by western blotting. Total protein of 1 .mu.g from each
RAB soluble fraction was loaded onto 4-12% NuPAGE Bis-Tris gels
(Invitrogen) under reducing conditions followed by transfer to
nitrocellulose membrane by using IBIot apparatus (Invitrogen). The
membranes were blocked with 5% milk in TBS with 0.05% tween 20
(TBST) followed by incubation with primary antibodies (HJ8.5, HJ9.3
and HJ9.4). HRP-conjugated donkey anti-mouse IgG (1:2000, Santa
cruz) was used as secondary antibody and membranes were developed
using Lumigen TMA6 (GE Healthcare).
[0215] ELISA to detect free HJ8.5B and HJ8.5B bound to tau The
concentration of free HJ8.5B was determined in serum and CSF of
mice 48 hrs after IP or ICV administration. Ninety-six well ELISA
plates were coated with 50 ng/ml of recombinant human tau at
4.degree. C. ELISA plates were blocked with 4% BSA at 37.degree. C.
for 1 hr. Plates were then washed 5 times followed by incubating
with serum and CSF samples diluted in sample buffer (0.25% BSA in
PBS, 300 nM Tris pH 7.4 supplemented with protease inhibitors) and
incubated at 4.degree. C. overnight. The next day, plates were
washed 8 times with PBS followed by the addition of
streptavidin-poly-horseradish peroxidase-40 (1:6000, Fitzgerald),
for 1.5 hr, in the dark, at room temperature. Plates were then
washed 8 times with PBS and developed with Super Slow ELISA TMB
(Sigma) and read at 650 nm. Different concentration of HJ8.5B was
used to create a standard curve that was run in each plate in
addition to serum and CSF samples.
[0216] The concentration of HJ8.5B bound to tau was measured by
coating 96 well ELISA plates with 20 .mu.g/ml of HJ8.7 antibody at
4.degree. C. ELISA plates were blocked with 4% BSA at 37.degree. C.
for 1 hr. Plates were then washed 5 times followed by incubating
with serum and CSF samples diluted in sample buffer and incubated
at 4.degree. C. overnight. The next day, plates were washed 8 times
with PBS and plates were incubated with
streptavidin-poly-horseradish peroxidase-40 (1:6000, Fitzgerald),
for 1.5 hr, in the dark, at room temperature. Plates were then
washed 8 times with PBS and developed with Super Slow ELISA TMB
(Sigma) and read at 650 nm. Different dilutions of purified HJ8.5B
complexed with recombinant tau were used to create a standard curve
in each plate.
[0217] Tau sandwich ELISA assay To determine total tau levels,
ELISA half 96 well plates (Costar) were coated with HJ8.7 antibody
(20 .mu.g/ml) in carbonate buffer pH 9.6 and incubated at 4.degree.
C., overnight on a shaker. ELISA plates were washed 5 times with
PBS with a BioTek ELx405 plate washer and blocked with 4% BSA in
PBS for 1 hr at 37.degree. C. Plates were then washed 5 times
followed by incubating wells with RAB, RIPA, or 70% FA
biochemically extracted soluble brain tissue fractions diluted in
sample buffer (0.25% BSA in PBS, 300 nM Tris pH 7.4 supplemented by
protease inhibitor) and incubated at 4.degree. C. 70% FA fractions
were neutralized by diluting 1:20 with 1 M Tris pH 11 followed by
diluting with sample buffer. The next day, plates were washed 8
times with PBS followed by the addition of the biotinylated mouse
monoclonal anti-tau antibody BT-2 antibody (0.3 .mu.g/ml, Pierce)
in 0.5% BSA in PBS for 1.5 hr at 37.degree. C. Plates were then
washed 8 times in PBS followed by addition of
streptavidin-poly-horseradish peroxidase-40 (1:4000), for 1.5 hr,
in the dark, at room temperature. Plates were then washed 8 times
with PBS, developed with Super Slow ELISA TMB (Sigma) and
absorbance read at 650 nm on BioTek Synergy 2 plate reader.
Recombinant human tau was used to create a standard in each plate.
Negative control wells included omission of primary antibody in
each plate. The longest recombinant human (hTau40, 441 aa) and
mouse tau (mTau40, 432aa) isoforms produced in the laboratory of
Eva-Maria Mandelkow were used as standards in the ELISA assays.
[0218] To determine the levels of human tau in 70% FA fractions,
ELISA 96 well plates were coated with mouse monoclonal antibody
Tau5 (20 .mu.g/ml) and mouse monoclonal anti-human tau specific
biotinylated HT7 antibody (0.2 ug/ml, Thermo Scientific) for
detection. For mouse tau levels in the 70% FA fraction, ELISA 96
well plates were coat with monoclonal anti-mouse tau specific HJ9.2
antibody (20 .mu.g/ml) and monoclonal biotinylated HJ8.7 was used
for detection. Recombinant human and mouse tau were used for
standards on each plate. To determine phospho tau levels at
positions Ser202 and Thr205, ELISA half 96 well plates were coated
with mouse monoclonal HJ8.7 antibody (20 .mu.g/ml) and biotinylated
AT8 antibody (0.2 ug/ml, Thermo Scientific) was used as detection
antibody.
[0219] Immunohistochemistry To detect the presence of abnormally
phosphorylated tau in the brain, three 50 .mu.m coronal brain
sections spaced 300 .mu.m apart from all treated mice were
assessed. The brain sections were blocked with 3% milk in
Tris-buffered saline (TBS) and 0.25% (vol/vol) Triton-X followed by
incubation at 4.degree. C. overnight with the biotinylated AT8
antibody (Thermo Scientific, 1:500) which recognizes tau
phosphorylated at ser202 and thr205. Biotinylated PHF1 antibody
(1:200) which recognizes abnormally phosphorylated tau at residues
ser396 and ser404 was also used to determine the correlation
between AT8 and PHF1 antibody staining. For correlation studies,
mice (N=6) were randomly selected from the HJ8.5, HJ9.3, and
PBS-treated groups. The stained tissues were scanned using the
NanoZoomer digital pathology system. To determine the correlation
between the AT8 staining and activated microglial staining, brain
sections from selected mice of all the treated groups (N=6), were
blocked with 10% normal goat serum in TBS with 0.25% (vol/vol)
Triton-X was incubated with a rat anti-mouse CD68 antibody (AbD
SeroTec, 1:500) at 4.degree. C. overnight. The sections were then
incubated with biotinylated goat anti-rat IgG antibody, mouse
adsorbed (Vector, 1:2000). All sections were scanned with a
NanoZoomer slide scanner (Hamamatsu Photonics). All images were
exported by using NDP viewer software and quantified by using
ImageJ software (National Institutes of Health). For AT8 staining,
3 brain sections from each mouse separated by 300 .mu.m,
corresponding approximately to sections at Bregma coordinates -1.4,
-1.7, and -2.0 mm in the mouse brain atlas were used. These
sections were used to determine the percentage of area occupied by
abnormally phosphorylated biotinylated AT8 antibody staining. All
converted images were uniformly thresholded to quantify AT8
staining and the average of all three sections was used to
determine the percentage of area covered by abnormally
phosphorylated tau staining for each mouse. For PHF-1 and CD68
staining, two brain sections from each mouse were used, separated
by 300 .mu.m and correspond to bregma coordinates -2.3 and -2.6 mm
in the mouse brain atlas. To determine ThioS staining, brain
sections from randomly selected mice from all the treated groups
(N=6) were stained in ThioS in 50% ethanol (0.25 mg/ml) for 3 min,
followed by washing in 50% ethanol and distilled water. Slices were
then mounted, dried and images were assessed by microscopy with the
Nanozoomer. Two brain sections from each mouse were used as
described adjacent to those used for PHF-1 and CD68 staining.
[0220] Semi Denaturing-Agarose Gel Electrophoresis (SDD-AGE) For
separation of tau species present in the different RAB soluble
fractions of 3 month old tau knockout (KO), 3 months old wild type
(WT), 3 months old P301 S and 9 month old PBS-treated P301 S mice,
the previously described Semi-Denaturing Detergent-Agarose Gel
Electrophoresis (SDD-AGE) method was employed with minor
modifications. Samples were run on horizontal 1.5% agarose gels in
Buffer G (20 mM Tris, 200 mM Glycine) with 0.2% SDS. Samples were
incubated in the sample buffer (60 mM Tris-HCl pH 6.8, 0.2% SDS, 5%
glycerol, and 0.05% bromphenol blue) for 7 min at RT. After the
electrophoresis, proteins were transferred from gels to Immobilon-P
PVDF sheets (Millipore) at 4.degree. C. in Laemmli Buffer (Buffer
G/0.1% SDS). Membranes were blotted using an anti-tau specific
rabbit polyclonal antibody (Abeam) at 1:2000. Blots were developed
using the GE ECL Plus system.
[0221] Immunofluorescence HEK293 cells were plated at 75,000
cells/well in 24 well plates coated with poly D-lysine. To
determine whether anti-tau antibodies used can detect tau species
taken up by the HEK293 cells, the cells were treated with P301 S
brain lysates for 2 hrs, followed by washing 3.times. with PBS,
fixed with 4% paraformaldehyde for 15 min at room temperature
followed by washing 3 times with PBS at room temperature. Cells
were permeabilized with 0.1% Triton X-100 for 10 min, washed 3
times with PBS, then blocked with 0.25% Triton X-100 in PBS
containing 10% normal goat serum and 20 mg/ml BSA. Then cells were
incubated with anti-mouse secondary antibody conjugated with
Alexa-fluor 546. To determine whether antibody can enter the cells,
P301 S brain lysates were pre-incubated with and without the
different anti-tau antibodies HJ8.5, HJ9.3, and HJ9.4 or the HJ3.4
antibody to AP. The lysates were then added to HEK293 cells for 2
hrs, fixed and permeabilized. Secondary antibody conjugated with
Alexa-fluor 546 was used to identify the antibodies. 4',
6'-diamidino-2-phenylindole (DAPI; shown in blue) was used for
nuclear stain. All the images were captured by using a Zeiss LSMS
confocal microscope (Zeiss).
[0222] Statistical analysis of pathological and biochemical data
All data are presented as mean.+-.SEM, and different conditions
were compared using one-way ANOVA followed by Dunnett's post hoc
test to compare controls with treatment groups. Statistical
significance was set at P<0.05. Statistics were performed using
Graph Pad Prism 5.04 for Windows (GraphPad Software Inc.). For
quantitative assessment of AT8 staining, gender is a significant
factor so results were adjusted by gender using SAS version 9.2
software.
[0223] Statistical analysis applying treatment and gender as
factors The control group (PBS and HJ3.4) mean was compared with
each treatment group, (mean of PBS+mean of HJ3.4)/2 VS mean of
treatment). Two-way ANOVA was used to test whether gender and
treatment are significant factors, which is achieved by PROC GLM in
SAS Version 9.2 and their p Values are shown in Table 7. A contrast
statement was used in PROC GLM of SAS Version 9.2 to access all
comparisons. Gender as an adjustment factor in the two-way ANOVA
was applied and p Values before/after the adjustment are shown in
FIG. 38D.
[0224] Behavioral tests Mice were assessed on locomotor activity
and exploratory behaviors and on a battery of sensorimotor measures
and the rotarod to provide additional control data for interpreting
the results of the conditional fear test, which was used to
evaluate cognitive function. The conditioned fear test was
conducted last in the series of tests to preclude effects of brief
footshocks on other behavioral indices.
[0225] Holeboard exploration, sensorimotor battery and rotarod. All
mice were evaluated on the holeboard exploration test where total
ambulations (whole body movements) and hole pokes were quantified
over a 30-min period and provided indices of locomotor activity and
exploration. The protocol involved the use of a computerized
holeboard apparatus (41.times.41.times.38.5 cm high) containing 4
corner and 4 side holes, with a side hole being equidistant between
the corner holes (Learning Holeboard; MotorMonitor, Kinder
Scientific, LLC, Poway, Calif.). Photobeam instrumentation was used
to quantify total ambulations and exploratory hole pokes during the
test session. This procedure has served as the habituation
component of our general holeboard exploration/olfactory preference
test. The mice were also tested on a battery of seven sensorimotor
measures that were used to assess balance (ledge, platform),
coordination (pole, 60.degree. and 90.degree. inclined screens),
strength (inverted screen), and initiation of movement out of a
small circumscribed area (walking initiation). This battery was
used in previous publications and greater procedural details may be
found in (Wozniak et al. (2004; Neurobiol Dis 17, 403-414). The
rotarod test was similar to previously-published methods and
included three types of trials: 1) stationary rod (60 s maximum; 2)
constant speed rotarod (2.5 rpm for 60 s maximum; and 3)
accelerating rotarod (2.5-10.5 rpm over 0-180 s). Our protocol
consisted of testing each mouse on one stationary rod trial, two
constant speed rotarod trials, and two accelerating rotarod trials
for each of three test sessions that were separated by 3 days to
limit motor learning.
[0226] Conditioned fear. Mice were evaluated on the conditioned
fear test, which was the last behavioral measure conducted.
Briefly, the mice were trained and tested in two Plexiglas
conditioning chambers (26 cm.times.18 cm, and 18 cm high)
(Med-Associates, St. Albans, Vt.) with each chamber containing
distinct and different visual, odor, and tactile cues. Each mouse
was placed into the conditioning chamber for a 5-min trial and
freezing behavior was quantified during a 2-min baseline period.
Beginning at 3 min and at 60-s intervals thereafter, the mice were
exposed to 3 tone-shock pairings where each pairing included a 20-s
presentation of an 80 dB tone (conditioned stimulus; CS) consisting
of broadband white noise followed by a 1.0 mA continuous footshock
(unconditioned stimulus; CS) presented during the last second of
the tone. Broadband white noise was used instead of a
frequency-specific tone in an effort to avoid possible auditory
deficits that might occur with age. The mice were placed back into
the conditioning chamber the following day and freezing behavior
was quantified over an 8-min period to evaluate contextual fear
conditioning. Twenty four hours later, the mice were placed into
the other chamber containing different cues and freezing behavior
was quantified during a 2-min "altered context" baseline and over
the subsequent 8 min, during which time the auditory cue (tone; CS)
was presented. Freezing was quantified using FreezeFrame image
analysis software (Actimetrics, Evanston, Ill.), which allowed for
simultaneous visualization of behavior while adjusting a "freezing
threshold," which categorized behavior as freezing or not freezing
during 0.75 s intervals. Freezing was defined as no movement except
for that associated with normal respiration, and the data were
presented as percent of time spent freezing. To assess the extent
of contextual fear conditioning, we conducted analyses within each
treatment group which involved comparing the percent time spent
freezing averaged over the 2-min baseline on day 1 with the
averaged percent time spent freezing during the first 2 min of the
contextual fear test on day 2, as well as with freezing levels
averaged across the entire 8-min session. Shock sensitivity was
evaluated following completion of the conditioned fear testing,
according to previously described procedures in Khuchua et al.
(2003; Neuroscience 1 19, 101-111).
[0227] Statistical Analyses of behavioral data Analysis of variance
(ANOVA) models were typically used to analyze the behavioral data
(Systat 12, Systat Software, Chicago, Ill.). The conditioned fear
data were analyzed using repeated measures (rm) ANOVA models
containing one between-subjects variable (Treatment) and one
within-subjects (repeated measures) variable (Minutes). The
Huynh-Feldt adjustment of alpha levels was utilized for all
within-subjects effects containing more than two levels to protect
against violations of sphericity/compound symmetry assumptions
underlying rmANOVA models. Planned comparisons between the
PBS+HJ3.4 control group and each of the three other antibody
treatment groups (i.e., HJ8.5, HJ9.3, HJ9.4) were conducted within
ANOVA models for testing certain key hypotheses. In other
instances, pair-wise comparisons were conducted following relevant,
significant overall ANOVA effects, which were subjected to
Bonferroni correction when appropriate. Pearson's correlation
coefficient (r) was also calculated between the total ambulations
recorded during the holeboard test and the percent time spent
freezing during the contextual fear test on day 2.
Example 16. Tau ELISA Assay
[0228] An ELISA assay was developed in order to detect the presence
of pathological tau aggregates in plasma samples of patients.
Antibodies used in this assay include mouse monoclonal anti-tau
HJ9.3 and HJ9.2. HJ9.3 is biotinylated using One-step Antibody
Biotinylation Kit (HJ9.3-Bio). This sandwich ELISA utilizes HJ9.3
and HJ9.2, at equivalent concentration, as capture antibodies.
96-well half area plates (Costar 3690) are coated with 20 .mu.g/ml
of HJ9.2/HJ9.3 prepared in bicarbonate buffer pH 9.6 (50
.mu.I/well) and incubated at 4.degree. C. overnight. Following a
blocking step using 4% BSA/PBS, plasma samples (diluted 1:4 in
sample buffer: 0.25% BSA/PBS, 300 nM Tris PH 7.4-8.0,
1.times.protease inhibitors) are applied in triplicates to wells
(50 .mu.I/well). Plates are then incubated at A-C overnight. For
detection, HJ9.3-Bio prepared in 0.5% BSA/PBS at 0.3 .mu.g/was
added to wells for 1.5 hr at 37.degree. C. A secondary
streptavidin-polyHRP40 antibody at 1:4,000 dilution in 0.5% BSA/PBS
(50 .mu.I/well and 1.5 hr in dark on a shaker at RT) is used for
final detection through an enzymatic reaction using TMB super slow
substrate. The ELISA has been designed to optimize detection of
rare species in plasma. Initial embodiments included coating the
surface of the ELISA plate with antibody pairs to optimize trapping
of aggregates. However it would be equally plausible to use
antibody coated beads from larger volumes of fluid samples to
increase the sensitivity of the assay. Negative plasma collected
from healthy young participants was used to calculate the
background signal of the assay. Tau seeds presence in the
experimental samples is reported as fold induction over signal from
negative plasma.
[0229] A set of plasma samples from pre-clinical and Alzheimer's
Disease (AD) patients previously tested with the disclosed seeding
assay were used to validate the sandwich tau ELISA assay. 12
control patients (CDR 0) with no seeding activity (Negative) and 12
patients (CDR>0) with seeding activity (Positive) were tested
using the newly developed ELISA assay. These patients were
previously determined to have seeding activity or not in CSF and
plasma based on a biosensor cellular assay. In this cellular assay,
RD fragments of the tau protein containing the AK280 mutation are
fused to cyan or yellow fluorescent protein. This enables detection
of aggregation by measuring fluorescence resonance energy transfer
via FRET. Extracellular aggregates are brought into the cell and
trigger intracellular aggregation of the tau FRET reporter
proteins.
[0230] As shown in FIGS. 45A-45C, no tau aggregates were detected
in the plasma of patients with negative seeding activity compared
to the clear tau presence of seeds in the plasma of AD patients
with positive seeding activity. This cell-free based assay could be
used in a more clinical setting as a non-invasive diagnostic tool
for many tauopathies including Alzheimer's Disease. Further, it
could allow detection of those with incipient pathology who are
destined to develop dementia, facilitating clinical trial design by
enriching a sample population. Finally, it could be used to monitor
efficacy of anti-tau or other anti-dementia therapies.
Example 17
[0231] A cellular propagation assay was set up to measure the
propagation of tau aggregates from one population to another. A
fragment of tau comprised of the repeat domain (RD) was used either
as an untagged form with two disease-associated mutations (LM: P301
L/V337M) to promote aggregation of the CFP-tagged form, or one
disease-associated mutation (.DELTA.K: .DELTA.K280). One group of
cells was transfected with RD(LM) and RD(.DELTA.K280)-CFP, and
another was transfected with RD(.DELTA.K280)-YFP. FRET was recorded
on a fluorescence plate reader from cells grown in quadruplicate in
a 96-well format. FRET signal derives from RD-CFP aggregates
transferring to cells containing RD-YFP, and vice-versa. Multiple
antibodies were added to the medium at various dilutions indicated.
The starting concentration of antibody was .about.1 mg/ml. For
example, a 10.sup.-3 dilution indicates a final concentration of
.about.1 .mu.g/ml. After 24 h the cells were fixed and FRET
measurements recorded. Data for individual antibodies are presented
in FIGS. 47A-47F. Some antibodies were very potent at preventing
trans-cellular propagation of aggregation (e.g. HJ8.2, HJ9.1).
Others were effective in a more intermediate fashion (e.g. HJ9.3),
and some were essentially not effective (HJ8.7).
In each graph, the first bar represents medium without added
antibody, representing baseline efficiency of propagation.
[0232] To test for synergy of antibodies, effects on propagation
were determined in the setting of individual antibodies diluted
over an indicated concentration range, or antibodies were mixed at
an equimolar ratio and then titrated over the same range. Some
pairs were strongly synergistic (e.g. HJ9.3/9.4), while others
interfered with one another (HJ8.5/9.1) (FIG. 48).
[0233] The effect of an antibody on tau aggregate uptake may also
be measured by flow cytometry. Cells were exposed to recombinant RD
fibrils that were chemically labeled with a fluorescent dye. After
trypsinization and dispersion, the cells were counted using a flow
cytometer. HJ9.3 dose-dependently reduces the number of
fluorescently labeled cells, indicating inhibition of aggregate
uptake (FIG. 50).
Sequence CWU 1
1
21113PRTArtificial SequenceSYNTHESIZED 1Asp Arg Lys Asp Gln Gly Gly
Tyr Thr Met His Gln Asp 1 5 10 27PRTArtificial SequenceSYNTHESIZED
2Lys Thr Asp His Gly Ala Glu 1 5 37PRTArtificial
SequenceSYTHENSIZED 3Pro Arg His Leu Ser Asn Val 1 5
44PRTArtificial SequenceSYNTHESIZED 4Glu Pro Arg Gln 1
55PRTArtificial SequenceSYNTHESIZED 5Ala Ala Gly His Val 1 5
616PRTArtificial SequenceSYNTHESIZED 6Thr Asp His Gly Ala Glu Ile
Val Tyr Lys Ser Pro Val Val Ser Gly 1 5 10 15 77PRTArtificial
SequenceSYNTHESIZED 7Glu Phe Glu Val Met Glu Asp 1 5
810PRTArtificial SequenceSYNTHESIZED 8Gly Gly Lys Val Gln Ile Ile
Asn Lys Lys 1 5 10 911PRTArtificial SequenceSYNTHESIZED 9Ser Lys
Ile Gly Ser Thr Glu Asn Leu Lys His 1 5 10 106PRTArtificial
SequenceSYNTHESIZED 10Thr Asp His Gly Ala Glu 1 5 116PRTArtificial
SequenceSYNTHESIZED 11Lys Thr Asp His Gly Ala 1 5
12333DNAArtificial SequenceSYNTHESIZED 12gacattgtgc tgacacagtc
tcctgcttcc ttagctgtat ctctgggaca gagggccacc 60atctcatgca gggccagcca
aagtgtcagt acatctagat atagttatat acactggtac 120caacagaaac
caggacagcc acccaaactc ctcatcaagt atgcatccaa cctagaatct
180ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct
caacatccat 240cctctggagg aggaggatgc tgcaacatat tactgtcacc
acagttggga gattccgctc 300acgttcggtg ctgggaccaa gctggagctg aaa
33313345DNAArtificial SequenceSYNTHESIZED 13gaagtgaagg ttgaggagtc
tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60tcctgtgttg tctctggatt
cactttcagt aactactggg tgaactgggt ccgccagtct 120ccagagaagg
ggcttgagtg ggttgctcaa attagattga aatctgataa ttatgcaaca
180cattatgagg agtctgtgaa agggaggttc accatctcaa gagatgattc
caaaagtagt 240gtctatctgc aaatgaacaa cctaagggct gaagacagtg
gaatttatta ctgcactaac 300tgggaagact actggggcca aggcaccact
ctcacagtct cctca 34514111PRTArtificial SequenceSYNTHESIZED 14Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30 Arg Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser
Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Asn Ile His 65 70 75 80 Pro Leu Glu Glu Glu Asp Ala Ala
Thr Tyr Tyr Cys His His Ser Trp 85 90 95 Glu Ile Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 15115PRTArtificial
SequenceSYNTHESIZED 15Glu Val Lys Val Glu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Val Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Val Asn Trp Val Arg Gln
Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu
Lys Ser Asp Asn Tyr Ala Thr His Tyr Glu Glu 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val
Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Gly Ile Tyr 85 90
95 Tyr Cys Thr Asn Trp Glu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110 Val Ser Ser 115 1615PRTArtificial SequenceSYNTHESIZED
16Arg Ala Ser Gln Ser Val Ser Thr Ser Arg Tyr Ser Tyr Ile His 1 5
10 15 177PRTArtificial SequenceSYNTHESIZED 17Tyr Ala Ser Asn Leu
Glu Ser 1 5 189PRTArtificial SequenceSYNTHESIZED 18His His Ser Trp
Glu Ile Pro Leu Thr 1 5 195PRTArtificial SequenceSYNTHESIZED 19Asn
Tyr Trp Val Asn 1 5 2019PRTArtificial SequenceSYNTHESIZED 20Gln Ile
Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Glu Glu Ser 1 5 10 15
Val Lys Gly 214PRTArtificial SequenceSYNTHESIZED 21Trp Glu Asp Tyr
1
* * * * *